Nanoemulsion formulations composed of isopropyl myristate and Tween 80 encapsulating a fluorescent dye were applied topically on porcine nipples using a Franz diffusion cell. A combination of dye extraction and fluorescence image analysis was used to quantify the total amount of dye retained within the nipple and to characterize the penetration routes. After diffusion for 6 hours, the amount of dye deposited in the nipple was proportional to the formulation’s water concentration. The 90% water formulation deposited significantly more dye via both the stratum corneum and mammary ducts, while the 80% and 70% water formulations moderately increased ductal penetration, but minimally altered stratum corneum penetration as compared to the control solution. Similar trends were found after diffusion for 48 hours; however, the overall impact was diminished, likely due to the nanoemulsion’s topical instability. This study indicates that drug delivery vehicles, nanoemulsions specifically, enhance delivery of encapsulated molecules via the stratum corneum and mammary ducts in a formulation-dependent basis.While the feasibility of transpapillary drug delivery has previously been established, localized transport via the mammary ducts may be improved with tailored drug delivery formulations. The objective of this study was to investigate the impact of nanoemulsion encapsulation on transpapillary delivery  An even higher incidence of women are diagnosed with non-obligate precursor lesions such as atypical hyperplasia5. Despite an increased incidence of localized disease and a better understanding of its biology, the available treatment approaches, including a combination of surgery, radiation, chemotherapy or hormone therapy, could better be tailored for this patient population. The long- and short-term psychological and physical side effects associated with these treatments are important considerations for the 60–70% of women whose disease will not progress to invasive breast cancer6. Therefore, the estimated 160,000 women in the United States diagnosed annually with pre-malignant or non-invasive breast cancer5 may benefit from a localized treatment approach that avoids first-pass metabolism and thus systemic effects, while maximizing drug exposure within the breast.The incidence of non-invasive breast cancer, namely ductal carcinoma 7, but are the cell population that most commonly malignantly transforms, accounting for 95% of breast cancer cases8. We and others have demonstrated that diffusion through and retention within the mammary canals varies depending on a molecule’s and drug carrier’s physicochemical properties11. Specifically, lipophilic small molecules preferentially diffuse through the conduits, capitalizing on the ductal orifices as an entry point and transport pathway. Furthermore, drug carriers with diameters of at least 5 nm injected intraductally remain concentrated within the mammary ducts12, thereby limiting exposure within the surrounding breast stroma and distant extraneous organs. Enhanced targeting of the ductal orifices and passive diffusion through the canals may therefore be accomplished through design of tailored drug delivery vehicles.An optimal approach would directly expose the epithelial cells that line the mammary ductal network to a chemopreventive agent. These epithelial cells represent only 8% of the total cellular makeup of the mammary gland13 and have more recently become a promising approach for cancer prevention and treatment14. Their two-phase composition of immiscible liquids, oil and water, and a surfactant stabilizer yields droplets that are typically <1 μm in size, isotropic, and thermodynamically (microemulsions) or kinetically (nanoemulsions) stable16. The selection and proportion of each component drives spontaneous formation of one of three different microemulsion types: water-in-oil, oil-in-water, or bicontinuous17. For transfollicular applications, microemulsions with greater water content preferentially target the hair follicles more so than vehicles with lower water content or a control solution18. The advantages of micro- and nanoemulsions for topical/skin delivery has been reviewed by Sutradhar and Amin19 and Nastiti et al.20, both of which highlight the advantages of emulsion-based delivery and the influence of emulsion formulation on transport properties. The ability of emulsion systems to enhance penetration into a skin orifice suggests their potential advantage for transpapillary delivery as well.Microemulsion and nanoemulsion delivery platforms are the most commonly used system for topical skin technologies21. A 5% water formulation significantly decreased drug retention within the nipple without impacting permeation as compared to an ethanolic solution, while a formulation with 35% water significantly increased both drug retention and permeation. However, the specific routes of penetration were not addressed21. Therefore, the targeting capabilities within the ductal channels of emulsion-based formulations remain unknown. Our own previous investigation of transpapillary delivery, in which we used qualitative and quantitative assessments to analyze permeation at both a micro and macro scale, indicates that permeation occurs via two distinct pathways, transepidermal and transductal9. Herein, we sought to use similar techniques to elucidate the role of emulsion-encapsulation on transport via the mammary papillae.A previous study of microemulsion diffusion through the nipple indicated varied tissue retention and permeation depending on the microemulsion formulationBased on the ability of microemulsions to enhance transport via hair follicles and the mammary papillae on a formulation-dependent basis, we hypothesized that an emulsion-based formulation with high water content would improve the efficiency of ductal transport. The objective of the current study was to evaluate the impact of nanoemulsion encapsulation on transpapillary drug delivery, and in particular, transport into the mammary ducts using three nanoemulsion formulations of varying water concentrations. Each formulation encapsulated nile red (NR), a model fluorescent lipophilic dye, for ease of quantification and visualization of the penetration pathways.23. Three formulations with water percentages ranging from 70–90% along the 1:9 oil:surfactant dilution line were chosen for characterization and in vitro transpapillary diffusion , the nanoemulsions were assumed to be of oil-in-water type17.To determine the relative water, oil, and surfactant concentrations yielding a nanoemulsion using isopropyl myristate (IPM) and Tween 80 (T80) as the oil phase and surfactant, respectively, we systematically varied the concentrations of each component. A limited nanoemulsion domain was found in the area of high water and surfactant content, which is consistent with previous findings24. The droplets in each formulation were considered nanoemulsions, rather than microemulsions, as the size distributions were more heterogeneous (PDI > 0.126) and instability was observed following long-term storage at room temperature  were characterized for size, dispersity, and zeta potential. Each formulation had similar average diameters ranging from 9.57 to 12.30 nm Table . The polTo understand the impact of nanoemulsion encapsulation on dye penetration into the nipple, NR was extracted from tissue following diffusion of nanoemulsions or the control solution. After diffusion for 6 hours, only 0.06% of NR from the control IPM solution was deposited within the nipple Fig. . The nan28.When assessing dye retention as a function of tissue depth, the main differences between treatments were found in the uppermost tissue sections Fig. , where t21. Similarly, skin penetration and permeation of numerous lipophilic compounds from oil-in-water microemulsions with high aqueous percentages is more pronounced than from microemulsions with lower water content31, thereby increasing drug retention in both the stratum corneum and viable skin layers up to 3.7-fold30. The ability of microemulsions to increase skin hydration29 resulting in ultrastructural changes in the tissue that make stratum corneum transport more favorable32 coupled with copious amounts of the penetration enhancer water33 likely contributes to the improved deposition of drugs within tissue.Among the nanoemulsion formulations, a direct relationship was found between the percentage of water in the formulation and the amount of deposited NR following 6 hours of diffusion Fig. . The amo28. To microscopically evaluate the penetration differences between the nanoemulsion formulations and assess distinct penetration routes, image analysis was performed on fluorescence micrographs of nipple cross-sections. The plot profile tool in ImageJ was used to generate fluorescence intensity as a function of distance from either the stratum corneum edge (stratum corneum penetration) or the mammary duct edge  into the connective tissue  was higher for the nanoemulsion formulations, particularly for the 90% water formulation, than the IPM solution at nearly every tissue depth Figs  and 4a. 9. Image analysis kinetics of an ethanolic NR solution or sulforhodamine B solution in phosphate buffered saline (PBS) showed increased penetration distances over time with varied ductal edge fluorescence intensities, yet both solutions had a substantial reduction in the plot profile slope, indicative of the concentration gradient, between 30 minutes and 8 hours when diffusing via the ductal penetration pathway.Furthermore, between 6 and 48 hours of diffusion, the peak fluorescence intensities at the ductal edge either notably decreased (90% water formulation), remained relatively unchanged (80% and 70% water formulations) or increased (IPM solution)  markedly improved dye penetration via both the mammary ducts and the stratum corneum after diffusion for 6 hours. Meanwhile, the 70% and 80% water formulations moderately improved dye deposition via the ductal penetration route only, suggesting their targeting capabilities. The ability of nanoemulsion formulations to rapidly promote transport into the mammary ducts is an important practical consideration for drug delivery applications. Thus, future studies will broaden the nanoemulsion formulations considered with a focus on optimizing their stability and efficacy for transpapillary drug delivery.Nanoemulsions, consisting of IPM and T80, containing lipophilic fluorescent dye NR, successfully enhanced dye penetration into porcine nipple tissue Acetone, cytoseal60, ethanol, isoamyl alcohol, isopropyl myristate (IPM), Tween 80 (T80), nile red (NR) were purchased from Thermofisher. Abdominal porcine tissue was supplied from a local market and stored at −20 °C until use.22. IPM and T80 were used as the oil phase and surfactant, respectively. Isopropyl myristate and Tween 80 emulsion formulations have been used in transdermal delivery because of their ability to enhance skin permeation20, suggesting the potential advantage of their use for transpapillary delivery. First, IPM and T80 were mixed at varying w:w ratios, e.g. 1:9, 2:8, 3:7 etc. Then, distilled water was added incrementally to the oil and surfactant mixture. The samples were vortexed for one minute and kept at room temperature overnight before the next water titration. At each titration, the emulsion mixtures were visually inspected and categorized as a nanoemulsion when they appeared transparent and fluid.The nanoemulsion region was determined by constructing a pseudoternary phase diagram using the water titration methodFirst, varying concentrations of NR were solubilized in IPM, in some instances aided by vortexing and heating. Appropriate amounts of IPM containing NR and T80 were weighed into a glass vial and vortexed for one minute. Then, water was added drop-wise to the oil-surfactant mixture and again vortexed. The final nanoemulsion formulations , and zeta potential were measured by dynamic light scattering using a Nanobrook 90Plus PALS at room temperature (25 °C). The refractive index of water (1.331) was used for measurements considering the nanoemulsion formulations contained 70–90% water. The reported data is an average of the particle intensity values from quintuplicate measurements of three different preparations. CryoTEM was used to confirm the droplet size. A 4 μL sample was applied