Objectives: The transition from acute low back pain (aLBP) to chronic LBP (cLBP) results from a variety of factors, including epigenetic modifications of DNA. The aim of this study was to (1) compare global DNA (gDNA) methylation and histone acetylation at LBP onset between the aLBP and cLBP participants, (2) compare mRNA expression of genes with known roles in the transduction, maintenance, and/or modulation of pain between the aLBP and cLBP participants, (3) compare somatosensory function and pain ratings in our participants, and (4) determine if the aforementioned measurements were associated.Methods: A total of 220 participants were recruited for this prospective observational study following recent onset of an episode of LBP. We retained 45 individuals whose gDNA was of sufficient quality for analysis. The final sample included 14 participants whose pain resolved within 6 weeks of onset (aLBP),15 participants that reported pain for 6 months (cLBP), and 16 healthy controls. Participants were subjected to quantitative sensory testing (QST), blood was drawn via venipuncture, gDNA isolated, and global DNA methylation and histone acetylation, as well as mRNA expression of 84 candidate genes, were measured.Results: Individuals that develop cLBP display multimodal somatosensory hypersensitivity relative to aLBP participants. cLBP participants also had significantly lower global DNA methylation, which was negatively correlated with interleukin-2 (IL2) mRNA expression.Discussion: cLBP is characterized by somatosensory hypersensitivity, lower global DNA methylation, and higher IL2 expression level compared to those whose pain will resolve quickly (aLBP). These results suggest potential diagnostic and therapeutic relevance for global DNA methylation and IL2 expression in the pathology underlying the transition from acute to chronic LBP. Low back pain (LBP) is a global health concern that affects nearly 1 in 10 people worldwide and ranks highest in terms of disability , 3, makiTRPA1 gene as well as lower TRPA1 mRNA expression . LBP status was tracked over a 6 month period, and only participants whose pain resolved within the 1st month (aLBP) or for whom pain was still present at 6 months (cLBP) were compared to healthy controls in the present analyses. Somatosensory functioning was assessed in all participants using multiple quantitative sensory testing (QST) measurements. We hypothesized that during the acute pain phase, individuals that eventually transition to cLBP would display differences in epigenetic markers, candidate gene expression levels, and somatosensory function (QST measures) compared to those whose pain would resolve (aLBP) and healthy controls. Indeed, we found participants that would go on to develop cLBP differed in global DNA methylation status, candidate gene expression, and somatosensory function at the time of pain onset compared to aLBP participants. Identifying factors predictive of the transition to cLBP, can shed light on novel precision pain medicine interventions targeting specific pathological processes early on in order to prevent the transition all together.Men and women between the ages of 18–50 years of age with recent onset of non-specific LBP who could read and write in English were invited to participate. The criteria for the LBP episode was pain anywhere in the region of the low back bound superiorly by the thoraco-lumbar junction and inferiorly by the lumbo-sacral junction, which had been present for >24 h but <4 weeks duration and was preceded by at least 1 pain free month. Recruitment took place at two urban university health systems from 2014 to 2018 following approval from the Institution Review Board.Advertisements at primary healthcare clinics, college campuses, and in the general community were used to recruit participants. All participants provided written consent prior to study participation. We previously reported preliminary reports for baseline demographic, psychological, and somatosensory measures as well as mRNA expression of candidate genes in a subsample of the participants presented here , 27, 28.Exclusion criteria for low back pain patients included: pain at another site or associated with a prior medical diagnosis of a painful condition , previous spinal surgery, presence of neurological deficits, history of comorbidities that affect sensorimotor function , pregnant or within 3-months postpartum, taking opioid or anticonvulsant medication, and history of diagnosed psychological disorders . Eligibility for the healthy no-pain control group  included men and women (a) between 18 and 50 years of age; (b) capable of reading and writing in English; (c) with no known medical, psychological diagnoses or prescribed medications; (d) not pregnant or breastfeeding; and, (e) no recent history of pain at any location.Participants who continued to have pain ≥ 2 on the numeric rating scale were followed up every 6 weeks until either their pain had resolved or until the end of the study (24 weeks). Participants whose pain had resolved as indicated by a McGill Pain Questionnaire Present Pain Intensity rating ≤ 1 by the 6-week time point were classified as aLBP and participants whose pain had not resolved (pain ≥ 2) at the 6-month visit were classified as cLBP.After obtaining informed consent, participants were scheduled to undergo baseline data collection as soon as possible but no longer than 1 week from the time of consent. Data collection took place in a private research suite; participants completed questions about age, gender, socioeconomic status, education, lifestyle behaviors , comorbidities, and past episodes of LBP. Following completion of the questionnaires, participants underwent venipuncture for collection of blood samples and quantitative sensory testing (QST). The sequence of data collection was followed for all patients. Whole blood was collected by venipuncture into one 5-mL EDTA vacutainer and one 10-mL Paxgene blood RNA tube , labeled with a unique study identification label, and transported directly to the laboratory for processing or storage.Participants completed both the Brief Pain Inventory (BPI) and the McGill Pain Questionnaire Short Form (MPQ-SF). BPI subscales assess the severity of pain, location of pain, pain medications, amount of pain relief in the past 24 h and the past week, and the impact of pain on daily functions. While there are instances where a summary score is used, investigators are encouraged to use the subscales . The BPIQST uses standardized stimuli to assess both nociceptive and non-nociceptive systems . FollowiTM  to increase pressure at a rate of 30 kPa/s until the participant indicated first pain sensation. Pressure pain threshold was determined after repeating the procedure at the same site until either the two values were recorded within 20 kPa of one another or three trials were administered. The mean of the two closest values was reported. Mechanical detection and pain threshold as well as mechanical pain sensitivity were determined using a standard set of von Frey hairs . The final threshold is calculated as the geometric mean of 5 series of ascending and descending stimuli intensities. Windup ratio was assessed as the mean pain rating of the stimulus trains divided by the mean pain rating to a single stimuli. Dynamic mechanical allodynia was measured with a standardized brush applied five times with a single stroke and the pain rating of each stroke was recorded. Vibration detection threshold was performed using a Rydel-Seiffer tuning fork  placed on the skin surface. Participants were asked to report when the vibration was no longer felt, and this number is recorded.QST measures included pain pressure threshold, mechanical detection threshold, mechanical pain threshold, mechanical pain sensitivity, dynamic mechanical allodynia, windup ratio, vibration detection threshold, cold and warm detection thresholds, and cold and heat pain threshold. Pain pressure was measured with an algometer (range from 50 to 600 kPa) attached to a Medoc Pathway SystemTM. A series of thermal testing procedures were carried out: cold detection threshold, warm detection threshold, cold pain threshold, and heat pain threshold. The mean threshold temperature of 3 consecutive measurements were calculated. The temperature stimuli were ramped at 1C/second and were terminated when the participant pressed a button.Thermal thresholds were determined with the Medoc Pathway SystemBlood samples were stored at 4°C for a minimum of 2 h, mixed with cold 1% fetal bovine serum and centrifuged at 3,000 rpm for 15 min to isolate the buffy coat. Buffy coat was isolated using a 200 μL pipette and stored at −80°C for subsequent extraction and purification of genomic DNA (gDNA) using the QiAmp DNA Blood Mini Kit according to manufacturer's instructions . gDNA concentration was determined by biospectrophotometer with 1.0 μL samples .