Chromosomal microarray analysis (CMA) is recommended as the first-tier clinical diagnostic test for individuals with developmental disabilities. In addition to detecting copy number variations, CMA platforms with single nucleotide polymorphism probes can detect large homozygous regions within the genome, which represent potential risk for recessively inherited disorders.To determine the frequency in which pathogenic or likely pathogenic variants can be detected in these regions of homozygosity, we performed whole exome sequencing (WES) in 53 individuals where homozygosity was detected by CMA. These patients were referred to our clinical laboratory for a variety of neurodevelopmental conditions including autism spectrum disorder, developmental delay, epilepsy, intellectual disability and microcephaly.GJB2, TPP1, SLC25A15, TYR, PCCB, and NDUFV2 which are implicated in a variety of diseases. The evaluation of heterozygous variants with autosomal dominant inheritance, compound heterozygotes and variants with X-linked inheritance revealed pathogenic or likely pathogenic variants in PNPLA4, CADM1, HBB, SOS1, SFTPC, OTC and ASMT in 15.1% (8/53) of cases. Two of these patients harbored both homozygous and heterozygous variants relevant to their phenotypes .In 11.3% (6/53) of cases, the analysis of homozygous variants revealed pathogenic or likely pathogenic variants in Our study highlights the clinical utility of WES in individuals whose CMA uncovers homozygosity. Importantly, we show that when the phenotype is complex and homozygosity levels are high, WES can identify a significant number of relevant variants that explain neurodevelopmental phenotypes, and these mutations may lie outside of the regions of homozygosity, suggesting that the appropriate follow up test is WES rather than targeted sequencing.The online version of this article (10.1186/s12881-018-0555-3) contains supplementary material, which is available to authorized users. Neurodevelopmental disorders (NDDs) are characterized by brain dysfunction, which results in the impairment of one or more features including motor skills, learning, cognition, speech and language and personal or social development. NDDs include developmental delay (DD), autism spectrum disorder (ASD), intellectual disability (ID), learning disability, attention-deficit hyperactivity disorder (ADHD) and others. Genetic causes contribute significantly to the etiology of these disorders, and in recognition of this, the American College of Medical Genetics and Genomics (ACMG) has published practice guidelines recommending CMA as the first-tier clinical diagnostic test for individuals with certain NDDs and congenital anomalies . Many geIn this study, we performed whole exome sequencing (WES) on 53 patients with ROH that were identified by CMA, with the goal of determining the diagnostic utility of WES in these patients. These individuals were referred to our laboratory for clinical CMA testing for a variety of clinical features including ASD, DD, epilepsy, anxiety, ID, microcephaly and encephalopathy. We investigated variants throughout the exome and identified homozygous pathogenic or likely pathogenic variants that explain at least part of the indicated phenotype in 11% (6/53) of cases. An additional analysis of heterozygous mutations with autosomal dominant inheritance as well as X-linked variants identified pathogenic/likely pathogenic variants in 15% (8/53) cases. Two of these cases (ROH44 and ROH52) harbored both homozygous and heterozygous pathogenic/likely pathogenic variants. This study strongly supports the use of WES in individuals with ROH detected by CMA and further highlights the clinical utility of WES in these individuals.All patient DNA samples were collected as part of standard clinical genetic testing by Lineagen, Inc. We obtained a waiver of consent from Western Institutional Review Board (IRB) Protocol No. 20162032, for this retrospective study. All patient data submitted to us for the purposes of clinical testing are covered under this IRB. Data was analyzed in a coded fashion in compliance with our IRB protocol. DNA isolated from buccal swabs  using the PureGene extraction kit  was used for all genetic analyses described in this study.Clinical CMA was performed using an optimized, ultra-high-resolution custom Affymetrix microarray that includes all functional content present on the Affymetrix CytoScan-HD microarray with probes added to improve detection of copy number variants associated with pediatric neurodevelopmental disorders . DNA samExome capture was performed using the Life Technologies AmpliSeq Exome RDY kit  and sequencing was performed on Life Technologies Ion Proton sequencers with 200 bp reads using a PIV3 chip. Whole exome sequencing for all the patients was performed on a research basis. Sequences were aligned with and compared to the Genome Reference Consortium Human Build 37  using the Ion Torrent Suite software v4.2 (ThermoFisher Scientific).https://www.wuxinextcode.com/). Homozygous and heterozygous likely protein-altering variants  were analyzed and were assessed for predicted deleterious effects by Variant Effect Predictor (VEP) (score ≥ 0.9) from WuXi NextCODE [Sequence data were analyzed using the default settings of the Clinical Sequence Analyzer (CSA) tool from WuXi NextCODE  and 20 females (38%) with ages ranging from 6 months to 22 years. The male-to-female ratio in this cohort is similar to that of our overall clinical testing population. The percentage of the genome that is homozygous in this cohort varied from 1.4% to 25%. The patient (ROH12) with a ROH of 1.4% of the genome has a large ROH of 26 Mb on chromosome 3 and it may harbor genes for autosomal recessive disorders. This region of 26 Mb is not a common ROH in normal control populations and thus was included in this cohort. In total 16/53 of our patients have 1.4–3.0% ROH, 28/53 of patients have 3–10% ROH, 7/53 of patients have 10–20% ROH, and 2/53 of patients have 20–25% ROH. Two patients were identified by CMA (ROH36 and ROH38) with ROH in a single chromosome, 6 (3.49% ROH) and 16 (1.53% ROH) respectively, suggesting uniparental isodisomy. In the remaining patients, we identified long contiguous stretches of homozygosity on multiple chromosomes. In addition to ROH, 3/53 patients had pathogenic CNVs: patient ROH40 had a gain of the entire Y chromosome , patient ROH34 had a 2.54 Mb 22q11.21 deletion (DiGeorge syndrome) and patient ROH39 had a 637 kb 16p12.2 deletion. In 40/53 patients, no clinically reportable CNVs were identified and in 10/53 patients, a CNV of uncertain clinical significance was detected using CMA. Detailed phenotypic information including percentage of the genome that in homozygous in these patients as well as the CMA findings are provided in Additional file % and 20 We analyzed homozygous, protein-disrupting variants and as expected we observed a strong linear correlation (Correlation coefficient of 0.85) between the percent of the genome with homozygosity and the number of predicted or known likely protein disrupting homozygous variants . Late-infantile NCL (LINCL), also known as Jansky-Bielschowsky disease, typically presents between two and four years of age with seizures, and clinical features progress as the patient ages. In a study of 74 patients with LINCL, the p.R208* variant observed in our patient accounted for ~ 28% of the mutant alleles in TPP1, including five samples that were homozygous for this variant [ClinicalTrials.gov Identifier: NCT01907087).Patient ROH44 was referred to our laboratory with indications of encephalopathy, severe DD, lack of speech, frequent brief absences and drop spells, seizures and intellectual disability. Parents reported themselves as first cousins, and 25% of the autosomal genome was found to be homozygous using CMA. No clinically relevant CNVs were present. Using WES, we identified a homozygous known pathogenic stop-gain mutation  in  as CLN2 . Mutatio variant . A diagnSLC25A15, the gene encoding the mitochondrial ornithine transporter. This variant has been classified as pathogenic for hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome [Patient ROH26 was referred for CMA testing due to delayed milestones, speech and language deficits, and autism spectrum disorder (ASD). There was not a prior report of parental relatedness, and 4.34% ROH as well as a 9p24.3 duplication classified as a variant of uncertain clinical significance were identified on CMA. Using WES, we identified a homozygous variant  in syndrome . HHH synsyndrome . Becausesyndrome . Our patsyndrome .NDUFV2, the gene encoding the NADH: ubiquinone oxidoreductase subunit of the inner mitochondrial membrane complex. This mutation was observed previously in three siblings of a consanguineous family who presented in the first year of life with hypertrophic cardiomyopathy, truncal hypotonia, feeding difficulties, and growth delay [NDUFV2 variant is causative for this patient’s features. Since this mutation in NDUFV2 is known to cause hypertrophic cardiomyopathy [Patient ROH31 was referred to our laboratory for clinical testing based on delayed milestones and hypotonia. Parents reported themselves as second cousins. No clinically relevant CNVs were identified by CMA, but 5% of the autosomal genome was homozygous. Using WES, we identified a homozygous four base pair deletion in a splice site in intron 2 (c.120 + 5_120 + 8delGTAA) of th delay . Functiomyopathy , monitormyopathy . Severalmyopathy .NDUFV2 variant, predicted deleterious homozygous variants were identified in INO80  and PLA2G6 . Aberrations in these genes have been implicated in intellectual disability and neurodegeneration in the first two years of life, respectively. While these variants are of uncertain clinical significance, the diseases associated with the affected genes are consistent with the phenotype of this patient. Our laboratory subsequently learned that this patient had clinical WES at an alternative laboratory and the same homozygous NDUFV2 variant was identified and the family was counseled accordingly. Because this child passed away in infancy, it is unclear if his clinical course was dictated strictly by the NDUFV2 variant or if the INO80 and/or PLA2G6 variants also contributed.In addition to the In addition to the pathogenic or likely pathogenic variants described above, in 74% (39/53) of the patients we identified several homozygous variants that potentially underlie the phenotypes of other patients . KCNAB2 encodes a voltage-gated potassium channel and could be involved in regulating neurotransmitter release and neuronal excitability [KCNAB2 (which resides in the deleted region) also had epilepsy or epileptiform activity on electroencephalogram [KCNAB2 was suggested as a significant risk factor for epilepsy. In another study, a de novo heterozygous mutation  in KCNAB1 was reported as disease causing for epilepsy in a patient with early infantile epileptic encephalopathy [KCNAB1 resulted in a premature termination codon. KCNAB1 and KCNAB2 encode proteins that are members of the cytoplasmic β subunit protein (kvβ) KCNAB family and are paralogous to each other. The β subunits stabilize the α subunits of the protein complex and a decrease in β subunits could reduce K+ currents in neurons resulting in fewer functional channels in the membrane. This could result in increased Ca2+ entry and neurotransmitter release, causing a hyperexcitable and seizure-prone circuit [KCNAB2 (Kcnab2−/−) resulted in deficits in associative memory and amygdala hyperexcitability, suggesting that loss of KCNAB2 contributes to the cognitive and neurological impairments observed in patients with 1p36 deletion syndrome [Patient ROH05 was referred to our laboratory with developmental delay and epilepsy. There was no prior report of parental relatedness, but 5.33% of the autosomal genome was found to be homozygous using CMA. No clinically relevant CNVs were present. Using WES, we identified a homozygous missense variant in tability . Dysfunctability . In a sthalogram . Therefoalopathy . This mu circuit . Furthersyndrome . While pAlmost all the patients in our cohort have phenotypes related to neurodevelopment and we know that neurodevelopmental disorders are a clinically and genetically highly heterogeneous and complex group of disorders. There are multiple genetic factors involved in the etiology of these disorders. Therefore, in addition to the homozygous mutations, we also evaluated heterozygous variants throughout the exome to determine if variation outside the regions of homozygosity might play a role in our patient cohort. In 8/53 (15%) cases, we identified a pathogenic/likely pathogenic variant that explains at least part of the phenotype. Two of these patients (ROH44 and ROH52) also harbor a pathogenic homozygous genetic change relevant to their phenotype, consistent with the idea that multiple genes could be contributing to their clinical symptoms. In our cohort, heterozygous variants relevant to clinical symptoms were more common than homozygous variants found in the ROH. The relevant heterozygous variants are shown in Table TPP1 gene, we identified an X-linked likely pathogenic hemizygous variant in the OTC gene . The gene OTC encodes a mitochondrial matrix enzyme, ornithine carbamoyltransferase. Genetic variants in this gene including missense, nonsense and frameshift mutations as well as deletions and duplications in this gene results in ornithine transcarbamylase deficiency, which causes hyperammonemia [In the patient ROH44 where we identified a homozygous variant in mmonemia –28. Anotmmonemia . Functiommonemia . The typHomozygosity in the genome raises the possibility of conditions that exhibit autosomal recessive inheritance; therefore, clinical laboratories typically proceed with targeted sequencing by selecting candidate genes within the homozygous regions based on a patient’s phenotype. This approach can be used in cases where the patient’s clinical presentation strongly suggests a specific genetic disorder and the number of genes consistent with the phenotype is limited. However, in many cases CMA may not detect all the regions of homozygosity and the complex phenotype may not allow for identification of strong candidate genes. Investigating the ROH detected by CMA to identify genes that exhibit an autosomal recessive inheritance pattern