to a carbon mesh 200 copper grid, blotted and then flash frozen in liquid ethane. Images were acquired using a FEI Tecnai G2 F30 transmission microscope.36. The receptor compartment was filled with 5 mL of PBS or IPM, stirred using a magnetic stir bar, and maintained at 37 °C. Different solutions were used in the receptor compartment based on NR’s solubility and potential to diffuse across the entire tissue and reach the receptor chamber. Because numerous studies indicate that the lag time across the nipple is minimally 11 hours11, the receptor compartment was filled with PBS for the 6-hour treatments. For the 48-hour experiments, during which it was more likely that the NR would diffuse across the entire length of the nipple and into the receptor chamber, the receptor chamber was filled with IPM. After specified treatment times, the nipple was removed from the Franz diffusion cell and the surface was rinsed with 3 mL of ethanol before storage at −20 °C until further processing.Porcine nipple tissue was supplied from a local market and stored at −20 °C. Prior to use, tissue was thawed and underlying dermal fat was removed. The nipple was then sandwiched between the donor and receptor compartments of a Franz diffusion cell for 6 or 48 hours. The donor compartment was filled with 400 μL of the nanoemulsion formulation or control solution (IPM) incorporating NR at 0.04 mM. IPM was chosen as the control solvent due to its relevance as the nanoemulsion’s oil phase as well as NR’s limited solubility in an aqueous solution28. Briefly, frozen nipples were sectioned on a cryostat from tip to base in 100-μm increments. Every 1 mm, tissue was collected in a storage tube and the tissue weight was determined. Dye was then extracted by adding 350 μL of 6:1 isoamyl alcohol:acetone (v:v) to each tube and incubated at room temperature for at least 30 min. The dye concentration was determined by interpolation from a standard curve following fluorescence measurement on a Synergy H1 fluorimeter at 550/630 nm excitation/emission.NR was extracted from 1-mm tissue sections as previously reported37 was then used to calculate the fluorescence intensity as a function of distance  at 650 nm was measured in triplicate using a UV spectrophotometer (NanoDrop2000). The percent transmittance (T) was then calculated using the equation T = 10(2-A). To determine the droplet size, samples of the donor solution were collected, diluted 1:10–1:25 (v:v) with distilled water, and immediately analyzed using dynamic light scattering (NanoBrook 90Plus PALS).Nanoemulsion stability during topical application on the nipple was studied as a function of transmittance and droplet size. Nipples were arranged in a Franz diffusion cell for up to 48 hours as described in the GraphPad Prism software version 7  was used for all statistical analysis. A one-way ANOVA or two-way ANOVA with Tukey’s multiple comparison post-test was used for analysis of total retained dye or analysis of treatment by depth, respectively. All results were deemed significant when p ≤ 0.05.Supplementary Information


The open microdiscectomy is the most common surgical procedure for the decompression of radiculopathy caused by lumbar disk herniation. To date, a variety of minimally invasive (MI) techniques have been developed. In the last decades, endoscopic techniques have been developed to perform discectomy. The transforaminal endoscopic discectomy (TED) with posterolateral access evolved out of the development of endoscopic techniques.A systematic literature search was performed using the PubMed, EMBASE, and Cochrane Library databases for trials written in English. The randomized trials and observational studies that met our inclusion criteria were subsequently included. Two reviewers respectively extracted data and estimated the risk of bias. All statistical analyses were performed using Review Manager 5.3.p value < 0.00001). However, there were no significant differences in the leg visual analog scale (VAS) scores, the Oswestry Disability Index (ODI) scores, and the incidence of complications and recurrence.Five prospective and four retrospective studies involving 1527 patients were included. The results of the meta-analysis indicated that there were significant differences between the two groups in length of hospital stay (MD = − 8.41, 95% CI − 10.26, − 6.56; The transforaminal endoscopic discectomy is superior to open microdiscectomy in the length of hospital stay. However, there were no differences in leg pain, functional recovery, and incidence of complications between TED and MD in treating LDH. Lumbar disk herniation (LDH) is a common medical condition with a pathological process that leads to spinal surgery. The fibrous ring of an intervertebral disk is fractured and allows the soft central portion, the nucleus pulposus, to bulge out beyond the damaged fibrous rings. LDH is considered to be the most prevalent spinal disk herniation and always causes a series of signs and symptoms. One of the most challenging medical problems is sciatica symptoms. Sciatica affects millions of individuals worldwide . The nerCurrently, early conservative treatment is used when the symptoms are not serious. However, surgery is adopted when conservative treatment fails, or complaints worsen over time , 5. In 1The indications for transforaminal endoscopic treatment are similar to classical open microdiscectomy procedures , 18. HowThe standards set by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were used to construct this systematic review. The 27-item checklist and 4-phase flow diagram of PRISMA were both consulted.The PubMed, EMBASE, and Cochrane Library databases were searched up to January 2017 to identify studies comparing transforaminal endoscopic discectomy with microdiscectomy for the treatment of lumbar disk herniation. The search terms included “transforaminal endoscopic discectomy,” “microdiscectomy,” “endoscopic,” “minimally invasive,” and “lumbar disk herniation.”References from each article directly comparing the two kinds of surgeries, in addition to review articles discussing the safety and efficacy of the two procedures, were cross-referenced to identify additional relevant studies.For inclusion in the systematic review, the articles were required to meet the following eligibility criteria: 1) patients suffering from lumbar disk herniation; (2) papers reporting the results of clinical studies evaluating transforaminal endoscopic discectomy and microdiscectomy; (3) patients followed for a minimum of 2 weeks; and (4) papers published in English prior to January 2017. Randomized controlled trials (RCTs) were identified as the primary studies for analysis. For inclusion in statistical analysis, the patients in a particular study must have been randomized to either TED or MD groups. Studies were excluded from the analysis if they included patients who had an infection, traumatic fracture, previous spinal surgery at the same disk level, and spinal stenosis among other conditions. The inclusion criteria for each study are listed in Table  patientsThe risk of bias of the Cochrane Handbook for Systematic Reviews of Interventions was evaluated by using the risk of bias tool implemented in Review Manager 5.3. The included RCTs were evaluated for the risk of bias, which included assessments of adequate sequence generation, allocation of concealment, blinding, incomplete outcome data, and freedom from other biases. The judgment of each entry involved assessing the risk of bias as “low risk,” “high risk,” or “unclear risk,” indicating either a lack of information or uncertainty over the potential for bias. Two reviewers independently assessed each RCT, and any disagreements were resolved by discussion and consensus.Two authors independently extracted the following data. Any disagreements were resolved via discussion among the three reviewers. The data extracted from the studies included the following: study characteristics, types of interventions, follow-up duration, and outcome parameters.The “degree of pain relief”  score) and the functional improvement (Oswestry Disability Index (ODI)) were the primary outcome measures of the effectiveness of the surgeries. The secondary outcome measures were average duration of surgery, complications, hospital stay, recurrence, and satisfactory outcome.t test under a random-effects model. Risk ratios (RRs) and 95% confidence intervals (CIs) were used to evaluate the dichotomous outcomes, such as the incidence of complications. The differences are displayed using a forest plot. The I2 statistic [I2 = 0–25%, no heterogeneity; I2 = 25–50%, moderate heterogeneity; I2 = 50–75%, large heterogeneity; and I2 = 75–100%, extreme heterogeneity). A p value < 0.05 was considered statistically significant.The data were collected and analyzed using Review Manager 5.3. Differences in pain, functional improvement, average duration of surgery, and hospital stay between the TED and MD groups were analyzed using the independent samples tatistic  , slightly better leg pain relief was observed in the TED group at 2 years and no differences were noted after 2 years of follow-up .Two retrospective studies , 26 repoFunction was measured using an ODI. Only two retrospective studies , 26, 28 The average duration of surgery was available in two of the RCT studies. Mayer and Brock  showed tp < 0.00001).Only two retrospective studies , 25 repop = 0.32).Both the RCT and retrospective studies recorded the postoperative complications Fig. a. The coThree retrospective studies , 25, 26 p = 0.26)  Fig. a.Fig. 8Ap = 0.02)  Fig. b.Lumbar open microdiscectomy is a popular procedure for the surgical treatment of lumbar disk herniation . HoweverWe summarized the results of studies comparing transforaminal endoscopic discectomy and open microdiscectomy and performed a meta-analysis to compare the effectiveness and safety of these two surgeries for treating lumbar disk herniation. We analyzed the effectiveness of these two procedures by evaluating improvements in patients’ pain, functional scores, average duration of surgery, and hospital stay. We also analyzed the safety of these two procedures by evaluating complications and recurrence of LDH. We included five prospective studies and four retrospective studies involving 1527 patients in our analysis (Table No significant difference in both leg pain and function recovery was observed between TED and MD. Both RCTs and retrospective studies support the evidence that the transmuscular approach to the transforaminal endoscopic discectomy is as effective as the conventional open microdiscectomy requiring paravertebral muscle retraction. It can be explained that the clinical symptoms were caused by the decompression of the nerve root due to the herniated disk . Both suThe results in operative times and length of hospital stay were difficult to interpret. Although the operative times of the TED group was slightly shorter than the MD group, no significant difference was observed between TED and MD groups. The differences in how operative time was defined are important. Whether the anesthesia time was considered into the operative time, it had a large influence on operative time. Moreover, the variability in the techniques used was also a factor . The lenIn this meta-analysis, no significant difference in rates of total complications was observed between the two groups. Some studies , 26 suggOne limitation of this study was the small number of RCTs. Although five RCTs were included in this article, different outcomes could only be extracted from a few studies. It made assessing the effectiveness and safety of the interventions on the different surgical approaches difficult. Another limitation of this review was that clinical heterogeneity, which cannot be resolved completely, may be associated with inconsistency of outcomes.Our study demonstrated that transforaminal endoscopic discectomy was superior to open microdiscectomy in the length of hospital stay. However, there was no difference in leg pain, functional recovery, and incidence of complications between TED and MD in treating LDH. Prior to selecting a surgical procedure for the management of LDH, the benefits and risks of the procedure discussed herein must be taken into consideration. Additional studies must be performed to guide the clinical decision-making process.