® Methylated DNA Quantification Kit, Sigma-Aldrich). DNA at a concentration of 150 ng was diluted with 30 μL of binding buffers and incubated at 37°C for 60 min. The samples were incubated with capture and detection antibodies and absorbance was read at 450 nm according to manufacturer's instructions. Quantification of global DNA methylation was obtained by calculating the amount of methylated cytosines in the sample relative to methylation in a positive control, which was provided by the manufacturer. All samples were run in duplicate and the global methylation level was measured and averaged to produce a single value for each participant.At the time of initial assessment, the gDNA cytosine methylation level for each participant was determined by using an enzyme-linked immunosorbent assay-based commercial kit  Quantification Kit ]. Histone extracts were first generated from each sample using the Histone Extraction Kit (abcam). Histone extracts were incubated with antibody buffer at room temperature according to the manufacturer's instructions. The samples were then incubated with detection solution for 60 min at room temperature followed by color developer and read at 450 nm according to the manufacturer's instructions. H4K8 acetylation was quantified relative to the total histone extract amount added and the standard control, which was provided by the manufacturer. All samples were run in duplicate and the global histone acetylation was determined and averaged to produce a single value for each participant.TM total RNA isolation system  according to the manufacturer's protocol and was reverse transcribed using RT2 cDNA kit (Qiagen USA). The mRNA expression of 84 genes involved in the transduction, maintenance, and modulation of pain was determined  using qPCR performed on the ABI StepOne Plus PCR machine. After an initial incubation step, 40 cycles (95°C for 15 s and 1 min at 60°C) of PCR were performed. Relative gene expression levels were quantified using the 2DD−CT method, which normalizes data of the genes of interest to the average of three housekeeping genes β-actin (ACTB), GAPDH and Beta-microtubulin (B2M), and expression level was determined as fold-change relative to healthy controls. Two participants were excluded from gene expression analyses for having undetermined values in their house keeping genes but were included for all other analyses for which their data sets were complete.RNA isolation was performed using the PAXgenep < 0.05 with the exception of the gene expression data, which was adjusted to p < 0.001 in order to correct for multiple comparisons. Stepwise linear regression analyses were used to explore the contributions of global DNA methylation or histone H4 acetylation (step 1) and our gene of interest, interleukin 2 , to each QST or BPI measure. Statistical significance was set at p < 0.05.All statistical analyses were performed using SPSS version 26 . Pearson correlation was used to examine correlations between global DNA methylation or histone acetylation and gene expression of 84 genes, QST parameters, and BPI subscales. Statistical significance was accepted at ClinicalTrials.gov). We have previously reported differences in demographic variables and the relationship between genetic variation of the fatty acid amide hydrolase (FAAH) and pain sensitivity (COMT) and brain-derived neurotrophic factor (BDNF) genes and their contribution to pain chronicity within this population , 15 participants who continued to have pain for 6 months , and 16 healthy controls who completed all study survey measures, QST, and whose mRNA and gDNA were of adequate quality and yield for mRNA expression analysis and the DNA methylation and histone acetylation assays, respectively . The remsitivity  as well t-tests revealed that cLBP participants reported significantly higher ratings of the BPI subscales  = −3.39, p < 0.05], Average Pain , Pain Now , and Pain Interference . BPI data from a larger cohort of these participants has been previously published (At the time of recruitment baseline pain thresholds were assessed using the Brief Pain Inventory (BPI) subscales. The participants that would go on to develop cLBP exhibited increased pain burden across multiple subscales compared to aLBP participants. Independent samples ubscales  for WorsFs>3.75, p < 0.05 (p < 0.05) compared to healthy controls, while no differences were observed between cLBP and aLBP participants, suggesting a moderate phenotype for aLBP participants. A similar battery of QST measures from a larger cohort of participants has been previously published  as well as on the non-dominant forearm as a control site. Univariate analysis of variance (ANOVA) revealed cLBP participants were more sensitive in a number of somatosensory function domains measured at the site of injury  but also showed a higher warm detection threshold (WDT) relative to aLBP participants and healthy controls [all p < 0.05 ]. Analysublished , 27.p < 0.05; t(df) = 2.270, p < 0.05; QST measurements were also evaluated in cLBP participants 6-months following study initiation. We used Pearson correlation to determine whether initial QST measurements were associated with measurements taken at the 6-month time point and found significant correlations in 17/24 measures , when both pain groups were combined, we observed a significantly higher amount of global histone H4 acetylation compared to healthy controls . At the control site, WUR10 and WDT were positively correlated with H4 acetylation  and MPT and CDT were negatively correlated with H4 acetylation .We assessed the potential relationship between H4 acetylation at time of pain onset and QST measurements. We found that MPS, WUR1, and WDT at the site of pain were positively correlated with H4 acetylation  = 213.40, p < 0.05; Post-hoc analyses revealed that cLBP participants had significantly lower global DNA methylation relative to aLBP participants and healthy controls (p < 0.05). Methylation levels did not differ between aLBP and healthy controls (p > 0.05).Whole blood was collected from each participant by venipuncture at the time of the first study visit. DNA (150 ng) was then isolated from blood samples and global DNA methylation was quantified for each participant. We found a significant group effect on global gDNA methylation levels [BDNF), Cx3C motif chemokine receptor 1 (CX3CR1), GTP cyclohydrolase 1 (GCH1), purinergic receptors, , prostaglandin E synthase 3 (PTGES3), and tumor necrosis factor (TNF) were positively correlated with global DNA methylation levels  was the only gene we found to be negatively correlated with global DNA methylation  with DNA methylation at the time of study initiation .Pearson correlations were also conducted to determine whether global DNA methylation taken at the start of the study correlated with gene expression level at the 6-month time point. As in the initial analysis, IL2 mRNA expression, we then directly compared levels of IL2 mRNA expression between each participant condition. Our analysis revealed that cLBP participants had significantly greater IL2 mRNA expression relative to aLBP participants and healthy controls [F = 16.937, p < 0.05; Because we found a significant inverse relationship between global DNA methylation and p > −0.407, p < 0.05; p = 0.302, p < 0.05). Our analysis also revealed that MPS, ALL, WUR1, and WUR10 at the control site were negatively correlated with global DNA methylation .Next, we explored the relationship between global DNA methylation and QST measurements. We found that MPS, ALL, WUR1, WUR10, and WDT at the site of pain were negatively correlated with global DNA methylation .We also correlated global DNA methylation with BPI self-reports of pain, and found that BPI worst, BPI least, BPI average, BPI now, and BPI interference were all negatively correlated with global DNA methylation  at the site of pain was positively correlated with IL2 expression .We found that the only gene whose expression was significantly negatively correlated with global DNA methylation in cLBP participants was IL2 expression. These analyses revealed that MPT, MPS, ALL, WUR1, WUR10, and WDT at the site of pain were all associated with global DNA methylation, all Fs > 4.3, r2 > 0.097, and p < 0.05  = 4.885, r2= 0.127, p < 0.05.We used multiple stepwise linear regression to further study the relationship between QST measurements, global DNA methylation, and p < 0.05 . AdditioIL2 expression using multiple stepwise linear regression. Our analysis revealed that BPI worst, BPI least, BPI average, BPI now, and BPI interference were significantly associated with DNA methylation  and healthy controls, indicating a potential role for hypomethylation in subsequent candidate gene expression changes contributing to pain chronicity. We further found that a single candidate gene, Histone acetylation occurs on the n-terminal of the histone tail and makes chromatin more accessible for transcription factors to bind , which uper se.cLBP participants had significantly lower global DNA methylation compared to aLBP and healthy controls, in line with other studies that report differences in DNA methylation patterns in individuals with chronic pain , 18, 37,IL2, whose expression was significantly negatively correlated with DNA methylation level at both the time of pain onset and after transition to chronic pain. Eight other candidate genes were significantly positively correlated with global DNA methylation status at the time of pain onset: BDNF, CX3CR1, GCH1, P2RX4, P2RX7, P2RY1, PTGES3, and TNF. Expression levels of each of these candidate genes have previously been associated with various chronic pain conditions in humans and preclinical models , R01 NR013932 (to AS), and NINR T32 NR013456 (mPIs) (to KMBC).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A better understanding of the transcriptomic modifications that occur in spina bifida may lead to identify mechanisms involved in the progression of spina bifida in utero and the development of new therapeutic strategies that aid in spinal cord regeneration after surgical interventions. In this study, RNA-sequencing was used to identify differentially expressed genes in fetal spinal cords from rats with retinoic acid-induced spina bifida at E15, E17, and E20. Gene ontology, KEGG, and protein–protein interaction analysis were conducted to predict pathways involved in the evolution of the disease. Approximately 3000, 1000 and 300 genes were differentially expressed compared to the control groups at E15, E17 and E20, respectively. Overall, the results suggest common alterations in certain pathways between gestational time points, such as upregulation in p53 and sonic hedgehog signaling at E15 and E17 and downregulation in the myelin sheath at E17 and E20. However, there were other modifications specific to gestational time points, including skeletal muscle development at E15, downregulated glucose metabolism at E17, and upregulated inflammation at E20. In conclusion, this work provides evidence that gestational age during spina bifida repair may be a significant variable to consider during the development of new regenerative therapeutics approaches. Myelomeningocele (MMC), the most significant form of spina bifida, is a devastating congenital malformation of the spinal cord associated with severe morbidity and mortality . This “tDespite the advances in surgical techniques for spina bifida repair in utero, effective regenerative