Phosphoproteome analysis of the crosstalk between sumoylation and phosphorylation in mouse spermatocytes.

Spermatogenesis is supported by various posttranslational modifications. There is growing evidence supporting a crosstalk between sumoylation and phosphorylation in different cell types. We have recently shown that inhibition of global sumoylation with a sumoylation inhibitor (Ginkgolic acid, GA) arrested purified mouse spermatocytes in vitro; the spermatocytes could not condense chromatin and disassemble the synaptonemal complex. Our data have also revealed that some kinases regulating the meiotic prophase (PLK1 and AURKB) were inhibited upon the inhibition of sumoylation. Nevertheless, specific phosphorylated targets affected by the inhibition of sumoylation have not been identified. To address this gap, in this study, we performed a comparative phospho-proteome analysis of the control spermatocytes and spermatocytes treated with the GA. Our analysis has narrowed down to several proteins implicated in the regulation of cell cycle and/or meiosis. Two of these targets, NPM1 and hnRNPH1, were studied further using western blotting in both cell lines and primary cells. Decrease in sumoylaion-dependend phosphorylation of NPM1 on Ser125 regulated by AURKB can be a contributing factor to the inability of spermatocytes to condense chromatin by the end of the prophase and should be studied further.

Sumoylation and its regulation in testicular Sertoli cells.

The molecular regulation of Sertoli cells and their crosstalk with germ cells has not been fully characterized. SUMO proteins are essential for normal development and are expressed in mouse and human Sertoli cells; However, the cell-specific role of sumoylation in those cells has only started to be elucidated. In other cell types, including granulosa cells, sumoylation is regulated by a SUMO ligase KAP1/Trim28. Deletion of KAP1 in Sertoli cells causes testicular degeneration; However, the role of KAP1 in those cells has not been identified. Here we show that both mouse and human Sertoli undergo apoptosis upon inhibition of sumoylation with a chemical inhibitor or via a siRNA technology. We have additionally detected changes in the Sertoli cell proteome upon the inhibition of sumoylation, and our data suggest that among others, the expression of ER/stress-related proteins is highly affected by this inhibition. Sumoylation may also regulate the NOTCH signaling which is important for the maintenance of the developing germ cells. Furthermore, we show that a siRNA-down-regulation of KAP1 in a Sertoli-derived cell line causes an almost complete inactivation of sumoylation. In conclusion, sumoylation regulates important survival and signaling pathways in Sertoli cells, and KAP1 can be a major regulator of sumoylation in these cells.

Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells.

Persistent hepatitis C virus (HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish CHC and cause its subsequent development into LC and HCC are poorly understood. We have identified a cytoplasmic double-stranded RNA binding protein, Stau1, which is crucial for HCV replication. In this study, Stau1 specifically interacted with the variable-stem-loop region in the 3' NTR and domain IIId of the HCV-IRES in the 5' NTR, and promoted HCV replication and translation. Stau1 coimmunoprecipitates HCV NS5B and a cell factor, protein kinase R (PKR), which is critical for interferon-induced cellular antiviral and antiproliferative responses. Like Stau1, PKR displayed binding specificity to domain IIId of HCV-IRES. Stau1 binds to PKR and strongly inhibits PKR-autophosphorylation. We demonstrated that the transport of HCV RNA on the polysomes is Stau1-dependent, being mainly localized in the monosome fractions when Stau1 is downregulated and exclusively localized in the polysomes when Stau1 is overexpressed. Our findings suggest that HCV may appropriate Stau1 to its advantage to prevent PKR-mediated inhibition of eIF2α, which is required for the synthesis of HCV proteins for translocation of viral RNA genome to the polysomes for efficient translation and replication.

Mental health problems raise the odds of cognitive impairment in COVID-19 survivors.

