Transcriptome Analysis Reveals Spermatogenesis-Related CircRNAs and LncRNAs in Goat Spermatozoa.

Mammalian spermatozoa comprises both coding and non-coding RNAs, which are traditionally believed to be a residual of spermatogenesis. The differential expression level of spermatozoal RNAs is also observed between fertile and infertile human, thereby anticipated as potential molecular marker of male fertility. This study investigated the transcriptome profile of goat (Capra hircus) spermatozoa. The sperm transcriptome was analyzed by three different methods viz. RLM-RACE, long-read RNA sequencing (RNAseq) in Nanopore™ platform, and short-read RNAseq in Illumina™ platform. The Illumina™ sequencing discovered 16,604 transcripts with 357 mRNAs having FPKM (fragments per kilobase per million mapped reads) of more than five. The spermatozoal RNA suite included mRNA (94%), rRNA (3%), miscRNA (1%), circRNA (1%), miRNA (1%), etc. This study also predicted circRNAs (127), lncRNAs (655), and imprinted genes (160) that have potential role in male reproduction. The gene ontology analysis revealed the involvement of spermatozoal RNA in regulating male meiosis (TET3, STAT5B), capacitation (ACRBP, CATSPER4), sperm motility (GAS8, TEKT2), spermatogenesis (ADAMTS2, CREB3L4), etc. The spermatozoal RNA were also associated with different biological pathways viz. Wnt signaling pathway, cAMP signaling pathway, AMPK signaling pathway, and MAPK signaling pathways having potential role in spermatogenesis. Overall, this study enlightened the suite of spRNA transcripts in goat and their relevance in male fertility for diagnostic approach.

Evidence mapping and review of long-COVID and its underlying pathophysiological mechanism.

PURPOSE: Apart from the global disease burden of acute COVID-19 disease, the health complications arising after recovery have been recognized as a long-COVID or post-COVID-19 syndrome. Evidences of long-COVID symptoms involving various organ systems are rapidly growing in literature. The objective was to perform a rapid review and evidence mapping of systemic complications and symptoms of long-COVID and underlying pathophysiological mechanisms. METHODS: Publications reporting clinical trials, observational cohort studies, case-control studies, case-series, meta-analysis, and systematic reviews, focusing on the squeal of the disease, consequences of COVID-19 treatment/hospitalization, long-COVID, chronic COVID syndrome, and post acute COVID-19 were reviewed in detail for the narrative synthesis of frequency, duration, risk factors, and pathophysiology. RESULTS: The review highlights that pulmonary, neuro-psychological, and cardiovascular complications are major findings in most epidemiological studies. However, dysfunctional gastrointestinal, endocrine, and metabolic health are recent findings for which underlying pathophysiological mechanisms are poorly understood. Analysis of the clinical trial landscape suggests that more than 50% of the industry-sponsored trials are focused on pulmonary symptoms. In contrast to the epidemiological trends and academic trials, cardiovascular complications are not a focus of industry-sponsored trials, suggestive of the gaps in the research efforts. CONCLUSION: The gap in epidemiological trends and academic trials, particularly concerning cardiovascular complications not being a focus of industry-sponsored trials is suggestive of the gaps in research efforts and longer follow-up durations would help identify other long-COVID-related health issues such as reproductive health and fertility.

Solid surface vitrification of goat testicular cell suspension enriched for spermatogonial stem cells.