LOXL1 SNPs  and XFS and/or XFG in a cohort of Iranian subjects.Pseudoexfoliation syndrome (XFS) is pathogenetically related to exfoliative glaucoma (XFG), which is the most common type of secondary glaucoma. We aimed to investigate the relationship between LOXL1 gene transcripts by Real-Time PCR technique. We analyzed the results for allele frequencies and haplotype association and investigated the relative gene expression.This cross-sectional study investigated possible association between LOXL1 gene polymorphisms and exfoliative glaucoma in Northeastern part of Iran between May 2014 and May 2015. Sixty unrelated XFS/XFG patients, as well as 40 control subjects, were studied by direct sequencing. In fifteen senile cataract patients without glaucoma and fifteen patients with coexisting XFG and cataract, capsulorhexis specimen of the anterior lens capsule was used to evaluate P<0.05 for both). The frequency of the G allele in the exonic SNP (rs1048661) appeared to be higher in XFS or XFG patients compared to control subjects (P= 0.0497). Moreover, in the rs3825942 SNP, the G allele was more frequent in XFS/XFG patients compared to control subjects (P=0.0016). The highest cumulative frequency was for the GG haplotype. GG haplotype was associated with increased risk of XFG compared to the rs1048661 G/T and rs3825942 G/A haplotypes. LOXL1 mRNA expression was not statistically significantly different between XFS/XFG and control subjects.Significant associations between the rs382594 SNP and XFG and between rs1048661 SNP and XFG were observed (LOXL1 gene polymorphism in an Iranian XFS/XFG cohort. Similar to many other ethnic groups and geographic regions, our results confirmed an association between LOXL1 gene variants and XFG in Iran.We reported the  Exfoliation syndrome (XFS) is an age-related disease of the extracellular matrix (ECM), characterized by gradual accumulation of abnormal fibrillary material in various ocular and extraocular tissues. Lindberg described the condition for the first time in 1917 .LOXL1 gene (LOXL1 has a prominent role in elastin fiber production (LOXL1 messenger RNA (mRNA), and decreased level of elastin fibers can contribute to optic nerve damage in much lower IOP in end-stage exfoliative glaucoma patients  males) and 40 healthy controls (18 (45.0%) males) were visited between May 2014 and May 2015 in Khatam Anbia Eye Hospital, Mashhad, Iran and included in this study. The mean ± SD age of XFG patients and control subjects were 66.0±1.7 and 70.7±1.4 yr, respectively. Detailed, informed consents were obtained from all subjects before participation in study.The Ethics Committee of Mashhad University of Medical Sciences authorized and granted approval to the current study.All patients and healthy controls went through an extensive ocular examination, including visual acuity testing, slit-lamp examination, Goldmann applanation tonometry, dynamic gonioscopy, evaluation of the optic disc and central corneal thickness measurement.The diagnosis of XFS was based on the presence of exfoliative material on the anterior lens capsule, the iris tissue, or the corneal endothelium along with multiple in-office IOP measurements of less than 21 mmHg and no evidence of glaucomatous optic nerve damage. To ensure detection of any exfoliative material, the pupil was dilated in all patients and a laser peripheral iridotomy was done in that subset of patients predisposed to pupillary block. Any patient with XFS finding and coexisting high/treated IOP, glaucomatous optic nerve cupping and visual field defect, was diagnosed as XFG. Individuals older than 55 yr with no apparent exfoliative material deposition on anterior segment structures and no evidence of glaucoma or ocular hypertension were selected as healthy control subjects. All of these selected healthy individuals had IOP readings of less than 21 mmHg as well as normal optic discs.2O. For all subjects, the DNA was amplified. The temperature and time for denaturation, annealing, elongation, and final extension were 95 °C for 5 min, 95 °C for 30 sec, 59 °C for 30 sec and 72 °C for 10 min, respectively. The ABI 3100 or ABI 3730 capillary sequencer  were used for sequencing.A DNA purification kit  was used for extraction of genomic DNA from tissue and peripheral. Primer sequences were designed by Primer Premier 5 program  . We usedLOXL1 and GAPDH gene expressions. Primer Premier 5 program was used to design the primer sequences in order to amplify the target genes  were used as negative control. RTPCR products were evaluated in 1% agarose gel. The standard curves were prepared for each gene and cDNA dilution and illustrated against respective cycle threshold (Ct) by an ABI 7300  system. As a mean to quantify the LOXL1 mRNA relative expression in patients' anterior lens capsule, we analyzed the qPCR outputs by the REST© 2009 program  to investigate any possible difference among the groups. In our study, GAPDH was applied as the reference gene for expression normalization.Anterior lens capsule specimens were obtained from 15 non-glaucomatous, senile cataract patients, and 15 patients with coexisting XFG and cataract. The anterior capsulorhexis tissue was used and in combined cases, the specimen was collected before proceeding to glaucoma surgery to reduce the risk of blood contamination of the specimen. The anterior capsule obtained during cataract surgery was immediately transferred into an RNAlater reagent  to preserve the RNA. Total RNA of the anterior lens capsules was isolated using the RNeasy mini kit plus . Next, cDNA was prepared using the AccuPower® RocketScript™ RT Pre-Mix, according to the manufacturer's protocol . For this purpose, cDNAs were synthesized by combining 500 μg of the anterior capsules RNA, 10 pmoles Oligo dT, 10 pmoles random hexamers and 200 U reverse transcriptase enzyme in a 20 μl reaction volume. The mixture was incubated at 42 °C for 1.5 h. We used Maxima SYBR Green/ROX qPCR Master Mix Kit  to measure human et genes . The PCRThe evaluation of RNA integrity for anterior lens capsule tissue samples was performed using 1% agarose gel. In addition, purification of the total RNA was done by Nanodrop .We used FinchTV  and BioEdit  programs to analyze and edit the results of sequencing . To estiSixty patients suffering from XFS/XFG and 40 control subjects were enrolled in this study. In our sample, CDR and age were not significantly different between those with and without XFS/XFG; however, IOP was significantly higher in the individual with XFG compared to control group .LOXL1 gene exon was found to be considerably associated with an increase in the risk of XFS/XFG under recessive models . In contrast to rs1048661 SNP, rs3825942 SNP had not a significant association with XFS/ XFG (P=0.997). The analysis of variance in both genotypes of rs3825942 indicated a significant effect of age, CDR, and IOP in both genotypes. However, no significant differences in age, CDR and IOP were found for rs1048661 SNP between the groups (P=0.278), however; it had significant association with CDR and IOP , compared to the GA and TG haplotypes  and for either SNP genotype. A normal amplification curve obtained in all samples  were associated with the prevalence of XFS/XFG in an Iranian population. To the best of our knowledge, the current study is the first research investigating the association of factors such as IOP, CDR, and age with LOXL1 SNPs (rs3825942 and rs1048661). However, the interactions between risk factors  and the LOXL1 genes are to be clarified in future research studies. These findings can encourage future studies to investigate the association between the LOXL1 SNPs and other possible risk factors of XFS/XFG.Polymorphisms of the Ethical issues  have been completely observed by the authors.


