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Hepatocellular carcinoma (HCC) is a heterogeneous, late-diagnosed, and highly recurrent malignancy that often affects the whole body's metabolism. Finding certain theranostic molecules which can address current concerns simultaneously is one of the priorities in HCC management. In this study, performing protein-protein interaction network analysis proposed Hepatocyte Nuclear Factor 4 alpha (HNF4α) as a hub protein, associating epithelial-mesenchymal transition (EMT) to reprogrammed cancer metabolism, formerly known as the Warburg effect. Both phenomena improved the compensation of cancerous cells in competitive conditions. Mounting evidence has demonstrated that HNF4α is commonly down-regulated and serves as a tumor suppressor in the HCC. Enhancing the HNF4α mRNA translation through a specific synthetic antisense long non-coding RNA, profoundly affects both EMT and onco-metabolic modules in HCC cells. HNF4α over-expression decreased featured mesenchymal transcription factors and improved hepatocytic function, decelerated glycolysis, accelerated gluconeogenesis, and improved dysregulated cholesterol metabolism. Moreover, HNF4α over-expression inhibited migration, invasion, and proliferation of HCC cells and decreased metastasis rate and tumor growth in xenografted nude mice. Our findings suggest a central regulatory role for HNF4α through its broad access to a wide variety of gene promoters involved in EMT and the Warburg effect in human hepatocytes. This essential impact indicates that HNF4α may be a potential target for HCC treatment.

Does pre-implantation uterine fluid lavage (UFL) of patients undergoing IVF and frozen embryo transfer (FET) affect implantation and clinical pregnancy rates? Which methods among ultracentrifugation, sucrose cushion and qEV column are suitable for isolating UFL extracellular vesicles?First, UFL was collected from 20 patients undergoing IVF and FET 2 days before embryo transfer as the case group. The control group consisted of 20 patients undergoing IVF and FET patients without lavage. All patients were monitored for 6 weeks. In the next step, the UFLs (n = 30) were collected and pooled. The UFL-derived extracellular vesicles were extracted by ultracentrifugation, sucrose cushion and qEV column methods and characterized.Preimplantation uterine lavage sampling did not affect implantation and clinical pregnancy rates. Extracellular vesicles were successfully isolated from UFL by all three methods. Scanning electron microscopy and dynamic light scattering analysis showed that the isolated vesicles were morphologically spherical. The qEV technique showed that they were smaller and homogenized in size. SDS-PAGE of extracellular vesicles showed a weaker albumin band in the qEV column. Western blot analysis indicated that the isolated extracellular vesicles by the qEV column were more immunoreactive for all the common extracellular vesicle markers (CD81, CD9, CD63, and TSG101). Six reference genes were compared by real-time polymerase chain reaction in the isolated extracellular vesicle subpopulations, and lowest cycle threshold value was observed for the 18SrRNA gene.The isolation of endometrial secretome extracellular vesicles is a minimally invasive procedure for individual assessment of endometrial receptivity and can be carried out during conception cycles along with transvaginal ultrasonography. Molecular analysis of UFL-derived extracellular vesicle components could suggest biomarkers to determine precise extracellular vesicle timing.

Diabetes mellitus may cause tau protein hyperphosphorylation and neurodegeneration, but the exact mechanism by which diabetic conditions induce tau pathology remains unclear. Tau protein hyperphosphorylation is considered a major pathological hallmark of neurodegeneration and can be triggered by diabetes. Various tau-directed kinases, including P38, can be activated upon diabetic stress and induce tau hyperphosphorylation. Despite extensive research efforts, the exact tau specie(s) and kinases driving neurodegeneration in diabetes mellitus have not been clearly elucidated. We herein employed different techniques to determine the exact molecular mechanism of tau pathology triggered by diabetes in in vivo and in vitro models. We showed that diabetes-related stresses and glucose metabolism deficiency could induce cis P-tau (an early driver of the tau pathology) accumulation in the midbrain and corpus callosum of the diabetic mice models and cells treated with 2-deoxy-D-glucose, respectively. We found that the active phosphorylated level of P38 was increased in the treated cells and diabetic mice models. We observed that oxidative stress activated P38, which directly and indirectly drove tau pathology in the GABAergic and glutamatergic neurons of the midbrain of the diabetic mice after 96 h, which accumulated in the other neighboring brain areas after 2 months. Notably, P38 inhibition suppressed tau pathogenicity and risk-taking behaviors in the animal models after 96 h. The data establish P38 as a central mediator of diabetes mellitus-induced tau pathology. Our findings provide mechanistic insight into the consequences of this metabolic disorder on the nervous system.

To find the gold standard out of three pre-established routine slow freezing (SF) methods, ovarian cortex tissues of nine transsexual individuals were cryopreserved and compared to each other, as well as the control (fresh) samples. Histological, genomic, and endocrinological effects of the SFs were assessed post-thawing and after a seven-day culture period. SF1 included 10% dimethyl-sulfoxide (Me2SO) in the base medium (BM), SF2 had 1.5 M/L ethylene-glycol (EG) and 0.1 M/L sucrose in the BM, and SF3 consisted of 6% Me2SO, 6% EG and 0.15 M/L sucrose in the BM. The cortical tissue strips went under a programmed cooling process and were stored in liquid nitrogen. Histological criteria (tissue damage and follicular quality), as well as gene expression levels, were assessed in the thawed and control tissues. Half of the thawed and control tissues were cultured for seven days and their histology, genetic profile, and hormonal status were examined as the reflection of the avascular tension effect. Post-thawing tissue damage was similar between all groups but significantly increased post-culture (P 0.05). The percentages of high-quality follicles diminished in all SFs after thawing and culture (P 0.05) except for the similarity of post-thawing SF3, compared to control. The genetic profile of the tissue after thawing and culture suggested quiescence/activation balance in SF1 and 2 and significant down-regulation in SF3, compared to the control specimens (P 0.05). Post-thawing BAX:BCL2 was higher than control in SF1 and SF3 (P 0.05), while this ratio in SF2 was similar to the control. However, after culture this ratio was similar to that of control in SF3 and diminished in SF1 and 2 (P 0.05). The expression levels of gap-junction genes showed dramatic pre- and post-thawing fluctuations in all groups. After culture, estradiol in SF3 was significantly higher than SF1 and 2 (P 0.05). In addition, progesterone in SF3 was similar to control but significantly lower in SF1 and 2 (P 0.05). In conclusion, all SFs showed advantages and disadvantages, and the follicular quality and its function depend on the type of cryoprotectant and the speed of thawing. The effects of freezing/thawing continue to appear during the seven days of culture. According to the results of this study, SF3 seems to be more promising in keeping the follicles functional and safe from cell damage during culture.

Background: Sperm-associated antigens (SPAGs) are 18 types of proteins, some of which play important roles in various biological functions associated with assisted reproductive technology outcomes,...

Breast cancer (BC) as a leading cause of cancer death among women, exhibits a wide range of genetic heterogeneity in affected individuals. Satisfactory management of BC depends on early diagnosis and proper monitoring of patients’ response to therapy. In this study, we aimed to assess the relation between the expression patterns of blood-based microRNAs (miRNAs) with demographic characteristics of the patients with BC in an attempt to find novel diagnostic markers for BC with acceptable precision in clinical applications. To this end, we performed comprehensive statistical analysis of the data of the Cancer Genome Atlas (TCGA) database and the blood miRNome dataset (GSE31309). As a result, 21 miRNAs were selected for experimental verification by quantitative RT-PCR on blood samples of 70 BC patients and 60 normal individuals (without any lesions or benign breast diseases). Statistical one-way ANOVA revealed no significant difference in the blood levels of the selected miRNAs in BC patients compared to any lesions or benign breast diseases. However, the multi-marker panel consisting of hsa-miR-106b-5p, −126-3p, −140-3p, −193a-5p, and −10b-5p could detect early-stages of BC with 0.79 sensitivity, 0.86 specificity and 0.82 accuracy. Furthermore, this multi-marker panel showed the potential of detecting benign breast diseases from BC patients with 0.67 sensitivity, 0.80 specificity, and 0.74 accuracy. In conclusion, these data indicate that the present panel might be considered an asset in detecting benign breast disease and BC.