followed by specific sequencing of those genes is a cumbersome and inefficient process and has the possibility to miss causative mutations. It is important to note that there is a variable size threshold for ROH in clinical settings with some labs reporting only regions larger than 10 Mbp . Most clFrom a clinical perspective, these data furnish genetic and other healthcare professionals with additional critical and objective information to communicate to families whose child has ROH observed on SNP-based CMA. Moreover, as reimbursement for genetic testing is limited by specific health plan policies and clinical guidelines, our results support a recommendation for the use of WES following detection of ROH from SNP-based CMA to gain a better understanding of the genetic cause of a patient’s condition. We highlighted some of the important management changes potentially enabled by our results in a number of these patients, and the less established findings are likely to lead to such changes in the future.In our cohort, the diagnostic yield of exome sequencing technology is 22% (12/53 cases had at least one pathogenic/likely pathogenic mutation) when we evaluate homozygous, heterozygous and X-linked variants across the whole exome. It is important to note that many of the causative mutations lie outside the regions of homozygosity, which is an important point to consider when a targeted or a whole exome sequencing test is being considered. In 41/53 (77%) of our patients, no diagnostic variant was identified. It is possible that in some of these patients, the causative mutation(s) lie in an exon not well covered by the exome capture methodology used, or in a non-coding/regulatory region, which are not targeted by WES. However in several of these patients, we identified homozygous rare variants that are classified as variants of uncertain significance  of patients in this cohort have homozygosity encompassing only 1.4–3% of their genomes. Some of the regions of homozygosity in these patients may reflect the presence of common haplotypes in a population. Because this study is focused on clinical samples where sequencing has only been performed on patients with neurodevelopmental phenotypes, we cannot estimate the burden of similar genetic variants in controls with similar number or size of ROH.Some of the patients in which we have identified pathogenic variants, for example Over the past few years, several studies have shown the diagnostic utility of exome sequencing in clinical diagnostic laboratories. One of the studies by Baylor College of Medicine was carried out on 250 consecutive probands and approximately 80% were children with phenotypes related to neurologic conditions . Prior gWES identified numerous variants that are relevant or potentially relevant to the phenotype of patients with ROH identified by SNP-based CMA. In (12/53) cases, we identified at least one pathogenic or likely pathogenic variant that explains at least part of the indicated phenotype of the patients. We identified an equal number of pathogenic/likely pathogenic variants outside the ROH, suggesting that WES is more appropriate than targeted sequencing in these patients. Importantly, the variants we identified provide definitive diagnoses that offer valuable information for future medical care as well as recurrence risk and family planning options. As similar cases are reported and as functional data become available, other variants identified in this study are likely to be re-classified as pathogenic or likely pathogenic. Therefore, this study demonstrates that whole exome sequencing, rather than targeted sequencing, has clinical utility in individuals with ROH identified on CMA, and should be considered for inclusion in clinical guidelines for the staged evaluation of individuals with neurodevelopmental disorders.Additional file 1:A detailed workflow showing the variants discovery and prioritization for the individuals with ROH. (DOCX 27 kb)Additional file 2:Clinical Phenotype information for the 53 ROH patients along with CMA findings. (XLSX 17 kb)Additional file 3:A scatter plot showing correlation of percent of homozygosity with the number of homozygous likely gene disrupting variants predicted to be deleterious. At this stage, no frequency filter was applied. (DOCX 124 kb)Additional file 4:Details of rare homozygous variants discovered in 74% (39/53) of the ROH cases that has variants relevant or potentially relevant to the phenotype of the patient. (XLSX 32 kb)


In this work, we report the transfer of graphene onto eight commercial microfiltration substrates having different pore sizes and surface characteristics. Monolayer graphene grown on copper by the chemical vapor deposition (CVD) process was transferred by the pressing method over the target substrates, followed by wet etching of copper to obtain monolayer graphene/polymer membranes. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and contact angle (CA) measurements were carried out to explore the graphene layer transferability. Three factors, namely, the substrate roughness, its pore size, and its surface wetting (degree of hydrophobicity) are found to affect the conformality and coverage of the transferred graphene monolayer on the substrate surface. A good quality graphene transfer is achieved on the substrate with the following characteristics; being hydrophobic (CA > 90°), having small pore size, and low surface roughness, with a CA to RMS (root mean square) ratio higher than 2.7°/nm. It is very strong (100 times stronger than steel) , highly  copper) , has a hr) [2/g) , is highr) [2/g) ,6, is hir) [2/g) ,8, and fr) [2/g) ,10,11.With the aforementioned unusual properties, graphene opens doors for many applications across disciplines. It is used in electronic applications as transistors , chemicaVarious fabrication routes exist for graphene production, including the mechanical ,28 and cAt present, no single fabrication route that produces graphene sheets is suitable for all potential applications, as every route has its advantages and disadvantages ,37. GrapCurrently, graphene transfers onto desired substrates using various methods are implemented in two ways: the wet transfer method and the dry transfer method. The most straightforward wet transfer method is to etch the metal away chemically to obtain free floating graphene composites that can be scooped onto chosen substrates. Other well-known wet etching methods include the standard transfer method , the dirIn all wet transfer methods, the Cu substrate is removed by a wet etching process using a copper etchant such as ferric chloride , iron niBunch et al. , revieweIn the present work, we examine the effect of substrate surface characteristics in terms of pore size, wettability (degree of hydrophobicity), and surface roughness on the graphene transferability experimentally. We selected eight polymeric substrates with different surface characteristics and transferred monolayer CVD grown graphene on copper foils onto these polymeric substrates. Copper was removed by wet etching to obtain monolayer graphene/polymer composite membranes. Two conditions related to the substrate surface were found to affect the quality of transferred graphene. Firstly, the substrate should have an adequate hydrophobicity (contact angle (CA) > 90°); if the first condition is achieved, then the ratio of the surface contact angle to the root mean square (RMS) value should be roughly higher than 2.5. Otherwise, poor graphene quality will be obtained. The quality of graphene was checked by Field Emission Scanning Electron Microscope (FESEM) and Atomic Force Microscopy (AFM) characterization, and an ionic transport study of potassium chloride (KCl) through the graphene/substrate composite membranes.We transferred monolayer graphene onto various polymeric substrates by modifying the direct transfer method developed by Regan et al. . It is aThe quality of transferred graphene was checked by studying the ionic transport through the graphene/substrate composite. To do so, a special Side-bi-Side glass diffusion cell procured from Permegear Inc., Hellertown, PA, USA was used . The celBoth cell chambers are throughly cleaned by de-ionized water and air dried before the composite graphene membrane is placed between them. The active side of the composite membrane (graphene side) faces the left chamber. The cell is then tightened using a rubber screw. Both sides of the membrane are then washed with ethanol to remove any water bubbles close to the membrane surfaces. To remove any entrapped ethanol from the previous stage, the left cell side was washed with 0.5 M KCl solution and the right side was washed three times with de-gassed de-ionized water.The ionic transport study was performed with 7 mL of 0.5 M KCl solution. KCl solution was introduced into the left chamber of the cell and 7 mL de-ionized water was introduced into the right chamber. Both solutions were magnetically stirred during the diffusion process to minimize concentration polarization effects. Potassium and chloride ions diffused through the graphene membrane towards the de-ionized water side. The diffusion rate of the ions was measured by monitoring the change in conductivity over time, using an eDAQ conductivity isoPod electrode  dipped in the de-ionized water side of the diffusion cell, as shown in FESEM, AFM, and water wettability characterizations for the as received polymer membranes and the graphene/polymer membranes were carried out to understand the role of the substrate surface characteristics on the graphene transferability as well as its coverage and quality.2. However, for PVDF 6 substrate, an area of 4 × 4 µm2 was used as AFM tip instability problems were encountered due to a rather rough surface for this particular substrate. Three sections were taken to explore the surface profile and to calculate the average root mean square (RMS) values. 3D surface profiles were also captured to check and qualitatively validate surface roughness values.The AFM results show that the PVDF 6 substrate with 100 nm pore size was found to have the roughest surface of RMS = 112 nm and appreciably high surface wettability of CA = 109° as shown in It is not always necessarily true that smaller pore size substrates should have smoother surfaces. PVDF 4 shown in Based on the contact angle measurements, four out of the eight substrates, namely, PES, PVDF 1, PVDF 2, and PVDF 3 are considered hydrophilic; (CA < 90°), and the remaining are considered hydrophobic.We transferred monolayer graphene onto polymeric substrates using the procedure (pressing method) shown in Graphene detached from four substrates  during copper etching as mentioned earlier (failed transfer). The contact angle of these substrates is lower than 90° and as such they can be considered hydrophilic. To prevent graphene layer detachment, a polymeric substrate with an adequate degree of hydrophobicity is required to prevent etchant penetration during the etching process that usually takes around 2 h.The other four substrates  have the required degree of hydrophobicity that prevents the detachment process; PVDF 2 has an average CA = 90° and PVDF 4 has a CA = 116°. Two of the four substrates (PP and PVDF 5) have smoother surfaces compared to the PVDF 4 and PVDF 6. The PP has a surface roughness with a root mean square (RMS) value = 42.4 nm; PVDF 5 has an RMS value = 23.8 nm. The other two substrates have a rougher surface, with 94.9 nm and 112.1 nm RMS values for PVDF 4 and PVDF 6, respectively .2 of monolayer graphene can be clearly seen by the naked eye at the upper right corner (inset). The FESEM images , the KCl ions are transported from the left side toward the right side through the graphene/polymer composite membrane. The rate of ion transport is calculated by monitoring the change of de-ionized water conductivity over time. Based on the fact that a defect free graphene is impermeable even to very small ionic species such as helium ,56, the So far, two conditions related to substrate surface characteristics should be controlled in order to have a good quality (low defect density) graphene transfer. First, the substrate surface should have a reasonable hydrophobicity (contact angle > 90°) and second, the contact angle (CA) to surface roughness  should be higher than 2.7 (CA/RMS > 2.7°/nm).A hydrophobic surface along with a high CA/RMS ratio means the substrate has the required hydrophobicity to prevent penetration of the etchant between the Cu/graphene and substrate, and the low roughness helps provide better contact between the transferred graphene and substrate, and minimize the unsupported graphene domains that could be detached during the etching process .Graphene transfer to a working substrate is the key to a wide range of graphene applications. This paper explores the effect of substrate surface characteristics, mainly the surface roughness and wettability (degree of hydrophobicity) on graphene transferability. To transfer graphene with high quality, the substrate surface should be smooth to allow the graphene to conform and adhere with minimum tears and cracks, and should have high hydrophobicity to prevent etchant penetration between the graphene and substrate during copper etching, which would otherwise cause graphene detachment. CVD monolayer graphene was transferred to eight different polymeric substrates having different surface characteristics by a simple pressing method, followed by wet etching of copper using an APS etchant. Graphene failed to transfer over four substrates  because they were hydrophilic, which facilitated etchant penetration between the Cu/graphene and the substrate and caused graphene detachment. Graphene successfully transferred to the other four substrates that were hydrophobic . PP and PVDF 5 exhibited better graphene transferability in terms of quality, due to the lower surface roughness as compared to PVDF 4 and PVDF 6. The quality of the graphene was checked by FESEM characterization and the simple diffusion of potassium chloride ions (KCl) through the transferred graphene/substrate membrane. To obtain high-quality graphene, the substrate should have an adequate hydrophobicity (contact angle (CA) > 90°); if this is attained, then the ratio of the contact angle value to the root mean square (RMS) value should be higher than 2.7 (CA/RMS > 2.7°/nm).