This study reports solid surface vitrification (SSV) of goat testicular cell suspensions (TCS) enriched for spermatogonial stem cells (SSCs). The TCS was isolated from pre-pubertal goat testis by enzymatic digestion, enriched for SSCs by filtration and differential plating, and were vitrified-warmed by SSV. The study showed that SSV could successfully vitrify goat TCS although the percentage of live cells in the vitrified-warmed group was lower (74.8 ± 4.1%) than in non-vitrified control (80.6 ± 6.27%). The vitrified-warmed TCS formed putative SSC colonies upon their in vitro culture, but the colony size of vitrified-warmed cells (24.3 ± 1.8 µm) was smaller than those of non-vitrified warmed cells (58.4 ± 2.5 µm). Mitochondrial activity (0.40 vs. 0.38 A U.), population doubling time (33.45 ± 1.25 h vs. 31.86 ± 1.90 h), and the cell proliferation rate (0.72 ± 0.10 vs. 0.75 ± 0.11 per day) of total cells (including putative SSCs and other somatic cells) did not differ (p > 0.05) between control and SSV vitrified-warmed groups. However, during in vitro culture for 96 h, vitrified-warmed cells showed significantly lower (0.75 vs. 1.33 A U.; p < 0.05) mitochondrial activity than non-vitrified controls. The DCFDA assay showed that ROS activity was significantly (p < 0.05) higher in vitrified-warmed cells (52.8 ± 4.1 A U) than non-vitrified control cells (32.8 ± 2.1 AU). In conclusion, our results suggest that SSC-enriched goat TCS could be successfully cryopreserved by SSV. However, ROS-induced damages to cell cytoplasmic components reduce their cellular proliferation and require further improvement in the protocol. To the best of our knowledge, this study is the first report on the SSV of SSC-enriched goat TCS.

Prediction of potential molecular markers of bovine mastitis by meta-analysis of differentially expressed genes using combined p value and robust rank aggregation.

Bovine mastitis causes significant economic loss to the dairy industry by affecting milk quality and quantity. Escherichia coli and Staphylococcus aureus are the two common mastitis-causing bacteria among the consortia of mastitis pathogens, wherein E. coli is an opportunistic environmental pathogen, and S. aureus is a contagious pathogen. This study was designed to predict molecular markers of bovine mastitis by meta-analysis of differentially expressed genes (DEG) in E. coli- or S. aureus-infected mammary epithelial cells (MECs) using p value combination and robust rank aggregation (RRA) methods. High-throughput transcriptome of bovine MECs, infected with E. coli or S. aureus, were analyzed, and correlation of z-scores were computed for the expression datasets to identify the lineage profile and functional ontology of DEGs. Key pathways enriched in infected MECs were deciphered by Gene Set Enrichment Analysis (GSEA), following which combined p value and RRA were used to perform DEG meta-analysis to limit type I error in the analysis. The miRNA-gene networks were then built to uncover potential molecular markers of mastitis. Lineage profiling of MECs showed that the gene expression levels were associated with mammary tissue lineage. The up-regulated genes were enriched in immune-related pathways, whereas down-regulated genes influenced the cellular processes. GSEA analysis of DEGs deciphered the involvement of Toll-like receptor (TLR), and NF-kappa B signaling pathway during infection. Comparison after meta-analysis yielded with genes ZC3H12A, RND1, and MAP3K8 having significant expression levels in both E. coli and S. aureus dataset, and on evaluating miRNA-gene network, 7 pairs were common to both sets identifying them as potential molecular markers.

Strategies for cryopreservation of testicular cells and tissues in cancer and genetic diseases.

Cryopreservation of testicular cells and tissues is useful for the preservation and restoration of fertility in pre-pubertal males expecting gonadotoxic treatment for cancer and genetic diseases causing impaired spermatogenesis. A number of freezing and vitrification protocols have thus been tried and variable results have been reported in terms of cell viability spermatogenesis progression and the production of fertile spermatozoa. A few studies have also reported the production of live offspring from cryopreserved testicular stem cells and tissues in rodents but their replication in large animals and human have been lacking. Advancement in in vitro spermatogenesis system has improved the possibility of producing fertile spermatozoa from the cryopreserved testis and has reduced the dependency on transplantation. This review provides an update on various cryopreservation strategies for fertility preservation in males expecting gonadotoxic treatment. It also discusses various methods of assessing and ameliorating cryoinjuries. Newer developments on in vitro spermatogenesis and testicular tissue engineering for in vitro sperm production from cryopreserved SSCs and testicular tissue are also discussed.

Comparison of spermatozoal RNA extraction methods in goats.

