The present study assessed sperm functional characteristics in the frozen-thawed semen of buffalo bulls and estimated their relationship with field fertility. Frozen semen samples from three ...different freezing operations each from nine Murrah buffalo bulls were used for the assessment of different sperm functions related to fertilizing potential. Bulls were classified into high (n = 2), medium (n = 5), and low (n = 2) fertile based on adjusted field fertility. The sperm functions estimated included membrane integrity using carboxyfluorescein diacetate-propidium iodide, acrosome reaction status using fluorescein isothiocyanate peanut agglutinine, status of apoptosis using Annexin-V, protamine deficiency using Chromomycin A3, membrane stability using Merocyanine 540 and lipid peroxidation status using 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene. The relationship between the proportion of live acrosome-intact spermatozoa and fertility was positive and significant (r = 0.59; P = 0.001). The proportion of moribund spermatozoa showed a significantly negative correlation with fertility (r = −0.50; P = 0.008). Similarly, the relationship of spermatozoa with unstable membrane (r = −0.51; P = 0.007), necrotic (r = − 0.42; P = 0.028), early necrotic (r = −0.42; P = 0.031), and apoptotic spermatozoa (r = −0.39; P = 0.046) with bull fertility was negative and significant. The correlation between the protamine-deficient spermatozoa and fertility was negative, but not significant. Among different combinations of tests, live acrosome-intact spermatozoa and lipid peroxidation status of spermatozoa revealed high positive correlation with buffalo bull fertility (adjusted R2 = 0.73, Cp = 0.80). These preliminary findings may help in developing tools for assessing fertility of buffalo bulls, once validated in more animals.
Seasonality in reproduction and effects of climatic variables on testicular cytology and semen quality in bucks reared under subtropical climatic conditions were not well understood. In the present ...study, using testicular cytology, semen evaluation and melatonin concentrations assessed over a period of 1 year, we report that bucks reared under subtropical climatic conditions did not show seasonality in reproduction. Climatic variables including temperature, relative humidity, temperature-humidity index (THI), sunshine hours and day length were recorded daily during the whole period of experimentation (one complete year). Ejaculates were collected from crossbred (Alpine X Beetal) males (
n
= 6) biweekly using artificial vagina, and semen quality (volume, mass activity, sperm concentration, motility, viability, membrane integrity and protamine deficiency) was assessed. To understand the seasonal influence at testicular level, using fine needle aspiration biopsy method, testicular cells were aspirated and different types of cells and testicular cytology indices were quantified. Blood was collected biweekly for estimation of melatonin concentrations. Mass activity was higher (
P
< 0.05) during rainy season while individual sperm motility and sperm concentration were higher (
P
< 0.05) during rainy and autumn seasons as compared to other seasons. Sperm functional parameters did not show any differences during different seasons. Sertoli cell count, spermatogenic cell count and testicular indices did not differ among the seasons. Melatonin concentrations also did not differ significantly among the four seasons studied. Among the climatic parameters, THI had significant (
P
< 0.05) influence on sperm quality. The proportion of Sertoli cell in the testicular cytology had a significant and positive relationship with RH, THI and day length. It was concluded that seasonal variations are less evident in terms of spermatogenesis and semen quality in Alpine X Beetal crossbred bucks reared under subtropical climatic conditions.
The present study compared the testicular cytology and histology between crossbred (Holstein-Friesian HF × Tharparkar) and purebred (HF and Tharparkar) bulls to find out differences if any.
Four ...peripubertal bulls from each breed were utilized for the study. Through percutaneous needle aspiration biopsy, Sertoli and spermatogenic cells were extracted, and morphometry was studied. For histological studies, testicular tissues obtained through unilateral castration were utilized. Sertoli cells specific GATA4 antibody was used to study the population of Sertoli cells in the seminiferous tubule through immunofluorescence.