Background Retained placenta (RP) is a prevalent disorder in cattle with many health-related and economic costs for the farm owners. Its etiology has not been clarified yet and there is no definite therapy for this disorder. In this study we conducted RNA-seq, hematologic and histologic experiments to survey the causes of RP development. Methods Blood samples were collected from 4 RP and 3 healthy cows during periparturtion period for hematological assessments followed by placentome sampling within 30 min after parturition. Cows were grouped as RP and control in case the placenta was retained or otherwise expelled, respectively. Total RNA was extracted from placentome samples followed by RNA-sequencing. Results We showed 240 differentially expressed genes (DEGs) between the RP and control groups. Enrichment analyzes indicated immune system and lipid metabolism as prominent over- and under-represented pathways in RP cows, respectively. Hormonal assessments showed that estradiol-17β (E2) was lower and cortisol tended to be higher in RP cows compared to controls at the day of parturition. Furthermore, histologic experiment showed that villi-crypt junctions remain tighter in RP cows compared to controls and the crypts layer seemed thicker in the placentome of RP cows. Complete blood cell (CBC) parameters were not significantly different between the two groups. Conclusion Overall, DEGs derived from expression profiling and these genes contributed to enrichment of immune and lipid metabolism pathways. We suggested that E2 could be involved in development of RP and the concentrations of P4 and CBC counts periparturition might not be a determining factor.

STUDY QUESTIONCan whole-exome sequencing (WES) reveal a shared pathogenic variant responsible for primary gonadal failure in both male and female patients from a consanguineous family?SUMMARY ANSWERPatients with primary ovarian insufficiency (POI) and non-obstructive azoospermia (NOA) were homozygous for the rare missense variant p. S754L located in the highly conserved MSH4 MutS signature motif of the ATPase domain. An oligozoospermic patient was heterozygous for the variant.WHAT IS KNOWN ALREADYMSH4 is a meiosis-specific protein expressed at a certain level in the testes and ovaries. Along with its heterodimer partner MSH5, it is responsible for double-strand Holliday junction recognition and stabilization, to ensure accurate chromosome segregation during meiosis. Knockout male and female mice for Msh4 and Msh5 are reportedly infertile due to meiotic arrest. In humans, MSH4 is associated with male and female gonadal failure, with distinct variations in the MutS domain V.STUDY DESIGN, SIZE, DURATIONThis was a retrospective genetics study of a consanguineous family with multiple cases of gonadal failure in both genders. The subject family was recruited in Iran, in 2018.PARTICIPANTS/MATERIALS, SETTING, METHODSThe proband who is affected by POI, an NOA brother, a fertile sister and their parents were subjected to WES. The discovered variant was validated in these individuals, and the rest of the family was also genotyped by Sanger sequencing. The variant was not detected in 800 healthy Iranian individuals from the Iranome database nor in 30 sporadic NOA and 30 sporadic POI patients. Suggested effect in aberrant splicing was studied by RT-PCR. Moreover, protein homology modeling was used to further investigate the amino acid substitution in silico.MAIN RESULTS AND THE ROLE OF CHANCEThe discovered variant is very rare and has never been reported in the homozygous state. It occurs in the ATPase domain at Serine 754, the first residue within the highly conserved MutS signature motif, substituting it with a Leucine. All variant effect prediction tools indicated this variant as deleterious. Since the substitution occurs immediately before the Walker B motif at position 755, further investigations based on protein homology were conducted. Considering the modeling results, the nature of the substituted amino acid residue and the distances between p. S754L variation and the residues of the Walker B motif suggested the possibility of conformational changes affecting the ATPase activity of the protein.LARGE SCALE DATAWe have submitted dbSNP entry rs377712900 to ClinVar under SCV001169709, SCV001169708 and SCV001142647 for oligozoospermia, NOA and POI, respectively.LIMITATIONS, REASONS FOR CAUTIONStudies in model organisms can shed more light on the role of this variant as our results were obtained by variant effect prediction tools and protein homology modeling.WIDER IMPLICATIONS OF THE FINDINGSIdentification of variants in meiotic genes should improve genetic counseling for both male and female infertility. Also, as two of our NOA patients underwent testicular sperm extraction (TESE) with no success, ruling out the existence of pathogenic variants in meiotic genes in such patients prior to TESE could prove useful.STUDY FUNDING/COMPETING INTEREST(S)This study was financially supported by Royan Institute in Tehran, Iran, and Institut Pasteur in Paris, France. The authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A

Missense variants in the RNA-helicase DHX37 are associated with either 46,XY gonadal dysgenesis or 46,XY testicular regression syndrome (TRS). DHX37 is required for ribosome biogenesis, and this subgroup of XY DSD is a new human ribosomopathy. In a cohort of 140 individuals with 46,XY DSD, we identified 7 children with either 46,XY complete gonadal dysgenesis or 46,XY TRS carrying rare or novel DHX37 variants. A novel p.R390H variant within the RecA1 domain was identified in a girl with complete gonadal dysgenesis. A paternally inherited p.R487H variant, previously associated with a recessive congenital developmental syndrome, was carried by a boy with a syndromic form of 46,XY DSD. His phenotype may be explained in part by a novel homozygous loss-of-function variant in the NGLY1 gene, which causes a congenital disorder of deglycosylation. Remarkably, a homozygous p.T477H variant was identified in a boy with TRS. His fertile father had unilateral testicular regression with typical male genital development. This expands the DSD phenotypes associated with DHX37. Structural analysis of all variants predicted deleterious effects on helicase function. Similar to all other known ribosomopathies, the mechanism of pathogenesis is unknown.

Familial hypercholesterolemia (FH, OMIM number #143890), a life-threatening monogenic disorder characterized by high levels of low-density lipoprotein cholesterol (LDL-C), is classified into dominant and recessive types (1). The dominant form of FH may result from mutations in the LDLR, APOB, and PCSK9 genes (2). However, mutations in the low-density lipoprotein receptor (LDLR) adaptor protein-1 (LDLRAP1) gene cause an autosomal recessive inheritance pattern of FH called autosomal recessive hypercholesterolemia (ARH, OMIM number #603813). The LDLRAP1 protein, encoded by the LDLRAP1 gene, is required for receptor-mediated endocytosis of LDL-C (2). ARH is a rare disorder with an estimated prevalence of less than 1 in a population of one million. This disease is considered a phenocopy of the most severe form of FH, homozygous familial hypercholesterolemia (HoFH; OMIM number 143890). Hence, most ARH patients are clinically indiscernible from HoFH, which is caused by two defective LDLR genes with an approximate prevalence of one individual per million (8). Considering that ARH patients may develop aggressive and premature atherosclerotic cardiovascular disease (ASCVD) due to hypercholesterolemia during early adulthood, lipid-lowering therapy must be initiated during childhood (3). Early identification of ARH patients through genetic analysis of the proband and their relatives can provide prognosis and subsequently appropriate, timely treatment. In this report, we describe a novel variant, c.649G>T, p.Glu217Ter, in the homozygous state in exon 7 of the LDLRAP1 gene, causing severe ARH.