RNA sequencing (RNAseq) has divulged newer role of spermatozoal RNA in male fertility. This study aimed to evaluate different sperm purification and RNA extraction methods for long-read RNA sequencing of poly(A) transcriptome in goat spermatozoa. Sperm samples were purified by swim-up separation using different purification medium. Spermatozoal RNA was extracted by seven different methods with additional supplementation of reducing agents in lysis buffer. poly(A) selected RNA was used for cDNA library preparation and long-read RNAseq in Nanopore sequencer. Sperm purification by 1 h swim-up resulted in higher recovery (89.20 ± 1.15%), motility (82.33 ± 1.53%), viability (88.10 ± 5.03%) and plasma membrane integrity (71.33 ± 4.51%) in sperm TALP (sp-TL) medium. A monophasic solution of GITC with phenol and DTT resulted in the highest yield of large sized RNAs (3.89 ± 0.46 ng/million cells) suitable for long-read RNAseq of poly(A) transcripts. RNAseq resulted in reads of length, ranging from 500bp to 2 Kb. A total of 123 transcripts were identified in goat spermatozoa by de novo assembly and included sperm-specific transcripts such as CATSPERG, PRM2, CYLC2, SPATA6, PLCZ1 etc. This study is the first report of long-read RNAseq of poly(A) transcriptome in goat spermatozoa.

Plasticized poly(vinylalcohol) and poly(vinylpyrrolidone) based patches with tunable mechanical properties for cardiac tissue engineering applications.

Polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) are the two most investigated biopolymers for various tissue engineering applications. However, their poor tensile strength renders them unsuitable for cardiac tissue engineering (CTE). In this study, we developed and evaluated PVA-PVP-based patches, plasticized with glycerol or propylene glycol (0.1%-0.4%; v:v), for their application in CTE. The cardiac patches were evaluated for their physico-chemical (weight, thickness, folding endurance, FT-IR, and swelling behavior) and mechanical properties. The optimized patches were characterized for their ability to support in vitro attachment, viability, proliferation, and beating behavior of neonatal mouse cardiomyocytes (CMs). In vivo evaluation of the cardiac patches was done under the subcutaneous skin pouch and heart of rat models. Results showed that the optimized molar ratio of PVA:PVP with plasticizers (0.3%; v-v) resulted in cardiac patches, which were dry at room temperature and had desirable folding endurance of at least 300, a tensile strength of 6-23 MPa and, percentage elongation at break of more than 250%. Upon contact with phosphate-buffered saline, these PVA-PVP patches formed hydrogel patches having the tensile strength of 1.3-3.0 MPa. The patches supported the attachment, viability, and proliferation of primary neonatal mouse CMs and were nonirritant and noncorrosive to cardiac cells. In vivo transplantation of cardiac patches into a subcutaneous pouch and on the heart of rat models revealed them to be biodegradable, biocompatible, and safe for use in CTE applications.

Comparison of two culture methods during in vitro spermatogenesis of vitrified-warmed testis tissue: Organ culture vs. hanging drop culture.

Solid surface vitrification (SSV) is a cost effective and simple method for testis tissue preservation. Vitrified-warmed testis tissue was successfully cultured using various organ culture methods. In this study, we compared two culture methods viz. hanging drop (HD) and organ culture (OC) methods for in vitro spermatogenesis of goat testis tissue vitrified-warmed by SSV. It was observed that OC method was superior (p < 0.05) to HD method in terms of post-warming metabolic activity of testicular tissue, as measured by MTT assay on Day 7 and Day 14 of culture, respectively. The size of the tissue also played an important role in post-warming metabolic activity and viability (4 mm3: 72.7 ± 1.2% vs. 9 mm3: 62.7 ± 1.3% vs. 16 mm3: 40.5 ± 1.7%) of vitrified tissues with smaller tissue resulting in better result. The vitrification-induced ROS activity significantly decreased during their in vitro culture. Histology and scanning electron microscopy (SEM) showed the rupture of basal membrane, surface morphology and, cell loss due to vitrification. However, histology and immunohistochemistry showed the progression of in vitro spermatogenesis and formation of elongated spermatozoa in both fresh and vitrified-warmed testis tissue cultured by OC method. Taken together, our results suggest that OC method is superior to HD method for culturing goat testis tissue vitrified-warmed by SSV.

Combinatorial ethanol treatment increases the overall productivity of recombinant hG-CSF in E. coli: a comparative study.