The testicular weight, volume, and scrotal circumference differed significantly among the breeds. The diameter and area of the seminiferous tubule was high in HF, followed by Karan Fries (KF), and Tharparkar bulls. However, the degree of compactness, based on qualitative evaluation, was high in Tharparkar followed by KF and HF bulls. The intensity of Leydig cells was higher in Tharparkar bulls followed by KF and HF. The proportion of Sertoli cells was higher (p<0.05) in HF and Tharparkar bulls compared to KF bulls.
It may be concluded that variations exist in testicular components of the breeds studied and the proportion of Sertoli cells in relation to spermatogenic cells was significantly lower in crossbred bulls compared to purebred bulls.
Sub-fertility is one of the most common problems observed in crossbred males, but the etiology remains unknown in most of the cases. Although proteomic differences in the spermatozoa and seminal ...plasma between breeds have been investigated, the possible differences at the sperm precursor cells and supporting/nourishing cells have not been studied. The present study reports the differential proteomic profile of spermatogenic and Sertoli cells in crossbred and purebred bulls. Testis was removed by unilateral castration of 12 peri-pubertal bulls (10 months age), four each from crossbred (Holstein Friesian × Tharparkar), exotic purebred Holstein Friesian (HF) and indigenous purebred Tharparkar (TP) bulls. Spermatogenic and Sertoli cells were isolated and subjected to proteomic analysis. Protein extracts from the Sertoli and spermatogenic cells of each breed were analyzed with 2-dimensional difference gel electrophoresis (2D-DIGE) and analyzed with Decyder™ software. Compared to HF, 26 protein spots were over expressed and 14 protein spots were under expressed in spermatogenic cells of crossbred bulls. Similarly, 7 protein spots were over expressed and 15 protein spots were under expressed in the spermatogenic cells of TP bulls compared to that of crossbred bulls. Out of 12 selected protein spots identified through mass spectrometry, Phosphatidyl ethanolamine binding protein was found to be over expressed in the spermatogenic cells of crossbred bulls compared to TP bulls. The protein, gamma actin was found to be over expressed in the Sertoli cells of HF bulls, whereas Speedy Protein-A was found to be over expressed in Sertoli cells of crossbred bulls. It may be concluded that certain proteomic level differences exist in sperm precursor cells and nourishing cells between breeds, which might be associated with differences in the fertility among these breeds.
“Silent Cerebral Hemorrhage” in a young alcohol-dependent individual can pose a significant challenge to the treating physician. The neuroimaging and clinical presentation maybe dichotomous to a ...critical extent.
Our aim was to explore the scientific understanding of “Silent Cerebral Hemorrhage” and share the wisdom gathered about the diagnosis and management of this rare entity.
A 32-year-old male presented with fever and episodes of vomiting alongwith urinary incontinence, in the backdrop of heavy alcohol abuse. Detailed neurological assessment revealed no abnormal findings. MMSE indicated towards no significant cognitive deficits. On MRI, an extensive intracerebral hemorrhage was observed.
The I.C.H. score was 2/6 and the FUNC score was 8/11; which was in stark contrast to NIH Stroke Scale score of 0/42.
Blood-panel showed deranged liver enzymes. Patient was managed conservatively. Irritability was controlled using Haloperidol. Prophylactic antiepileptics were also started.
Upon follow-up, patient showed drastic improvement. Patient was started on Acamprosate for maintenance of abstinence.
Through this case report we have tried to highlight that “Silent Cerebral Hemorrhage” can present as a curve-ball for clinicians and psychiatrists. The dichotomy in interpretation of stroke-related scales may cause a dilemma about the course of management.
“In this case, the absence of neurological deficits was indeed a stroke of luck for the patient.”
: “Silent Cerebral Hemorrhage” can present as a curve-ball for clinicians and psychiatrists. Hence, we should be vigilant about it in our clinical practice. The dichotomy in interpretation of stroke-related scales may cause a dilemma about the course of management.