treatments for the devastating neurotological alteration have not yet been developed. To develop better therapeutic approaches, it is extremely important to understand the molecular changes present in the neural tissue once the defect has occurred and during this progressive degeneration in utero. The analysis of transcriptome studies at different time points throughout gestation would provide in-depth knowledge of the regulatory changes present in the neural tissue of the neurodegenerative progression in utero after spina bifida occurs. Through these studies, pathways could be identified as therapeutic targets to aid in spinal cord regeneration.In a follow-up study to our previous publication , we haveSprague Dawley rats weighing 200–250 g  were housed at 22 °C in a standard dark:light cycle (10:14 h) with access to water and standard food ad libitum. The mating date was defined as E-1 and plug day as E0. The experimental protocol was in agreement with the National Institutes of Health Guidelines for Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital and Medical Center (IACUC 2019-0081).On E10 at 10:00 a.m., pregnant dams were gavaged with 100 mg/kg trans-retinoic acid (RA)  solubilized in olive oil or an equal volume of olive oil only (vehicle). With this model, 70% of fetuses in each liter were diagnosed with spina bifida based on open spinal defect . At E15,At E15, E17, and E20, comparable size spinal cords from vehicle, control, and MMC fetuses were dissected from the lumbar region and snap-frozen and stored at −80 °C until used for gene expression analysis. In MMC fetuses, only the spinal cord from the open area were dissected. Frozen spinal cords were homogenized using an IkaT10 basic Ultra-Turrax homogenizer in RLT buffer and then RNA was extruded using the RNeasy Plus Mini Kit  following the manufacturer’s protocol. RNA quantity was assessed through spectrophotometric analysis using an Epoch Biotek spectrophotometer .® spectrophotometer . RNA concentration was measured using the Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer  and the RNA integrity was evaluated using the RNA Nano6000 Assay Kit for the Bioanalyzer 2100 system .Agarose gels (1%) were used to monitor RNA degradation and contamination. RNA purity was assessed using the NanoPhotometern = 2 animals per group per time point). Libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations. cDNA products were purified using the AMPure XP system and library quality was determined using the Agilent Bioanalyzer 2100 system. The cBot Cluster Generation System using the HiSeq PE Cluster Kit cBot-HS  was used to cluster the samples according to the manufacturer’s instructions. After this, the library products were sequenced on an Illumina HiSeq  and 125 bp/150 bp paired-end reads were generated per sample. Three micrograms of total RNA per sample were used to generate sequencing libraries  between control, vehicle, and MMC groups at E15, E17, and E20. Principal component analysis was conducted using the AltAnalyzer package v2.0  [p value less than 0.05 were considered statistically significant. RNAseq data were deposited in the Sequence Read Archive (SRA) and can be found via BioProject (PRJNA683230) and SRA(PRJNA683793). The protein–protein interaction network (PPI) was constructed and illustrated using the search tool for the retrieval of interacting genes/proteins (STRING)  database to reveal the relationships of the top 25 DEGs based on a minimum required interaction.Raw FastQ files were processed through in-house Perl scripts, where clean reads were obtained by removing reads containing adapter or poly(N) as well as low-quality reads. TopHat v2.0.12 was used to align clean reads to the reference genome, which was built using Bowtie v2.2.3. HTSeq v 0.6.1 was used to calculate the fragments per kilobase of exon per million base pairs mapped (FPKM) based on the gene’s length and reads mapped to that gene. Differential expression analysis was performed using the DESeqR software v1.18.0. The resulting ry 2021) ,14,15. T2 First Strand Kit , 1 µg RNA /sample was reverse transcribed into cDNA. Four to six samples from the MMC and vehicle groups were analyzed. A 1 µg cDNA sample was then used as a template for RT-qPCR employing TaqMan® gene expression assays    in the 7p value < 0.05 was considered statistically significant. Relative quantification of transcript expression from RT-qPCR was performed using the 2−ΔΔCt method comparing MMC and vehicle, where Ct represents the threshold cycle. Error bars indicate the standard error of the mean.All graphs were performed in GraphPad Prism9 software . A fold change > 1.5 and p < 0.05 for each comparison at each time point is found in To identify spinal cord changes in gene expression after MMC occurs in rat fetuses, we compared RNA-sequencing data between MMC, control, and vehicle groups at E15, E17, and E20. Principal component analysis indicates segregation of transcriptomes from the MMC group compared to the vehicle and control groups at each time point . The numHierarchical clustering analysis indicates that vehicle and control groups cluster together compared to the MMC group at each time point studied . These dThe 25 genes that are most significantly upregulated and downregulated between MMC and vehicle groups at E15, E17 and E20 are listed in Collectively, these results provide initial evidence that downregulated genes at E15 and E17 may lead to a decline in neural function and upregulated genes at E20 result in inflammation and disruption in myelin sheath development in rat fetuses with RA-induced MMC. The top 10 enriched GO biological processes, cellular components, and molecular functions from upregulated and downregulated DEGs between MMC and vehicle at E15 are listed in At E17, upregulated DEGs were enriched in biological processes, including proximal/distal pattern formation and negative regulation of cell fate specification, cellular components, including myosin complex and collagen, and molecular functions, such as extracellular matrix structural constituent, actin filament binding, and growth factor binding A. At E17At E20, upregulated DEGs were enriched in biological processes, such as cellular response to interferon-gamma, and regulation of cytokine production cellular components, such as keratin filament, and molecular functions, such as GTPase activity and cytokine receptor binding A. At E20The top 10 GO biological processes, cellular components, and molecular function enriched from DEGs that are upregulated or downregulated in MMC spinal cords, compared to the control at E15, E17, and E20, can be found in p < 0.05. Interestingly, there were several common pathways changed at E15 and E17 when comparing MMC and vehicle groups  were determined for each time point by submitting the top 25 up and downregulated DEGs between vehicle and MMC groups to the string. At E15, we identified a cluster of interactions between upregulated DEGs involved in structure and skeletal muscle development including Acta1, Actn2, and Myog B. AdditiTo validate the sequencing results, the gene expression of 7–8 genes was randomly measured using qRT-PCR. The Log2fold change was determined between spinal cords isolated from MMC and vehicle groups at each time point and analyzed compared to results from the RNA sequencing . While tIn this study, we employed RNA sequencing to characterize the chronology of the most significant gene expression alterations in the spinal cord after defect development and exposure to the amniotic fluid in the congenital retinoic acid rat model of spina bifida. Models created from single or double mutants in mice do not always match the phenotype of human neural tube defect with the same mutation . For exaOur results suggest that a spina bifida defect is associated with alterations in the spinal cord gene expression that regulates aspects of cell survival and positioning, neuron function, and skeletal muscle development during E15 and E17 gestational points. Compared to fetuses with normal development, genes involved in p53 signaling were upregulated in fetuses with MMC at E15, a time point similar to gestational weeks 5–6 in humans . This paInterestingly, p53 and hedgehog signaling were also upregulated in fetuses with retinoic acid-induced MMC at E17, a time point similar to gestational weeks 20–26 in humans ,30. In aThe dysregulation of processes involved in metabolic pathways and myelination was also apparent in fetuses with retinoic acid-induced MMC at E20, a time point similar to gestational week 34 in humans . In contWe hypothesize that the changes in gene expression we observed after the spina bifida defect is at least in part due to exposure of the spinal cord to the amniotic fluid. Studies on amniotic fluid supernatant collected from pregnant women at the time of open defect identified alterations in pathways similar to our results including those associated with neuronal development, axonal development, and synapse formation . InteresIn conclusion, through a comprehensive time-course transcriptomic analysis, this is the first study that characterized the progressive changes that occur in the neural tissue after exposure to the amniotic fluid in utero, using the congenital retinoic acid-induced spina bifida rodent model. Current standard surgical strategies only structurally repair the defect and inhibit further neurological damage due to amniotic fluid exposure. Likely due to limited understanding of the mechanisms that drive spina bifida degeneration in utero, there are no treatments available that regenerate healthy neural tissue and function after spina bifida diagnosis. Our results provide evidence that different mechanisms may play more important roles during specific periods throughout fetal progression. Therefore, it may be beneficial to tailor new therapeutic strategies to the gestational age at the time of treatment as well as to entertain an approach where a combination of pathways is targeted. Furthermore, future studies further elucidating the specific targets at different gestational ages during disease progression should be a focus in the spina bifida research community.