Background: COVID-19 survivors around the globe are suffering from mental health issues. While mental health problems can be an early warning sign of dementia, they may also increase the chances of developing the disease. In this study, we examined the mental health of COVID-19 survivors and mapped its associations with cognitive and demographic variables. Method: COVID-19 survivors listed in the databases of three tertiary care hospitals in Kolkata were contacted sequentially. 376 willing patients were interviewed over the telephone. 99 COVID-19 patients and 31 matched controls participated in the in-person interviews that were arranged for a more detailed investigation. The participants were administered standardized tests that are widely used for the assessment of cognitive functioning and mental health status. Result: 64.89% of COVID-19 survivors reported a deterioration in physical functioning. 44.95% reported a decline in mental health, whereas 41.49% reported a drop in cognitive performance. Detailed investigations revealed that they had an increased risk of having depression, anxiety, and poor sleep quality by 91%, 68%, and 140%, respectively. 6.1% of the patients had mild cognitive impairment, and 4% had dementia. COVID-19 patients who had depression and anxiety were 8.6 and 19.4 times more likely to have cognitive decline, respectively. Compared to the matched controls, COVID-19 patients had greater depression (p<.001), anxiety (p<.001), stress (p =.003), and insomnia (p <.001). They also scored significantly lower on Addenbrooke's Cognitive Examination-III (p =.009) and Picture Naming Test (p =.005) and took significantly longer to complete Trail Making Test-A (p =.002). Conclusion: COVID-19 survivors in this study had major mental health issues even one year after contracting the virus. They had significant cognitive deficits that might progress into dementia. Strict monitoring and systematic treatment plans should be implemented as soon as possible.

Opiate antagonist prevents µ- and δ-opiate receptor dimerization to facilitate ability of agonist to control ethanol-altered natural killer cell functions and mammary tumor growth.

In the natural killer (NK) cells, δ-opiate receptor (DOR) and µ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.

Squamous cell lung cancer producing bilateral air bronchograms on CT scan thorax.

A young lady presented with cough for three months and dyspnoea for one month along with clinical signs of bilateral consolidation of lung. On CT scan thorax there were bilateral air bronchograms with alveolar filling opacities predominantly in lower lobes. After bronchoscopy when there was presence of suspicious malignant cells on bronchoalveolar lavage fluid, transbronchial lung biopsy diagnosed the case as squamous cell lung cancer. Radiological presentation of squamous cell lung cancer as air bronchogram is not a very common phenomenon and can present a diagnostic challenge to the clinician.

Clinical outcome of orbital apex syndrome in COVID associated mucormycosis patients in a tertiary care hospital.

AIM: To share clinical pattern of presentation, the modalities of surgical intervention and the one month post-surgical outcome of rhino-orbito-mucormycosis (ROCM) cases. METHODS: All COVID associated mucormycosis (CAM) patients underwent comprehensive multidisciplinary examination by ophthalmologist, otorhinolaryngologist and physician. Patients with clinical and radiological evidence of orbital apex involvement were included in the study. Appropriate medical and surgical intervention were done to each patient. Patients were followed up one-month post intervention. RESULTS: Out of 89 CAM patients, 31 (34.8%) had orbital apex syndrome. Sixty-six (74.2%) of such patients had pre-existing diabetes mellitus, 18 (58%) patients had prior documented use of steroid use, and 55 (61.8%) had no light perception (LP) presenting vision. Blepharoptosis, proptosis, complete ophthalmoplegia were common clinical findings. Seventeen (19.1%) of such patients had variable amount of cavernous sinus involvement. Endoscopic debridement of paranasal sinuses and orbit with or without eyelid sparing limited orbital exenteration was done in most cases, 34 (38.2%) patients could retain vision in the affected eye. CONCLUSION: Orbital apex involvement in CAM patients occur very fast. It not only leads to loss of vision but also sacrifice of the eyeball, orbital contents and eyelids. Early diagnosis and prompt intervention can preserve life, vision and spare mutilating surgeries.

A Delphi consensus statement for the management of post-COVID interstitial lung disease.