Inactivation of tumor suppressor genes, such as RAP1GAP, by hypermethylation of their regulatory region can give rise to thyroid tumors. The aim of this study was to investigate the expression of the RAP1GAP gene and the DNA methylation patterns of its CpG74a, CpG74b, and CpG24 in an Iranian population with differentiated thyroid cancer (DTC). In this study, 160 individuals who underwent thyroidectomy in the Tehran Erfan Hospital between 2018 and 2020 were selected. DNA methylation patterns of selected CpG islands (CpG74a, CpG74b, and CpG24) were determined using methylation-specific PCR. The mRNA expression and protein level of ­RAP1GAP were also evaluated. SW1736 and B-CPAP cells were treated with 5-aza-2′-deoxycytidine (5-Aza) to demethylate these regions. The hypermethylation rates of CpG74a and CpG24 in DTC samples were significantly higher than in the control. The mRNA expression and protein level of ­RAP1GAP were significantly decreased in the DTC group. In the DTC group, hypermethylation in CpG74a was correlated with decreasing RAP1GAP expression (R2: 0.34; p = 0.043). CpG74a with a specificity of 86.4% has significant prediction power to distinguish between DTC and normal thyroid tissues. Additionally, hypermethylation of CpG74a was significantly associated with higher tumor stages (stage III-IV: 77%; stage I-II: 23%; p = 0.012). Increasing expression of RAP1GAP after demethylation with 15 µM of 5-Aza was observed in both cell lines. These results indicate that DNA hypermethylation in CpG74a can be considered as an epigenetic biomarker in DTC.

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide that is attributed to gradual long-term accumulation of both genetic and epigenetic changes. To reduce the mortality rate of CRC and to improve treatment efficacy, it will be important to develop accurate noninvasive diagnostic tests for screening, acute and personalized diagnosis. Epigenetic changes such as DNA methylation play an important role in the development and progression of CRC. Over the last decade, a panel of DNA methylation markers has been reported showing a high accuracy and reproducibility in various semi-invasive or noninvasive biosamples. Research to obtain comprehensive panels of markers allowing a highly sensitive and differentiating diagnosis of CRC is ongoing. Moreover, the epigenetic alterations for cancer therapy, as a precision medicine strategy will increase their therapeutic potential over time. Here, we discuss the current state of DNA methylation-based biomarkers and their impact on CRC diagnosis. We emphasize the need to further identify and stratify methylation-biomarkers and to develop robust and effective detection methods that are applicable for a routine clinical setting of CRC diagnostics particularly at the early stage of the disease.

Autophagy is responsible for degradation of non-essential or damaged cellular constituents and damaged organelles. The autophagy pathway maintains efficient cellular metabolism and reduces cellular stress by removing additional and pathogenic components. Dysfunctional autophagy underlies several diseases. Thus, several research groups have worked toward elucidating key steps in this pathway. Autophagy can be studied by animal modeling, chemical modulators, and in vitro disease modeling with induced pluripotent stem cells (iPSC) as a loss-of-function platform. The introduction of iPSC technology, which has the capability to maintain the genetic background, has facilitated in vitro modeling of some diseases. Furthermore, iPSC technology can be used as a platform to study defective cellular and molecular pathways during development and unravel novel steps in signaling pathways of health and disease. Different studies have used iPSC technology to explore the role of autophagy in disease pathogenesis which could not have been addressed by animal modeling or chemical inducers/inhibitors. In this review, we discuss iPSC models of autophagy-associated disorders where the disease is caused due to mutations in autophagy-related genes. We classified this group as "primary autophagy induced defects (PAID)". There are iPSC models of diseases in which the primary cause is not dysfunctional autophagy, but autophagy is impaired secondary to disease phenotypes. We call this group "secondary autophagy induced defects (SAID)" and discuss them. Graphical abstract.

Mouse embryonic fibroblasts (MEFs) accessibility coupled with their simple generation make them as a typical embryonic cell model and feeder layer for in vitro expansion of pluripotent stem cells (PSCs). In this study, a mechanical isolation technique was adopted to isolate MEFs and the efficiency of this technique was compared with enzymatic digestion method. The suspended MEFs were prepared either by mechanical method or 0.25% trypsin enzymatic digestion. The effect of tissue processing on cell apoptosis/necrosis, morphology, viable cell yield, population doubling time, surface marker expression, and the capacity to support PSCs were determined. The mechanical method yielded a significantly higher number of viable cells. However, it showed similar morphology and proliferation characteristics as compared to enzymatic digestion. The mechanical method induced slight apoptosis in MEFs; however, it did not exert the necrotic effect of trypsinization. Treatment of tissue slurry with trypsin solution caused cell lysis and subsequently cell clump formation. Mechanically isolated cells exhibited a higher expression of the MEF surface antigens Sca1, CD106, and CD105. The PSCs on mechanically isolated MEFs displayed a higher expression of pluripotency genes, and formed more compact colonies with a stronger tendency to crowding compared with those cultured on cells isolated by enzymatic digestion. The mechanical method based on tissue inter-syringe processing is relatively a rapid and simple method for MEF isolation. Compared to the enzymatic digestion, the cells obtained from this method show higher expression of embryonic fibroblasts markers and a more functional capacity in supporting PSCs culture.

BACKGROUND AND AIM Cancer stem cells (CSCs), a subpopulation of tumor cells, assess the capacity of self-renewal, metastasis, and therapeutic resistance. Regulation of CSCs and their epithelial to mesenchymal transition (EMT) potential is one of the promising strategies to eliminate cancer or to inhibit metastasis. Micro-RNAs (miRNAs) as regulators of several cell properties, such as self-renewal, metastasis, and resistance to the drug, could be proper targets in cancer diagnosis and therapy. The aim of the present study is to select common miRNAs targeting both self-renewal and metastasis in gastric cancer. METHODS Stemness-related and EMT-related genes were selected by literature mining. The common miRNAs targeting genes were chosen using different databases and r programming language. The expression pattern of selected miRNAs and genes was evaluated in gastrospheres-as a gastric CSC model-and gastric tumor biopsies. RESULTS Based on the integrated analysis, six miRNAs common to both stemness and metastasis were identified. miR-200c-3p and miR-520c-3p overexpressed in MKN-45 gastrospheres and grade III tumors. In AGS spheres, however, miR-520c-3p and miR-200c-3p upregulation and miR-34a-5p downregulation were similar to grade II tumors. Interestingly, miR-200c-3p and miR-520c-3p indicated a positive correlation with OCT4 and NOTCH1 expression in grade III tumors and MKN-45 spheres. Protein-protein network revealed that the EMT acquisition can be induced by stemness activation through intermediated core-regulatory genes, including CTNNB1, CTNND1, MAML1, KAT2A, and MAML3. CONCLUSION The upregulation of mir-200c-3p and mir-520c-3p could effect on stemness and metastasis in gastric cancer as well as gastric CSCs. Therefore, they can be used as diagnosis and prognostic factors.