We repeated the brushing protocol for another 21 days on demineralized enamel. Enamel vestibular surfaces were examined using a spectrophotometer  and a Scanning Electron Microscope . Differences were statistically significant for colour parameters L* and ΔE*. SEM evaluation reveals demineralized enamel mineral gain after brushing with selected toothpastes. Toothpastes with specific ingredients can represent a balance between aesthetic and mineralization, and an oral hygiene correct algorithm is able to preserve enamel characteristics during ortodontic treatement with fixed appliances.The aim of this in vitro study was to assess the effect of toothpastes, with different compositions, on optical and morphological features of sound and demineralized enamel. We selected twenty-five teeth, recently extracted for orthodontic purposes, for this in vitro study. The teeth were caries free, without stains, fissures, filling or hypoplasia observed at inspection under standard conditions. Teeth were brushed , with five different toothpastes  containing fluoride and hydroxyapatite. After that, teeth were demineralized with 37% orthophosforic acid (Ultra Etch The preference for bright white teeth and promotion of dental and facial aesthetics in the media has contributed to an increase in patient interest for healthy and beautiful teeth. Through adolescence, and especially in the course of orthodontic treatment, whitening procedures require caution and for this reason, dental specialists recommended the use of specific dentifrice and accurate brushing technique in order to preserve enamel structural characteristics and appearance .For the duration of orthodontic treatment with fixed appliances, tooth enamel is often at an elevated risk of stains, white spot lesions, caries and dental sensitivity, due to carbonated beverage and food stagnation, facilitated by the appliance and deficient oral hygiene. These factors act on sound enamel and on enamel subjected to adhesives techniques for orthodontic bonding. In line with this, many oral hygiene strategies and products need to address various problems: remineralisation, elimination of dental plaque and preservation of tooth colour ,2.Studies showed that the levels of cariogenic biofilm in the oral cavities of orthodontic patients might be 2–3 times higher than in normal individuals experiencing from high rates of biofilm formation ,5. DentaA toothpaste is defined as a semi-solid material for removing naturally occurring deposits from teeth when used with a toothbrush ,7. The tThe basic ingredients used in toothpastes  are completed in selected toothpastes composition with remineralising agents and higher amounts of abrasives that are capable of removing or preventing the deposition of stains on the tooth’s surface. Their action consists mainly of removing extrinsic pigmentations without affecting tooth colour, because real whitening action demands the existence of bleaching agents, which consists of free radicals that act on pigments of discoloured teeth ,10.The thickness and translucency of the overlying enamel adapt the natural colour of permanent teeth, mainly determined by dentine. The aspect of the teeth whiteness is aesthetically important to many individuals and tooth discoloration is a common patient complaint ,7. ToothThe CIELAB colour system is widely used in dentistry for the determination of colour differences. We described the CIELAB values as “chromaticity coordinates”: L* value refers to ‘lightness’ , a* shows red colour on positive values and green colour on negative values, and b* shows yellow colour on positive values and blue colour on negative values ,10. ChroColour preservation is important during orthodontic treatment; however, we have to take into account other elements: enamel integrity, dental sensibility and caries risk.10 (PO4)6(OH)2. When demineralization of enamel occurs, the enamel hydroxyapatite will dissolve. This is represented with a simplified chemical reaction: Ca10 (PO4)6(OH)2 + H+ = Ca2+ + HPO42− + H2O. The left to right direction is demineralization. When calcium (Ca2+), phosphate (PO43−), and hydroxyl (OH−) ions are accumulated, demineralization slows down to the moment when the saliva reaches saturation. When the pH goes up, re-deposition of minerals  will occur and the reaction shifts from right to left 0.5 at baseline (T0) and after treatment 1 (T1-T0), 2 (T2-T1) and 3 (T3-T1) respectively. Values that are the clinically acceptable for colour changing and perceived by human eye are around 3.3 for ΔE* and 2 for ΔL* [ΔE*ab = [(ΔL*) for ΔL* ,12.® Scanning Electron Microscope  to assess morphological changes. Enamel surfaces were examined by one examiner, in order to reduce human variation, at X50 and X300 magnification.Scanning Electron Microscopy (SEM) observations were carried out by means of Inspect Sp > 0.05 for all analyses. All collected data were statistically analysed using IBM SPSS . Repeated-measures analysis of variance (ANOVA) was performed to analyse colorimeter parameters , luminosity (ΔL*), alteration on the green-red axis (Δa*) and alteration on the blue-yellow axis (Δb*)) with employed toothpaste as a repeated factor. Bonferroni correction was performed. In this study, the statistical significance was set at According to literature, human eye is more sensitive for L*, b* and ΔE* changes ,12.The L* value increased after the first brushing protocol T1;p < 0.05) for lacalut_es, lacalut w_r and biomed_s;Significant differences for L* parameter between toothpastes at T0 and T1 (p < 0.05);After demineralization (T2) we notice significant changes for L* parameter between toothpastes  L* parameter express values less important compared to T2, but the changes are noticeable when compared to T1.Average values and standard deviations of L* during experimental phases are presented in We obtained important differences for ΔL* parameter especially for lacalut_es, aslamed_st and experim_tp .p < 0.05);b* parameter present significant differences for the duration of the study, between toothpastes (p < 0.05);After T1 significant differences for b*parameter were noticed for lacalut_wr and experim_tp (p < 0.05).Biomed_s values for b* were significantly different (T2-T1 and T3-T1)  variation of yellow–green axis and influence tooth colour perception .b* paraAfter we complete all the treatments on selected teeth, we notice ΔE* variation, with statistical significant differences .p < 0.05);After demineralization ΔE* values appear higher than 3 for all evaluated toothpastes, and the differences were statistically significant (T2-T1) (p < 0.05);After demineralization and brushing, ΔE* values tend to diminish, but the differences remain clinically acceptable and statistically significant . The demineralization procedure, by ortophosphoric acid 37% for 1 min., removes aprismatic enamel and exposes hydroxyapatite prisms became evident (b). After the second brushing protocol, enamel regain mineral content, via fluoride from toothpaste composition, and crystalline structure was detected (f).Nano-hydroxyapatite from toothpaste composition restored enamel crystalline structure (c).Natural ingredients from biomed_s exert a limited protection for enamel structure .The enamel surface did not present particular changes, like granular structures, after T3.Fluoride and natural ingredients from aslamed_st ensure mineral gain after demineralization .The enamel surface is completely smooth (a) and the aprismatic surface layer is uniform (b). SEM evaluation exposed pores and superficial irregularities (c).Nano-hydroxyapatite from experimental_tp can be an alternative to fluoride to preserve enamel integrity .The demineralization procedure by 37% ortophosphoric acid, exposed hydroxyapatite prisms (e). After T3, enamel surface became smooth .The tooth colour outcomes by their intrinsic colours and the presence of any extrinsic stains . In our During tooth brushing, a three-phase system is formed by the tooth surface, the toothbrush bristles, and the abrasives between these is responsible for dental plaque and discoloration removal. Dependent on toothpaste composition, tooth brushing may perform not only a mechanical action on the tooth surface but also produce modifications in colour and act as a balance for mineral enamel loss ,20.The physical characteristics of the minerals included in toothpaste composition appear to be the major determinant for the mechanical effect, not their quantity or whitening capacity, or rather, their ability to remove enamel surface stains . Our resIn our study, all toothpastes produced alteration in L*, b* and E* parameters when brushing sound and demineralized enamel. For L* parameter variation, it is reasonable to presume that during the brushing protocol, teeth became lighter; changes that would be associated with an increase in perceptual whiteness. A decrease in b* value is most likely to be the result of a reduction in yellowness of the teeth during the demineralization process.ΔE* parameter variation was greater than 3.3 for the evaluated toothpastes; this feature expresses visible changes for the human eye in enamel appearance. This spreading of the results can be expected as no selections of teeth used in this in vitro study were made on the basis of tooth colour. We can assume that the lighter the teeth, the less the change would be and vice versa, and this element can influence the effect of toothpaste ingredients ,26,27. AThe experimental model used in the present study for the demineralization of sound enamel was by means of ortophosphoric acid. We used the 37% phosphoric acid in dentistry for etching the enamel and dentine in order to enhance the adhesion of the composite resins. This generates a porous surface, due to the selective dissolution of apatite crystals from the enamel prisms even if the enamel is the most acid-resistant substance in the human body. Apart from this “controlled dental procedure”, the demineralization take place constantly when the environmental acidity (pH) drops below a critical pH level. The main component of enamel is the hydroxyapatite crystal, which is an element of the enamel prism. The space between the columnar prisms is filled with organic components and water, components that are lost when dissolution of the enamel happens ,32. p < 0.05). The enamel, due to the prisms’ arrangements, has the ability to transmit light to the underlying dentin, which features several nuances and concede three-dimensional aspects of colour. There is a correlation among the tooth shade and the size of the hydroxyapatite enamel crystals. The demineralization procedure by ortophosphoric acid 37% for 1 min, removes the aprismatic enamel, and hydroxyapatite prisms became evident. From our results, the enamel underwent an augmentation in lightness (differences notable for human eye) when orthophosforic acid act on vestibular surface, a factor that contributes to teeth appearance improvement, performing an artificial sense of whitening, with subsequent mineral loss [L* parameter modify after demineralization for all toothpastes, with statistically significant differences (ral loss ,34. Successive control of caries risk and dental sensitivity of demineralized enamel required the action of the remineralizing elements, in order to restore the structure and ensure enamel mechanical features preservation. Assessed toothpastes have fluoride and/or nano hydroxyapatite that can supplement the minerals dropped after demineralization. These features play an important role during orthodontic treatment, because removing dental plaque from areas around orthodontic attachments is more difficult. We have to take into account that during orthodontic treatment, detached braces re-bonding necessitates an additional adhesive protocol, a supplementary risk factor for enamel surfaces. In line with these, toothpaste is the simplest way to deliver mineral content and act as first barrier against demineralisation. The fluoride and/or hydroxyapatite incorporated in toothpastes composition can compensate mineral loss, but the effect is dependent on patient cooperation; therefore, its outcomes cannot be relied for noncompliant patients. Oral hygiene routine for orthodontic patients with a multivalent product is beneficial for enamel appearance and structure. Studies also reveal that whitening dentifrices containing hydrogen peroxide and carbamide may produce lesions on the enamel surface and, for this particular reason; they are used with caution during and after orthodontic treatment or in young patients . There are a few studies addressing the clinical efficacy of whitening dentifrices and, for this particular reason, our results emphasize the equilibrium between cosmetic effects and enamel protection when recommending a toothpaste ,37. The Within the limitations of this in vitro study, we assumed that brushing with the evaluated toothpastes has effects on dental enamel appearance. Analysing the ∆E values, we establish a colour alteration that is visible for the patient. Nano-hydroxyapatite and fluoride can ensure a mineral regain for demineralized enamel, a protective factor during orthodontic treatment. It is important to evolve from traditional dentifrice formulations and adopt more biocompatible and bioactive ingredients with multiple actions. Further clinical studies should be performed to evaluate the real effect of constituents used in toothpaste compositions to reach a definite conclusion concerning this matter.