INTRODUCTION: As millions of people worldwide recover from COVID-19, a substantial proportion continue to have persistent symptoms, pulmonary function abnormalities, and radiological findings suggestive of post-COVID interstitial lung disease (ILD). To date, there is limited scientific evidence on the management of post-COVID ILD, necessitating a consensus-based approach. AREAS COVERED: A panel of experts in pulmonology and thoracic radiology was constituted. Key questions regarding the management of post-COVID ILD were identified. A search was performed on PubMed and EMBASE and updated till 1 March 2022. The relevant literature regarding the epidemiology, pathophysiology, diagnosis and treatment of post-COVID ILD was summarized. Subsequently, suggestions regarding the management of these patients were framed, and a consensus was obtained using the Delphi approach. Those suggestions which were approved by over 80% of the panelists were accepted. The final document was approved by all panel members. EXPERT OPINION: Dedicated facilities should be established for the care of patients with post-COVID ILD. Symptom screening, pulmonary function testing, and thoracic imaging have a role in the diagnosis. The pharmacologic and non-pharmacologic options for the management of post-COVID ILD are discussed. Further research into the pathophysiology and management of post-COVID ILD will improve our understanding of this condition.

COVID-19 associated rhino-orbital-cerebral mucormycosis: An observational study from Eastern India, with special emphasis on neurological spectrum.

AIMS: 1: Describe the epidemiology and determine risk factors for COVID-19 associated mucormycosis. 2: Elaborate the clinical spectrum of Rhino-Orbital-Cerebral Mucormycosis (ROCM), pattern of neuroaxis involvement and it's radiological correlates. METHODS: Observational study. Consecutive, confirmed cases of mucormycosis (N = 55) were included. A case of mucormycosis was defined as one who had clinical and radiological features consistent with mucormycosis along with demonstration of the fungus in tissue via KOH mount/culture/histopathological examination (HPE). Data pertaining to epidemiology, risk factors, clinico-radiological features were analysed using percentage of total cases. RESULTS: Middle aged, diabetic males with recent COVID-19 infection were most affected. New onset upper jaw toothache was a striking observation in several cases. Among neurological manifestations headache, proptosis, vision loss, extraocular movement restriction; cavernous sinus, meningeal and parenchymal involvement were common. Stroke in ROCM followed a definitive pattern with watershed infarction. CONCLUSIONS: New onset upper jaw toothache and loosening of teeth should prompt an immediate search for mucormycosis in backdrop of diabetic patients with recent COVID-19 disease, aiding earlier diagnosis and treatment initiation. Neuroaxis involvement was characterized by a multitude of features pertaining to involvement of optic nerve, extraocular muscles, meninges, brain parenchyma and internal carotid artery.

Gene expression profiling of human primary astrocytes exposed to manganese chloride indicates selective effects on several functions of the cells.

Exposure of adult humans to manganese (Mn) has long been known to cause neurotoxicity. Recent evidence also suggests that exposure of children to Mn is associated with developmental neurotoxicity. Astrocytes are critical for the proper functioning of the nervous system, and they play active roles in neurogenesis, synaptogenesis and synaptic neurotransmission. In this report, to help elucidate the molecular events underlying Mn neurotoxicity, we systematically identified the molecular targets of Mn in primary human astrocytes at a genome-wide level, by using microarray gene expression profiling and computational data analysis algorithms. We found that Mn altered the expression of diverse genes ranging from those encoding cytokines and transporters to signal transducers and transcriptional regulators. Particularly, 28 genes encoding proinflammatory chemokines, cytokines and related functions were up-regulated, whereas 15 genes encoding functions involved in DNA replication and repair and cell cycle checkpoint control were down-regulated. Consistent with the increased expression of proinflammatory factors, analysis of common regulators revealed that 16 targets known to be positively affected by the interferon-gamma signaling pathway were up-regulated by Mn(2+). In addition, 68 genes were found to be similarly up- or down-regulated by both Mn(2+) and hypoxia. These results from genomic analysis are further supported by data from real-time RT-PCR, Western blotting, flow cytometric and toxicological analyses. Together, these analyses show that Mn(2+) selectively affects cell cycle progression, the expression of hypoxia-responsive genes, and the expression of proinflammatory factors in primary human astrocytes. These results provide important insights into the molecular mechanisms underlying Mn neurotoxicity.