Background Vitiligo is an acquired pigmentary skin disorder characterized by depigmented macules and patches which brings many challenges for the patients suffering from. For vitiligo severity assessment, several scoring methods have been proposed based on morphometry and colorimetry. But, all methods suffer from much inter- and intra-observer variations for estimating the depigmented area. For all mentioned assessment methods of vitiligo disorder, accurate segmentation of the skin images for lesion detection and localization is required. The image segmentation for localizing vitiligo skin lesions has many challenges because of illumination variation, different shapes and sizes of vitiligo lesions, vague lesion boundaries and skin hairs and vignette effects. The manual image segmentation is a tedious and time-consuming task. Therefore, using automatic image segmentation methods for lesion detection is necessarily required. Materials and methods In this study, a novel unsupervised stack ensemble of deep and conventional image segmentation (SEDCIS) methods is proposed for localizing vitiligo lesions in skin images. Unsupervised segmentation methods do not require prior manual segmentation of vitiligo lesions which is a tedious and time-consuming task with intra- and inter-observer variations. Results Our collected dataset includes 877 images taken from 21 patients with the resolution of 5760*3840 pixels suffering from vitiligo disorder. Experimental results show that SEDCIS outperforms the compared methods with accuracy of 97%, sensitivity of 98%, specificity of 96%, area overlapping of 94%, and Dice index of 97%. Conclusion The proposed method can segment vitiligo lesions with highly reasonable performance and can be used for assessing the vitiligo lesion surface.

International audience; Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1 Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor β-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

Lithium remains the first-line treatment for bipolar disorder (BD), but patients respond to it variably. While a myriad of studies have attributed many genes and signaling pathways to lithium responsiveness, a comprehensive study with an integrated conclusion is still lacking. We aim to present an integrated mechanism for the therapeutic actions of lithium in BD. First, a list of lithium responsiveness-associated genes (LRAGs) was collected by searching in the literature. Thereafter, gene set enrichment analysis together with gene–gene interaction network analysis was performed, in order to find the cellular and molecular events related to the LRAGs. Gene set enrichment analyses showed that the chromosomal regions 3p26, 4p21, 5q34 and 7p13 could be novel associated loci for lithium responsiveness in BD. Also, expression pattern analysis of the LRAGs showed their enrichment in adulthood stages and different cell lineages of brain, blood and immune system. Most of the LRAGs exhibited enriched expression in central parts of human brain, suggesting major contribution of these parts in lithium responsiveness. Beside the prediction of several biological processes and signaling pathways related to lithium responsiveness, an interaction network between these processes was constructed that was found to be regulated by a set of microRNAs. Proteins of the network were mainly classified as transcription factors and kinases, which also highlighted the crucial role of glycogen synthase kinase 3β (GSK3β) in lithium responsiveness. The predicted cellular and molecular events in this study could be considered as mechanisms and also determinants of lithium responsiveness in BD.

The major risk factor for kidney stone disease is idiopathic hypercalciuria. Recent evidence implicates a role for defective calcium reabsorption in the renal proximal tubule. We hypothesized that claudin-2, a paracellular cation channel protein, mediates proximal tubule calcium reabsorption. We found that claudin-2–null mice have hypercalciuria due to a primary defect in renal tubule calcium transport and papillary nephrocalcinosis that resembles the intratubular plugs in kidney stone formers. Our findings suggest that a proximal tubule defect in calcium reabsorption predisposes to papillary calcification, providing support for the vas washdown hypothesis. Claudin-2–null mice were also found to have increased net intestinal calcium absorption, but reduced paracellular calcium permeability in the colon, suggesting that this was due to reduced intestinal calcium secretion. Common genetic variants in the claudin-2 gene were associated with decreased tissue expression of claudin-2 and increased risk of kidney stones in 2 large population-based studies. Finally, we describe a family in which males with a rare missense variant in claudin-2 have marked hypercalciuria and kidney stone disease. Our findings indicate that claudin-2 is a key regulator of calcium excretion and a potential target for therapies to prevent kidney stones.

Cardiomyopathies are a group of common heart disorders that affect numerous people worldwide. Left ventricular non-compaction (LVNC) is a structural disorder of the ventricular wall, categorized as a type of cardiomyopathy that mostly caused by genetic disorders. Genetic variations are underlying causes of developmental deformation of the heart wall and the resultant contractile insufficiency. Here, we investigated a family with several affected members exhibiting LVNC phenotype. By whole-exome sequencing (WES) of three affected members, we identified a novel heterozygous missense variant (c.1963CA:p.Leu655Met) in the gene encoding myosin heavy chain 7 (

Histone-to-protamine transition is an essential step in the generation of fully functional spermatozoa in various mammalian species. In human and mouse, one of the two protamine-encoding genes produces a precursor pre-protamine 2 (pre-PRM2) protein, which is then processed and assembled. Here, we design an original approach based on the generation of pre-PRM2-specific antibodies to visualize the unprocessed pre-PRM2 by microscopy, flow cytometry and immunoblotting. Using mouse models with characterized failures in histone-to-protamine replacement, we show that pre-PRM2 retention is tightly linked to impaired nucleosome disassembly. Additionally, in elongating/condensing spermatids, we observe that pre-PRM2 and transition protein are co-expressed spatiotemporally, and their physical interaction suggests that these proteins act simultaneously rather than successively during histone replacement. By using our anti-human pre-PRM2 antibody, we also measured pre-PRM2 retention rates in the spermatozoa from 49 men of a series of infertile couples undergoing ICSI, which shed new light on the debated relation between pre-PRM2 retention and sperm parameters. Finally, by monitoring 2-pronuclei embryo formation following ICSI, we evaluated the fertilization ability of the sperm in these 49 patients. Our results suggest that the extent of pre-PRM2 retention in sperm, rather than pre-PRM2 accumulation per se, is associated with fertilization failure. Hence, anti-pre-PRM2 antibodies are valuable tools that could be used in routine monitoring of sperm parameters in fertility clinics, as well as in experimental research programmes to better understand the obscure process of histone-to-protamine transition.

Proper assembly of the synaptonemal complex is essential for successful meiosis, and impairments in the process lead to infertility. Meiotic transverse filament proteins encoded by the SYCP1 (synaptonemal complex protein 1) gene are one of the main components of the synaptonemal complex and play an important role in correct synapsis and recombination. Family-based whole-exome sequencing revealed a rare homozygous SYCP1 frameshift mutation (c.2892delA: p.K967Nfs*1) in two men with severe oligozoospermia, followed by validation and segregation through Sanger sequencing. This single nucleotide deletion not only changes lysine 967 (K) into asparagine (N) but also causes a premature stop codon, which leads to deletion of 968–976 residues from the end of the C-tail region of the SYCP1 protein. Although, sycp1 knockout male mice are reported to be sterile with a complete lack of spermatids and spermatozoa, to date no SYCP1 variant has been associated with human oligozoospermia. HADDOCK analysis indicated that this mutation decreases the ability of the truncated SYCP1 protein to bind DNA. Immunodetection of ϒH2AX signals in SYCP1 mutant semen cells, and a 40% DNA fragmentation index might indicate that a small number of DNA double-strand breaks, which require SYCP1 and/or synapsis to be repaired, are not efficiently repaired, resulting in defects in differentiation of germline cells and appearance of the oligozoospermia phenotype. To our knowledge, this is the first report of a homozygous SYCP1 mutation that decreases sperm count. Further studies are required to determine the function of the SYCP1 mutation, which is potentially associated with human oligozoospermia.