Firmicutes (67.8%), Proteobacteria (18.4%), Cyanobacteria (8.9%), and Bacteroidetes (3.1%). Regardless of wildlife species, sample condition, season, or region, the microbiome is diverse across all samples, with no significant difference in alpha or beta diversity. The findings also suggest the presence of DNA signatures of potentially dangerous zoonotic pathogens, including those from the genus Bacillus, Brucella, Coxiella, and others, in bushmeat. Together, this investigation provides a better understanding of the microbiome associated with this major food source in samples collected from the Western Serengeti in Tanzania and highlights a need for future investigations on the potential health risks associated with the harvesting, trade, and consumption of bushmeat in Sub-Saharan Africa.Bushmeat, the meat and organs derived from wildlife species, is a common source of animal protein in the diets of those living in sub-Saharan Africa and is frequently associated with zoonotic spillover of dangerous pathogens. Given the frequent consumption of bushmeat in this region and the lack of knowledge about the microbial communities associated with this meat, the microbiome of 56 fresh and processed bushmeat samples ascertained from three districts in the Western Serengeti ecosystem in Tanzania was characterized using 16S rRNA metagenomic sequencing. The results show that the most abundant phyla present in bushmeat samples include  In Tanzania, hunting is not allowed without proper permits. However, illegal bushmeat hunting is conducted all year-round and increases during the period when migrating large herbivores, such as wildebeest (Connochaetes taurinus) are in close proximity to the villages contributing to the annual harvesting of ~70,000–120,000 wildebeest alone3. For many households, bushmeat represents an important source of animal protein, and a recent survey in regions surrounding the Greater Serengeti ecosystem suggests between 2 and 5 bushmeat meals is consumed per week per household4. The meat is sold as either fresh or processed (dried meat) depending on multiple factors, including the animal availability, region, taste preference, and migration season5. In the Serengeti ecosystem, bushmeat is primarily acquired through house-to-house sales and via middlemen traders or hunters to the end-consumers7. Notably, bushmeat consumption and trade has been increasing during recent decades due to several socio-economic factors, including growing food insecurity, demographic shifts, low cost (compared to other meat products), cultural practices, taste preferences, perceived medicinal value and prestige13.“Bushmeat” refers to the meat and organs derived from wildlife species that are frequently hunted as a food source in many countries in Africa, including Tanzania14. These practices provide an opportunity for the transmission of dangerous pathogens and a means for the emergence of zoonotic diseases17. According to the World Health Organization, over 50% of the new infectious diseases in humans are caused by pathogens originating from animals or animal products, of which 70% have originated from wildlife18. Therefore, it is not surprising that recent studies have indicated that the handling and consumption of bushmeat contributes to the transmission of pathogens from animals to humans17.While the majority of bushmeat is consumed locally in the regions where the wildlife is hunted, a considerable amount is illegally transported to larger cities in Africa. Bushmeat is also smuggled into the United States and Western Europe, which poses a potential global health and biosecurity risk if this meat is harbors dangerous pathogens. For instance, reports suggest that up to 5 tons of bushmeat is smuggled through a single airport in Western Europe each weekDue to the potential global health risks associated with the consumption and transportation of bushmeat, here, we examined the microbial diversity associated with bushmeat samples harvested from the Greater Serengeti Ecosystem in Tanzania to better understand the distribution and prevalence of different bacterial communities, including possible dangers pathogens that may affect human health.Connochaetes taurinus, n = 27), buffalo , as well as some less common wildlife species including eland (Tragelaphus oryx), Thomson’s Gazelle-gazelle (Eudorcas thomsonii), zebra (Equus burchellii quagga), giraffe , rabbit (Lepus microtis), sheep (Ovis aries), topi , porcupine (Hystrix cristata), and warthog (Phacochoerus africanus)  , Proteobacteria (18.4%), Cyanobacteria (8.9%), and Bacteroidetes (3.1%). No significant differences were found in the phyla-level alpha diversity in samples collected from different species  representing samples with the lowest diversity (an average of 95% Firmicutes) and included primarily processed samples collected during dry season  has a total of eight wildebeest samples, of which, seven are processed samples collected during dry season, mostly from the Serengeti district. These samples contain on average about 72% Firmicutes and 20% Cyanobacteria  and IV (n = 10) include samples that contain about 58% Firmicutes, 36% Proteobacteria and a small percentage of Cyanobacteria and Bacteroidetes. These two clusters include about half of the total samples  has fresh samples collected mostly during dry season. These samples have high diversity, with an average of 37% Firmicutes, 29% Bacteroidetes, and 16% Proteobacteria  and low in Proteobacteria (3%) , a fresh warthog sample collected during rainy season from Tarime (Other_F06), and a processed wildebeest sample collected during dry season from Serengeti (WB_P16)  and bootstrap probability (BP) values of the branching patterns were computed Fig. . The samson Fig. . Clusterles Fig. . The betles Fig. . There iles Fig. . The PColes Fig. .Figure 3https://www.selectagents.gov/selectagentsandtoxinslist.html), in the collected bushmeat samples. The relative abundance of the select agent genera, including Bacillus, Brucella, Burkholderia, Clostridium, Coxiella, Francisella, Mycoplasma, Ralstonia, Rickettsia, Staphylococcus, Xanthomonas, and Yersinia are plotted as a heatmaps , Staphylococcus (0.72%), Bacillus (0.30%), and Ralstonia (0.05%). Interestingly, the analyses showed that processed wildebeest samples collected during dry season contained a high abundance of Clostridium species (>78%). While this data demonstrates the possible presence of select agents, it is important to note that genus-level analysis cannot confirm the presence of specific select agents in these samples. This analysis provides preliminary evidence for much needed future investigations on the presence of select agents in bushmeat samples using more directed molecular techniques to better assess public health risks associated with bushmeat consumption.The data were screened for the presence of DNA signatures of potential select agents , the feeding habits of the different wildlife species  and the social organization of the species (e.g. solitary or gregarious), and the interactions of wildlife with other domestic and wild species. The microbiome present in the samples could likewise be affected by the conditions during the processing of the bushmeat, including the microbiota composition of the environment and shared butchering tools23. For this study, the sample collection was performed on incidental basis, which reflected the variability of conditions in which bushmeat is handled and demonstrates the microbial communities present in bushmeat at the point of consumption. The samples were collected by different enumerators who might have obtained them from different sources and followed different methods of wildlife handling and sample processing prior to storing the samples for our study. Another factor that might contribute to the microbiota profile of these samples is the condition of which the samples were processed. For instance, freshly harvested bushmeat may be processed, through laying the meat on the grassy areas for weeks to dry out under the sun where these samples may come in contact with soil borne- organisms, which is consistent with our observation of the relatively higher abundance of Cyanobacteria (a photosynthetic microorganism) in wildebeest samples24.Overall, the samples have high diversity irrespective of the origin of the species, season, or sample condition. This could be dependent on several factors related to the conditions of pre-processing or post-processing of the samples. Since the samples were collected along different points of the supply chain, it is difficult to speculate if these bacterial species may be endogenous to the host or have been transferred from the environment during the process of hunting, butchering, and transportation. These processes are conducted under unhygienic conditions which affects the microbiota profile of the meat as well. For instance, human skin microbiota is also mostly composed of 25.The Shannon Diversity Index was used to compare the alpha diversity between wildlife species Fig. , season Firmicutes and at the family level, the samples include Staphylococcaceae, Clostridiaceae, and Bacillales  and adjacent game reserves and game-controlled areas. There are 27 species of large mammal herbivores inhabiting the Serengeti ecosystem35, of which wildebeest, zebra, Thomson’s gazelle, buffalo, topi, and eland are the most abundant grazing species36. There are also close to 1.5 million white-bearded wildebeest, 250,000 plains zebra, and 500,000 Thomson’s gazelle that seasonally migrate between the Serengeti ecosystem and the Maasai Mara in southern Kenya4. The climate of the Serengeti is considered subtropical, with a bimodal rainfall pattern consisting of two rainy and two dry seasons. There is a “short rainy season” from November to December and a “long rainy season” from March to May, with the average annual rainfall varies from 600 mm to 1100 mm per year, depending on the region35. To ensure a proper temporal representation of the presence of microbes in bushmeat, samples were collected throughout the year, including during both rainy and dry seasons.The ~25,000 km6. Most of the communities where the samples were collected from are adjacent to the game reserves. The human population densities vary amongst the districts; Bunda District has the highest (91.3 persons per km2) followed by Serengeti District (16.1 persons per km2)35.Spatially, samples were collected from the Bunda, Serengeti, and Tarime districts located in the Western part of the Serengeti ecosystem Fig. . The BunSample collection occurred between September 2016 and March 2017. Bushmeat samples were collected and classified as either “fresh” or “processed”. Fresh samples were those taken from raw, uncured bushmeat, while processed samples were those which were dried by the hunters, a preservative measure to allow them to last longer. Sample identification sheets were completed for all collected samples and the metadata, including information regarding sample condition, the collection site (villages using GPS coordinates), seasonality, and date of collection were recorded  and stored in −20 °C vehicle freezers until arrival at Serengeti wildlife veterinary laboratory where they were transferred to −20 °C solar freezers. For larger tissues, double zip lock freezer bags containing non-toxic Silica Gel