Diabetic nephropathy (DN), the single strongest predictor of mortality in patients with type 2 diabetes, is characterized by initial glomerular hyperfiltration with subsequent progressive renal function loss with or without albuminuria, greatly accelerated with the onset of overt proteinuria. Experimental and clinical studies have convincingly shown that early interventions retard disease progression, while treatment if started late in the disease course seldom modifies the slope of GFR decline. Here we assessed whether the negligible renoprotection afforded by drugs in patients with proteinuric DN could be due to loss of glomerular structural integrity, explored by scanning electron microscopy (SEM). In diabetic patients with early renal disease, glomerular structural integrity was largely preserved. At variance SEM documented that in the late stage of proteinuric DN, glomerular structure was subverted with nearly complete loss of podocytes and lobular transformation of the glomerular basement membrane. In these circumstances one can reasonably imply that any form of treatment, albeit personalized, is unlikely to reach a given cellular or molecular target. These findings should persuade physicians to start the putative renoprotective therapy soon after the diagnosis of diabetes or in an early phase of the disease before structural integrity of the glomerular filter is irreversibly compromised. Diabetic Kidney Disease (DKD), the most devastating complications of type 2 diabetes, is the single leading cause of end-stage renal disease (ESRD) in the industrialized world3.Type 2 diabetes is a public health concern and predictions regarding its future effects on human and social costs, as well as health economic impact are staggering6. Progressive renal function loss can be noted even in patients with normoalbuminuria as well as in those with microalbuminuria, but the onset of macroalbuminuria or overt proteinuria is invariably associated with accelerated rate of renal disease progression4.The natural history of DKD in type 2 diabetes has been recently depicted as a sequence of stages characterized initially by normal or elevated (hyperfiltration) glomerular filtration rate followed by progressive renal function decline to end-stage renal disease7. In a proteinuric rat model of streptozotocin-induced diabetes the administration of an ACE-inhibitor normalized the urinary protein excretion when treatment was started early in the course of the disease, but not at later time, when proteinuria was elevated8. The effect of ACE inhibition on proteinuria was associated with protection against the development of glomerular structural changes only early, but not later in the course of disease8. Consistent with these findings in the animal model, in the early 2000s large clinical trials in type 2 diabetic patients showed that RAS inhibition provided some degree of protection against renal disease progression in the course of proteinuric DKD, but early interventions  were more beneficial than late intervention  in delaying ESRD13. Moreover, in the advanced nephropathy of RENAAL and IDNT trials consistent renoprotection was only achieved in those patients who reported a reduction in urinary protein excretion12. More recent clinical trials in type 2 diabetic patients with overt proteinuria have also largely failed to provide significant renoprotection independently of the novel treatments investigated14. Collectively, these observations highlight the variability of response to treatment during the course of the disease with negligible benefit in later stages of proteinuric DKD.Current strategies to prevent or slow DKD progression of type 2 diabetes focus on metabolic and blood pressure control, as well as on reducing protein trafficking through the glomerular capillary barrier, mainly through blockade of the renin-angiotensin system (RAS) with angiotensin-converting enzyme inhibitors (ACEi) and/or angiotensin II type 1 receptor blockers (ARBs)One of the principal obstacles to the development of improved therapeutic interventions remains our limited in depth understanding of the structural abnormalities at glomerular level in patients with proteinuric advanced DKD.15, which provides a global visualization of the actual three-dimensional appearance of the glomerular tuft surface and podocyte cytoarchitecture. SEM allows a more in depth information on glomerular injury than that already available in diabetic patients with a single cross-section at light as well as transmission electron microscopy (TEM)16.Thus, in the present study we sought to investigate whether failure to renoprotection by the available treatments in DKD with overt nephropathy could be related to loss of glomerular structural integrity. To this purpose, we took advantage of scanning electron microscopy (SEM), an imaging approach rarely used for patientsIn order to establish the reliability of our scanning electron microscopy (SEM) procedure, we first examined kidney specimens of healthy tissue obtained from non-diabetic controls undergoing nephrectomy for renal adenocarcinoma without histological evidence of glomerular disease. SEM analysis showed well preserved glomerular architecture with normal peripheral capillary loop ultrastructure , and compared the three-dimensional appearance of glomeruli with that of biopsy specimens from six patients with late type 2 DKD and overt proteinuria, all receiving RAS therapy as part of their current renoprotective treatment.17.As shown in Fig. SEM evaluation provided more relevant findings in a 70-year-old man with microalbuminuric diabetic nephropathy. In this setting, the examination of the renal cortex surface suggested a glomerular adaptative growth, with a consequent mismatch between the expansion of the tuft and the hypertrophy of podocytes, which displayed stretched and flattened cell bodies Fig. . In addiBiopsy specimens from six patients aged 50 to 73) with DKD and overt proteinuria, despite therapy with ACEi and ARB, were also evaluated Figs . In all 0 to 73 w18  demonstrated less severe glomerular injury by light microscopy compared with the other five patients with nephrotic range proteinuria. TEM and SEM findings were consistent with that, revealing a segmental pattern of GBM denudation, with some regions of the capillary loop surface partially covered by highly damaged podocytes. The fact that residual podocyte foot processes were clearly detectable by SEM, along with some fragments of cellular bodies, further support the hypothesis that GBM denudation in the other patients with overt nephropathy was not an artifactual issue. Notably, the presence of glomerular filtration pores in the rare preserved areas, further ruled out artifactual damage and suggested that, despite the massive structural damage clearly visible with SEM, the glomerulus was likely not globally sclerotic, but areas of the surface of the glomerular capillary tuft were still structurally intact.31. There is evidence that mechanical forces may have a role in podocyte detachment under physiologic and pathophysiologic conditions32. Thus, in DKD, long-lasting intraglomerular mechanical challenges, including glomerular hypertension, hyperfiltration, hypertrophy and outflow of filtrate from subpodocyte spaces, could promote progressive podocyte loss through detachment from the GBM. In addition, many other complex pathways contribute to the long-lasting podocyte injury and detachment in type 2 diabetes, including hyperglycemia itself, oxidative stress and inflammatory cytokines, as well as the activation of vasoactive hormonal systems35. Although SEM does not provide insight into the long-term mechanisms of podocyte injury in the diabetic setting, we hypothesize that, as a consequence of the intraglomerular hemodynamic derangement and mechanical stress as well as hyperglycemic and oxidative/inflammatory challenges persisting for years, ultimately complete loss of podocytes may occur in advanced DKD with overt proteinuria. Is this catastrophic process really ineluctable? In this regard, previous studies suggest that reversal of DKD can been achieved in humans and mice, but only rarely and under special circumstances36. In an experimental mouse model of DN, the leptin-deficient BTBR ob/ob mouse, it has been shown that leptin replacement resulted in near-complete reversal of both structural and functional measures of advanced DKD37. Immunohistochemical labeling with specific podocyte markers identified parietal epithelial cells as a possible source of new podocytes, supporting the hypothesis that podocytes may regenerate from parietal epithelial cell progenitors29. In our study the occasional finding of isolated podocytes, with stretched and elongated primary processes, forming bridges between the Bowman’s capsule and the GBM  were reviewed to identify all renal biopsy specimens from six patients with diabetic nephropathy and overt proteinuria. We also identified two autopsy cases of type 2 diabetic patients with early stage of diabetic nephropathy  in the Unit of Pathology at the same institution. Patients’ medical records were reviewed for demographics, clinical features of diabetic nephropathy, medication history, parameters of renal function, treatment, and outcome.A 61-year-old woman with a history of type 2 diabetes since 1994 and obesity, on oral antidiabetic therapy , good blood pressure control with two antihypertensive agents including the ARB Valsartan (80 mg/d), died suddenly for cardiovascular event and an autopsy was performed with tissue sampling 24 hours later. The most recent available laboratory test reported normal renal function (s. creat. 0.8 mg/dl), fasting blood glucose 152 mg/dl, and normoalbuminuria (5 µg/min).2). All of them had near normal fasting blood glucose on oral antidiabetic agents, and good blood pressure control (130-140/70–80 mmHg) with at least two antihypertensive agents, including ACEi or ARBs. Additionally, two female diabetic patients (aged 50 and 55) underwent a renal biopsy in the beginning of 2017 due to overt proteinuria (Supplementary Table 2). Patient 5 had a longstanding insulin-dependent type 2 diabetes with poor glycaemic control (glycated haemoglobin of 101 mmol/mol) and uncontrolled hypertension (150–160/80–90 mmHg). At the time of renal biopsy she was taking a calcium antagonist and an ACEi, which was discontinued due to iperkalemia. Patient 6 was diagnosed with type 2 diabetes in 2014, but she already had severe micro- and macro-vascular complications (retinopathy and multiple diabetic foot ulcers). At the time of renal biopsy she was on a single oral antidiabetic agent (metformin 1 gr. t.i.d) with good glycaemic control (glycated haemoglobin of 43 mmol/mol) and she was receiving dual blockade of the renin-angiotensin-aldosterone system. In addition, two non-diabetic controls undergoing nephrectomy for kidney adenocarcinoma without histological evidence of glomerular disease and no history of renal disease, were included. All experimental protocols involving human subjects are carried out in accordance with the Declaration of Helsinki and good clinical guidelines, and approved by the Ethical Committee of the Azienda Socio Sanitaria Territoriale (ASST) Papa Giovanni XXIII. Written informed consent was obtained from patients enrolled in the study.A 70-year-old man with type 2 diabetes since 1998, on oral antidiabetic treatment with metformin (400 mg/die), glibenclamide (5 mg t.i.d), with a history of smoking and hypertension, developed microalbuminuria (102 µg/min) beginning in 2012. At that time, he was on a low dose of ACEi ramipril (2.5 mg/day), then increased to 5 mg/day, which lowered urinary albumin excretion (UAE). A few years later, he died suddenly for a cerebrovascular event and an autopsy was performed with tissue sampling 24 hours after death. At the most recent clinical evaluation UAE was still in the microalbuminuric range (53.5 µg/min), with normal renal function (s.creat. 