Background: Autosomal recessive hypercholesterolemia (ARH) is a rare monogenic disorder resulting from mutations of the LDLRAP1 gene, which leads to elevated LDL-C levels. Here, using whole exome sequencing (WES), we describe a 22-year-old Iranian female who carries a novel nonsense mutation in LDLRAP1. Methods: Genetic investigations were performed for the patient and her family. She showed LDL-C level of 720 mg/dL since the age of 11 years. At the age of 13 years old, aortic valve repair surgery was performed due to severe aortic valve stenosis (AVS). At the age of 17, along with prescription of rosuvastatin plus ezetimibe, coronary angiography displayed the presence of serious stenotic lesions of the coronary arteries and also aortic valve, making the patient eligible for coronary artery bypass grafting (CABG) and aortic valve replacement (AVR). Results: Genetic analysis showed the presence of a previously unreported homozygous LDLRAP1 gene variant, c.649G>T, generating a nonsense mutation at amino acid 217, shortening the ARH protein from 308 to 217 amino acid, which removes AP-2 binding domain of ARH, as an important part in LDL uptake.Conclusion: During a 10-year treatment, we observed a 74% reduction in LDL-C level. Despite the treatment with maximal dose of rosuvastatin plus ezetimibe, the results of coronary angiography demonstrated severe supravalvular aortic stenosis (SVAS) resulted in significant stenotic lesions of the coronary arteries and aortic valve. This highlights the importance of WES in early diagnosis of ARH, and it is proposed to prevent or at least delay the onset of the cardiovascular events.

Background EIF1AY is one of the genes essential for normal spermatogenesis and is located in azoospermic factors region. Objective The present study was designed to investigate the EIF1AY gene nucleotide variations, and correlate it with spermatogenic maturation arrest and azoospermia in Iranian population. Methods A total number of 30 Iranian idiopathic non-obstructive azoospermic patients were selected as case group and 30 fertile men served as a control group who had at least 1 child. Nucleotide variation was analyzed in exon 3 and exon 5 in EIF1AY gene of both groups. DNA extraction from peripheral blood samples of selected individuals was done followed by amplification by PCR and sequencing with Sangar method. Results Totally 3 single nucleotide variations were identified: one in the intronic region of exon 3, next one in non-coding transcript exon variant (rs13447352) and the third one in the exonic region of exon 5, all were registered in NCBI-Gene database. Conclusion There was no statistically significant difference in the incidence of nucleotide variation between 2 study populations (p > 0.05). Further studies are required to specify the effects of Y: T20588295G variation on modification of protein structure, as well as the expression pattern of the gene and its association with azoospermia.

Pancreatic ductal adenocarcinoma (PDAC) arises through accumulation of multiple genetic alterations. However, cancer cells also acquire and depend on cancer-specific epigenetic changes. To conclusively demonstrate the crucial relevance of the epigenetic programme for the tumourigenicity of the cancer cells, we used cellular reprogramming technology to reverse these epigenetic changes. We reprogrammed human PDAC cultures using three different techniques – (1) lentivirally via induction of Yamanaka Factors (OSKM), (2) the pluripotency-associated gene OCT4 and the microRNA mir-302, or (3) using episomal vectors as a safer alternative without genomic integration. We found that induction with episomal vectors was the most efficient method to reprogram primary human PDAC cultures as well as primary human fibroblasts that served as positive controls. Successful reprogramming was evidenced by immunostaining, alkaline phosphatase staining, and real-time PCR. Intriguingly, reprogramming of primary human PDAC cultures drastically reduced their in vivo tumourigenicity, which appeared to be driven by the cells’ enhanced differentiation and loss of stemness upon transplantation. Our study demonstrates that reprogrammed primary PDAC cultures are functionally distinct from parental PDAC cells resulting in drastically reduced tumourigenicity in vitro and in vivo. Thus, epigenetic alterations account at least in part for the tumourigenicity and aggressiveness of pancreatic cancer, supporting the notion that epigenetic modulators could be a suitable approach to improve the dismal outcome of patients with pancreatic cancer.

This study aims to prepare intermediate mesoderm-like cells from mouse embryonic fibroblasts (MEFs). In the first step, intermediate mesoderm-like cells (IMLCs) and renal epithelial-like cells (RELCs) were extracted from mouse embryonic stem cells (mESCs) in a specified media that contained two small molecules, CHIR99021 and TTNPB, along with growth factors, FGF9and BMP7. Then, MEFs were directly converted into IM by genes for the pluripotency factors, which encode the transcription factors; Oct4, Sox2, Klf4, and c-Myc (OSKM). These unstable intermediate cells were quickly encouraged to form IM with the assistance of CHIR99021 and TTNPB. The results showed that exogenous expression of OSKM factors for four days was adequate to generate partially reprogrammed cells (SSEA1+/Nanog-). Real-time PCR and immunocytochemistry analysis confirmed the presence of the MEF-derived IMs. This study introduced a method for mESCs differentiation to RELCs followed by MEF conversion in an attempt to generate IM by circumventing pluripotency.

Study question Can whole exome sequencing (WES) reveal a novel pathogenic variant in asthenozoospermia in a multiplex family including multiple patients? Summary answer Patients were discovered to be homozygous for a rare 2-bp deletion in the ADCY10 coding region (c.1205_1206del, rs779944215). What is known already ADCY10 encodes for soluble adenylyl cyclase (sAC), which is the predominant adenylate cyclase in sperm. It is already established that proper sAC activity and a constant supply of cAMP are crucial to sperm motility regulation, and knockout mouse models have been reported as severely asthenozoospermic. ADCY10 is a susceptibility gene for dominant absorptive hypercalciuria (OMIM#143870); however, no ADCY10 variations have been confirmed to cause human asthenozoospermia to date. Study design, size, duration This was a retrospective genetics study of a highly consanguineous pedigree of asthenozoospermia. The subject family was recruited in Iran in 2016. Participants/materials, setting, methods The two patients were diagnosed as asthenozoospermic through careful clinical investigations. Both patients, respective parents, and an unaffected brother were subjected to WES. The discovered variant was validated by Sanger sequencing and segregated with the phenotype. To confirm the pathogenicity of the variant, sperm samples from both patients, 10 normozoospermic men and 10 asthenozoospermic patients not representing the variation, were treated with a cAMP analogue dissolved in human tubal fluid medium, followed by computer-assisted sperm analysis and statistical analyses. Main results and the role of chance The discovered homozygous variant occurs at 10 amino acids upstream of the ADCY10 nucleotide binding site leading to a premature termination (p.His402Argfs*41). Treatment of the patients' sperm samples with a cell-permeable cAMP analogue resulted in a significant increase in sperm motility, indicating the pathogenic role of the variant. Moreover, absorptive hypercalciuria, segregating within the family, was also associated with the same variant following a dominant inheritance. Limitations, reasons for caution Though nonsense-mediated decay is highly likely to occur in the mutated transcripts, we were not able to confirm this due to low RNA levels in mature sperm. Wider implications of the findings Our finding enlarges the phenotypic spectrum associated with the ADCY10 gene, previously described as a susceptibility gene for dominant absorptive hypercalciuria. Study funding/competing interest(s) This study was supported by grants from the Royan Institute, Tehran, Iran, and San Raffaele Hospital, Milan, Italy. The authors have no conflict of interest. Trial registration number N/A.

Sox2 transcription factor has been frequently used for reprograming starting cells to neural progenitor/stem cells. In vivo administration of Sox2 in the adult mouse brain reprogrammed the transduced astrocytes to neurons. In searching for adequate cell source for repairing the myelin insults, here, we studied the possible conversion of astrocytes to oligodendrocyte lineage cells by Sox2, while an extensive demyelination exists in animal brain. Lentiviral particles expressing Sox2-GFP were injected into the corpora callosa of animals fed with cuprizone diet for 12 weeks. Transduced cells were mainly astrocytes that changed their fate to oligodendrocyte lineage cells by time. For further conformation astrocytes received the vector in culture and then transplanted to the animal brains. Tracing the fate of transplanted cells showed their conversion to oligodendrocyte lineage cells. In vitro transduced cell were also maintained in the oligodendrocyte progenitor cell (OPC) induction medium. Produced OPC-like cells were positive for specific markers. This study provides a new strategy for endogenous production of myelinating cells. After optimizing the experimental conditions for safety and feasibility, this approach may contribute into future cell based therapies in patients with white matter insults as like as those with multiple sclerosis.