Human granulocyte colony-stimulating factor (hG-CSF) is a cytokine that regulates the proliferation, maturation, and differentiation of precursor cells to neutrophils. In the present study, we report the feasibility of inducing recombinant hG-CSF expression (rhG-CSF) in a pET vector system by combinatorial induction using low-concentration ethanol, IPTG, and lactose and auto-induction media (AIM). The coding sequence of hG-CSF transcript variant 2 was expressed in pET14 vector, and the effect of combinatorial induction was analyzed on inclusion body (IB) formation, biomass, protein purification, and bioactivity. Results showed that there was an inverse relationship between the temperature and soluble expression of rhG-CSF. Three-step washing with Triton-X, 2 M, and 5 M urea resulted in the maximum recovery of IBs. Combinatorial single-spike induction with IPTG, ethanol, and lactose in a batch culture led to a 3-fold increase in the expression of rhG-CSF. It was also observed that low concentration of ethanol (1-3% v/v) could be used in lieu of IPTG for inducing the rhG-CSF protein expression without adversely affecting biomass production. A 2.4-fold increase in productivity was obtained in LB-AIM media with combinatorial ethanol induction, and the overall yield of 2.8 g/L rhG-CSF was found. The purified rhG-CSF was bioactive and increased the cellular proliferation of umbilical cord blood-derived mesenchymal stem cells (U-MSC) by 29%. In conclusion, our study shows that combined ethanol induction can enhance the expression of rhG-CSF with three-step washing for recovery of the proteins from IBs and a single-step purification of rhG-CSF by affinity chromatography. KEY POINTS: • Low concentration of ethanol (1-3%) could be used in lieu of IPTG for inducing rhG-CSF expression. • Combinatorial single-spike induction with IPTG, ethanol, and lactose improved rhG-CSF expression. • Purified rhG-CSF was bioactive and increased the proliferation of U-MSC.

Spontaneous bilateral extraperitoneal ureteroinguinal herniation.

We describe the clinical and imaging findings of a 53-year-old male who presented with recurrent urinary tract infections with bilateral inguinoscrotal swelling, diagnosed as spontaneous bilateral extraperitoneal ureteroinguinal herniation.

Arteriovenous malformation with a renal mass: A rare association.

Renal arteriovenous malformation can be rarely associated with a renal mass. A vigilant approach and careful planning is required to tackle both the pathologies in form of preoperative coil embolization followed by a minimally invasive radical nephrectomy.

Prolonged natural history of a cystic renal cell carcinoma: A case report.

We describe the successful management of a 50-year male who presented with gradually progressive abdominal swelling for over 20 years. The highlights of the case are giant renal mass occupying the whole abdomen and the absence of metastasis despite a long history.

Cryopreservation of murine testicular Leydig cells by modified solid surface vitrification with supplementation of antioxidants.

Reports on the vitrification of somatic cells are scarce. Here, we show that Leydig cells (murine cell line TM3) could be successfully vitrified by both open vitrification [plastic straw (PS) and plastic vials (PV)] and closed ultravitrification [microdrop (MD) and solid surface vitrification (SSV)], after protocol optimization. However, open ultravitrification resulted in better post-warming viability than closed systems of vitrification with highest success obtained in modified SSV (84.8 ±â€¯1.86%; p < 0.05). Leydig cells vitrified-warmed by modified SSV also showed superior (p < 0.05) cell growth, mitochondrial activity and cytoplasmic esterase enzyme activity, than MD, PS and PV, respectively. It was also observed that vitrified-warmed cells had higher level of ROS activity than non-vitrified control cells (41.6 ±â€¯4.0 vs. 16.7 ±â€¯1.0; p < 0.05). Treatment of cells with glutathione (GSH) or 2-mercaptoethanol (2-ME) (0, 10, 50, 100 µM) significantly (p < 0.05) reduced the ROS activity but had no significant (p > 0.05) effect on post-warm viability. Nevertheless, antioxidant-treated cells had improved mitochondrial activity, cytoplasmic esterase activity and cell growth during in vitro culture (p < 0.05). In conclusion, our results suggest that modified SSV offers a viable method for vitrifying single cell suspension of Leydig cells. To the best of our knowledge, this is the first report on cryopreservation of Leydig cells by vitrification.

Deep vein thrombosis in medical and surgical Intensive Care Unit patients in a Tertiary Care Centre in North India: Incidence and risk factors.