desiccant moisture absorber/dehumidifier were used to store the samples. The samples were then transported to the Nelson Mandela African Institute of Science and Technology (NM-AIST) in Arusha via vehicle freezers, where they were stored at −80 °C until processing. Cold chain maintenance was ensured through temperate trackers included with the samples throughout the entire process.This study and all the methods were approved and performed in accordance with the relevant guidelines and regulations with permits from the appropriate institutions, including the Tanzania Wildlife Research Institute , Tanzania National Parks Authority , Ngorongoro Conservation Area Authority  and Tanzania Wildlife Authority; permit number CB.517/519/01/14.From each sample, three small sections, weighing approximately 120 mg total, were dissected from both superficial and deep muscle tissue, using a sterile, disposable Rapid Core punch  or sterile disposable safety scalpels  and placed in the MagMAX™ Lysis/Binding Solution Concentrate buffer . Homogenization was performed using the company’s recommended procedure with slight modifications. In brief, using the Bead Ruptor 24 Bead Mill Homogenizer , fresh samples were processed for 45 second at 5.5 m/s using 2.3 MM zirconia beads  in the MagMAX™ Lysis/Binding buffer. Processed samples were pre-soaked in UltraPure DNase/RNase-Free Distilled Water  at 4 °C overnight, followed by beating for three 30 second intervals at 5.5 m/s with 2.3 MM zirconia beads in the MagMAX™ Lysis/Binding buffer. Nucleic acid extractions were performed using MagMAX™ 96 DNA Multi-Sample Kit  and KingFisher Flex automated DNA purification system  from tissue samples per manufacturer’s instructions with minor modifications. If the yield and purity of extractions were not adequate for a specific sample, such as the DNA concentration was less than 4 ng/μl and/or if the quality of the amplicons analyzed by the Bioanalyzer did not pass the quality control, DNA was extracted manually using DNeasy PowerSoil Kit  per manufacturer’s instructions. Extracted DNA was quantified using Qubit™ 3.0 Fluorometer . To ensure purified DNA was of high-quality, DNA was also visualized through agarose gel electrophoresis.38, the V4 hyper-variable region of the 16S rRNA gene was amplified from the DNA extracts during the first PCR step using the universal 515 F forward primer (5′ GTGCCAGCMGCCGCGGTAA 3′) and uniquely barcoded 806 R reverse primers (5′ GGACTACHVGGGTWTCTAAT 3′) for each sample. The PCR was performed using 25 μl Bioneer premix . The premix was complemented with 1.5 μl each of 10 μM forward and reverse primers  and 5 μl genomic DNA in a final volume of 50 μl. The PCR reaction was carried out on a Thermal Cycler  as follows: an initial denaturation step (95 °C for 1 min), 35 cycles of amplification  and a final elongation step at 72 °C for 5 min. Amplicons were then purified using the QIAquick PCR purification kit  following the manufacturer’s instructions. The concentration of the purified amplicons was analyzed using Qubit dsDNA BR Assay kit .The purified DNA was transported on dry ice to the Biosciences eastern and central Africa-International Livestock Research Institute (BecA-ILRI) Hub in Nairobi, Kenya for the microbiome analysis. Using previously published primers40, which was added during the PCR step. The quality of a set of amplicons (per sequencing run) was tested using Agilent DNA 7500 chips using the Bioanalyzer 2100 . 40 ng of each sample library was pooled in order to normalize the libraries. The final pool was denatured (NaOH 0.2 N) and diluted to 12 pM The PhiX Control V3 was added to the pool at 15% of the final concentration as described in the Illumina procedure for sequencing of low diversity libraries. Six hundred μl of this treated pool mixture was loaded onto the Illumina MiSeq cartridge according to the manufacturer’s instructions and sequenced on the Illumina MiSeq System generating 2 × 151 bp Paired-End Reads.Sample multiplexing was performed using a unique reverse primer per sample39. Analysis of demultiplexed paired-end 16S rRNA gene reads was completed with QIIME (v1.9.1)40 incorporated into the EDGE Bioinformatics platform (v2.3.0)39. EDGE implementation is based on QIIME v1.9.1 and includes demultiplexing and quality filtering, operational taxonomic unit (OTU) clustering, taxonomic assignment, phylogenetic reconstruction, diversity analyses and visualizations of each of these analyses. QIIME started the pre-processing step with 18,452,934 paired-end reads (in FASTQ format) by removing reads with a PHRED quality score lower than Q20. Data were further filtered to remove reads with more than one ambiguous base. If less than 50% of a read contained consecutive high-quality bases, the read was discarded. The forward and reverse reads were merged before analysis. A 94% nucleotide identity sequence clustering threshold (approximately genus level) was used to generate OTUs.To produce a high-quality data set, microbiome analysis was performed using an open source platform, Empowering the Development of Genomics Expertise (EDGE)41 for taxonomic classification assignment. The depth filter was set to 1000 reads – any barcoded sample that had less than 1000 assembled reads did not undergo taxonomic classification or further alpha- and beta-diversity analyses. Next, the integrated phylotype command generated the consensus taxonomy using phylotype- based approach, where taxonomy-linked sequences were assigned to OTUs based on similarity.The representative sequence for each OTU was then queried against the Greengenes database42. Results show that we were able to consistently identify all the detectable OTUs present in every sample with a minimum reading depth of 6,470 sequences per sample. This suggests that the variation of the microbiota among samples is not likely caused by low sequence coverage  was calculated using the Shannon Diversity Index (R package: Vegan) at the phylum-, family-, and genus-level43. Statistical analysis between the groups was performed using a t-test in R44. Hierarchical clustering of the samples based on relative abundance of phyla was performed using the R, pvclust, with 1000 bootstrapped replications of the clusters45. To examine the beta diversity of the samples, PCoA was performed using the Bray-Curtis Matrix at the phylum level using the R package, Vegan43. Groups were clustered drawing ellipses estimated as the 95% confidence region of the joint distribution of the two components considered. Statistical analysis of the PCoA data was performed using the R package, Vegan43  in relation to the different characteristics of the samples (fresh versus processed; dry versus rainy season; buffalo versus wildebeest, versus https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP151593.The data is publicly available at: Additional Text file 1Supplementary FiguresSupplementary TablesSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4


Hydroxysafflor yellow A (HSYA) is the major active chemical component of the safflower plant flower, which is widely used in Chinese medicine for cerebrovascular and cardiovascular disease treatment. Recent studies have demonstrated that HSYA exerts neuroprotective effect on cerebral ischemia, such as neuronal anti-apoptosis, antioxidant activity and oxygen free radical-scavenging. However, whether and how HSYA has a protective effect on cognitive impairment induced by cerebral ischemia reperfusion remains elusive. In the present study, by using the middle cerebral artery occlusion (MCAO) model, we found that 8 mg/kg and 16 mg/kg HSYA administration by common carotid artery (CCA) injection improved impaired cognitive function in Morris water maze (MWM) and passive avoidance tasks, but not 4 mg/kg HSYA treatment, suggesting that HSYA treatment in a certain concentration can improve cognitive impairment in MCAO rats. Furthermore, we found that 8 mg/kg HSYA treatment rescued the impaired long-term potentiation (LTP) in hippocampus of MCAO rats. Taken together, these results for the first time demonstrate that HSYA has the capacity to protect cognitive function and synaptic plasticity against cerebral ischemia-reperfusion injury, and provide a new insight that HSYA may be a promising alternative for recovery of cognitive dysfunction after brain ischemic injury. Stroke is the third leading cause of death and adult disability worldwide, particularly of the elderly  model, which is considered to be a convenient, reproducible, and reliable rodent model of cerebral ischemia in humans, to investigate the effects of HSYA on cognitive function and hippocampus synaptic plasticity in cerebral ischemia reperfusion-injured rats. We found that HSYA significantly improved the learning and memory of rats subjected to MCAO model in the Morris water maze (MWM) and passive avoidance tasks. Furthermore, HSYA recovered the impaired LTP at hippocampus while leaving basal synaptic transmission unaffected.ad libitum access to food and water. All animal experiments described in this study were conducted according to Animals Act  and approved by the Institutional Animal Care and Use Committee  of the East China Normal University. All efforts were made to minimize animal suffering and reduced the number of animals used. In addition, HSYA was produced by Shanghai Yuanye Pharmaceutical Co., Ltd. , and its purity as analyzed by high performance liquid chromatography peak area normalization was 98.8%. Solution of drugs was always prepared afresh before use.Sprague-Dawley male rats  were obtained from Shanghai Laboratory Animal Center of Chinese Academy of Sciences. The rats were housed in temperature (22–26°C) and humidity (40%–70%) controlled conditions with a 12/12 h light/dark cycle, and the rats had Rats were randomly divided into five groups: (1) sham operated rats; (2) MCAO (vehicle-treated) group; (3) 4 mg/kg HSYA group; (4) 8 mg/kg HSYA group; and (5) 16 mg/kg HSYA group. Focal cerebral ischemia/reperfusion injury was induced by MCAO, based on the method developed by Longa et al. . The ratAssessment of neurological deficits was performed on rats 24 h after reperfusion by using the Garcia test, which is a neurological examination that utilizes an 18-point scale  staining was used to measure the infarct volumes 24 h after reperfusion. Rats were killed under deep anesthesia using 10% chloral hydrate and brains were rapidly took out and cut into 2-mm thick coronal sections using the brain matrix . The fresh slices were incubated away from light in 2% 2,3,5-triphenyl-tetrazolium chloride solution at 37°C for 30 min to visualize in infarctions. Normal and damaged tissue were stained red and white, respectively. The brain slices were photographed with a digital camera and the size of the infarct area (unstained) was assessed by ImageJ software. The percentage of the infarct volume was calculated according to the following formula: {[contralateral volume − ]/contralateral volume}·100%.After modeling and drug treatment, the rat was first tested by open field to assess its locomotor and exploratory activity. The apparatus  test Morris, . All of The consecutive training days were commenced from the 4th day after the termination of the HSYA treatment described above. All of rats were trained through four trial sessions in the afternoon each day for five sequential