In recent years there has been a growing interest in the use of proteins as biocompatible and environmentally friendly biomolecules for the design of wound healing and drug delivery systems. Keratin is a fascinating protein, obtainable from several keratinous biomasses such as wool, hair or nails, with intrinsic bioactive properties including stimulatory effects on wound repair and excellent carrier capability. In this work keratin/poly(butylene succinate) blend solutions with functional properties tunable by manipulating the polymer blending ratios were prepared by using 1,1,1,3,3,3-hexafluoroisopropanol as common solvent. Afterwards, these solutions doped with rhodamine B (RhB), were electrospun into blend mats and the drug release mechanism and kinetics as a function of blend composition was studied, in order to understand the potential of such membranes as drug delivery systems. The electrophoresis analysis carried out on keratin revealed that the solvent used does not degrade the protein. Moreover, all the blend solutions showed a non-Newtonian behavior, among which the Keratin/PBS 70/30 and 30/70 ones showed an amplified orientation ability of the polymer chains when subjected to a shear stress. Therefore, the resulting nanofibers showed thinner mean diameters and narrower diameter distributions compared to the Keratin/PBS 50/50 blend solution. The thermal stability and the mechanical properties of the blend electrospun mats improved by increasing the PBS content. Finally, the RhB release rate increased by increasing the keratin content of the mats and the drug diffused as drug-protein complex. Nowadays, one of the main challenges facing biomedicine is the need to be able to administer drugs and active ingredients in a controlled and targeted manner. Therefore, precision design principles have been recently adopted to develop new methods of administration, which can be varied according to the treatment required, and limit any side effects as much as possible.Currently, in the wide spectrum of biomolecules used for biomedical applications, natural proteins such as gelatin, collagen, keratin or silk fibroin are fascinating biomolecules owing to their availability and favorable properties such as biocompatibility, biodegradability and capability to mimic the in vivo environment of the human body . Among tHowever, the processing of keratin into structured materials is very challenging. Indeed, this protein is characterized by relatively low molecular weights and wide molecular weight distributions which confer keratin solutions with poor rhological properties for their processing. The blending of keratin with other both natural or synthetic biopolymers is a common strategy used to overcome the aforementioned drawback and to improve its processing in order to fabricate biomaterial systems and devices with tunable chemical, mechanical, biological and biomimetic properties according to the desired application ,7. As toAs examples, processing keratin solutions blended with other polymers e.g., poly-ethylene-oxide (PEO) , polyvinWithin this framework, recently proposed  electrosThis work is the continuation of the previous one, since the keratin/PBS blend solutions with different polymer blending ratios are electrospun in order to obtain nanofibers with functional properties tunable by acting on the polymer mixture composition. To this end, the stability of keratin in HFIP was studied by comparing the molecular weight distribution of the pristine protein with that of the protein regenerated from the solvent. Moreover, the relationships between keratin/PBS blending ratios and the rheological behavior of the processing solutions as well as the mechanical, thermal and adhesion properties of the electrospun mats were investigated. Finally, in order to understand the potential of such mats as drug delivery systems, electrospun keratin/PBS blend nanofibers loaded with rhodamine B (RhB) were prepared and the drug release mechanism and kinetics as function of blend composition was studied. RhB was chosen as model drug since it is easily detectable by UV-visible measurements.n) of 50,000 g/mol, was synthesized from dimethyl succinate (DMS) and 1,4-butanediol (BD) as described in a previous work [High molecular weight keratin powder (~50 kDa) extracted from raw wool was kindly donated by Kerline Srl . Poly(butylene succinate) (PBS), with a molecular weight , and left under stirring for at least one additional hour, still covered. The final solutions were then loaded into a 5 mL syringe connected to a needle  and electrospun towards a static collector (10 × 10 square centimeters) using an applied field of 1 kV/cm  and a flow rate of 1.8 mL/h. For each kind of sample, three production processes were repeated in order to validate the reproducibility of the process.The solutions for electrospinning were prepared first by separately dissolving PBS (520 mg) and keratin (520 mg) in HFIP (4 mL) containing RhB (3.9 mg/mL), in order to obtain solution at a polymer concentration of 13% ® Tetra Vertical Electrophoresis Cell . The samples were dissolved in a buffer solution of Tris-Hcl  containing DTT (140 mM) and urea (8 M). These keratin solutions were added to the 2XLaemmli sample buffer in a 1:1 ratio and boiled at 90 °C for 5 min in order to denature the protein. The separation was performed in a 4–20% gradient gel at 200 V for 45 min by using 20 μL of sample solution (10 μg/μL), for each well. When the separation was finished, the gel was rinsed with ultrapure water and stained overnight with a Coomassie Brilliant Blue R-250 staining solution. Destaining was done overnight in diluted acetic acid (5%).The molecular weight distribution of keratin powder and keratin regenerated from HFIP, were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), using a Mini-PROTEAN−1. Data were acquired and elaborated with the RheoCompass Software (Anton Paar).Rheological properties of the polymer blends solutions were determined carrying out shear rate-dependent viscosity measurements, using a MCR 102 Compact Rheometer  equipped with a PDT 200/56/l Peltier temperature control device, set at 25 (±0.1) °C, using a cone plate geometry , in controlled shear rate mode. The shear rate was ranged from 0.1 to 1000 sThe electrospun nanofiber morphology was investigated by scanning electron microscopy (SEM) using an EVO LS 10 LaB6 scanning electron microscope  with an acceleration voltage of 5 kV and working distances of 4.3 mm and 5.2 mm, respectively. Samples were gold-sputtered for 1 min before the analysis. The fiber diameters were obtained by using GIMP software . Two SEM images of the samples obtained from each production process were acquired. The mean diameter and the diameter distribution of nanofibers were evaluated from 150 measurements randomly gathered from all the acquired SEM images for each sample.−1 region, with 26 scans and a resolution of 4 cm−1. A TGA4000 instrument  was used to carry out thermogravimetric analysis (TGA). Measurements were performed heating each sample (≈5 mg) at 10 °C/min from 40 to 800 °C, under pure nitrogen flux (40 mL/min). The temperature of initial degradation (Tid) and the one corresponding to maximum degradation rate (Tmax) were calculated.Infrared spectra were acquired using the attenuated total reflectance technique (ATR) with a Vertex 70 interferometer  equipped with a diamond crystal single reflection platinum ATR accessory, in the 4000–600 cmg), denaturation/degradation temperature (Td) and melting temperature (Tm), together with the heat associated to these transitions, calorimetric analysis was performed by means of a Perkin Elmer DSC6 instrument, calibrated with indium and zinc standards. Weighed samples of about 10 mg were subjected to a I scan by heating at 20 °C/min from −70 °C to about 40 °C above melting temperature in case of PBS, up to 380 °C for the samples containing keratin.In order to determine the main thermal transitions, i.e., glass transition temperature  equipped with a 1 KN load cell. At least six rectangular specimens (5 × 50 suqre millimeters) were tested for each electrospun mat, adopting a 10 mm/min crosshead speed and a gauge length of 20 mm. Stress-strain curves were obtained from load- displacement data directly measured by the software. Stress (σB) and strain (εB) at break were calculated, together with elastic modulus (E), this last extrapolated from the initial linear slope of the stress-strain curves.Tensile tests were performed using a 5966 machine (InstronS), corresponding to maximum stress reached before the mat and cellulose acetate detached from each other, was measured. The same tests were also conducted in dry conditions: the specimens were prepared using the procedure described above, but they were stored at room temperature until complete drying before proceeding with the measurements.Adhesion tests were carried out at room temperature on electrospun mats obtained from PBS and from Keratin/PBS 50/50 blend, using a cellulose acetate membrane to simulate the human skin. More in details, rectangular strips with dimensions of 5 × 50 square millimeters were obtained from both electrospun mats and cellulose acetate membranes. Electrospun strips as well as cellulose acetate ones were immersed in distilled water for one minute, and dripped on filter paper, to simulate the humidity of human skin. Then, they were overlapped each other for a length of 2 cm. The so-obtained specimens, 6 for each sample, were then stretched at a constant speed (10 mm/min). The mean value of shear stress  was cut from each sample loaded with RhB and put in 6 mL of phosphate buffer with a pH of 7.4 and a temperature of 37 °C. Aliquots of 100 μL were removed at the specific time intervals and replaced with fresh buffer; the released RhB was detected at 554 nm using a UV-Vis spectrophotometer , after the determination of a calibration curve  is plotted as function of shear rate (η0 (Pa s) is the zero-shear n is the power law flow behavior index. The rheological parameters and the correlation coefficients (R2) of the fitting are shown in In n values are lower than 1 indicating that all the blend solutions exhibited well-behaved shear thinning properties [As shown, for all the blending ratios, the operties .Among the Keratin/PBS blend solutions, the 50/50 blend showed the highest zero-shear viscosity and the lowest infinite-shear viscosity, thereby representing a d stress . The lowAll the keratin/PBS blend solutions doped with RhB were successfully electrospun into free standing membranes as shown in B) and elongation (εB) at break are reported in Tensile tests have been carried out only on PBS, Keratin/PBS 30/70 and Keratin/PBS 50/50 electrospun mats. Indeed, although the blend richest in keratin (Keratin/PBS 70/30) was electrospinninable, the obtained electrospun mat was too fragile to be subjected to these tests with the tools provided. The values of elastic modulus (E), stress (σS) are reported in S values (0.7 MPa for PBS vs. 1.5 MPa for Keratin/PBS 50/50): the shear resistance was therefore more than doubled in the case of the blend, thanks to the very high adhesion conferred by keratin. Moreover, the detachment of keratin/PBS 50/50 electrospun mat occurs after a slight elongation, indicating that this material is more prone to deform rather than detach from the synthetic skin (S *) although slighlty lower than the one obtained in wet conditions, is a further proof of how keratin contributes to enhance the adhesion properties of the blend electrospun mat.In order to check the adhesion ability of the materials under investigation, in vitro adhesion tests on PBS and Keratin/PBS 50/50 mats were performed, in both wet and dry conditions, using membranes of cellulose acetate as synthetic skin model . The valtic skin . Adhesioid) and of maximum weight loss rate (Tmax) are collected in The thermal stability of keratin, PBS and their blend nanofibers has been analyzed by TGA measurements. The temperatures of initial degradation  are shown in d) and the transitions typical of PBS (Tg and Tm), with intensities consistent with the composition of the blends.Conversely, keratin shows three endothermic phenomena, which differ both in shape and intensity: the first one, located at 88 °C, is related to evaporation of water bounded with the protein, while the second at 226 °C, is attributed to the denaturation secondary structures of keratin, in particular α-helix and the one at higher temperature (282 °C), ascribed to the protein degradation in agreement with TGA data. The DSC traces of the mats containing both PBS and keratin show both the phenomena characteristic of pure keratin ):nofibers .In order to better understand the polymeric matrix behaviour during RhB release, FTIR spectra of the samples before and after the release tests were compared .−1). The FTIR spectra of the starting samples (pre-release) show the typical adsorption peaks associated to the amide I (1650 cm−1) and amide II (1590 cm−1) of keratin [In amide I 150 cm−1 aIn this study, keratin/PBS blend solutions with different blending ratios were successfully electrospun into nanofibrous mats using HFIP as common solvent. All the blend solutions displayed a shear thinning behavior which is more pronounced in the Keratin/PBS 50/50 blend solution. Moreover, the Keratin/PBS 30/70 and 70/30 blend solutions displayed a higher attitude of the polymer chains to orient along the applied shear, giving rise to nanofibers with narrower diameter distributions. The addition of PBS contributed to improve the mechanical properties of the blend electrospun mats, making possible to obtain free-standing and processable mats, even if it has been impossible to measure the mechanical parameters of the sample richest in keratin. Conversely, compared to PBS electrospun mat, the Keratin/PBS 50/50 blend one was characterized by a better adhesion to cellulose acetate membranes, used to mimic human skin, both in wet and in dry conditions, confirming the unaltered adhesion capability of keratin in the blend. The thermal stability of the blend nanofibers ranged from those of keratin and PBS, and it increased by increasing the PBS content. Furthermore, the RhB release strongly depended on the nanofibers’ composition. In particular, a faster RhB release was obtained for the samples richer in keratin. Finally, the release occurred through a drug-protein complex and by means of a Fickian diffusion mechanism.The results obtained for the single electrospun mats are meaningful for the design of patches with desired and tailored properties such as thermal and mechanical behavior, adhesion properties as well as drugs loading and drug release profiles. These patches can be engineered simply by exploiting the additive manufacturing of the electrospinning process through a layer-by-layer deposition of electrospun mats.