Abstract
Background: Colorectal carcinoma (CRC) is the second leading cause of cancer related deaths in the United States. The five-year survival rate of patients diagnosed with distant stages ...declines to 14%, which is of concern when compared to 90% for localized-stage and 71% for regional stage disease. This necessitates the need for early diagnostic biomarkers, to minimize poor drug response/resistance, recurrence, metastasis and mortality. Disproportionate biochemical stressors increase the risk of developing CRC and its progression by influencing molecular drivers within the coding and noncoding parts of the genome. Thus, understanding the biological mechanism of these stress factors on the molecular drivers of this disease can provide pivotal information pertinent to CRC development and progression. Recently, our laboratory has identified a novel long noncoding RNA (lncRNA) namely, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), which is highly over-expressed in CRC and is involved in its pathogenesis and is regulated by transcription factor Nuclear Factor of Activated T cell 1 (NFATc1).
Methods: Archived human CRC tissues were stained using a recently standardized Z-probe technology. TCGA database of ~600 CRC patients was also analyzed using the bioinformatic approach. CRC cell lines were profiled for MALAT1 expression using RT-PCR. Lentiviral constructs were used to generate stable lncRNA MALAT1 expressing cell lines. CRISPR/Cas9 constructs were used to knockdown lncRNA MALAT1 and NFATc1. Mouse model was used to verify the stress induced expression of MALAT1 and NFATc1. ReCLP (Reversible Cross-Linked Precipitation) and iRAP (invitro RNA Antisense Proteomics) studies are in progress to identify the associated proteins and complexes.
Results: RNAScope analysis showed MALAT1 to be highly over-expressed in human CRC tissues. MALAT1 expression increased with stage and negatively correlated with the tumor size. TCGA database analysis confirmed our findings. Multiple NFATc1 binding site were identified by ChIPseq database. Overexpression of transcription factor NFATc1 upregulated lncRNA MALAT1 expression. CRISPR/Cas9 based knockdown of NFATc1 downregulated NFATC1 and lncRNA MALAT1, but vice versa was not true, indicating NFATc1 to be upstream of lncRNA MALAT1. Also, expression of MALAT1, NFATc1 and IL-6 were highly upregulated by biochemical stressor cortisol in mouse colonic tissues. Further studies are in progress for direct association of NFATc1 on MALAT1 promoter and mechanism by which the stress factor regulate NFATc1 expression and hence lncRNA MALAT1 expression.
Conclusion: This study helps to understand influence of biochemical stress factors on long noncoding RNA MALAT1 and transcription factor NFATc1 etiology. Early diagnosis of these molecular markers will help in designing novel preventive/therapeutic strategies to reduce CRC progression, metastasis and hence mortality.
Citation Format: Kyle Doxtater, Chidi Zacheaus, Radhika Sekhri, Utkarsh K. Mishra, Zachary E. Stiles, Nitish Mishra, Chittibabu Guda, Nadeem Zafar, Mahul Amin, Pradeep Shukla, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan, Manish K. Tripathi. Stress regulated role of lncRNA Malat1 in colorectal cancer progression and metastasis abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3142.
Senescent cells exacerbate COVID-19
Cellular senescence is a state elicited in response to stress signals and is associated with a damaging secretory phenotype. The number of senescent cells ...increases with advanced age and this in turn drives age-related diseases. Camell
et al.
show that senescent cells have an amplified inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (see the Perspective by Cox and Lord). This response is communicated to nonsenescent cells, suppressing viral defense mechanisms and increasing the expression of viral entry proteins. In old mice infected with a SARS-CoV-2–related virus, treatment with senolytics to reduce the senolytic cell burden reduced mortality and increased antiviral antibodies.
Science
, abe4832, this issue p.
eabe4832
; see also abi4474, p.
281
SARS-CoV-2 amplifies the damaging senescent cell secretory state, and drugs that selectively clear senescent cells reduce mortality in infected aged mice.