0.96 mg/dl), fasting blood glucose was 174 mg/dl and the patient was given two antihypertensives to control blood pressure. Four male patients (aged 60 to 73) with a history of type 2 diabetes were referred to our Nephrology Unit due to nephrotic proteinuria, and underwent a renal biopsy between 2011 and 2016 . Ultrathin sections (60 to 100 nm) were cut on an EM UC7 ultramicrotome , stained with uranyl acetate and lead citrate, and examined with TEM .Biopsy specimens were immersed in ice-cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) immediately after their extraction and sectioning, and fixed for 4 h at 4 °C, carefully handling samples to avoid ex vivo artefacts15. SEM analysis was performed on the remaining tissue  of the fixed biopsy sample used for TEM diagnosis. Sections were repeatedly washed in cacodylate buffer and postfixed in 1% osmium tetroxide for 1 hour. Fixed specimens were dehydrated through increasing concentrations of ethanol, starting with 10, 50, 70 and 90% of ethanol. Once the tissues were equilibrated in anhydrous 100% ethanol, they were rinsed with liquid carbon dioxide with a Bal-Tec 030 critical point dryer . Kidney specimens from non-diabetic controls, serving as method control, were processed at the same time and with same solutions used for samples from normoalbuminuric, microalbuminuric and severe DKD patients. Samples were mounted on stubs, and coated with a thin layer of atomic gold particles in a sputter coater . Coated specimens were observed through SEM using secondary electron detection . Acceleration voltage was set to 0.4 to 1.5 kV, working distance to 4 to 6 mm and enlargement up to 20 kx.For SEM analysis, as for TEM samples, all the analyzed kidney biopsies were immersed in ice-cold 2.5% glutaraldehyde  immediately after their extraction and sectioning, and fixed for 4 h at 4 °C, carefully handling samples to avoid All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


Here, we discuss renal organoids, their development, and how they can be employed to further understand kidney development and disease.The kidney is a complex organ whose excretory and regulatory functions are vital for maintaining homeostasis. Previous techniques used to study the kidney, including various animal models and 2D cell culture systems to investigate the mechanisms of renal development and regeneration have many benefits but also possess inherent shortcomings. Some of those limitations can be addressed using the emerging technology of 3D organoids. An organoid is a 3D cluster of differentiated cells that are developed  Renal diseases currently affect epidemic numbers of people worldwide, and have continued to escalate in their prevalence globally in recent years, thus representing a significant public health problem for both developing and developed countries. Renal diseases pose immediate, life-threatening medical consequences because the kidneys are needed to carry out a battery of essential tasks that cooperatively maintain the daily homeostasis of the body. Humans have a pair of bean-shaped kidney organs that are responsible for vital functions that include (i) the excretion of metabolic wastes and other toxins, most notably being urea, (ii) the regulation of body fluids, which entails the precise control of osmolarity, acid-base balance, and electrolyte levels, and (iii) the production of various hormones and enzymes that regulate such processes as the control of blood pressure and erythrocyte maturation within the bone marrow . As suchDuring embryogenesis there are three major germ layers: the endoderm, mesoderm, and ectoderm. Together these layers give rise to the initial tissues and structures that are further refined during development to create an organism. The mammalian kidney develops from the mesoderm, specifically presomatic mesoderm (PSM), also termed primitive streak, in a spatiotemporal manner  3,4]. C,4. C3,4]As development progresses, the MM responds to elaborate signals from the UB that induce its condensation and mesenchymal-to-epithelial transition (MET) . ProgresBiomedical research has largely employed 2D cell culture and animal models to interrogate development and perform drug testing. Currently, each model has limitations with regard to determining the toxicity and efficacy of a drug before it reaches clinical trials . Partly in vivo system, and usually needs extremely high concentrations to result in any toxic effect [In the nephrology field, there remains a tremendous need in particular to understand more about renal toxins, as they cause 30–50% of acute kidney injury cases ,14. Howec effect –17. Althc effect .Specific examples of these models and their shortcomings can be illustrated. A study completed by Jenkinson and colleagues 2012) highlights some weaknesses of using cell lines to test toxicity in the nephron . Several12 highliin vivo development, and replating the cells in 3D culture where spontaneous organoid formation is observed  to induce the formation of mature glomeruli within murine embryonic kidney derived organoids . First, c Nestin .These chimeric organoids hold great promise for future studies of diseases, cell autonomous processes, and clinical uses because they do not need continuous treatment with exogenous chemical factors. However, questions remain based upon the method and animal model used in these studies. One of these questions involves the use of embryonic serum in media, which contains a number of growth factors, whose exact role in the differentiation and survival of organoids remains unknown. These questions can also be applied to signals, which may originate from the host environment, but as of yet remain unidentified. Secondly, these studies were performed in athymic rats, which fail to produce T-cells and therefore do not have adverse reactions to cells from other exogenous sources. The use of induced pluripotent stem (iPS) cells to produce renal organoids would provide further benefit and circumvent the need for anti-rejection medications in clinical settings.in vitro [As previously described, specific renal lineages arise during ontogeny when the IM differentiates into MM, from which nephron cell types emerge, or into the UB lineage which forms the collecting duct system. Xia et al. reported a method to create UB in vitro . This prin vitro .PAX2 expression in CHIR treated cells. Combining FGF2 with RA results in +LHX1+PAX2 cells, both markers of IM. After optimizing the protocol with these added factors, they tested the procedure on three human ES cell lines and two human iPS cell lines where they found all but one iPS line had over 80% of +LHX1+PAX2 cells. RT-PCR results showed expression profiles of these cells were consistent with data of high IM gene expression on day three, which then decreased on day five. Inversely, WT1 expression increased from day three to day five. Similarly, both +LHX1+PAX2 iPS cell lines and ES cell lines show decreased expression of IM markers, while epithelial tubules form that stained positive for LTL, KSP, and N-cadherin. Other markers including those of the podocytes, collecting duct, proximal tubules, and loop of Henle were identified by immunostaining.Lam et al. (2013) developed a technique to efficiently differentiate human ES and human iPS cells into cells that express IM and renal tubular structure markers . After uSIX2 expression, a marker of CM. Staining for two other CM markers, SALL1 and WT1, further supported this increase in CM. During kidney development, CM induction is caused by Wnt signaling. Lam et al. mimicked this phenomenon using SIX2+ cells and adding CHIR on day six of differentiation. The CHIR treatment resulted in decreased SIX2 expression and increased LTL expression indicating mature cells. While Lam and colleagues did complete a reaggregation study, similar to previously mentioned studies, they did not test the functionality of their products [To further developing their protocol to increase CM levels, Lam et al. screened a number of growth factors as well . Using iproducts . Testingproducts .+TBX6T+ late primitive streak cells that were treated with Activin from day four to day seven developing into +HODX11+PAX2−LHX1WT1− cells, indicative of IM.In a follow up study, Morizane et al. (2015) describes their system for differentiating human iPS and ES cells into what they term ―nephron progenitor cells‖ (NPCs) via mimicking metanephric development . These NSIX2+ cells [OSR1 levels remained constant in these cells from days 7–9 differentiation. However, SIX2 expression decreased between days 10–14 as these NPCs differentiated into clusters of +LHX1PAX8+ cells, defined by the authors as renal vesicles. At this point Morizane et al. kept some cells in 2D culture while also replating some NPCs in 3D round bottom wells and removed FGF9. Both 2D and 3D cells formed clusters that were +NPHS1+PODXLLTL+. The removal of FGF9 shows these cells were intrinsically programmed to differentiate into nephron structures. Initially this process was not efficient, but upon further optimizing the procedure they were able to produce 76% +LHX1PAX8+ cells that organized into clusters. With no additional factors added, by day 21 of differentiation the clusters formed nephron structures that stained positive for markers of distinct cell types including glomerulus, proximal tubules, distal tubules, and Loop of Henle, with collecting duct noticeably absent. The absence of collecting duct shows this method is specific for NPCs, the result of MM. However, the study does not illustrate the ability to form a complete kidney with all cell types.Next, the researchers wanted to create MM and found the addition of FGF9 results in 2+ cells . They usKIM-1). Using γH2AX, a DNA damage marker, the researchers found cisplatin treatment also damaged proximal and distal tubules in a dose dependent manner [Interestingly, all organoids formed lasted for a minimum of 56 days and they were over 20 times more efficiently constructed compared to the lab’s previous protocol . These ot manner .in vivo. The second technique relied upon stimulation of canonical Wnt signaling with the chemical CHIR. Both approaches resulted in robust formation of primitive streak based upon expression of MIXL1, T, and SOX17 and led to spontaneous differentiation of the primate streak tissue into IM. However, while robust expression of OSR1 was noted, other markers such as PAX2 and LHX1 were absent based on PCR and immunofluorescence, indicating the need for further refinement of the differentiation method.In a similar study completed by Takasato et al. (2014) human ES cells (hESCs) were used to generate kidney organoids . In ordePAX2 and LHX1 after four days of culture. A direct role for FGF signaling was shown by knockdown of FGFR1 and FGFR3 that inhibited IM induction. To further direct differentiation potential to a nephrogenic fate, IM was treated with FGF9, BMP7, and RA for an additional 12 days. This allowed for differentiation into MM, which was identified based on SIX2, WT1, GDNF, and HOXD11 indicating the presence of tubular progenitors, renal stroma, and nephric duct. At day 14 +PAX2ECAD+ epithelial structures were surrounded by +WT1+SIX2 mesenchyme [SNPO, NPHS1 and WT1), proximal tubules (AQP1 and SLC3A1), as well as collecting ducts (AQP2 and SCNNB1). At this point, RA and FGF treated posterior streak generated from treatment with CHIR created robust UE at the expense of MM.Based on its physiological role, posterior streak