AMER1, INVS and OCRL variants were found in unrelated probands and were first reported in a BA cohort. Liver specimens and blood samples showed identical variants, suggesting that somatic variants were unlikely to occur during morphogenesis. Consistent with earlier attempts, this study implicated genetic heterogeneity and non-Mendelian inheritance of BA.Biliary atresia (BA) is a progressive inflammation and fibrosis of the biliary tree characterized by the obstruction of bile flow, which results in liver failure, scarring and cirrhosis. This study aimed to explore the elusive aetiology of BA by conducting whole exome sequencing for 41 children with BA and their parents . We exclusively identified and validated a total of 28 variants  in 25 candidate genes from our BA cohort. These variants were among the 10% most deleterious and had a low minor allele frequency against the employed databases: Kinh Vietnamese (KHV), GnomAD and 1000 Genome Project. Interestingly,  The disease occurs in the early stage of neonates and can be treated by hepatic portoenterostomy or Kasai operation2. After surgical treatment, however, approximately 50% of affected infants require liver transplantation, while the rest would sustain their own liver up to the age of 5–10 years3. A study on Vietnamese BA patients reported that 84% and 71% of Kasai-treated patients survived after 1–2 years, respectively. Additionally, the respective ratios were 52% and 28% for the group without Kasai treatment4. It is estimated that after hepatic portoenterostomy operation, 70–80% of patients with BA still require liver transplantation by adulthood due to the progressive development of liver scarring, failure and cirrhosis6.Biliary atresia is a progressive inflammation and fibrosis of the biliary tree that consequently results in the development of cholestatic liver disease. BA was first described by surgeon John Thomson in 18927. Regarding genetic aspects, debate over the Mendelian mechanism of the disease has been raised due to a lack of familial BA and a discordant presentation of BA in the monozygotic twin8. Nevertheless, some cases with familial BA have been reported, suggesting that either a recessive autosomal inheritance or a combination of genetic and acquired factors might contribute to the disease’s aetiology12. In addition, some studies have examined an association between BA and microchimerism, where the genetic trait is maternally transferred from the mother and later contributes to phenotypic heterogeneity and non-Mendelian inheritance14. More specifically, a heterozygous transition CFC1:c.433G > A in 5 BA patients with polysplenia syndrome implies a genetic predisposition to BA splenic malformation15. In a mouse model, inactivation of the hepatocyte nuclear factor 1 beta gene (Hnf1β) causes abnormalities of the gallbladder and intrahepatic bile ducts, resulting in severe jaundice16. Observations of an increased incidence of BA in some groups, such as Asian and Polynesian populations, suggest that genetic and environmental factors might cause the disease. Recent genetic studies have revealed a linkage between cholestatic jaundice and genetic predispositions in both nuclear DNA and mitochondrial DNA20.Although BA has been extensively studied, its aetiology and pathogenesis remain elusive. Several hypotheses explaining the cause of the disease, including viral infection, autoimmune-mediated bile duct destruction, biliary toxin, and genetic abnormality, have been proposed21. The disease occurs more frequently in Southeast Asia and the Ocean Pacific22. It is approximately 1 in 5000 live births in Taiwan compared to 1 in 14,000–20,000 in North America or Western Europe24. To our knowledge, there is no epidemiological study of BA in Vietnamese, as is the prevalence of this fatal disease. The prevalence of BA in Vietnam is estimated to be as high as 1 in 2400 live births as equal to that of the Ocean Pacific regions22. Although BA and BA-related liver diseases are often observed in Vietnamese infants and are life-threatening diseases, few studies have been reported thus far26. To date, the Kasai portoenterostomy procedure has been introduced as a routine surgical practice and offers a better opportunity to patients25. However, a number of patients still require liver transplantation after the operation or have a low quality of life due to the disease’s complications. Recently, next-generation sequencing (NGS), particularly whole exome sequencing (WES), has been increasingly applied for detecting variants in patients with cholestasis27. It appears to be a powerful tool to aid diagnosis and to provide timely and accurate therapeutic treatments. Therefore, we aimed to investigate the genetic pattern of BA by conducting a family-based WES for children with BA in hope of exploring new and characterized causative variants, which would shed light on the aetiology of the deadly disease.The prevalence of BA is 1 in 8000–18,000 live births and varies among countries and groups, with a dominance of females over malesBA diagnosis was based on intraoperative findings and liver biopsy. Patients with confirmed BA and their parents were recruited at Vinmec International Hospital and Vietnam National Hospital of Pediatrics in Hanoi from May 2019 to May 2020. A written informed consent form was provided to the parents for their participation. The study was approved by the Ethics Committee of the hospitals in accordance with the Declaration of Helsinki.Approximately 2 mL of peripheral blood from patients and their parents was collected in an EDTA anticoagulant tube and stored at − 80 °C. Liver wedge specimens were collected from the Kasai operation, snapped frozen in liquid nitrogen and stored at − 80 °C. Genomic DNA was extracted by using a DNA Mini Blood Isolation Kit based on the manufacturer’s protocol . DNA samples were quantified by fluorescence using a Qubit BR Quantification Kit . Extracted DNA samples were preserved at − 80 °C for future uses.Exome sequencing libraries were prepared by using a Nextera Rapid Capture Kit  based on the manufacturers’ protocol with slight modifications. The library concentration was quantified by a Qubit dsDNA Broad Range Assay Kit . Library size was measured by using a LabChip 3 K Hisense Kit . Paired-end exome sequencing with 150 bp cycles was performed on a HiSeq 4000 , targeting an averaged depth of 100X.28. Reads with low quality, adapters and noise were removed prior to the downstream analysis by using FastQC and Trimmomatic. Reads were aligned with the reference genome GRch38 version29. Bowtie2, BWA and Qualimap were used for quality control30. To minimize the false-positive rate, multiple variant calling tools, including the Genome Analysis Toolkit (GATK)31, SAMtools mpileup32 and Freebayes33, were mutually used.Variant calling and annotation were performed based on highly regarded tools34, GnomAD (https://gnomad.broadinstitute.org/) and 1000 Genome Project29; (ii) inclusion of variants with 3 types of inheritance modes: X-linked, homozygous and putative de novo; and (iii) variants with a CADD Phred score of > 10, indicating the 10% most deleterious variants in the genome35. In silico prediction tools, including SIFT36, PolyPhen-237, Mutation Taster38, I-Mutant39 and HOPE40, were employed to predict the impact of genetic changes. Molecular Signatures Databases (MSigDB v7.2) were used to compute the candidate genes with the gene sets of human phenotype ontology42.A stringent strategy was applied for variant classification, including (i) inclusion of rare and nonsynonymous variants with a minor allele frequency (MAF) < 1% against three databases: the Kinh Vietnamese population (KHV) obtained from our previous study on the Vietnamese human genome databaseSelected variants were then confirmed by bidirectional Sanger sequencing. Proper primers were designed for these variants, followed by PCR amplification and sequencing on an ABI 3500 DX system using a BigDye Terminator v3.1 .The study was approved by the Ethics Committee of Vinmec International General Hospital JSC (Decision No. 48/2019/QD-VMEC).A written informed consent form was provided to the parents prior to their participation.The participants provided consent for publication of