INTRODUCTION
The COVID-19 pandemic revealed enhanced vulnerability of the elderly and chronically ill to adverse outcomes upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Senescence is a cell fate elicited by cellular stress that results in changes in gene expression, morphology, metabolism, and resistance to apoptosis. Senescent cells (SnCs) secrete pro-inflammatory factors, called the senescence-associated secretory phenotype (SASP). SnCs accumulate with age and drive chronic inflammation. In human cells and tissues and using a new infection paradigm, we asked whether SnCs are a cause of adverse outcomes of infection with aging. This is relevant because SnCs can be selectively eliminated in vivo with a new class of therapeutics called senolytics, potentially affording a new approach to treat COVID-19.
RATIONALE
We hypothesized that SnCs, because of their pro-inflammatory SASP, might have a heightened response to pathogen-associated molecular pattern (PAMP) factors, resulting in increased risk of cytokine storm and multi-organ failure. To test this, we treated senescent and nonsenescent human cells with the PAMPs lipopolysaccharide (LPS) and SARS-CoV-2 spike protein (S1) and measured the SASP and its effect on non-SnCs. Similarly, old and progeroid mice were challenged with LPS, and we measured the SASP. Previously, we created a “normal microbial experience” (NME) for mice by transmitting environmental pathogens to specified-pathogen–free (SPF) mice through exposure to pet store mice or their bedding. The first pathogen transferred was mouse hepatitis virus (MHV), a β-coronavirus closely related to SARS-CoV-2. NME rapidly killed aged SPF mice known to have an increased burden of SnCs compared with young SPF mice, which survive NME. This afforded an experimental paradigm to test whether senolytics blunt adverse outcomes in β-coronavirus infection.
RESULTS
Human endothelial SnCs became hyperinflammatory in response to challenge with LPS and S1, relative to non-SnCs. The PAMP-elicited secretome of SnCs caused increased expression of viral entry proteins and reduced expression of antiviral genes in nonsenescent human endothelial and lung epithelial cells, and the proximity of these events was established in human lung biopsies. Treatment of old mice with LPS significantly increased SASP expression in several organs relative to young mice, confirming our hypothesis in vivo. Similarly, old mice exposed to NME displayed a significant multi-organ increase in SnCs and the SASP, impaired immune response to MHV, and 100% mortality, whereas inoculation with antibodies against MHV before NME afforded complete rescue of mortality. Treating old mice with the senolytic fisetin, which selectively eliminates SnCs after NME reduced mortality by 50%, reduced expression of inflammatory proteins in serum and tissue and improved the immune response. This was confirmed with a second senolytic regimen, Dasatinib plus Quercetin, as well as genetic ablation of SnCs in aged mice, establishing SnCs as a cause of adverse outcomes in aged organisms exposed to a new viral pathogen.
CONCLUSION
SnCs amplify susceptibility to COVID-19 and pathogen-induced hyperinflammation. Reducing SnC burden in aged mice reduces mortality after pathogen exposure, including a β-coronavirus. Our findings strongly support the Geroscience hypothesis that therapeutically targeting fundamental aging mechanisms improves resilience in the elderly, with alleviation of morbidity and mortality due to pathogenic stress. This suggests that senolytics might protect others vulnerable to adverse COVID-19 outcomes in whom increased SnCs occur (such as in obesity or numerous chronic diseases).
SnCs that accumulate with age or chronic disease react to PAMPs such as SARS-CoV-2 S1 by amplifying the SASP, which increases viral entry protein expression and decreases viral defense IFITMs in normal cells.
Old mice exposed to pathogens such as the β-coronavirus MHV have increased inflammation and higher mortality. Treatment with a senolytic decreased SnCs, inflammation, and mortality and increased the antiviral antibody response.