Regulation of phosphorylation of tau by cyclin-dependent kinase 5 and glycogen synthase kinase-3 at substrate level.

Microtubule associated protein tau, which is expressed in six alternatively spliced molecular isoforms in human brain, is abnormally hyperphosphorylated in Alzheimer disease and related tauopathies. Here, we show (i) that GSK-3alpha and neither GSK-3beta nor cdk5 can phosphorylate tau at Ser262 and phosphorylation at Ser235 by cdk5 primes phosphorylation at Thr231 by GSK-3alpha/beta; (ii) that tau isoforms with two N-terminal inserts (tau4L, tau3L) are phosphorylated by cdk5 plus GSK-3 at Thr231 markedly more than isoforms lacking these inserts (tau4, tau3); and (iii) that Thr231 is phosphorylated approximately 50% more in free tau than in microtubule-bound tau, and the phosphorylation at this site results in the dissociation of tau from microtubules. These findings suggest that the phosphorylation of tau at Thr231 and Ser262 by cdk5 plus GSK-3, which inhibits its normal biological activity, is regulated both by its amino terminal inserts and its physical state.

The common insecticides cyfluthrin and chlorpyrifos alter the expression of a subset of genes with diverse functions in primary human astrocytes.

Given the widespread use of insecticides in the environment, it is important to perform studies evaluating their potential effects on humans. Organophosphate insecticides, such as chlorpyrifos, are being phased out; however, the use of pyrethroids in household pest control is increasing. While chlorpyrifos is relatively well studied, much less is known about the potential neurotoxicity of cyfluthrin and other pyrethroids. To gain insights into the neurotoxicity of cyfluthrin, we compared and evaluated the toxicity profiles of chlorpyrifos and cyfluthrin in primary human fetal astrocytes. We found that at the same concentrations, cyfluthrin exerts as great as, or greater toxic effects on the growth, survival, and proper functioning of human astrocytes. By using microarray gene expression profiling, we systematically identified and compared the potential molecular targets of chlorpyrifos and cyfluthrin, at a genome-wide scale. We found that chlorpyrifos and cyfluthrin affect a similar number of transcripts. These targets include molecular chaperones, signal transducers, transcriptional regulators, transporters, and those involved in behavior and development. Further computational and biochemical analyses show that cyfluthrin and chlorpyrifos upregulate certain targets of the interferon-gamma and insulin-signaling pathways and that they increase the protein levels of activated extracellular signal-regulated kinase 1/2, a key component of insulin signaling; interleukin 6, a key inflammatory mediator; and glial fibrillary acidic protein, a marker of inflammatory astrocyte activation. These results suggest that inflammatory activation of astrocytes might be an important mechanism underlying neurotoxicity of both chlorpyrifos and cyfluthrin.

A rare case of isolated testicular tuberculosis and review of literature.

Testicular tuberculosis (TB) is a rare form of genitourinary TB. It is usually presented as painful or painless testicular swelling with or without scrotal ulceration or discharging sinus. Infertility may occur. Epididymal involvement is usually seen in testicular TB. In most cases, genital TB is associated with TB involvement of kidneys or lower urinary tract. Ultrasound (USG) and USG-guided fine needle aspiration cytology of testicular swelling confirm the diagnosis. Anti-TB chemotherapy is the mainstay of treatment to ensure the complete resolution of the lesion. However, in very few cases, orchidectomy is required for both diagnosis and treatment. Here, we report a very rare case of left sided isolated testicular TB in a 20-year-old male who was completely cured with 6 months regimen of anti-TB chemotherapy.