After lifting the COVID-19 lockdown restrictions and opening businesses, screening is essential to prevent the spread of the virus. Group testing could be a promising candidate for screening to save time and resources. However, due to the high false-negative rate (FNR) of the RT-PCR diagnostic test, we should be cautious about using group testing because a group’s false-negative result identifies all the individuals in a group as uninfected. Repeating the test is the best solution to reduce the FNR, and repeats should be integrated with the group-testing method to increase the sensitivity of the test. The simplest way is to replicate the test twice for each group (the 2Rgt method). In this paper, we present a new method for group testing (the groupMix method), which integrates two repeats in the test. Then we introduce the adaptive version of both the groupMix and the 2Rgt methods. We compare these methods analytically regarding the sensitivity and the average number of tests. The tradeoff between the sensitivity and the average number of tests should be considered when choosing the best method for the screening strategy. We applied the non-adaptive groupMix method to screening 263 people and identified 2 infected individuals by performing 98 tests. This method achieved a 63% saving in the number of tests compared to individual testing. This method is currently applied to COVID-19 screening in the Clinical Genetic Laboratory at the Royan Institute, Tehran, Iran. Our experimental results show that in COVID-19 screening, the viral load can be low, and the group size should not be more than 6; otherwise, the FNR increases significantly. A web interface of the non-adaptive groupMix method is publicly available for laboratories to implement this method.

Autosomal recessive hypercholesterolemia (ARH) is a rare monogenic disorder caused by pathogenic variants in the low-density lipoprotein receptor (LDLR) adaptor protein 1 (LDLRAP1) gene, encoding for the LDLRAP1 protein, which impairs internalization of hepatic LDLR. There are variable responses of ARH patients to treatment and the pathophysiological mechanism(s) for this variability remains unclear. This is in part caused by absence of reliable cellular models to evaluate the effect of LDLRAP1 mutations on the LDLRAP1 protein function and its role in LDLR internalization. Here, we aimed to validate patient-specific induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) as an appropriate tool to model ARH disease. Fibroblasts from an ARH patient carrying the recently reported nonsense mutation, c.649G>T, were reprogrammed into hiPSCs using Sendai viral vectors. In addition, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create an LDLRAP1 gene (also known as ARH) knockout in two different human iPSC lines. ARH patient-derived iPSCs, ARH-knockout iPSC lines, and control iPSCs were efficiently differentiated into HLCs. Western blot analysis demonstrated the absence of LDLRAP1 in HLCs derived from patient and knockout iPSCs, and this was associated with a decreased low-density lipoprotein cholesterol (LDL-C) uptake in ARH-mutant/knockout HLCs compared to control HLCs. In conclusion, we determined that the recently described c.649G>T point mutation in LDLRAP1 induces absence of the LDLRAP1 protein, similar to what is seen following LDLRAP1 knockout. This causes a decreased, although not fully absent, LDL-uptake in ARH-mutant/knockout HLCs. As knockout of LDLRAP1 or presence of the c.649G>T point mutation results in absence of LDLRAP1 protein, residual LDL uptake might be regulated by LDLRAP1-independent internalization mechanisms. Patient-specific iPSC-derived HLCs can therefore be a powerful tool to further decipher LDLRAP1 mutations and function of the protein.

Background: Autosomal recessive hypercholesterolemia (ARH) is a rare monogenic disorder resulting from mutations of the LDLRAP1 gene, which leads to elevated LDL-C levels. Here, using whole exome sequencing (WES), we describe a 22-year-old Iranian female who carries a novel nonsense mutation in LDLRAP1. Methods: Genetic investigations were performed for the patient and her family. She showed LDL-C level of 720 mg/dL since the age of 11 years. At the age of 13 years old, aortic valve repair surgery was performed due to severe aortic valve stenosis (AVS). At the age of 17, along with prescription of rosuvastatin plus ezetimibe, coronary angiography displayed the presence of serious stenotic lesions of the coronary arteries and also aortic valve, making the patient eligible for coronary artery bypass grafting (CABG) and aortic valve replacement (AVR).Results: Genetic analysis showed the presence of a previously unreported homozygous LDLRAP1 gene variant, c.649G>T, generating a nonsense mutation at amino acid 217, shortening the ARH protein from 308 to 217 amino acid, which removes AP-2 binding domain of ARH, as an important part in LDL uptake.Conclusion: During a 10-year treatment, we observed a 74% reduction in LDL-C level. Despite the treatment with maximal dose of rosuvastatin plus ezetimibe, the results of coronary angiography demonstrated severe supravalvular aortic stenosis (SVAS) resulted in significant stenotic lesions of the coronary arteries and aortic valve. This highlights the importance of WES in early diagnosis of ARH, and it is proposed to prevent or at least delay the onset of the cardiovascular events.

Background Intrauterine Insemination (IUI) outcome prediction is a challenging issue which the assisted reproductive technology (ART) practitioners are dealing with. Predicting the success or failure of IUI based on the couples' features can assist the physicians to make the appropriate decision for suggesting IUI to the couples or not and/or continuing the treatment or not for them. Many previous studies have been focused on predicting the in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) outcome using machine learning algorithms. But, to the best of our knowledge, a few studies have been focused on predicting the outcome of IUI. The main aim of this study is to propose an automatic classification and feature scoring method to predict intrauterine insemination (IUI) outcome and ranking the most significant features. Methods For this purpose, a novel approach combining complex network-based feature engineering and stacked ensemble (CNFE-SE) is proposed. Three complex networks are extracted considering the patients' data similarities. The feature engineering step is performed on the complex networks. The original feature set and/or the features engineered are fed to the proposed stacked ensemble to classify and predict IUI outcome for couples per IUI treatment cycle. Our study is a retrospective study of a 5-year couples' data undergoing IUI. Data is collected from Reproductive Biomedicine Research Center, Royan Institute describing 11,255 IUI treatment cycles for 8,360 couples. Our dataset includes the couples' demographic characteristics, historical data about the patients' diseases, the clinical diagnosis, the treatment plans and the prescribed drugs during the cycles, semen quality, laboratory tests and the clinical pregnancy outcome. Results Experimental results show that the proposed method outperforms the compared methods with Area under receiver operating characteristics curve (AUC) of 0.84 ± 0.01, sensitivity of 0.79 ± 0.01, specificity of 0.91 ± 0.01, and accuracy of 0.85 ± 0.01 for the prediction of IUI outcome. Conclusions The most important predictors for predicting IUI outcome are semen parameters (sperm motility and concentration) as well as female body mass index (BMI).

Non-obstructive azoospermia (NOA) and primary ovarian insufficiency (POI) present the most severe forms of male and female infertility. In the last decade, the increasing use of whole exome sequencing (WES) in genomics studies of these conditions has led to the introduction of a number of novel genes and variants especially in meiotic genes with restricted expression to gonads. In this study, exome sequencing of a consanguineous Iranian family with one POI and two NOA cases in three siblings showed that all three patients were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 (NM_001282717.1:c.1942G A: p.Ala648Thr; NM_001282717.1:c.1951_1953del: p. Leu652del). Both variants occur within a short proximity of each other affecting the relatively conserved armadillo-type fold superfamily feature. STAG3 is a specific meiotic cohesin complex component that interacts with the α-kleisin subunit through this feature. Protein homology modeling indicated that the in-frame deletion destabilizes kleisin biding by STAG3. Although the missense variant did not seem to affect the binding significantly, protein homology modeling suggests that it further destabilizes kleisin binding when in double homozygous state with the deletion. Our findings are in line with several other studies having associated deleterious variants affecting this region with male and female infertility in humans and mouse models. This is the first report associating an in-frame STAG3 variant with NOA and POI in a single family. SUMMARY SENTENCE: A patient with primary ovarian failure and her two brothers with non-obstructive azoospermia were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 that potentially disrupt the protein's meiotic functions.