BACKGROUND AND AIMS: Deep venous thrombosis (DVT) prophylaxis is underutilized, and there is a paucity of data reflecting the incidence of DVT in Indian Intensive Care Unit (ICU) population. We sought to evaluate the incidence and risk factors for DVT in medical and surgical ICU patients with DVT prophylaxis. MATERIAL AND METHODS: The ICU patients more than 18 years old, expected to be in the ICU for more than 48 h were enrolled and DVT prophylaxis were given as per risk and were observed for clinical signs of DVT along with duplex ultrasound until in ICU. The patients receiving anticoagulant for some other reasons were excluded along with those with pregnancy, congenital coagulation disorders and terminal illness. RESULTS: The incidence of DVT was 0.8% (95% confidence interval: 0.78-0.81) in mixed populations (1.6% in medical and 0.5% in surgical). The higher DVT score (DVT (+) 10.75 ± 2.06/DVT (-) 8.75 ± 1.7 P = 0. 0264), Acute physiology and chronic health evaluation (APACHE) IV score (DVT positive patient - DVT (+) 59.25 ± 15.06/DVT negative patients - DVT (-) 44.01 ± 13.74) P = 0. 0292), length of ICU stay ([DVT (+) 26.75 ± 12.87 days/DVT (-) 5.19 ± 6.18] P < 0.010), and inotropes (DVT (+) 50%/DVT (-) 12.3% P = 0. 023) were associated with DVT. CONCLUSION: The incidence of DVT was 0.8% with prophylaxis. High DVT and APACHE IV score were associated with DVT. Prolonged ICU stay and vasopressors were the risk factors.

Poly(ADP-ribosyl)ation is involved in pro-survival autophagy in porcine blastocysts.

Poly(ADP-ribosyl)ation (PARylation) prevents apoptosis through its involvement in pro-survival autophagy in cultured cells; whether or not the same is true for pre-implantation embryos has not yet been documented. In this study, we investigated the participation of PARylation and autophagy in in vitro porcine pre-implantation embryo development. The transcript levels of autophagy-related genes and poly(ADP-ribose) polymerase 1 (PARP1), an enzyme required for PARylation, were transiently up-regulated by fertilization, decreased at the late 1-cell stage, and maintained until the blastocyst stage. LC3, a marker of autophagosomes, and poly(ADP-ribose) (PAR) polymer were present in all stages of pre-implantation development. Exposure of embryos to 3-methyladenine, an autophagy inhibitor, or 3-aminobenzamide, a PARP inhibitor, suppressed the development of blastocysts. Pharmacological inhibition of PARylation further suppressed pro-survival autophagy by decreasing the expression of autophagy-related genes (ATG5, BECLIN1, and LC3) and decreasing LC3 protein abundance while increasing the rate of apoptosis in blastocysts. Deficiency in autophagy also induced abnormal accumulation of SQSTM1/p62 aggregates in porcine blastocysts. Collectively, these data suggest that PARylation is involved in selective autophagic degradation of ubiquitinated proteins, functioning in a pro-survival role, in porcine in vitro-produced embryos. These pro-survival regulatory mechanisms may be important for the control of embryo quality.

Open encapsulation-vitrification for cryopreservation of algae.

Vitrification offers a cost-effective solution for the preservation and management of genetic resources with, low-cost international movement of selected genetic materials and for long-term maintenance of stable stocks of a wide variety of microorganisms. However, its success is limited by the wide range of algal species. Here, we report a simple open encapsulation-vitrification protocol of cryopreservation. Results showed that ∼58% and ∼27% of Oocystis sp. survived vitrification-warming after the open and closed system of vitrification respectively when compared to non-cryopreserved controls. The improved success in an open system of vitrification was also observed in Anabaena sp. Furthermore, with the addition of 2-mercaptoethanol or glutathione the post-warming viability of vitrified algae in both open and closed system of vitrification was significantly improved (p < 0.05). The present case study aimed to develop a vitrification-based cryopreservation protocol and confirms an improvement in survival percentage over conventional encapsulation-vitrification method.

A Japanese Encephalitis Vaccine From India Induces Durable and Cross-protective Immunity Against Temporally and Spatially Wide-ranging Global Field Strains.