The COVID-19 pandemic has revealed the pronounced vulnerability of the elderly and chronically ill to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–induced morbidity and mortality. Cellular senescence contributes to inflammation, multiple chronic diseases, and age-related dysfunction, but effects on responses to viral infection are unclear. Here, we demonstrate that senescent cells (SnCs) become hyper-inflammatory in response to pathogen-associated molecular patterns (PAMPs), including SARS-CoV-2 spike protein-1, increasing expression of viral entry proteins and reducing antiviral gene expression in non-SnCs through a paracrine mechanism. Old mice acutely infected with pathogens that included a SARS-CoV-2–related mouse β-coronavirus experienced increased senescence and inflammation, with nearly 100% mortality. Targeting SnCs by using senolytic drugs before or after pathogen exposure significantly reduced mortality, cellular senescence, and inflammatory markers and increased antiviral antibodies. Thus, reducing the SnC burden in diseased or aged individuals should enhance resilience and reduce mortality after viral infection, including that of SARS-CoV-2.
Alzheimer's Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. ...Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 female mice, a translational model of AD. Treatment of symptomatic APP/PS1 mice with complex I inhibitor improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy in vivo was monitored using translational biomarkers FDG-PET,
P NMR, and metabolomics. Cross-validation of the mouse and the human transcriptomic data from the NIH Accelerating Medicines Partnership-AD database demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.
Abstract Multiple changes occur in hormonal regulation with aging and across various endocrine organs. These changes are associated with multiple age-related disorders and diseases. A better ...understanding of responsible underling biological mechanisms could help in the management of multiple endocrine disorders over and above hormone replacement therapy (HRT). Cellular senescence is involved in multiple biological aging processes and pathologies common in elderly individuals. Cellular senescence, which occurs in many older individuals but also across the lifespan in association with tissue damage, acute and chronic diseases, certain drugs, and genetic syndromes, may contribute to such endocrine disorders as osteoporosis, metabolic syndrome, and type 2 diabetes mellitus. Drugs that selectively induce senescent cell removal, “senolytics,”, and drugs that attenuate the tissue-destructive secretory state of certain senescent cells, “senomorphics,” appear to delay the onset of or alleviate multiple diseases, including but not limited to endocrine disorders such as diabetes, complications of obesity, age-related osteoporosis, and cancers as well as atherosclerosis, chronic kidney disease, neurodegenerative disorders, and many others. More than 30 clinical trials of senolytic and senomorphic agents have already been completed, are underway, or are planned for a variety of indications. Targeting senescent cells is a novel strategy that is distinct from conventional therapies such as HRT, and thus might address unmet medical needs and can potentially amplify effects of established endocrine drug regimens, perhaps allowing for dose decreases and reducing side effects.
The most generally spread neurotransmitter acetylcholine (Ach) is used as a chemical messenger assisting in conveying signals transversely through the nerve synapse. Herein, two enzymes ...acetylcholinesterase and choline oxidase were covalently immobilized over the gold nanoparticles (AuNPs) embedded graphene oxide (GO; 2D carbon material) nanocomposite modified ITO coated glass plate. The synergetic and unique properties of AuNPs and GO present in nanocomposite are used to detect the ultra-small concentration of analyte, Ach. The prepared nanocomposites were characterized using different techniques i.e. TEM, XRD, SEM, FTIR, UV–Vis and Raman Spectroscopy. All the electrochemical measurements were performed using 3 electrodes integrated electrochemical system by introducing Ach through varying its concentration from 100 pM to 1000 nM. Cyclic voltammetry curves for different concentrations of Ach indicate the facile charge transfer process over the working electrode. Square wave voltammetry curves indicate the good sensing measurements of the modified electrode at the fixed potential. The limit of detection was found to be as low as 100 pM. In addition to these, selectivity of the electrode towards Ach molecule was confirmed by measuring the response towards other interfering agents. Besides this, the present nano interface is capable of detecting Ach in biological fluid such as serum.
•Gold nanoparticles (AuNPs) embedded graphene oxide (GO) nanocomposite modified ITO electrode was fabricated.•Acetylcholine esterase (AChE) and choline oxidase (ChO) were co-immobilized onto modified ITO electrode.•Fabricated electrochemical biosensor showed excellent detection limit and storage stability.•The present biosensor is also tested for the real sample analysis.
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