Bilateral injection of isoproterenol into hippocampus induces Alzheimer-like hyperphosphorylation of tau and spatial memory deficit in rat.

The abnormal hyperphosphorylation of tau protein is one of the hallmarks of Alzheimer disease and other tauopathies; as yet the exact role of various tau kinases in this pathology is not fully understood. Here, we show that injection of isoproterenol, an activator of cAMP-dependent kinase (PKA), into rat hippocampus bilaterally results in the activation of PKA, calcium/calmodulin-dependent kinase II and cyclin-dependent kinase-5, inhibition of protein phosphatase-2A, hyperphosphorylation of tau at several Alzheimer-like epitopes and a disturbance of spatial memory retention 48 h after the drug injection. These findings suggest the involvement of PKA and PKA-mediated signaling pathway in the Alzheimer-like tau hyperphosphorylation and memory impairment.

Heme deficiency suppresses the expression of key neuronal genes and causes neuronal cell death.

Defective heme synthesis may cause acute porphyrias, which are associated with a wide array of neurological disturbances involving both the central and peripheral nervous systems. Thus, the understanding of the roles of heme in neuronal cell function may provide insights into the molecular events underlying the pathogenesis of neuropathies associated with defective heme synthesis. In this report, we use rat pheochromocytoma (PC12) clonal cells as a model system for studying the role of heme in neuronal cell survival. We examined the effects of inhibition of heme synthesis on signaling pathways and gene expression in nerve growth factor (NGF)-induced PC12 cells. We found that succinyl acetone-induced heme deficiency selectively caused apoptosis in NGF-induced PC12 cells. Further, we found that in succinyl acetone-treated, NGF-induced cells, the pro-survival Ras-ERK1/2 signaling pathway was inactivated and the pro-apoptotic JNK signaling pathway was activated. In these cells, the activation of caspase and the cleavage of nuclear poly (ADP-ribose) polymerase (PARP) were also evident. Importantly, microarray gene expression analysis showed that more than 20 key neuronal genes that were induced by NGF were suppressed by succinyl acetone. These genes include those encoding survival motor neuron protein, synaptic vesicle protein SVOP, and neural cell adhesion molecule NCAM. These results indicate that heme is important for neuronal cell signaling and the proper functioning of neuronal cells.

Substrate Stiffness Regulates Proinflammatory Mediator Production through TLR4 Activity in Macrophages.

Clinical data show that disease adversely affects tissue elasticity or stiffness. While macrophage activity plays a critical role in driving disease pathology, there are limited data available on the effects of tissue stiffness on macrophage activity. In this study, the effects of substrate stiffness on inflammatory mediator production by macrophages were investigated. Bone marrow-derived macrophages were grown on polyacrylamide gels that mimicked the stiffness of a variety of soft biological tissues. Overall, macrophages grown on soft substrates produced less proinflammatory mediators than macrophages grown on stiff substrates when the endotoxin LPS was added to media. In addition, the pathways involved in stiffness-regulated proinflammation were investigated. The TLR4 signaling pathway was examined by evaluating TLR4, p-NF-κB p65, MyD88, and p-IκBα expression as well as p-NF-κB p65 translocation. Expression and translocation of the various signaling molecules were higher in macrophages grown on stiff substrates than on soft substrates. Furthermore, TLR4 knockout experiments showed that TLR4 activity enhanced proinflammation on stiff substrates. In conclusion, these results suggest that proinflammatory mediator production initiated by TLR4 is mechanically regulated in macrophages.

Unexplained dyspnea in a patient of chronic arsenicosis: A diagnostic challenge and learning curve for physicians.