BACKGROUND: Japanese encephalitis (JE) is a vaccine-preventable acute disease. We report the results of a phase 2/3 trial of JENVAC, a Vero cell-derived vaccine developed using an Indian strain of JE virus (JEV). METHODS: JENVAC was administered in 2 doses 28 days apart, and immunogenicity was compared to that from a single dose of SA-14-14-2, the only approved JE vaccine and regimen at the time in India. RESULTS: After both the doses, seroconversion and seroprotection were >90% for JENVAC. For SA-14-14-2, seroconversion and seroprotection were 57.69% and 77.56%, respectively, on day 28 and 39.74% and 60.26%, respectively, on day 56. The geometric mean titers at day 28 and day 56 were 145.04 and 460.53, respectively, for JENVAC and 38.56 and 25.29, respectively, for SA-14-14-2. With a single dose of JENVAC, seroprotection titers lasted at least 12 months in >80% of the subjects. Following receipt of 2 doses, 61.17% of subjects retained seroprotection titers at 24 months, and immunogenicity criteria were higher than that for SA-14-14-2 at 12, 18, and 24 months each. Sera from JENVAC subjects neutralized JEV genotypes I, II, III, and IV equally well. Adverse events were not significantly different between the 2 vaccines. CONCLUSIONS: JENVAC elicits long-lasting, broadly protective immunity. CLINICAL TRIALS REGISTRATION: CTRI/2011/07/001855.

Supplementation of insulin-transferrin-selenium to embryo culture medium improves the in vitro development of pig embryos.

Insulin, transferrin and selenium (ITS) supplementation to oocyte maturation medium improves the post-fertilization embryonic development in pigs. ITS is also commonly used as a supplement for the in vitro culture (IVC) of embryos and stem cells in several mammalian species. However, its use during IVC of pig embryos has not been explored. This study investigated the effect of ITS supplementation to IVC medium on the in vitro development ability of pig embryos produced by parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). We observed that ITS had no significant effect on the rate of first cleavage (P > 0.05). However, the rate of blastocyst formation in ITS-treated PA (45.3 ± 1.9 versus 27.1 ± 2.3%), IVF (31.6 ± 0.6 versus 23.5 ± 0.6%) and SCNT (17.6 ± 2.3 versus 10.7 ± 1.4%) embryos was significantly higher (P < 0.05) than those of non-treated controls. Culture of PA embryos in the presence of ITS also enhanced the expansion and hatching ability (29.1 ± 3.0 versus 18.2 ± 3.8%; P < 0.05) of blastocysts and increased the total number of cells per blastocyst (53 ± 2.5 versus 40.9 ± 2.6; P < 0.05). Furthermore, the beneficial effect of ITS on PA embryos was associated with significantly reduced level of intracellular reactive oxygen species (ROS) (20.0 ± 2.6 versus 46.9 ± 3.0). However, in contrast to PA embryos, ITS had no significant effect on the blastocyst quality of IVF and SCNT embryos (P > 0.05). Taken together, these data suggest that supplementation of ITS to the IVC medium exerts a beneficial but differential effect on pig embryos that varies with the method of embryo production in vitro.

Nanofibrous composite from chitosan-casein polyelectrolyte complex for rapid hemostasis in rat models in vivo.

Bleeding causes ∼5.8 million deaths globally; half of the patients die if rapid hemostasis is not achieved. Here, we report a chitosan-casein (CC)-based nanofibrous polyelectrolyte complex (PEC) that could clot blood within 10 s in the rat femoral artery model in vivo. The nanofiber formation by self-assembly was also optimized for process parameters (concentration, mixing ratio, pH, and ultrasonication). Results showed that increasing the concentration of chitosan from 10 % to 90 % in the formulation increased the productivity (r = 0.99) of PECs but led to increased blood clotting time (r = 0.90) due to an increase in zeta potential (r = 0.98), fiber diameter (r = 0.93), and decreased surface porosity (r = -0.99), absorption capacity (r = -0.99). The pH also influenced the zeta potential of PEC, with an optimized pH of 8.0 ± 0.1 yielding clear nanofibers. Sonication improved the segregation of nanofibers by promoting water removal. The optimized PECs containing chitosan and casein in the ratio of 30:70 (CC30) at a pH of 8.0 and dehydration under sonication could clot the blood within 9 ± 2 s in vitro and 9 ± 2 s in rat femoral artery puncture model. The CC30 formulation did not cause any irritation or corrosion on rat skin. Histopathology and immunohistochemistry of various organs showed that CC30 was biocompatible and non-immunogenic under in vivo conditions.