induced hESCs were subsequently treated with fibroblast growth factors (FGFs) . FGF2 orsenchyme . This resenchyme . At day PAX2++CALB), renal vesicle (+JAG1CDH6+) and progenitor mesenchyme (+WT1+SIX2). However, hESCs that had undergone differentiation with the sequential treatment of BMP4 and Activin A, followed by FGF9 treatment, and finally with exposure to FGF9, BMP7, and RA only contributed to UE and MM. Taken together, these results illustrate the ability of hESCs to successfully differentiate and form nephrogenic and mature nephron structures [In order to determine the ability of single cells to reproduce nephrogenic structures, mouse embryonic kidneys were dissociated and the cell suspensions mixed with hESCs that had undergone the differentiation protocol to day 12 . Upon seructures . An advaructures .Takasato et al. (2015) adapted their technique from the previously described study (2014) to accomplish kidney differentiation from human iPS and ES cells ,38. Theiz-stack images, the authors illustrate correct 3D positions of the developing nephrons with collecting duct at the bottom and glomerulus at the top. Furthermore, RNA sequencing on whole organoids was performed at days 0, 3, 11, and 18. While observing the expected inverse relationship of renal progenitors and segment markers, the authors also noted that organoids from days 11 and 18 were comparable to the RNA transcript profile of a first trimester human embryonic kidney [Using this information, Takasato et al. altered their procedure to increase the MM formed in both iPS and ES cells . After f+) exhibiting the functionality of these organoids. Another example of a conserved physiological response was testing the apoptotic result of cisplatin treatment. As expected, the mature proximal cells underwent apoptosis after being treated with cisplatin. Taken together, these two tests illustrate that these organoids are a potential tool for future uses such as renal toxicity assays, modeling kidney development and diseases, and cell therapies [Next, Takasato et al. showed that the developing kidney organoids displayed unique characteristics . Some ofherapies .In summary, the findings reported by several independent research groups to date have indicated very similar steps in the directed formation of renal organoid structures from iPS cells, thus cumulatively suggesting a shared working model for this procedure  3,36–38–3836–38.The complexity of the kidney offers tremendous difficulty to the field trying to study this vital integral organ. With advances in technology such as organoids, researchers have a new opportunity to explore critical questions that may not be appropriately addressed or answered with currently existing models. As the refinement of these organoids evolves, the number of possible applications grows. The easily accessible nature of organoids positions them for use in therapeutic studies.in vivo nephrotoxic results, many believe organoids can provide a better model than using 2D cell culture and animal models [One major application of kidney organoids will be their use for nephrotoxicity assays. Due to the similarities between 3D kidney cell clusters and l models ,40. Anotl models . Kidney l models .Disease modeling with organoids has shown promise as well. Batchelder et al. (2015) were able to sustain 3D organoids of human renal cell carcinoma in culture for 21 days. This enables a new avenue for testing patient specific cancer treatments . Also, uin vivo. For example, how will this transplanted organoid respond to blood pressure? Does the anatomical position in the human body affect signaling within the kidney? While these questions and many more will need to be answered before kidney organoids could be applied in clinical settings, there is no doubt that organoid technology has endless possibilities. These possibilities are sure to occupy the field of kidney research for the near future.One important goal in developing kidney organoids is the prospect that this technology could be used for personalized medicine. For example, a patient’s own cells could be converted to iPS cells, and then differentiated down a lineage with the intention of kidney replacement or individualized nephrotoxicity assays . This wo


Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of approximately 7,600 km2 on the island. C. russula is normally distributed in Northern Africa and Western Europe, and was previously absent from the British Isles. Whilst invasive species can have dramatic and rapid impacts on faunal and floral communities, they may also be carriers of pathogens facilitating disease transmission in potentially naive populations. Pathogenic leptospires are endemic in Ireland and a significant cause of human and animal disease. From 18 trapped C. russula, 3 isolates of Leptospira were cultured. However, typing of these isolates by standard serological reference methods was negative, and suggested an, as yet, unidentified serovar. Sequence analysis of 16S ribosomal RNA and secY indicated that these novel isolates belong to Leptospira alstonii, a unique pathogenic species of which only 7 isolates have been described to date. Earlier isolations were limited geographically to China, Japan and Malaysia, and this leptospiral species had not previously been cultured from mammals. Restriction enzyme analysis (REA) further confirms the novelty of these strains since no similar patterns were observed with a reference database of leptospires. As with other pathogenic Leptospira species, these isolates contain lipL32 and do not grow in the presence of 8-azagunaine; however no evidence of disease was apparent after experimental infection of hamsters. These isolates are genetically related to L. alstonii but have a novel REA pattern; they represent a new serovar which we designate as serovar Room22. This study demonstrates that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira.The greater white-toothed shrew ( Leptospira are excreted in urine from asymptomatic carrier hosts which facilitates disease transmission to new hosts. To date, there are 10 species of pathogenic leptospires which comprise more than 200 serovars. Disease transmission of these strains is maintained by a wide range of domestic and wild animal species. In this work, we discovered that an invasive mammalian species, the greater white toothed shrew, which was first identified in Ireland in 2007, acts as a carrier for a species of leptospires never before identified in Ireland. Results demonstrate that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira.Leptospirosis is a global zoonotic disease. Pathogenic species of  Crocidura russula) is an exotic species to Ireland first recorded in 2007[Sorex minutus)[The greater white-toothed shrew  which carries squirrelpox virus that severely adversely affected native red squirrels (Sciurus vulgaris) in Britain and Ireland[Nyctereutes procyonoides) in Europe, which has an expanding range, and which can facilitate the spread of infectious diseases including echinococcosis, trichinellosis and rabies[C. russula) in Ireland is a reservoir host for a novel strain of pathogenic Leptospira.There is clear evidence that invasive species act as vectors for pathogens and parasites that can have environmental conservation, and human health, implications. Globalization has facilitated the movement of exotic and invasive species, and a range of associated pathogens e.g. mosquitoes and West Nile Virus. The comd Ireland, 30; thend rabies. In thisGreater white-toothed shrews (GWTS) were live-trapped and euthanized by cervical dislocation. All animal experimental procedures were performed in accordance with relevant guidelines and regulations, and as approved by the National Parks and Wildlife Service (NPWS) in Ireland and the Animal Research Ethics Committee in University College Dublin (AREC-13-24).−3) into 1% BSA and 500μl of this mixture was used to inoculate the surface of 10ml EMJH medium containing 200μg 5-Fluoruracil and 0.2% noble agar. Cultures were transported back to the laboratory and maintained at 29°C. Cultures were examined at weekly intervals by dark-field microscopy.Kidneys were removed from GWTS at time of euthanasia and immediately processed for the culture of leptospires. In brieL. alstonii Serogroup Ranarum Serovar Pingchang Strain 80–412 and L. alstonii Serogroup Undesignated Serovar Sichuan Strain 79601 were sourced from the WHO/OIE Leptospirosis Reference Laboratory at the Royal Tropical Institute, The Netherlands. L. alstonii strains MS267, MS311 and MS316 were kindly provided by Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Japan.Leptospira biflexa (ATCC® 23582™) was used as a positive control. Cultures were incubated at 30°C for 14 days. The cultures were counted by dark-field microscopy at days 1, 3, 5, 7 and 14 using a Cellometer® disposable cell counting chamber (Nexcelom Bioscience).Growth assessment in the presence of 8-azaguanine was performed as previously described; in brieLeptospira serogroups[Leptospira serogroups tested included Australis , Autumnalis, Ballum, Canicola, Celledoni, Cynopteri, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Louisiana, Mini, Pomona (serovar Pomona and Altodouro), Pyrogenes, Sejroe, Semaranga and Tarassovi. In addition, rabbit sera generated against each of the three shrew isolates were then tested against the panel of Leptospira antigens from the 17 serogroups mentioned above, and additionally against a panel of 9 antigens from serogroups comprised of: Andamana, Semaranga, Hursbridge, Sarmin, Lyme, Louisiana, Shermani (serovar Shermani and Aquaruna), Bataviae, Ranarum, and against one undesignated serogroup (serovar Sichuan).Serological strain identification was initially attempted by cross-agglutination. In this procedure, the Microscopic Agglutination Test (MAT) was carried out using a panel of 19 reference antisera against the 17 major pathogenic erogroups–36. The Leptospira was harvested and whole cell leptospiral DNA purified as previously described[EcoRI, electrophoresis and gel analysis were carried out as previously described[Four hundred ml culture grown from each shrew isolate of described. DNA condescribed.8 per ml. The weekly injected doses were 5, 10, 15, and 20 ml respectively. Rabbits were bled by cardiac puncture one week after the last injection.Rabbit sera were prepared as previously described with slight modification and as lGenome sequencing was performed by the Centre for Genomic Research at the University of Liverpool. Genomic DNA material was purified with 1x cleaned Ampure beads (Agencourt) and the quantity and quality was assessed by Nanodrop and the Qubit assay. In addition, the Fragment Analyser (using a high sensitivity genomic kit) was used to determine the average size of the DNA and the extent of degradation. This procedure was also used at the steps indicated below to determine average fragment size of the DNA. DNA was sheared using Covaris G tubes by centrifugation at 7,000 rpm in an Eppendorf 5415R centrifuge. The fragment size was checked as before. DNA was purified with 0.5x ampure beads and treated with Exonuclease VII at 37°C for 15 minutes. The ends of the DNA were repaired as described by Pacific Biosciences protocol. Each sample was incubated for 20 minutes at 37°C with DNA Damage Repair Mix supplied in the SMRTbell library kit (Pac Bio). This was followed by 5 minutes incubation at 25°C with End Repair Mix. DNA was cleaned using 0.5x ampure and 70% ethanol washes. DNA was ligated to adapter sequences overnight at 25°C. Ligation was terminated by incubation at 65°C for 10 minutes followed by exonuclease treatment for 1 hour at 37°C. The SMRTbell library was purified with 0.5x ampure