all relevant data and this manuscript.We recruited a total of 42 children who had been diagnosed with BA based on intraoperative findings and liver biopsy. All patients showed typical BA symptoms, such as prolonged jaundice, acholic stool and abnormalities of the biliary tract at early infantile. The patients, including 23 males and 19 females born from 2009 to 2019, but the majority of patients were born in recent years. All patients underwent Kasai surgery immediately after birth (mostly after their 2 months of life), but the concentrations of bilirubin and serum enzymes indicating liver function, such as ALP, ALT, AST and γ-GT, remained high at the time of enrolment  , INVS (BA014 and BA041), and OCRL (BA032 and BA041). Noticeably, proband BA014 carried an INVS de novo variant, while proband BA041 carried an INVS homozygous variant . Additionally, we did not detect any significant variants based on our rationales for variant classification (Table The detected variants showed extremely low MAFs against three employed databases: Kinh Vietnamese (KHV), GnomAD and 1000 Genome Project Table . We notiof HACE1, PHKA1, XIAP, and AMER1 (c.A1075 > T), POF1B, MAOA, BCAR1, FOCAD, ARSF and OCRL variant, while the rest varied from tools , indicated that the candidate genes felt into various human phenotype gene sets, ranging from gonosomal inheritance and X-linked recessive inheritance to involuntary movements Table . However43, we did not identify any variants in some genes that have been previously suggested to be associated with BA or BA-related diseases, such as PKD2 , CFC1 (polysplenia), JAG1 (Alagille syndrome) and PKD1L1 . We also did not find significant variants in the susceptibility loci of ADD3, XPNPEP1, GPC1, ARF6 and EFEMP1, as suggested by GWAS44.Similar to a previous study45.Similar to other previous studies, we attempted to reveal the genetic pattern of BA disorder by conducting trio-based exome sequencing for 40 families involving 41 children with BA. Going beyond this establishment in a genetic study for such a rare and complex disorder, we further tested our hypothesis of whether the detected variants occurred in somatic or germline cells by sequencing both blood and available liver specimens obtained from our BA cohort. Due to the complexity of BA, we applied a stringent bioinformatics pipeline and tight quality control to determine either the rarest variants or putative de novo events from our BA cohort, which would avoid a huge number of variants as often experienced from mass sequencing. Taking this straightforward principle enabled us to end up with a total of 28 variants in 25 respective genes. Identical variants detected from blood and liver samples allowed us to rule out the occurrence of somatic variants in the development of the disease as previously hypothesizedAMER1, INVS and OCRL. While the aetiology of BA remained unclear and was unlikely to follow the Mendelian model, our results implicated their role in the disease's development. Overlapping findings of BCOR, INVS and OCRL in the Vietnamese BA cohort with a large comprehensive ciliopathy and biliary genes of interest in the previous study43 further supported the possibility of the causative role of these genes in BA. AMER1 (MIM#300647) encodes APC membrane recruitment protein 1, which acts as an inhibitor of the canonical Wnt/beta-catenin signalling pathway46 and controls hepatobiliary development during embryogenesis. In mature healthy liver cells, it is mostly inactive, and the abnormal Wnt/beta-catenin signalling pathway can promote the development of liver diseases47. AMER1 associates with osteopathia striata with cranial sclerosis48 and Wilms tumour development51. The gene is involved in the activation of the Wnt/beta-catenin signalling pathway, which drives hepatocarcinoma and cholangiocarcinoma52. In addition, analysis of the effect of genetic predispositions of AMER1 variants indicated that they were damaging because the alternated residues were located in highly conserved positions. The alternations might lead to destabilization of the local conformation and a loss of protein interaction  was de novo, and a loss-of-function variant with a CADD score of 37 and its allele frequency was absent from all employed databases. INVS encodes inversin protein, which plays a role in primary cilia function and is involved in the cell cycle. Intriguingly, inactivation of INVS in a mouse model shows a significant increase in bilirubin levels compared to that of the wild-type and pathogenic changes in ductal plate malformation in the intrahepatic biliary of the mutant mouse53. The association of INVS with BA had not been previously established due to an absence of INVS variants detected in BA patients55. However, INVS is associated with infantile nephronophthisis type 258. In our study, we detected an INVS heterozygous de novo variant and a homozygous variant from 2 BA unrelated patients (BA014 and BA041). To our knowledge, this novelty is first reported in BA patients, although future studies are needed to clearly explore the role of INVS in BA development. Similar to BCOR and INVS, OCRL encodes inositol polyphosphate 5-phosphatase, which might be involved in primary cilia assembly. OCRL has been widely reported to be linked to Lowe and Dent syndrome, where clinical manifestations often overlap with Zellweger spectrum disorders, characterized by low muscle tone, feeding difficulty, seizures and liver dysfunction61. Likewise, a lack of an association of OCRL and BA or liver diseases remains a gap for future investigation.Our study highly suggested 62. In BA studies, mitochondrial DNA has been found to associate with BA, suggesting the role of mitochondria in underling the pathogenic mechanism17. WES has revealed dozen candidate genes either encode ATP-binding cassette transporters (the ABC superfamily)19 or are involved in the Notch signalling pathway, such as JAG163 and NOTHC220. GWAS have highlighted a strong association between BA and some variants in the ADD3 gene located on 10q24.264. Another subsequent study on 171 BA patients and 1,630 controls of European descent found the strongest signal at rs7099604 in the ADD3 gene65. A significant association was found between variant rs17095355 on the XPNPEP1 gene and the disease66. Taken together, the aetiology of BA remains challenging due to the involvement of multiple genes and complex mechanisms. Being encouraged by the pioneers, we provided a concrete genetic aspect obtained from an exome trio-based study of a Vietnamese BA cohort. The findings add to our knowledge of the genetic heterogeneity and complexity of BA disorder.As a result of a rapidly declining cost of DNA sequencing, dozens of rare and previously undiagnosed genetic disorders are currently detectable. For the last 10 years, NGS technology has revolutionized our understanding of human genetics with a high level of accuracy, cost effectiveness and high throughput capability. NGS is steadily becoming a standard in routine diagnostic practicesAMER1, INVS and OCRL. Identical variants detected from blood and liver wedge specimens from each BA individual suggested that somatic variants in the liver cells were unlikely to occur during morphogenesis. Taken together, we highlighted the genetic heterogeneity of BA and ruled out the Mendelian model. Future studies are needed to further explore the roles of these genes in the development of BA.The aetiology of BA remains challenging because there is a lack of conclusive evidence despite extensive research and medical practices for hundreds of years. However, the recent development of NGS technology and its application in studies of BA and liver diseases have gradually revealed the hidden genetic picture of BA aetiology, where dozens of BA-associated genes have been found. Our study identified 28 variants in 25 genes  from 41 children with BA. These variants were in the 10% most deleterious and were either rare or extremely rare in the population genome database. A combination of functional prediction and analysis of biological processes enabled us to suggest these candidate genes for the development of BA, particularly with those detected in unrelated BA individuals, including Supplementary Information.