days, and the southwest quadrant was maintained as the target quadrant in all acquisition trials. Facing against the maze wall, the rats were released into the pool randomly in one quadrant  for each trial without repetition. They should find the hidden platform according to the program’s instructions. For memory acquisition, the location of the platform remained stable and the rats were given a maximum of 60 s to find the hidden platform. Once a rat climbed onto the platform and remained on it for 20 s, the trial was terminated. If the rat failed to reach the platform within 60 s, it would be gently guided to the hidden platform and allowed to stay on it for 20 s. The average swimming speed was recorded and escape latency time, the time required for the rats to climb the platform was recorded as well. After 5-day acquisition trial, the rat was subjected to the retrieval test in the 6th day.Memory retention was evaluated during a probe test. On the 6th day, the platform was removed from the pool. Each rat was placed in water maze and allowed to swim for 60 s to explore it. Every rat was subjected to one such trial, and each trial was started by placing the rat in the quadrant farthest from where the platform had previously been placed. The platform crossings and the time spent in the target quadrant for searching the missing platform were recorded. The experimenter always stood at the same position. Throughout the study, experimenter must be careful not to disturb the relative location of the pool with respect to other objects in the laboratory, which could be served as prominent visual clues.Passive avoidance test (PAT) which was conducted in another batch of rats used for evaluating learning and memory  modified artificial cerebrospinal fluid (section ACSF) containing (in mM): choline chloride, 110; KCl, 2.5; CaCl2, 0.5; MgSO4, 7; NaHCO3, 25; NaH2PO4, 1.25; and D-(+)-glucose, 25; pH 7.4. The slices were recovered in an incubation chamber with normal ACSF (incubation ACSF) containing (in mM): NaCl, 119; CaCl2, 2.5; KCl, 2.5; MgSO4, 1.3; NaHCO3, 26.2; Na2HPO4, 1.0; and Dglucose, 11; pH 7.4, 95% O2 and 5% CO2 for 60 min at 31°C before recording.After the behavioral tests, rats were anesthetized using chloral hydrate and decapitated. The protocols were similar to those described previously . A typical experiment began with an input-output curve ranging from subthreshold to maximal response. Fiber volley amplitude was measured from peak negative voltage to baseline. Paired-pulse facilitation (PPF) was assessed among inter-stimulus intervals ranging from 0.02 s to 0.4 s. The PPF was defined as the ratio of the amplitude of the second to that of the first fEPSP amplitude elicited by pairs of stimuli (pulse2/pulse1 × 100). Synaptic responses were monitored with stimuli consisting of constant current pulses of 0.05 ms duration at 0.033 Hz.For field potential recording, slices were transferred to a recording chamber filled with the oxygenated incubation ACSF, and the rate of ACSF superfusion was 0.5 mL/min. An unipolar tungsten stimulating electrode was placed in the stratum radiatum to activate the Schaffer-collateral pathway projecting to CA1 and the field excitatory post-synaptic potentials (fEPSPs) were recorded using a glass microelectrode filled with 0.1 M CHAfter obtaining a stable baseline response for at least 15 min, LTP was induced by theta burst stimulation . fEPSP continued to be recorded for 60 min after TBS stimulation. Data were recorded using a Multiclamp 700B amplifier and digitized with a Digidata 1322A . The fEPSP amplitude was used to measure the synaptic efficacy and LTP induction was expressed as the percentage of the increase of fEPSP amplitude compared with the average amplitude of the baseline period. If the change of fEPSP amplitude in sham group of rats exceeded 20%, it was defined as a successful induction of LTP.t-test was used for comparison of two groups. For comparisons with multiple data sets, one-way analysis of variance was used. For input-output curve and PPF analysis, two-way repeated measures ANOVA followed was used. Differences were considered statistically significant at p < 0.05.All data were shown as mean ± SEM. Student’s n = 8; MCAO, 9.25 ± 0.16, n = 8; Student’s t-test, P < 0.0001) was significantly decreased in MCAO rats. Brain water content  was significantly increased in MCAO rats. Moreover, MCAO rats displayed an obvious manifestation of cerebral infarction , whereas rats in the sham group did not show any signs of cerebral injury, indicating the success of the MCAO model.The neurological scores, brain edema and infarct volume were evaluated at 24 h of MCAO. The results showed that neurological scores  and distance  in the open field test. Moreover, after HSYA treatment, it also showed that HSYA no matter in which dosage  has no effect on the spontaneous locomotor activity of control rats .To investigate whether HSYA can improve cognitive impairment after MCAO. We began by exploring modifications of the spontaneous locomotor activity of the different groups of rats. As shown in Figure P > 0.05). However, the escape latency of 8 mg/kg HSYA group and 16 mg/kg HSYA group significantly decreased compared to that of MCAO group , but not of 4 mg/kg HSYA group .To determine the effects of HSYA on the rats’ spatial learning acquisition and memory retention, MWM test was used. All rats learned to locate the hidden platform through 5 days of acquisition training. During this training, we found that the average swimming speed of rats in each and every group was similar, which suggested that every groups of rats have normal sensory-motor function and motivation for survival . After HSYA treatment, 8 mg/kg HSYA group and 16 mg/kg HSYA group produced a significant increase in the retention phase, measured as platform crossings , but there was no difference between 4 mg/kg HSYA group and MCAO group . In addition, we found that with the increasing dose of HSYA, rats spent more time in the target quadrant. Target quadrant time in 8 mg/kg HSYA group and 16 mg/kg HSYA group was significant increased compared with that in MCAO group, and even reached values observed in the sham group . There was still no difference between 4 mg/kg HSYA and MCAO group in time spent in the target quadrant . These results suggest that 8 mg/kg HSYA group and 16 mg/kg HSYA can improve the reference memory after brain ischemic injury.During the retrieval trial, platform crossings and time spent in the target quadrant were calculated for each rat to evaluate memory retention. As shown in Figures n = 3; sham, 327.90 ± 27.79 s, n = 6; one-way ANOVA, P < 0.001), and 8 mg/kg HSYA group and 16 mg/kg HSYA group produced a significant increase of STL in comparison with MCAO group , but not 4 mg/kg HSYA group . These data indicate that 8 mg/kg HSYA group and 16 mg/kg HSYA can improve the fear memory after brain ischemic injury.To further investigate whether HSYA can improve the cognitive dysfunction after MCAO, passive avoidance task was used. We found that STL significantly declined in MCAO group compared with sham group  and PPF  between sham and MCAO group of rats. In addition, after 8 mg/kg HSYA treatment, there was also no significant difference was observed in input-output curves  and PPF  between HSYA treatment and MCAO group of rats. These results suggest that HSYA have no effect on the basal synaptic transmission after brain ischemic injury.The cognitive facilitation exhibited by HSYA prompted us to investigate how HSYA facilitates the learning and memory after brain ischemic injury. Given that synaptic plasticity is one of the important cellular foundations of learning and memory, we first examined the basal excitatory synaptic transmission in Schaffer collaterals-CA1 synapse of hippocampus by n = 6; MCAO, 111.7 ± 4.86%, n = 9; Student’s t-test, P < 0.01). After 8 mg/kg HSYA treatment, the LTP was significantly improved in the HSYA treatment group compared to that in the MCAO group ; and, there was no statistically difference of LTP between sham group and HSYA treatment group . These data suggest 8 mg/kg HSYA can significantly improve the synaptic plasticity after brain ischemic injury.We then investigated whether synaptic plasticity in the hippocampus was improved by HSYA treatment after brain ischemic injury. As shown in Figure Post-stroke cognitive dysfunction is one of the major consequences after stroke and chronic cerebral hypoperfusion can induce vascular cognitive impairment (Back et al., To study whether MCAO model induces cognitive impairment as reported and HSYA has a protective effect on cognitive impairment induced by cerebral ischemia reperfusion, we first carried out a serious behavioral experiment. Using the open field test, we found brain ischemia induced by MCAO did not influence spontaneous locomotor activities on the rats which was consistent with previous study (Chang et al., MWM is one of the classic behavioral test to study spatial learning and memory (Loureiro et al., Passive avoidance is a behavioral test to study fear learning and memory. Using this PAT, we found that the fear learning and memory was significantly impaired on MCAO rats, which was in agreement with previous study (Yang et al., in vitro fEPSP recording. No distinguishable difference was observed between MCAO and sham groups in input-output curves and PPF, suggesting that basal synaptic transmission was unchanged after brain ischemic insult. After HSYA treatment, whether basal synaptic transmission in MCAO rats was effected by HSYA, we found that 8 mg/kg of HSYA which could recover the cognitive impairment after MCAO has no effect on both input-output curves and PPF in MCAO rats. Impairment of synaptic plasticity has been thought of as the mechanism underlying cognitive impairment after stroke (Feng et al., Through behavioral experiments, we found HSYA in a certain concentration improved cognitive function in MCAO rats. The cognitive improvement exhibited by HSYA treatment prompted us to further investigate the mechanism underlying the improvement by HSYA treatment in MCAO rats. Given that synaptic plasticity is one of the important cellular foundations of learning and memory (Esmaeili Tazangi et al., in vitro field potential recording, we found that LTP at hippocampus was impaired in MCAO group of rats, which was consistent with previous reports (Li et al., It’s known that PPF is an indirect index for measurement of Glutamate release from pre-synaptic terminals (Dobrunz and Stevens, In summary, we found that MCAO model of brain ischemia induced cognitive impairment and injection with a certain concentration of HSYA into the CCA promoted learning and memory after MCAO. One of the underlying mechanisms of HSYA’s neuroprotective effect is about its capacity of recovery on synaptic plasticity impairment. Thus, our findings of the present study demonstrated that HSYA treatment may be a promising alternative to protect cognitive and synaptic function against brain ischemic injury.LY carried out experiments and wrote the manuscript. YD carried out experiments and revised the manuscript. WH carried out experiments. ZZ analyzed experimental results. MX assisted with data analysis. QZ designed experiments. XC designed and instructed experiments.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.