beads. The quantity of library and therefore the recovery was determined by Qubit assay and the average fragment size determined by Fragment Analyser. SMRTbell library was annealed to sequencing primer at values predetermined by the Binding Calculator (Pac Bio) and a complex made with the DNA Polymerase (P6/C4 chemistry). The complex was bound to Magbeads and this was used to set up 3 SMRT cells for sequencing. Sequencing was done using 240 minute movie times.The 16S rRNA gene sequence identified within the newly sequenced organism described herein was used to retrieve 108 similar sequences from the Ribosomal Database Project (RDP) via the SeqMatch tool. SequencsecY gene sequence identified within the newly sequenced organism described herein was compared with other sequences of secY from the genus Leptospira, as retrieved from GenBank[secY were aligned with CLUSTAL W[The m GenBank. SequencCLUSTAL W. PhylogeCLUSTAL W and the CLUSTAL W.7 of GWTS isolate #1, #2 or #3 IP respectively. Three hamsters acted as negative controls and received media alone. All animal experimental procedures were performed in accordance with relevant guidelines and regulations, and as approved by USDA Institutional guidelines.Golden Syrian hamsters were inoculated by intraperitoneal (IP) injection as previously described. Groups The microscopic agglutination test was performed as previously described according to OIE guidelines.The fluorescent antibody test was performed as previously described.L. alstonii serovar Room22 strain GWTS#1 is available in GenBank under the accession numbers CP015217 (Chromosome I) and CP015218 (Chromosome II).The annotated assembly for Culture of leptospires was attempted from a single kidney in each of 18 trapped GWTS. Kidneys from three of the GWTS were culture positive as confirmed by dark-field microscopy and the isolates were named GWTS Isolate #1, #2 and #3 respectively.Leptospira was tested against a standard panel of reference antisera, representing 19 serovars from 17 serogroups and representative of the geographical locale, for typing purposes, Leptospira, representing 9 serogroups, one undesignated serogroup, and 13 serovars, Leptospira santarosai. However, the lack of a consistently high MAT titre detected between GWTS isolate-specific antisera and reference antigen indicated an inconclusive serological typing classification of any of the GWTS isolates, and suggesting that they were of an as yet unidentified serovar.Each GWTS isolate of Leptospira isolates using reference antisera and reference antigens indicates that the GWTS Leptospira isolates are atypical compared to those previously identified in Western Europe. Therefore, whole genome sequencing was performed on a single strain, GWTS isolate #1. The gene sequence for 16S rDNA was extracted from the complete genome and compared to 108 16S rDNA sequences available for Leptospira from the Ribosomal Database project (https://rdp.cme.msu.edu/). Phylogenetic analysis indicated that GWTS isolate #1 clustered among 4 strains of Leptospira recently isolated from soil samples in Fukuoka, Japan , Fig 1  in China; serogroprovided .L. alstonii that have been cultured to date, L. alstonii isolates. Analysis of REA patterns compared with a reference database of Leptospira strains held in the OIE Reference Laboratory  did not identify any similar REA patterns.Restriction enzyme analysis was performed on DNA purified from each GWTS isolate #1, 2 & 3 for comparison with 5 of the 6 available isolates of L. alstonii serogroup Undesignated serovar Room22.Collectively, these results provide evidence of the unique and novel molecular attributes of each of the GWTS isolates, which we designate as Leptospira alstonii is considered to be a member of the pathogenic complex of Leptospira, as defined by DNA-DNA relatedness, 16S rDNA and secY sequence. In addition to these criteria, the genome sequence of GWTS#1 contains lipL32, which to date has only been identified in pathogenic leptospires  which were subdivided into serovars, a division based on shared agglutinating lipopolysaccharide antigens and for which more than 200 serovars have been described[in silico hybridization of whole genome sequences, and include Leptospira alexanderi, L. alstonii, L. borgpetersenii, L. interrogans (sensu stricto), L. kirschneri, L. kmetyi, L. mayottensis, L. noguchii, L. santarosai and L. weilii [L. interrogans or L. borgpetersenii. Nevertheless, the serologic classification of leptospires continues to play an important role in the epidemiology of leptospirosis and is the basis for the current “gold standard” serologic diagnostic assay, the microscopic agglutination test (MAT). In the MAT, serum from a patient  is incubated with a panel of serovars of leptospires to test for a positive agglutination reaction, with the selected panel being representative of a geographical region; one of the obvious limitations of this assay is the composition of the diagnostic panel which will remain negative if tested with serum from a patient that is infected with a serovar not represented in the panel. Such is the case in our studies; when L. alstonii serovar Room22 was used to inoculate hamsters, all hamsters seroconverted and were MAT positive when tested against serovar Room22; but all were negative, with no cross-reactivity, when tested against six common pathogenic serovars, as typically found in Ireland. Nor was specific antiserum for L. alstonii serovar Room22 reactive with a range of pathogenic leptospires  to detect leptospires in host infected tissue using polyclonal antibodies which cross reacts with L. alstonii serovar Room22  confirmed that GWTS#3 has an identical pattern to that of GWTS#1, which differed slightly to that of GWTS#2. All REA patterns were different to that of other strains of L. alstonii cultured to date  during its evolution, whilst no gene loss was observed. Such observations are interpreted to facilitate the adaptation by Leptospira to different hosts and an expanding range of environments.Our results suggest that the GWTS acts as a reservoir host for  to date . Similarvironment, 61. Unionditions. Alterna in China; resultsTyto alba) and kestrels . Barn owls are susceptible to leptospirosis[L. alstonii serovar Room22, or indeed if the decline of the native pygmy shrew in those areas inhabited by the GWTS is due in part to incidental infection with serovar Room22. There is little information available to assess the implications of the GWTS and associated pathogens on domestic animals and wildlife.The GWTS was originally identified in Ireland from skeletal remains in the pellets of barn owls .(PDF)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.


Nerve regeneration is a serious clinical challenge following peripheral nerve injury. Lycium barbarum polysaccharide (LBP) is the major component of wolfberry extract, which has been shown to be neuroprotective and promising in nerve recovery in many studies. Electrospun nanofibers, especially core-shell structured nanofibers being capable of serving as both drug delivery system and tissue engineering scaffolds, are well known to be suitable scaffolds for regeneration of peripheral nerve applications. In this study, LBP was incorporated into core-shell structured nanofibrous scaffolds via coaxial electrospinning. Alamar blue assays were performed to investigate the proliferation of both PC12 and Schwann cells cultured on the scaffolds. The neuronal differentiation of PC12 cells was evaluated by NF200 expression with immunostaining and morphology changes observed by SEM. The results indicated that the released LBP dramatically enhanced both proliferation and neuronal differentiation of PC12 cells induced by NGF. Additionally, the promotion of Schwann cells myelination and neurite outgrowth of DRG neurons were also observed on LBP loaded scaffolds by LSCM with immunostaining. In summary, LBP, as a drug with neuroprotection, encapsulated into electrospun nanofibers could be a potential candidate as tissue engineered scaffold for peripheral nerve regeneration. Although the peripheral nervous system (PNS) has a capacity for axonal regeneration after injury, axonal reconnection and functional recovery by spontaneous peripheral nerve repair are nearly always incomplete.Peripheral nerve injury (PNI) caused by accidents, physical conflict and surgical intervention is a common global clinical problem which can significantly affect the patients’ quality of life. Over 200 thousand procedures were performed in the US alone annually to repair PNI, and it cost 150 billion dollars annually in the US, which cause an enormous socioeconomic burden2. However, when the defect is larger 3, retracts after injury and tensionless repair is impossible4. In this case, graft between nerve stumps is required to bridge the gap and support axonal regrowth. The implantation of autologous nerve graft is regard as the gold standard therapy for peripheral nerve gap repair5. However, several drawbacks of this therapy such as the need for multiple surgeries, and donor site morbidity limit its widespread clinical use, especially for longer nerve gaps. With the progress in tissue engineering, recent advances in neural tissue engineering have shown great promise for neural regeneration. Various biological and artificial nerve grafts have been used as supplement and even substitution for autologous nerve grafts. A typical tissue engineered nerve graft is composed a biomaterial-based scaffold and a multitude of cellular and/or molecular components4.In recent hundred years, various types of medical therapy have been used to repair nerve lesions. Normally, the surgical reconnection is possible for bridging small PNI gaps where the disconnected nerve stumps can be sutured end-to-endGenerally, the scaffolds are capable of guiding the regeneration of axons and function as a bridge to restore the gap. Additionally, the biomaterial-based scaffold should mimic the native extracellular matrix (ECM) structure, which can provide enough space and adhesion sites for the growth of cells and extension of axons. What’s more, the scaffold should have the capacity to deliver drugs and/or signaling factors which can direct the growth and extension of regenerating axons.2. It provides a conducive environment for cellular functions including adhesion, migration, proliferation and differentiation due to its close imitation of native neural ECM. On the other hand, core-shell structured nanofibers fabricated via coaxial electrospinning has frequently been served as a carrier for drug release application7. Proteins, drugs, and nucleic acids all have been delivered by core-shell nanofibers. The drug release behavior can be controlled by turning the core-sheath compositions, while retaining their bioactivity. Meanwhile, nanofibers exhibit much higher surface area-to- volume ratio which will facilitate the release the release of drugs and increase the contact area between cells and fibers, thereby enhancing drug uptake by cells10. Hence, electrospun nanofibers are potential candidates for nerve regeneration applications. Poly (lactic-co-glycolic acid) (PLGA) has been used in many tissue engineering application due to its biodegradability and biocompatibility13. Electrospun PLGA nanofibers have also been used to enhance the sciatic nerve regeneration in rat models14.Electrospun nanofibers have been used extensively as potential scaffold in neural tissue engineering15. Plant extracts provide an alternative choice which always have multi-target and less side effects. And the plant extracts used for nerve repair should be effective in axonal regeneration and functional recovery. Wolfberry extracts is made up of a group of components