Autologous transplantation of epidermal cells has been used increasingly to treat vitiligo patients and is a simple, safe, and relatively efficient method. However, the outcome is not always satisfactory, and some patients show less or no response to this treatment. This study was evaluated to identify genes expressed differently among responders and non-responders to cell transplantation to find potential markers that could predict 'patients' responses to this type of cell therapy.Eleven stable vitiligo patients who received autologous epidermal cell transplantation were included in this clinical trial study. Before cell transplantation, skin samples were obtained from the recipient's vitiligo lesions. After epidermal cell transplantation, patients were followed for at least six months to assess the response to epidermal cell injection. RNA sequencing was used to determine potential gene expression profile differences between responder and non-responder vitiligo patients.The RNA sequencing results showed differences in expression levels of 470 genes between the skin specimens of responder versus non-responder patients. There were 269 up-regulated genes and 201 down-regulated genes. Upregulated genes were involved in processes, such as Fatty Acid Omega Oxidation. Down-regulated genes were related to PPAR signaling pathway, and estrogen signaling pathway. Among the most differentially expressed genes (DEGs) with the most altered RNA expression levels in responders versus non-responder patients, we selected three genes (up-regulated genesBased on our findings, it is estimated that proposed genes might predict the response of vitiligo patients to cell therapy. However, further studies are required to clarify the role of these genes in pathogenesis and to characterize gene expression in a larger number of vitiligo patients in the context of epidermal cell transplantation therapy (registration number: IRCT201508201031N16).

Inactivation of tumor suppressor genes, such as RAP1GAP, by hypermethylation of their regulatory region can give rise to thyroid tumors. The aim of this study was to investigate the expression of the RAP1GAP gene and the DNA methylation patterns of its CpG74a, CpG74b, and CpG24 in an Iranian population with differentiated thyroid cancer (DTC). In this study, 160 individuals who underwent thyroidectomy in the Tehran Erfan Hospital between 2018 and 2020 were selected. DNA methylation patterns of selected CpG islands (CpG74a, CpG74b, and CpG24) were determined using methylation-specific PCR. The mRNA expression and protein level of -RAP1GAP were also evaluated. SW1736 and B-CPAP cells were treated with 5-aza-2'-deoxycytidine (5-Aza) to demethylate these regions. The hypermethylation rates of CpG74a and CpG24 in DTC samples were significantly higher than in the control. The mRNA expression and protein level of -RAP1GAP were significantly decreased in the DTC group. In the DTC group, hypermethylation in CpG74a was correlated with decreasing RAP1GAP expression (R2: 0.34; p = 0.043). CpG74a with a specificity of 86.4% has significant prediction power to distinguish between DTC and normal thyroid tissues. Additionally, hypermethylation of CpG74a was significantly associated with higher tumor stages (stage III-IV: 77%; stage I-II: 23%; p = 0.012). Increasing expression of RAP1GAP after demethylation with 15 µM of 5-Aza was observed in both cell lines. These results indicate that DNA hypermethylation in CpG74a can be considered as an epigenetic biomarker in DTC.

Background: Medullary thyroid cancer (MTC) is a rare type of neuroendocrine tumor. This study aimed to investigate the gene and protein expression of RAP1GAP and DNA methylation patterns of its CpG74a , CpG74b , and CpG24 in an Iranian population with MTC. Methods: In this case-control study, we selected 55 individuals who underwent thyroidectomy in Erfan hospital, Tehran, between 2018 and 2020. Samples were divided into normal thyroid tissues (control; n=20), benign nodule (n=20), and MTC (n=15). DNA methylation patterns were investigated using MSP (methylation-specific PCR). The protein level and mRNA expression of RAP1GAP were also evaluated using western blotting and real-time PCR, respectively. Results: The hyper-methylation rates of CpG24 and CpG74a in the MTC samples were considerably higher than the controls (83% versus 15% and 74% versus 17%, respectively; P

Abstract Ovarian cancer is the eighth most commonly occurring cancer in women. Clinically, the limitation of conventional screening and monitoring approaches inhibits high throughput analysis of the tumor molecular markers toward prediction of treatment response. Recently, analysis of liquid biopsies including circulating tumor DNA (ctDNA) open new way toward cancer diagnosis and treatment in a personalized manner in various types of solid tumors. In the case of ovarian carcinoma, growing pre-clinical and clinical studies underscored promising application of ctDNA in diagnosis, prognosis, and prediction of treatment response. In this review, we accumulate and highlight recent molecular findings of ctDNA analysis and its associations with treatment response and patient outcome. Additionally, we discussed the potential application of ctDNA in the personalized treatment of ovarian carcinoma. Graphical abstract ctDNA-monitoring usage during the ovarian cancer treatments procedures.

Objective Recurrent pregnancy loss (RPL) is a common infertility-related complication that affects approximately 1–3 % of women worldwide. Known causes of etiology are found in approximately half the cases but the other half remain unexplained. It is estimated that several thousands of genes contribute to reproductive success in mammals and the genetic causes of RPL cannot be fully addressed through targeted genetic tests. In recent years, massive parallel sequencing technologies has helped discovering many causal mutations in hereditary diseases such as RPL. Study design Using whole-exome sequencing (WES), we studied a large multiplex consanguineous family with multiple cases of RPL and hydatidiform moles (HM). In addition, targeted Sanger sequencing was applied to 40 additional non-related individuals with RPL. Results The use of WES permitted to identify the pathogenic variant in KHDC3L (c.322_325delGACT) in related who experienced RPL with or without HM. Sanger sequencing confirmed the segregation of the mutation throughout the pedigree and permitted to establish this variant as the genetic cause responsible for RPL and HM in this family. Conclusion KHDC3L is well established as a susceptibility gene for HM but we confirmed here that KHDC3L deleterious variants can also induce RPL. In addition, we observed a genotype-phenotype correlation, demonstrating that women with a truncating KHDC3L homozygous variant could not sustain a pregnancy and often had pregnancy losses mainly due to HM while those with the same heterozygous variant could have children but often endured RPL with no HM.

Parkinson disease (PD) is the second most common neurodegenerative disorder, whose prevalence is 2~3% in the population over 65. α-Synuclein aggregation is the major pathological hallmark of PD. However, recent studies have demonstrated enhancing evidence of tau pathology in PD. Despite extensive considerations, thus far, the actual spreading mechanism of neurodegeneration has remained elusive in a PD brain. This study aimed to further investigate the development of α-synuclein and tau pathology. We employed various PD models, including cultured neurons treated with either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or with recombinant α-synuclein. Also, we studied dopaminergic neurons of cytokine Interferon-β knock-out. Moreover, we examined rats treated with 6-hydroxydopamine, Rhesus monkeys administrated with MPTP neurotoxin, and finally, human post-mortem brains. We found the α-synuclein phosphorylation triggers tau pathogenicity. Also, we observed more widespread phosphorylated tau than α-synuclein with prion-like nature in various brain areas. We optionally removed P-tau or P-α-synuclein from cytokine interferon-β knock out with respective monoclonal antibodies. We found that tau immunotherapy suppressed neurodegeneration more than α-synuclein elimination. Our findings indicate that the pathogenic tau could be one of the leading causes of comprehensive neurodegeneration triggered by PD. Thus, we can propose an efficient therapeutic target to fight the devastating disorder.