Chronic arsenic exposure causes cutaneous effects like hyperkeratosis, peripheral vascular disease, hypertension, ischemic heart disease, non-cirrhotic portal hypertension, hepatomegaly, peripheral neuropathy, respiratory involvement, bad obstetrical outcome, hematological disturbances, and diabetes mellitus. Here we present a case of a 24-year-old lady, with chronic exposure to arsenic, presenting to us with progressive dyspnea. We found pulmonary arterial hypertension (PAH) as a cause of her dyspnea. PAH can occur in arsenicosis, secondary to arsenic-induced chronic obstructive pulmonary disease (COPD), lung fibrosis, and portal hypertension, which we excluded by appropriate investigations in our case. We also excluded a familial or heritable form of PAH. Thus, with the exclusion of all these secondary causes of PAH, as well as a hereditary cause, we came to a conclusion that this PAH might be due to chronic arsenic exposure. To the best of our knowledge, no case of PAH in chronic arsenicosis has been reported to date.

Memantine inhibits and reverses the Alzheimer type abnormal hyperphosphorylation of tau and associated neurodegeneration.

Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, reduces the clinical deterioration in moderate-to-severe Alzheimer disease (AD) for which other treatments are not available. The activity of protein phosphatase (PP)-2A is compromised in AD brain and is believed to be a cause of the abnormal hyperphosphorylation of tau and the consequent neurofibrillary degeneration. Here we show that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation and accumulation of tau in organotypic culture of rat hippocampal slices. Such restorative effects of memantine were not detected either with 5,7-dichlorokynurenic acid or with D(-)-2-amino-5-phosphopentanoic acid, NMDA receptor antagonists active at the glycine binding site and at the glutamate binding site, respectively. These findings show (1) that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation of tau/neurofibrillary degeneration and (2) that this drug might be useful for the treatment of AD and related tauopathies.

Regulation of microtubule-associated proteins, protein kinases and protein phosphatases during differentiation of SY5Y cells.

Regulation of expression and function of microtubule-associated proteins (MAPs) is critical for neurons to maintain normal cytoskeletal architecture and functions. We have shown previously that in differentiated human neuroblastoma SY5Y cells, the expression of tau, a major neuronal MAP, is dramatically increased, and tau phosphorylation is differentially regulated. In the present study, we investigated the expression, the subcellular distribution and the microtubule-binding activities of several MAPs in SY5Y cells upon differentiation. We also studied the activities of protein kinases and phosphatases that are involved in regulation of tau phosphorylation during cell differentiation. We found that the expression of MAP1b in addition to tau was upregulated upon differentiation. Tau, MAP1a, MAP1b and MAP2 had distinct immunocytochemical staining patterns in differentiated SY5Y cells, suggesting differential biological functions. The microtubule-binding activity of tau increased after cell differentiation, whereas the activities of MAP1a and MAP2 decreased. Upon differentiation, the phosphorylation of tau at Ser198/Ser199/Ser202 and Ser396/Ser404 was increased, but that at Ser262/Ser356 was decreased. These changes in tau phosphorylation were accompanied by an upregulation of activities of several protein kinases (cdk5, MAPK, PKC and CK-1) as well as protein phosphatases PP-1 and PP-2A. These results suggest that the expression, post-translational modifications and biological activities of various MAPs are differentially regulated to meet the biological needs during cell differentiation.

Squamous cell carcinoma lung: Presented with bilateral lower limb deep venous thrombosis with gangrene formation.

Bilateral venous thrombosis due to underlying malignancy is a rare entity. It is worthy to search for malignancy in patients of bilateral venous gangrene. Our patient presented with severe bilateral leg pain as a result of venous gangrene. There was associated left sided massive pleural effusion with scalp nodule. Fine needle aspiration cytology of scalp nodule revealed metastatic squamous cell carcinoma and fiber optic bronchoscopy guided biopsy from growth at left upper lobe bronchus confirmed the case as squamous cell carcinoma lung. It was rare for squamous cell carcinoma lung to present as bilateral venous gangrene with anticardiolipin antibody negative.