Sus scrofa pigs. Keratin–alginate composite sponges supported the enhanced proliferation of human dermal fibroblasts compared to alginate-only sponges and exhibited decreased contraction in vitro when compared to keratin only sponges. As dermal substitutes in vivo, the sponges supported the expression of keratin 14, alpha-smooth muscle actin, and collagen IV within wound sites, comparable to collagen sponges. Keratin–alginate composite sponges supported the regeneration of basement membranes in the wounds more than in collagen-treated wounds and non-grafted controls, suggesting the subsequent development of pathological scar tissues may be minimized. Results from this study indicate that crosslinked keratin–alginate sponges are suitable alternative dermal substitutes for clinical applications in wound healing and skin regeneration.Deep partial-thickness burns damage most of the dermis and can cause severe pain, scarring, and mortality if left untreated. This study serves to evaluate the effectiveness of crosslinked keratin–alginate composite sponges as dermal substitutes for deep partial-thickness burns. Crosslinked keratin–alginate sponges were tested for the ability to support human dermal fibroblasts in vitro and to support the closure and healing of partial-thickness burn wounds in  Interest in the use of materials derived from natural sources for clinical applications has been consistently high, primarily due to the rationale that materials derived from natural sources offer better cell and tissue compliance. The processing of natural materials through decellularization and removal of antigenic elements ensures that these materials can be implanted without stimulating significant immune response . MateriaHuman hair keratin presents exciting new possibilities to the field of biomedical science as an abundant, bioactive, affordable, sustainable, and potentially autologous source of human-compatible material . In rece4β1 integrins while also being critical contributors to cutaneous wound healing and skin regeneration . M. M13]. MIn vitro cytocompatibility of the crosslinked keratin–alginate sponges was evaluated based on their ability to support the viability, proliferation, and colonization of HDFs . QuantitLive–dead staining of colonized sponges revealed that HDFs form small colonies with increased cell densities in all groups from day 7 to 14. Collagen sponges supported more proliferation of HDFs after 7 and 14 days than all other substrates, which is consistent with the dsDNA quantification assay results. Notably, collagen also supported the typical elongated spindle morphology of fibroblasts b. HDFs gClassical hematoxylin and eosin (H&E) staining was used to evaluate the overall cell distribution and anatomy of the scaffolds. It was evident that HDFs were uniformly distributed in collagen, keratin, and keratin–alginate sponges c. At higIn addition, the de novo biosynthesis of extracellular matrix was evaluated, using immunohistochemistry to identify and localize collagen type III and fibronectin a. The anThe long-term interests encompass using the crosslinked keratin–alginate sponges for wound-healing applications; thus, it was important to evaluate how dermal cells might respond to the sponges and support wound healing. Analysis of cytokines in conditioned media recovered from HDF cultivated on different sponge materials was done. In particular, cytokines that contribute to angiogenesis were evaluated based on our previous observation that keratin–alginate sponges supported more effective angiogenesis in vivo, such as Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), and Placenta Growth Factor (PlGF) b 13]. P. P13]. PGraft contraction is one of the important considerations of the suitability of a material as dermal substitutes. Therefore, the change in shape and size of the four types of sponges after 14 days colonization by HDFs was measured. Intriguingly, it was determined that keratin and keratin–alginate sponges displayed less contraction compared to collagen sponges a. CollagPrevious studies have evaluated the biocompatibility of the keratin–alginate sponges relative to commercially obtained collagen sponges using mice . HoweverEvidence for substantial cell infiltration into the grafts was evident within a few days of injury in all sample groups . Wounds The efficacy of re-epithelialization was corroborated by the positive keratin 14 immunohistochemistry results reported in Biomaterials can interact with cells and induce changes in cell phenotype and response such as cell proliferation, differentiation, and ECM production . It is kIn terms of ECM production, it was reported that fibroblasts have the tendency to spread anisotropically and the extent of spreading increased with fibronectin density . In the The contraction observed by the four groups in In the proliferation stage of wound healing, infiltrated monocytes differentiate into macrophages, which secrete and release multiple growth factors and cytokines to promote angiogenesis and stimulate fibroblast migration . EndotheRe-epithelialization occurs during the proliferative phase, and this happens via the migration of keratinocytes from the wound edges and from viable skin appendages in the dermis into the wound area. A basement membrane will form between the dermis and epidermis after re-epithelialization is complete. The basement membrane is important, as it anchors the epidermis for mechanical stability and forms a communication interface between the dermal fibroblasts and epidermal keratinocytes ,36. AlteKeratin was extracted from human hair in reducing conditions, as previously reported . Rat taiThe microarchitecture of the samples was examined with Scanning Electron Microscope  using secondary electron imaging (SEI) mode. Prior to being imaged, samples were snap-frozen in liquid nitrogen and cut to expose their cross-sections. Samples were mounted onto sample holders using carbon tape and sputter-coated with gold at 18 mA for 15 s. SEM images were recorded at an accelerating voltage of 5 kV and spot size of 8 nm.The keratin–alginate sponges were analysed using micro computed tomography (microCT) . The microCT operated with a cone beam originating from a 4 µm focal-spot X-ray tube. Photons were detected using an area-based charge-coupled device (CCD) detector and rendered as a 3400 × 3400 image matrix. Physical parameters were calculated using the maximum fitted spheres method .3, and cultured at 37 °C in 5% CO2, 95% air; media was changed every 3 days. Evaluation of cell viability and cell proliferation were determined using Live/Dead® staining and Picogreen® assay , respectively, following the manufacturer’s guidelines.Primary HDFs  were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 nM non-essential amino acids, and 100 U/mL penicillin and streptomycin . HDFs were harvested when 90% subconfluent using 0.25% trypsin, 0.9 mM EDTA, and counted using a hemocytometer. Sponges (4 mm in thickness and 16 mm in diameter) were seeded with HDF applied to the top surface at a density of 50,000 cells/cmAfter selected periods, samples were harvested and gently rinsed 3 times with phosphate-buffered saline (PBS), before being fixed in 4% paraformaldehyde in PBS overnight at 4 °C. The samples were dehydrated through a graded series of 70, 80, 90, and 100% ethanol, before clearing in 100% xylene. After clearing, the samples were infiltrated with paraffin wax at 65 °C for two 2-h cycles; 5 µm thick sections were collected on glass slides and processed for staining with hematoxylin and eosin  using the manufacturer’s recommended protocol.The biosynthesis of ECM by HDFs cultured in the 3D sponges was evaluated using immunohistochemistry. The sections were heated to 65 °C for 30 min, dewaxed in 100% xylene, and rehydrated using graded ethanols of 100, 90, 80, and 70%, followed by water. Rehydrated sections were subjected to heat-induced epitope retrieval by heating to 120 °C in Dako Target Retrieval Solution  in a pressure cooker  . Slides The presence of cytokines was assayed using the Proteome Profiler™ Human Angiogenesis Antibody Array from R&D Systems, Minneapolis, MN, USA. Briefly, supernatants from individual samples were recovered after 14 days of culture, centrifuged briefly to remove cell debris, applied to the nitrocellulose array (blocked with buffer supplied by the manufacturer), and incubated at 4 °C overnight, according to the manufacturer’s instructions. Subsequently, each array was washed 3 times with the supplied washing buffer and then incubated with the streptavidin–horseradish peroxidase (HRP) working solution (1:200) for 30 min at room temperature. The arrays were washed 3 times with the washing buffer and placed onto individual plastic sheet protectors. A Chemi reagent mixture (chemiluminescent reagent) was applied onto each membrane, incubated for 1 min, and chemiluminescence was recorded for 2 min using an image analyzer . Images were analyzed for signal intensity using Image Studio™ Acquisition software . Signal intensities were normalized to the cell numbers and used for the direct comparison of cytokine expression levels.The contraction of the collagen, keratin, keratin–alginate, and alginate sponges were measured manually, using a ruler placed onto the upper surface of each sample. Measures were recorded at the start and after 14 days of culture, and contractions were calculated as percentage change from the original diameter.Sus scrofa male pigs  were used in the study. Anaesthesia was induced via intramuscular administration of ketamine and diazepam at a dose of 15 mg/kg body weight and maintained with inhalation of 2–3% isoflurane. At day 0, the pigs were shaved, and six 8 cm diameter wounds were created on the flanks of each pig using a modified Pyrex bottle filled with hot water and applied for 40 s to cause a deep partial-thickness burn , PELNAC™ (referred to as collagen sponge)  , keratin–alginate sponge ). Prior to use, the collagen sponge was immersed in PBS, leaving the protective silicone layer intact. These were applied onto the wounds with the silicone layer facing outwards. Keratin–alginate sponges were placed on the wound bed and dressed with Tegaderm, which were additionally sutured in place. All wounds were dressed externally with Vetrap™  and non-stick Urgotul , tulle gras, Opsite, gamgee, crepe bandage, and a protective garment to prevent the animals from disturbing their dressings. For dressing changes, the animals were sedated with an intramuscular dose of ketamine/xylazine . Every wound was inspected and photographed during each external dressing change to record wound parameters. Punch biopsies were collected during these time points from each sample group for histological assessment. The biopsies collected were fixed in 10% formalin and embedded in paraffin. Dressings were changed at days 6, 14, 16, 22, 30, 37, 44, and 51 post-injury. Animals were euthanized on day 51.The animal study was approved by the SingHealth Institutional Animal Care and Use Committee (IACUC 2016/SHS/1209) and performed under the Guidelines on the Care and Use of Animals for Scientific Purposes developed by the National Advisory Committee for Laboratory Animal Research (NACLAR). Two ess burn . The wouTM Mounting Medium . Images were captured using an Olympus IX53 microscope .In order to evaluate re-epithelialization, vascularization, and the formation of granulation tissue, immunohistochemistry was done on formalin-fixed, paraffin-embedded tissue sections acquired from each wound. After deparaffinization and rehydration, samples were subjected to heat-induced epitope retrieval; slides were immersed in Dako Target Retrieval Solution  and heated to 120 °C for 20 min in a pressure cooker  . Residuap < 0.05.Quantitative results are represented as mean ± standard deviation. Comparisons of means were carried out using ANOVA and Tukey’s post hoc analysis. Statistical significance was accepted when As proven in the previous study, the presence of alginate in the keratin–alginate sponges helped to increase the tensile strength, tensile modulus, and compression modulus compared to keratin alone . In this