including polysaccharides, betaine, zeaxanthin, beta-carotene, groups of vitamin, 19 kinds of amino acid and trace minerals16. It is believed that the polysaccharides of wolfberry, also named as Lycium barbarum polysaccharides (LBP), are the active components responsible for its various pharmacological properties including anti-aging, anti-tumor, anti-oxidation and immune modulation effects18. Increasing lines of evidence discloses the therapeutic effects of LBP on neuroprotection, which raises the possibility of using LBP as a neuroprotective agent after nerve injuries17. Studies involving primary cultured cortical neurons showed that LBP treatment reduced cellular death induced by amyloid-beta peptides and homocysteine insults21. Chan et al. reported that intragastric administration of LBP can reduce the death of retinal ganglion cells induced by experimental ocular hypertension22. Lau et al. found that LBP could promote the neuronal proliferation, differentiation of neural precursor cells23. Studies of Gao et al. also found that LBP could effectively protect PC12 cells and primary neurons against 6-hydroxydopamine-induced cell death24. Thus, introduction of bioactive chemicals such as LBP in neural tissue engineering may accelerate nerve regeneration. In our present study, we intended to investigate the effects of LBP along with PLGA nanofibrous scaffold on nerve cells behaviors such as proliferation, neuronal differentiation, neurite outgrowth and myelination in vitro. For the first time, we evaluated the effect of LBP on PC12 cells and Schwann cells proliferation and the differentiation of PC12 cells. In order to further confirm the potential role of LBP in peripheral nerve regeneration, core-shell structured PLGA-LBP nanofibers loading different dosage of LBP were fabricated via coaxial electrospinning. The topography of nanofibers was characterized by Scanning electron microscope (SEM), while the core-shell structure was confirmed by Transmission electron microscope (TEM) and fluorescent images. The surface hydrophilicity were evaluated by water contact angle. The mechanical properties of the scaffold were measured with tensile mechanical test. The in vitro release behavior of LBP from core-shell structured PLGA-LBP nanofibers was also determined. Then biocompatibility of the scaffold and the influence of scaffolds with diverse amount of LBP on nerve cells growth were tested in terms of proliferation and morphology of PC12 cells and Schwann cells. Furthermore, the neuronal differentiation of PC12 cells were performed on the scaffold in the absence and presence of Nerve growth factor (NGF). In order to explore the potential application of the PLGA-LBP nanofibrous scaffold in peripheral nerve repair, the neurite outgrowth of Dorsal root ganglion (DRG) neurons and the myelination of Schwann cell were further investigated.Bioactive molecule is another important factor in tissue engineering, which can improve neurogenesis in nerve injury site. However, most bioactive molecules such as growth factors and cytokines are expensive and instable in clinic efficacy, sometimes have side effectsThe objective of this research was to develop a coaxial electrospun nanofibrous scaffold containing LBP, which might improve both the differentiation of neurons and myelination of Schwann cells for peripheral nerve regeneration. The preparation process and working hypothesis of the scaffold are schematically illustrated in Fig. Coaxial electrospinning was utilized to fabricate PLGA-LBP nanofibrous scaffold during this study. PLGA-LBP10, PLGA-LBP30, and PLGA-LBP50 were fabricated with LBP aqueous solution of three different concentration as the core solution. PLGA-LBP0 was fabricated with pure PLGA solution by conventional electrospinning. The fabrication process is schematically demonstrated in Fig. In nerve tissue, the topographical features of fibers play a significant role towards the behavior of cells. In Fig. The core-shell structure of LBP encapsulated nanofibers was confirmed by TEM and fluorescent images which were shown in Fig. 25, so mechanical properties of nanofibers would play critical roles in peripheral nerve applications. The potential scaffolds should provide sufficient mechanical support for cells and axon regeneration. From Table et al.26 reported that stress of fresh rat sciatic nerve was 2.72 ± 0.97 MPa, therefore the PLGA-LBP nanofibers had sufficient tensile stress to be utilized as a nerve graft. As shown in Fig. 27. Even the mechanical properties decreased with the addition of LBP, the PLGA-LBP nanofibrous scaffolds were still suitable for nerve tissue engineering applications.Peripheral nerves are specific viscoelastic tissues with high mechanical propertiesWater contact angle of nanofibrous scaffold reflects its surface hydrophilicity, which were shown in Table in vitro release behavior of drugs is affected by several factors including the degradation rate of the carriers, hydrophilicity of drugs and the drug distribution. In our study, coaxial electrospinning was used to fabricate core-shell structured nanofibers, which could encapsulate drugs in the core. The shell part could alleviate burst release and preserve the bioactivity of drugs as a barrier. Stimulating nerve growth at the injury site is most important during the first 10–15 days of repair28, and approximately 4 weeks were also very crucial for the peripheral nerve regenerations, which has been verified in rat sciatic nerve29. Hence, 4 weeks release of the LBP incorporated in the fibers would be favorable. The Ultra-violet absorption spectra of LBP solution with various concentration and the calculated standard line of LBP were shown in Figs The The morphologies of PLGA, PLGA-LBP10, PLGA-LBP30 and PLGA-LBP50 nanofibrous scaffolds after 1, 3, 6, 8 weeks degradation were shown in Fig. 30. Therefore, the proliferation of PC12 cells should be considered in peripheral nerve tissue engineering. The ability of a tissue engineered scaffold to support the proliferation of cells is one of the major criteria to determine its application in target tissue regeneration. As shown in Fig. In various studies, PC12 cells have been used as neuron precursors–NGF showed elongated shape, while on PLGA-LBP10 and PLGA-LBP30 nanofibrous scaffolds with Differentiation Medium+NGF, there were more cells showed a phenotype with elongated morphologies. Additionally, some cells on PLGA-LBP0 nanofibrous scaffolds with Differentiation Medium+NGF also projected the neurites, while with Differentiation Medium−NGF, the cells were still showed undifferentiated phenotype. The phenotype of elongated morphologies confirmed the differentiation of PC12 cells. From all the aforementioned results, we concluded that NGF was essential in stimulating PC12 cells differentiation, but the stimulation of NGF could be enhanced with moderated amount of LBP.PC12 cells are the most widely used cells for the neurogenesis study. The morphology changes of PC12 cells will reflect its differentiation directly. Figure 31. Based on the results of PC12 cells proliferation and morphology of differentiated PC12 cells, we found that PLGA-LBP30 nanofibrous scaffolds should be more effective on nerve regeneration. Therefore, the expression of NF200 was evaluated to further confirm the neuronal differentiation of PC12 cells on PLGA-LBP30, and PLGA-LBP0 was used as control. The immunostaining of NF200 also enabled the comparison of cell phenotype and neurite outgrowth on different nanofibrous scaffolds. It can be seen from Fig. Neurofilaments (NF) are the major groups of intermediated filaments which are found predominantly in cells and tissues of neuronal origin. Neurofilament proteins are synthesized in the neuronal perikarya, assembled to form filaments and then slowly transported within the axons towards the synaptic terminals33. Additionally, the proliferation of Schwann cells can form columns of cells, known as brands of Büngner, which serve as a physical guide for regenerating nerve axons34. Therefore, the proliferation of Schwann cells should also be considered in peripheral nerve tissue engineering. According to Fig. Schwann cells play a critical role in neuron survival and regeneration. They can provide neurotropic growth factors such as NGF and neurotrophin 3 (NT3) for neurite outgrowth as well as axonal nerve regeneration37. Thus to further confirm the interactions of Schwann cells with electrospun nanofibrous scaffolds, the morphology and phenotype of cells were evaluated after 7 days in vitro culture by SEM and immunostaining. The SEM images in Fig. It has been known that Schwann cells play a critical role via the synergetic effect with neurons and macrophages during the regeneration process of injured PNSIn order to corroborate the results obtained by SEM observation, immunostaining with S-100 antibody, which was a specific protein for Schwann cells was conducted. According to the aforementioned results, PLGA-LBP30 and PLGA-LBP0 scaffolds were used for this evaluation, and tissue culture plate (TCP) was chosen as control. Confocal images presented in Fig. All these results indicate that the PLGA-LBP30 scaffolds were the most favorable for both PC12 and Schwann cells growth. Therefore PLGA-LBP30 scaffold was selected for further study, and PLGA-LBP0 scaffold was used as control.39. To investigate the myelinating of Schwann cells, the expression of marker of Schwann cells myelination, such as myelin basic protein (MBP) was measured with immunostaining. As shown in Fig. Myelination of Schwann cells is critical for the success of peripheral nerve regeneration. The matured myelinating Schwann cells are able to secrete various neurotrophins such as NGF, brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), which can promote the neurogenesis and guide axon elongationWe have demonstrated that LBP loaded PLGA nanofibrous scaffolds promoted proliferation and neurite outgrowth of PC12 cells. For further study, we performed evaluation of neurite outgrowth behavior of the rat primary DRG neurons. The expression of Neurofilament 160 protein (NF160) was evaluated by immunostaining and observed by LSCM. The results in Fig. 17. Electrospun nanofibers have been widely used as scaffold for peripheral nerve tissue engineering application. A variety of natural and synthetic polymers have been fabricated into nanofibers to support neural tissue engineering development. PLGA, as a biocompatible and biodegradable polymer material, has been widely used in peripheral tissue engineering40. The present study was intended to fabricate PLGA nanofibers loaded with LBP via coaxial electrospinning. Thus, a synergistic effect was produced by the combination of nerve tissue engineering and drug therapy to accelerate the nerve regeneration.Wolfberry has been used as functional food as well as medicinal herbs in Asian countries for more than thousands of years. LBP as the active component extracted from Wolfberry has been shown to be neuroprotective in many studies41. The LBP was indeed encapsulated within the polymer shell and could be released slowly from the fibrous scaffold  and double-stranded-RNA-dependent protein kinase (PKR)20. Additionally, Ho et al. reported that cell death of Rat primary cultured cortical neurons treated with Aβ25–35 and Aβ1–42 was reduced by LBP through the improve of phosphorylation of Akt21. Consequently, the application of LBP loaded nanofibrous scaffolds in nerve injured sites may inhibit the apoptosis of neurons. The differentiation of PC12 cells induced by NGF was also improved on LBP loaded nanofibers, as evidenced by morphological evaluation and immunofluorescence staining 43. It has been known that the process of phosphorylation of