Background: Diabetes mellitus may cause neurodegeneration, but the exact mechanism by which diabetic conditions induce neuronal cell death remains unclear. Tau protein hyperphosphorylation is considered to be a major pathological hallmark of neurodegeneration and can be triggered by diabetes. Various tau-directed kinases, including P38, can be activated upon diabetic stress and induce tau hyperphosphorylation. Despite extensive research efforts and the known importance of tau pathology in neurodegeneration, the exact tau specie(s) and kinases driving neurodegeneration in diabetes mellitus have not been clearly elucidated. Methods: We herein employed protein expression data analysis as well as immunofluorescence and immunoblotting techniques to determine the exact molecular mechanism of tau pathology triggered by diabetes in both in vitro and in vivo systems.Results: We found that P38, a major tau kinase, was increased in Glutamatergic & GABAergic neuron subtypes under diabetic conditions. This rendered them more responsive to oxidative stress caused by diabetes. We observed that oxidative stress activated P38, which in turn directly and indirectly drove tau pathology in the brainstem (enriched by Glutamatergic & GABAergic neurons), which gradually spread to neighboring brain areas. Notably, P38 inhibition suppressed tau pathogenicity and neurodegeneration in diabetic mouse models. Conclusion: The data establish P38 as a central mediator of diabetes mellitus induced tau pathology. Furthermore, the inhibition of P38 at early stages of diabetes-induced stress can inhibit tau pathology. Our findings provide mechanistic insight on the consequences of this metabolic disorder on the nervous system.

Background: Infertility is one of the common health issues around the world. The prevalence of male factor infertility among infertile couples is approximately 30%- 35%, of which genetic factors account for 15%. The family-based whole-exome sequencing (WES) approach can accurately detect novel variants. However, selecting an appropriate sample for data generation using WES has proven to be challenging in familial male infertility studies. The aim of this study was to identify types of pathogenic male infertility in cases of familial asthenozoospermia. Case: Two families with multiple cases were recruited for the purpose of WES. The study population included two affected cases in pedigree I and three affected cases in pedigree II. Two different variant callers (SAMtools and GATK) with a single-sample calling strategy (SSCS) and a multiple-sample calling strategy (MSCS), were applied to identify variant sites. Conclusion: In this study, we represented the results for variant prioritization of WES data without sequencing fertile siblings in the same pedigree by applying two different pipelines (homozygosity and linkage-based strategy). Using the aforementioned strategies, we prioritized annotated variants and generated a logical shortlist of private variants for each pedigree. Key words: Male infertility, Whole-exome sequencing, GATK, SAMtools.

Significance Sex development involves a precise spatiotemporal expression and interactions of numerous genetic factors, including the WT1 (Wilms tumor 1) gene. Complete and partial loss-of-function WT1 variants are associated with 46,XY disorders/differences of sex development (DSD). Some 46,XX individuals develop testis in absence of the testis-determining gene SRY. We describe a genotype/phenotype association where variants impacting the C-terminal zinc finger (ZF4) of WT1 cause testis development in 46,XX individuals. XX mice carrying a pathogenic variant of ZF4 display masculinization of the fetal gonads. Testis formation may be due to inappropriate interaction between the mutated WT1 and an essential ovarian determinant β-CATENIN. These data show that variants affecting a specific domain of a developmental transcription factor can switch organ fate., Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms’ tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10−6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10−4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor β-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

Background: Oxidative stress is commonly accrued in thyroid tissue during hormone synthesis. Objectives: We aimed to examine oxidative stress in patients with thyroid cancer, benign thyroid nodules, and healthy individuals. Methods: In this study, 138 individuals were involved. Among the selected participants, 108 had thyroid nodules, including 30 papillary thyroid cancer (PTC), 30 follicular thyroid cancer (FTC), six anaplastic thyroid cancer (ATC), 12 medullary thyroid cancer (MTC), and 30 benign nodules. In addition, 30 individuals were selected as a healthy control group. The levels of total antioxidant capacity (TAC) and total oxidant status (TOS) of thyroid tissue were measured using the ELISA method, and the oxidative stress index (OSI) was calculated. Results: The TAC level was significantly lower in MTC and FTC subtypes than in controls. The TOS level was considerably higher in the MTC group than in the control and benign nodule groups. The TOS level was not changed in other groups. The OSI was considerably higher in MTC and FTC subtypes. The TAC and OSI in benign nodules were significantly lower and higher than those of controls, respectively. The OSI was higher in female patients than in males. Conclusions: The OSI can not be considered a diagnostic biomarker for benign nodules and MTC. The diverse oxidative stress status between genders may be related to the elevated cancer incidence in females.

The novel coronavirus has been spreading since December 2019. It was initially reported in Wuhan, Hubei province of China. Coronavirus disease 2019 (COVID-19) has currently become a pandemic affecting over seven million people worldwide, and the number is still rising. Wenzhou, as the first hit city out of Hubei Province, achieved a remarkable success in effectively containing the disease. A great record was also observed in Wenzhou for the clinical management of COVID-19 patients, leading to one of the lowest death rates in China. Researchers and clinical specialists proposed and formulated combined approaches such as computerized tomography (CT)- scans and molecular assays, as well as using both allopathic and traditional medications to mitigate its effects. Iranian and Chinese specialists and scientists had a communication in clinical, molecular and pharmaceutical aspects of COVID-19. A proper guideline was prepared according to the experiences of Chinese clinicians in managing the full spectrum of COVID-19 patients, from relatively mild to highly complex cases. The purpose of this guideline is to serve a reference in the hospital for specialists so that they may better diagnose cases and provide effective therapies and proposed antiviral and anti-inflammatory drugs for patients.

Background: Intrauterine Insemination (IUI) outcome prediction is a challenging issue with which the assisted reproductive technology (ART) practitioners are dealing. Predicting the success or failure of IUI based on the couples' features can assist the physicians to make the appropriate decision for suggesting IUI to the couples or not and/or continuing the treatment or not for them. The large number of studies that have been focused on predicting the IVF and ICSI outcome by machine learning algorithms. But, to the best of our knowledge, a few studies have been focused on predicting the outcome of IUI. The main aim of this study is to develop an automatic classification and feature scoring method to predict intrauterine insemination (IUI) outcome and ranking of the most significant features, based on patients' cycle’s characteristics under IUI treatment.Methods: For this purpose, a novel hybrid approach combining complex network-based feature engineering and stacked ensemble (CNFE-SE) is proposed. Three complex networks are extracted considering the patients' data similarities. The feature engineering step is performed on the complex networks. The original feature set and/or the features engineered are fed to the proposed stacked ensemble to classify and predict IUI outcome for couples per IUI treatment cycle. Our study is a retrospective study of a 5-year couples' data undergoing IUI. Data is collected from Reproductive Biomedicine Research Center, Royan Institute for 8,360 couples who underwent 11,255 IUI cycles were included. Results: Experimental results show that the proposed method outperforms the compared methods with AUC of 0.84 ± 0.01, sensitivity of 0.79 ± 0.01, specificity of 0.91 ± 0.01, and accuracy of 0.85 ± 0.01 for the prediction of IUI outcome. Conclusions: The most important predictors for predicting IUI outcome are semen parameters (sperm motility and concentration) as well as female BMI.