The alteration of the phospholipid composition within the cell, in particular the ratio between saturated and unsaturated fatty acids, can serve as an important biomarker to prognosis of the disease progression . Major techniques for lipid analysis in biological samples require a lipid extraction procedure that is not compatible with live cell studies. To address this challenge, we apply microRaman-Biomolecular Component Analysis (BCA) for comparative analysis of phospholipid composition and sensing the saturation degree of fatty acid lipid chain in live HeLa cells and lipids extracted from HeLa cells. After processing raw Raman data, acquired in lipid droplets (LDs) free cytoplasmic area, LDs and extracted lipids with BCA, the lipid component was isolated. Despite the similarity in general profiles of processed Raman spectra acquired in live cells and extracted lipids, some clear differences that reflect diversity in their phospholipids composition were revealed. Furthermore, using the direct relation between the number of double bonds in the fatty acid chain and the intensity ratio of the corresponding Raman bands, the saturation degree of fatty acids was estimated. It is known that lipids play vital roles in establishing cellular architecture and maintaining cellular processes, including but not limited to energy storage, signaling reactions, protection against some forms of cellular stress, and many others ,2,3. FurThese evidences evoke increasing interest in the development of analytical techniques for lipid analysis. Currently, a number of advanced techniques are frequently used as tools for chemically selective imaging or quantitative analysis of lipid content in biological samples ,23,24,25From this perspective, microRaman-BCA approach offers an attractive alternative for analysis of phospholipid composition in a label-free and nondestructive way at the single-cell level without lipid extraction procedure required for most methods in lipidomics. The effectiveness of the micro-Raman-BCA assay for quantification of major classes of biomolecules, including lipids, in different organelles of live cells was demonstrated a number of times ,31,32,33In this study, we employed a microRaman-BCA approach for comparative analysis of phospholipids composition in live HeLa cells and lipids that were extracted from HeLa cells. The raw Raman spectra acquired in live cells are very complex and contain a lot of bands associated with different macromolecules that often overlap or partially overlap. Therefore, it can be very challenging to analyze and interpret the obtained results. Here, we demonstrated the analytical capability of the BCA approach to analyze the phospholipids composition from the Raman spectra acquired from LDs’ free cytoplasmic area, LDs of live cells, and lipids extracted from HeLa cells.Cell culture and fluorescence staining. HeLa cells were plated onto glass bottom dishes . HeLa cells were cultured in advanced DMEM , supplemented with 2.5% fetal calf serum (FBS) , 1% glutamax (Life Technologies), and 1% antibiotic antimycotic solution (Sigma) at 37 °C in a humidified atmosphere, containing 5% CO2.Extraction of lipid from HeLa cells. The total lipids were extracted directly from the cell dishes using ethanol. The extracted lipids were transferred to another glass dish and dried. The dried lipids were further analyzed.Raman microspectrometry. The confocal Raman microspectrometer system consists of an inverted Nikon TE200 microscope equipped with single frequency laser diode  excitation source, fiber-input MS3501i imaging monochromator/spectrograph , and HS101H−2048/122-HR2 series CCD  cooled down to −30 °C. The spectral resolution for the fixed diffraction grating position  was ~1.5 cm−1. The excitation laser beam from a single frequency laser diode  of power ~70 mW was focused onto the sample in a spot of diameter ~0.8 μm, using a 100× Nikon oil-immersion objective lens with NA = 1.3. A 100 μm pinhole provides for confocal acquisition of the Raman signal, which corresponded to a confocal parameter of ~1.8 μm (in FWHM for λ = 638 nm). A series of three Raman spectra were acquired with an accumulation time of 60 s per each spectrum and then averaged, giving a total spectral integration time of 180 s per measurement.Biomolecular Component Analysis. The lipid component was isolated and analyzed, as described in detail in previous publications [−1. These lipid components were used for subsequent analysis.ications ,32,33. BDuring the Raman measurements, intracellular LDs and the cytoplasm without LDs inclusions were identified by their characteristic dense appearance in the transmitted light. Aimed to figure out the difference in phospholipid composition between different sites in live cells as well as to explore the impact of extraction of lipids procedure on phospholipids content compared with live cells, the Raman data from various sites in live cells and extracted lipids were processed and analyzed.Raman spectra were acquired in different cells from 28 different areas in cytoplasm, 22 areas in LDs, and 12 areas from lipid extract. After applying preprocessing routine and BCA to raw Raman data, the lipid component was isolated using previously established techniques .−1  of fatty acids are significantly higher for extracted lipids  and 1440 cm−1 .For the next step, the processed spectra were used to determine the saturation degree of intracellular fatty acids in different areas of cytoplasm and extracted lipids from cells. Information about the degree of saturation of fatty acids can be obtained from the ratio of Raman intensity of specific bands that correspond to the C=C stretch and C–Hondingly ,38,39,40The Raman ratio I(1665)/I1440) for every measurement from the lipid extracted from HeLa cells and from the lipids in cytoplasm and LDs in live cells are plotted on the same chart . As a re for everThese results demonstrate that the degree of intracellular fatty acid saturation varies significantly not only in cytoplasmic LDs and LDs free cytoplasmic area, but also vary from one measurement to another. For example, the Raman intensity ratio varies from 0.48 to 0.72 for LDs free cytoplasmic area, from 0.34 to 0.49 for LDs, and from 0.30 to 0.51 for extracted lipids. Interestingly, on average, the variation in degree of saturation in LDs free cytoplasmic area is more diverse compared with the variation in different LDs. Moreover, this data suggests that cellular cytoplasm contain higher relative unsaturated lipid concentrations than in LDs or extracted lipids. Specifically, the average value of I(1665)/I(1440) for cytoplasm is 0.59 ± 0.06 (mean ± s.d.) while it is 0.42 ± 0.04 for LDs and 0.41 ± 0.06 for extracted lipids. These data also revealed that the degree of saturation of fatty acids in LDs and extracted lipids is almost the same. This finding reflects the fact that the EtOH lipid extraction procedure leads to preferential LDs lipids preservation, while the major fraction of cytoplasmic lipids is washed out from the extract or remains in the cell.A comparative analysis of processed Raman spectra of LDs, LDs free cytoplasmic area, and extracted lipids revealed that phospholipid content is significantly different in live cells and extracted lipids. Although the extracted lipids show all major lipid Raman bands, which are also present in the spectra measured in live cells, the BCA approach revealed that the lipid extraction procedure results in noticeable changes in fatty acid composition compared with live cells. In particular, it was shown that the degree of intracellular fatty acids saturation differs significantly for LDs and LDs free cytoplasmic area and varies between measurements. Furthermore, this data demonstrates that the cellular cytoplasm contains higher relative unsaturated lipid concentrations (0.59 ± 0.06) than in LDs (0.42 ± 0.04) or extracted lipids (0.41 ± 0.06). Altogether, these findings demonstrate that in contrast to traditional lipidomics approaches which involve cell destruction with subsequent biochemical analysis, the microRaman-BCA approach is capable of performing noninvasive analysis of phospholipid composition in situ. This capability expands the list of available analytical tools in the emerging field of lipidomics for multiple applications.


Autoimmune metaplastic atrophic gastritis is a chronic progressive inflammatory condition. The clinical spectrum includes pernicious anemia, atrophic gastritis, antibodies to parietal cell antigens and intrinsic factor, achlorhydria, hypergastrinemia and carcinoma. It is rare in paediatric cohorts.FOXP3 gene mutation. The patient was referred to the hospital at the age of 3 years with recurrent emesis, diarrhoea and malnutrition. His elder brother died at 9 years of age from acute respiratory distress syndrome and renal tubular acidosis. The patient was allergic to cow milk formula and noodles. Oesophagegastroduodenoscopy revealed redness, erosion and edema throughout the stomach; whitish granules in the duodenal bulb; and edema in the second part of the duodenum. Biopsies showed extensive villous atrophy and goblet cell depletion in the duodenum. He was diagnosed with type-1 diabetes mellitus (T1DM) during the treatment of methylprednisolone. Serum antibodies against glutamic acid decarboxylase and pancreatic islets were detected. The patient’s FOXP3 gene was sequenced; this identified that the patient was hemizygous for a pathogenic variant [NM_014009.3:c.748_750del (p.Lys250del)].We present the case of a boy with metaplastic atrophic gastritis in whom immune dysregulation, polyendocrinopathy, enteropathy, X-linked(IPEX) syndrome was confirmed by Metaplastic atrophic gastritis is rarely reported in patients with IPEX. Clinical gastroenterologists should be aware of IPEX syndrome when facing the complex syndromes of metaplastic atrophic gastritis and endocrinopathy. FOXP3) gene, which is a master transcriptional regulator for the development and function of CD4+ regulatory T (Treg) cells  which is associated with IPEX. The mother was subsequently tested and identified as a heterozygous carrier for this variant.Now he is 5 years and 9 months of age. His weight for age reaches the 5th percentile. His height for age is the 2nd percentile. The diagnosis of IPEX was confirmed by molecular analysis using EDTA whole blood, which revealed a pathogenic variant in the leucine zipper of The distinctive symptoms of IPEX syndrome present as early onset of intractable diarrhoea, T1DM, and eczema . The onsFOXP3 mutation identical to that of our patient [In previous reports, there were three patients with IPEX syndrome who had a  patient –10. HoweMetaplastic atrophic gastritis and duodenal atrophy are atypical manifestations in IPEX syndrome. To our knowledge, gastritis has rarely been reported in patients with IPEX , 11, 12.FOXP3 mutation needs to be further studied.The presentation of our patient is not classic IPEX syndrome because of late-onset symptoms and metaplastic atrophic gastritis. The mechanism of the variable phenotype of the same