ERK is the necessary requirement of PC12 cells differentiation induced by NGF46. Thus, we supposed that the LBP could facilitate the PC12 cells differentiation induced by NGF through the up regulation of phosphorylation of ERK. Additionally, the present study found that the expression of MBP in Schwann cells showed significant enhancement on PLGA-LBP30 group , which is a key protein kinase enzyme involved in other signal pathways such as mitogen-activated protein kinase (MAPK) to indirectly influence Schwann cells myelination51. Rat DRG neurons showed better viability and neurite out growth on LBP containing nanofibers, which could be seen in Fig. In the present study, four different groups of nanofibers were prepared according to the different concentration of LBP. The diameters of fibers gradually decreased from about 900 nm to about 300 nm with the increase of LBP concentration Fig. . Early sold Fig. . The feaoup Fig. . Gao et in vitro. There was a beneficial synergistic effect between NGF and LBP in promoting PC12 cells differentiation and axonal extension of neurons. To allow regeneration following peripheral nerve injury, the microenvironment must be permissive enough for neurite outgrowth. Therefore, the LBP loaded nanofibrous scaffolds, especially PLGA-LBP30, could be a promising substrate for target peripheral nerve injury repair.Taken together, these results demonstrated that LBP has a positive effect on nerve cells proliferation and differentiation The 75/25 Poly (lactic-co-glycolic acid) (PLGA) (inherent viscosity 0.61 dL/g) was purchased from Jinan Daigang Biomaterial CO., Ltd, China. Lycium barbarum polysaccharide was provided by Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFP), glutaraldehyde, Dulbecco’s modified eagle’s medium (DMEM/F12), were purchased from Sigma, Singapore. Nerve growth factor (NGF) was purchased from Millipore, Singapore. Rat pheochromocytoma (PC12) cells in the adherent type and Schwann cells were obtained from ATCC, USA, while fetal bovine serum (FBS), horse serum (HS) and trypsin/EDTA were purchased from GIBCO Invitrogen, USA. Alamar Blue (AbD Serotec) was purchased from Chemoscience, Singapore. Schwann cell medium was purchase from Gene-Ethics Asia Pte Ltd, Singapore.28% w/v PLGA solution was prepared by dissolving PLGA into HFP followed by a magnetic stirring at room temperature overnight. Meanwhile, LBP aqueous solution with 10 mg/ml, 30 mg/ml, and 50 mg/ml concentration were prepared separately. Core-shell structured PLGA-LBP nanofibers were fabricated via our lab’s electrospinning set-up as shown in Fig. The scanning electron microscope (SEM) was applied to observe the surface topography of fabricated nanofibrous scaffolds. The electrospun nanofibers were sputter-coated with gold  and then visualized using a SEM . The average diameter of the nanofibers was calculated from 100 random points chosen from the SEM images with analysis by Image J software .The core-shell structure of the nanofibers was verified using transmission electronic microscopy  at 200 kV. The samples for TEM observation were prepared by collecting nanofibers with carbon-coated copper grids. Laser scanning confocal microscopy  was used to further confirm the distribution of LBP in the core of nanofibers, and to do so, the LBP aqueous solution used for electrospinning was mixed with FITC.−1 was applied for all specimens during the process. Six specimens of each sample were measured.Tensile properties of the nanofibrous scaffolds were evaluated using a universal materials tester  at an ambient temperature of 20 °C and humidity of 65%. Tests were performed on rectangular specimens with dimensions of 30 × 10 mm. A cross-head speed of 10 mm minHydrophilicity of the electrospun nanofibrous scaffolds was measured by water contact angle measurement using VCA Optima Surface Analysis System . To prepare samples for water contact angle measurement, the nanofibers were collected on coverslips and placed on the testing plate. Then deionized water was dropped on the specimens and the data was recorded. Three different points of the same specimen were measured and each sample was performed in triplicate.in vitro. 10 mg of PLGA-LBP30 nanofibrous scaffolds were soaked in a 2 ml centrifuge tube with 1 ml of PBS. Subsequently, the centrifuge tubes with nanofibrous mats were all incubated in a continuous horizontal shaker at a speed of 150 rpm at 37 °C. At preset time points, the PBS was retrieved completely from the tubes and the equal volume of fresh PBS was replaced for continuous incubation. The LBP concentration was determined by UV-vis spectrophotometer at wavelength at 260 nm. The cumulative amount of released LBP was calculated. The samples for each time point were run in triplicate.PLGA-LBP30 nanofibrous scaffold was used to evaluate the release behavior of LBP 52. Briefly, the nanofiber meshes were cut into small square pieces. Then the specimens were immersed in 10 ml of PBS in 15 ml centrifuge tubes. Subsequently, the tubes were kept in a shaking incubator at 37 °C operating at 150 rpm. At each particular time point of study, the specimens were taken out from the tubes, rinsed several times with distilled water to remove residual buffer salts, and dried in a vacuum desiccator. The morphology was observed with SEM as described previously.The degradation of nanofibrous scaffolds were performed as described previously2 cell culture flask. Cells were maintained at 37 °C in a humidified incubator with 5% CO2, and the culture medium was changed once in every two days. The nanofibrous scaffolds for cell culture were collected with 15 mm cover slips. All the samples were sterilized for 5 h under UV light in biological safety cabinet  and then placed in a 24-well plate pressed with stainless steel rings. Subsequently all samples were washed thrice with PBS, and immersed in Growth Medium until cell seeded. After the confluence was sufficient, PC12 cells in flask were detached by trypsin/EDTA, counted by automated cell counter, and seeded on all nanofibrous scaffolds with a density of 1.0 × 104 cells/well.PC12 cells were cultured in normal Growth Medium composed of Dulbecco’s modified eagle’s medium (DMEM/F12) supplemented with 10% horse serum (HS), 5% fetal bovine serum (FBS), and 1% antibiotic/antimycotic solution in a 75 cmSimilarly, the Rat Schwann cells were grown in SCM medium with 10% FBS. And before seeding the cells, the scaffolds need coating with Poly-L-lysine. In detail, the scaffolds were washed with PBS for three times after sterilization under UV light, and then incubated with 200 μl Poly-L-lysine solution  for 2 h in 37 °C incubator. Subsequently, the scaffolds were washed again with PBS for three times, and placed in an incubator overnight to allow the scaffolds to be dry fully. All the other followed process was the same as PC12 cells.The proliferation of PC12 on different scaffolds was performed after 1, 3, 5, 7 days of culture by means of the Alamar Blue assay. At predetermined time points, medium was carefully removed and 1 ml of Alamar Blue solution  was added to each well followed by incubation in darkness for 4 h under 37 °C. After incubation, the solution was pipetted from each well into 96-well plate and read at 560 nm (excitation)/590 nm (emission) by a Varioskan flash reader .+NGF composed of DMED/F12 supplemented with 1% HS, 0.5% FBS, 1% antibiotic/antimitotic solution and 100 ng ml−1 NGF after cells seeded for 1 day. For comparison, cells were also cultured with the same medium above without NGF which is referred as Differentiation Medium−NGF.For differentiation study of PC12 cells, the medium was changed from Growth Medium to Differentiation MediumThe morphology of differentiated PC12 cells was different from its original ones. To evaluate the neuronal differentiation of PC12 cells, the morphological changes were observed with SEM after 10 days of incubation in Differentiation Medium. Meanwhile, the morphology of cells cultured on scaffolds with Growth Medium was also observed as control. After 10 days incubation, cells-scaffolds constructs were rinsed twice with PBS and then fixed with formalin for 20 min. After that, all samples were rinsed thrice with PBS and dehydrated with a series of graded ethanol  for 15 min each, followed by air-drying overnight in room temperature. Finally, the dry samples were sputter coated with gold and observed under SEM.The neuronal differentiation of PC12 cells was also evaluated by immunostaining of neuronal specific protein-neurofilament 200 (NF200). After 10 days of cell culture in Differentiation Medium, all scaffolds were rinsed with PBS, fixed in formalin for 20 min and permeabilized with 0.1% Triton-X100 for 5 min. Then 3% BSA solution was used to block nonspecific binding for 90 min. After that, the samples were stained with primary antibody, anti-NF200 produced in rabbit (Sigma) at a dilution of 1:100 for 2 h at room temperature. Subsequently, all samples were washed thrice with PBS and stained with secondary antibody, FITC conjugated goat anti rabbit (Sigma) at a dilution of 1:300 for 1 h. After rinsing with PBS for five times, the scaffolds were treated with mounting medium with DAPI . Finally, the immunostained samples were visualized under LSCM to observe the cell phenotype and neurite extension of differentiated PC12 cells.the proliferation of PC12 cells.The proliferation of Schwann cells on different scaffolds was performed after 1, 3, 5, 7 days of culture by means of the Alamar Blue assay. The detailed process was the same as what described in section of Neuronal differentiation of PC12 cells. For the fluorescent microscopy observation, immunostaining of specific protein for Schwann cells, namely S100, was carried out. And the followed process was similar with that described in section of Neuronal differentiation of PC12 cells. The primary antibody used in this study was rabbit anti-S100 .The morphology of Schwann cells on different scaffolds was observed by both SEM and fluorescent microscopy. For SEM observation, all the process was the same as what described in section of Neuronal differentiation of PC12 cells. Anti-MBP antibody produced in rabbit  was used as the primary antibody in this study.Myelination of Schwann cells is critical for the success of peripheral nerve regeneration. To evaluate the influence of PLGA-LBP nanofibrous scaffold on modulating myelination of Schwann cells, the expression of MBP of Schwann cell was determined with immunostaining. The process of immunostaining was the same as what described in section of −1 NGF. The culture medium with NGF was replaced every two days. After 7 days incubation, the DRG neurons were fixed for 30 min in a 4% paraformaldehyde at 4 °C and then washed three times with PBS. Subsequently, the DRGs were immunostained with the primary antibody solution overnight at 4 °C. After washing with PBS for three times, the samples were incubated with Goat anti-rabbit IgG Alexa 488  diluted 1:200 in PBS as the secondary antibody. The nuclei were stained with DAPI for 15 min finally (Thermo Scientific). The outgrowth of immunostained DRG neurons were visualized under LSCM.DRG neurons were isolated from the embryo of the 14 day pregnant rat. Before seeding the DRG neurons, the samples were exposed to UV light overnight for sterilization, followed by thrice rinse with PBS. The culture medium for DRG neurons was Neuralbasal medium with 1% Penicillin-Streptomycin Solution, 2% B27 supplement factors and 20 ng mlStatistical analyses were carried out using one-way ANOVA followed by Tukey’s test. All data were reported as mean ± standard deviation (SD). Differences were considered statistically significant when P < 0.05.Supplementary Information