Previous studies has shown that dry eye test abnormalities, meibomian gland dysfunction (MGD), may occur in psoriasis.The authors aimed to evaluate the dry eye disease (DED), MGD, in psoriasis patients with meibography which is a current, objective, noninvasive method for patients with meibomian gland diseases, to investigate the relationship between disease severity and ocular involvement.This study included 50 participants with psoriasis and 50 healthy individuals. All subjects were examined by the same dermatologist and referred for ophthalmological examination including meibomian gland obstruction, lid margin alterations assessment, ocular surface disease index assessment, tear film break-up time test, Schirmer test, corneal conjunctival fluorescein staining assessment. Additionally, upper and lower lids were evaluated for meibomian gland loss with meibography.MGD (28%), meibomian gland loss (MGL) (29.5%), upper meiboscore (0.61 ± 0.81), lower meiboscore (0.46 ± 0.61), DED (22%) were significantly higher in the psoriasis group compared with the control group . There was a significant relationship between MGD and psoriasis area severity index (PASI) . There was a significant positive relationship between MGL with PASI  and psoriasis duration .Smaller study group and inability to detect quality changes of meibum with meibography were limitations of the study.Psoriasis may affect the meibomian gland morphology, may cause structural changes in meibomian glands, and as a result of these may cause MGD and DED. Therefore, ophthalmologists and dermatologists should be aware of this situation and co-evaluate the patients in this respect. Psoriasis is a chronic inflammatory disease that affects 2%–5% of the world population with multiple extracutaneous manifestations.The meibomian glands (MG) are sebaceous glands located in the eyelids, and that are responsible for secreting the lipids which play an important role on the ocular surface by preventing the evaporation of tears.Meibomian gland dysfunction (MGD) is a chronic disease characterized by terminal duct obstruction and/or quantitative-qualitative changes in secretions.MGD can cause or exacerbate dry eye symptoms, and approximately two-thirds of patients with dry eye disease (DED) have MGD.21In this study, the authors aimed to evaluate DED and MGD using meibograpy as well as the current diagnostic criteria for patients with psoriasis.This study was performed with the Institutional Review Board protocol approval, date 19.04.2019 and number 03/59 in Aksaray University Research and Training Hospital, Department of Dermatology and Ophthalmology between May 2019 and August 2019. All study procedures were performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants before the study.This single-centered, prospective-controlled study included 50 participants (50 eyes) age range from 18 to 65 years, who were clinically and histopathologically diagnosed with psoriasis vulgaris. A parallel (by gender and age) healthy control group  without a family history of psoriasis was also included in the present study.Participants with ocular infection, allergy, ocular surface disorder, history of ocular surgery or injury, users of topical or systemic drugs which may affect the ocular surface, and users of contact lens were excluded from the present study. Patients who have any dermatologic disease other than plaque-type psoriasis vulgaris and who have systemic diseases and malignancies and patients with Psoralen Ultra-violet A (PUVA) treatment history were not included to the study.The disease severity was assessed using Psoriasis Area and Severity Index (PASI). The severity of plaque psoriasis was graded into mild and moderate to severe disease. The mild disease was defined as PASI ≤ 10, and moderate to severe disease was grouped as PASI > 10.Examinations and tests were performed sequentially as follows: Upper and lower eyelids were assessed with a microscope for obstruction, telangiectasia, irregular lid margins (notching), and a mucocutaneous junction shift. After the examination, fluorescein staining of the ocular surface, Tear Film Breakup Time (TFBUT) testing, Schirmer test, and meibography were performed, and the results of these tests were recorded.The diagnostic criteria recommended by the MGD Study Group in Japan were used for the MGD diagnosis.The dry eye disease diagnosis was ascertained using the modified Tear Film & Ocular Surface Society Dry Eye Workshop II (TFOS DEWS II) Criteria: OSDI > 13 plus one among TFBUT <10 s, Schirmer test score <10 mm, or corneal and conjunctival staining >0.t-test  and the Mann-Whitney test  were used to compare the means of numerical variables between the two groups. The Chi-Square test was used to compare the means of categorical variables between the two groups. Spearman’s correlation analysis was used to determine the relationship between the numerical variables that were normally distributed. Binary logistic regression analyses were applied to compute odds ratios for the associations between the explanatory variables; p-values of <0.05 were considered statistically significant.The statistical analysis was performed using SPSS version 23.0 for Windows software . The Shapiro-Wilk test was used to assess whether the distribution of the numerical data was normal. The independent sample Patients diagnosed with psoriasis (n = 50) in this study were 26 (52%) female and 24 (48%) males, and the mean age of 50 patients was 43.4 ± 14.1  years. In addition, 25 (50%) of the control group were female and 25 (50%) were male. The mean age of the control group (n = 50) was 41.2 ± 8.1  years. There were no statistically significant differences in age and gender between the two groups (p > 0.05).According to the clinical anamnesis of the patients, 4 of 50 patients (8%) had subjective ocular symptoms, such as itch, burning, and stinging.The frequency of MGD was 28% (n = 14) in the psoriasis group, and 6% (n = 3) in the control group (p = 0.008). A comparison of Meibomian gland occlusion, telangiectasia, mucocutaneous junction shifts, and lid margin irregularity (notching) is presented in MGL was 29.5% in the psoriasis group and 9.5% in the control group (p < 0.001). A comparison of the upper and lower meiboscores is shown in The frequency of DED in the psoriasis group was 22% (n = 11), whereas it was 6% (n = 3) in the control group (p = 0.044). The comparison of the OSDI, TFBUT, Schirmer test, and the corneal staining score is presented in Spearman’s correlation test was performed between MGL, PASI scores, and the duration of psoriasis. A significant positive correlation was found between MGL and PASI scores  as well as the duration of psoriasis .The relationship between MGD and age, gender, duration of psoriasis, and PASI scores were assessed with a binomial logistic regression test. A significant relationship was found between MGD and PASI scores . No significant relationship was observed between age, gender, and duration of psoriasis (p > 0.05) .Table 3RThe relationship between DED and age, gender, duration of psoriasis, and PASI scores were evaluated with a binomial logistic regression test. A significant relationship was found between DED and age . No significant relationship was observed between gender, duration of psoriasis, and PASI scores (p > 0.05) .The data from this present study represent the first report on higher MGL, MGD, and DED in the psoriasis patients than the healthy population with using meibography technique and their positive relationship with disease severity.In previous studies about psoriasis and MGD which were performed without meibography according to different diagnostic criteria, MGD was found high in patients with psoriasis.Dry eye test abnormalities in patients with psoriasis have been shown in several studies.The mechanism by which psoriasis induces MGD is not clear. Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperproliferation and hyperkeratinization.There are some limitations to this study. First of all, the study included a relatively small number of patients. Secondly, the meibography method only shows morphological changes in the Meibomian glands, so the authors did not determine quality changes in the meibum.According to the results of this study, the authors suggest that psoriasis may affect the Meibomian gland morphology, it may cause structural changes in Meibomian glands, and may cause MGD and DED. Meibography is a current objective method, a non-invasive approach which has been widely preferred by clinicians for patients with meibomian gland diseases, and it has allowed many studies about MGD to be carried out. This study is the first report which evaluated MGD in psoriasis patients using meibography technique and their positive correlation with disease severity. On the other hand, the dermatological literature has generally not adequately pointed these complications; however, a thorough understanding and management of ophthalmic involvement are important for the comprehensive care of patients with psoriasis.None declared.Funda Kemeriz: Approval of the final version of the manuscript; critical literature review; data collection, analysis and interpretation; effective participation in research orientation; intellectual participation in propaedeutic and/or therapeutic management of studied cases; manuscript critical review; preparation and writing of the manuscript; study conception and planning.Burcu Tuğrul: Approval of the final version of the manuscript; critical literature review; effective participation in research orientation; manuscript critical review; preparation and writing of the manuscript.Erdoğan Yaşar: Approval of the final version of the manuscript; critical literature review; data collection, analysis and interpretation; effective participation in research orientation; intellectual participation in propaedeutic and/or therapeutic management of studied cases; manuscript critical review; preparation and writing of the manuscript; statistical analysis; study conception and planning.None declared.