Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

Early rRNA processing is a stress-dependent regulatory event whose inhibition maintains nucleolar integrity.

The production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here, we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but is independently regulated. Failure to coordinately control ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils how the rapid translational shut-off in response to stress coordinates with rRNA synthesis production to maintain nucleolar integrity.

Expression of the DeaD RNA Helicase Is Regulated at Multiple Levels through Its Long mRNA 5' Untranslated Region.

DEAD-box proteins (DBPs) are a prominent class of RNA remodeling proteins that alter RNA structure, a process they typically perform through an ATP-dependent RNA helicase activity. Although many DBPs have been characterized at the structural and functional level in detail, much less is known about how they are regulated. We previously showed that the mRNA for the Escherichia coli DeaD DBP contains an unusually long 5' untranslated region (5' UTR) of 838 nucleotides (nt) and that it is the primary RNA determinant of DeaD autoregulation. We speculated that such a long and complex 5' UTR might regulate deaD expression in additional ways. Here, we show that the deaD mRNA 5' UTR regulates deaD expression at two additional levels, temperature-dependent expression and through a stem-loop structure overlapping the start codon. These results support the hypothesis that a long 5' UTR can regulate gene expression through multiple mechanisms. IMPORTANCE The expression of genes is frequently regulated by determinants in the 5' UTR. Although many different regulatory mechanisms that operate via the 5' UTR have been described, the functional relevance of genes with long UTRs is less clear. Here, we show that the 838-nt-long 5' UTR in the deaD mRNA regulates the expression of DeaD at multiple levels. We propose that long UTRs originate to provide precise control of gene expression through multiple regulatory mechanisms, and they are indicators of the importance of their associated gene products for cellular adaptation to different environments.

Dual-level autoregulation of the E. coli DeaD RNA helicase via mRNA stability and Rho-dependent transcription termination.

DEAD-box proteins (DBPs) are RNA remodeling factors associated with RNA helicase activity that are found in nearly all organisms. Despite extensive studies on the mechanisms used by DBPs to regulate RNA function, very little is known about how DBPs themselves are regulated. In this work, we have analyzed the expression and regulation of DeaD/CsdA, the largest of the DBPs in Escherichia coli (E. coli). We show that deaD transcription initiates 838 nt upstream of the start of the coding region. We have also found that DeaD is autoregulated through a negative feedback mechanism that operates both at the level of deaD mRNA stability and Rho-dependent transcription termination, and this regulation is dependent upon its mRNA 5' untranslated region (5' UTR). These findings suggest that DeaD might be regulating the conformation of its own mRNA through its RNA helicase activity to facilitate ribonuclease and Rho access to its 5' UTR.

eIF4G has intrinsic G-quadruplex binding activity that is required for tiRNA function.

As cells encounter adverse environmental conditions, such as hypoxia, oxidative stress or nutrient deprivation, they trigger stress response pathways to protect themselves until transient stresses have passed. Inhibition of translation is a key component of such cellular stress responses and mounting evidence has revealed the importance of a class of tRNA-derived small RNAs called tiRNAs in this process. The most potent of these small RNAs are those with the capability of assembling into tetrameric G-quadruplex (G4) structures. However, the mechanism by which these small RNAs inhibit translation has yet to be elucidated. Here we show that eIF4G, the major scaffolding protein in the translation initiation complex, directly binds G4s and this activity is required for tiRNA-mediated translation repression. Targeting of eIF4G results in an impairment of 40S ribosome scanning on mRNAs leading to the formation of eIF2α-independent stress granules. Our data reveals the mechanism by which tiRNAs inhibit translation and demonstrates novel activity for eIF4G in the regulation of translation.

Transforming the attitudes of young men about gender roles and the acceptability of violence against women, Bihar.

Although the importance of working with young men to transform traditional gender norms has been widely acknowledged, programmes for young men remain sparse in highly gender stratified settings such as India, and those that have been implemented have not reached those in rural areas and those out-of-school. Drawing on data from a cluster randomised controlled trial with panel surveys, of a gender-transformative life skills education and sports-coaching programme conducted among young men aged 13-21 who were members of youth clubs, this paper examines the extent to which it transformed the gender role attitudes of young men and instilled in them attitudes rejecting violence against women and girls. The intervention succeeded in changing gender role attitudes and notions of masculinity, attitudes about men's controlling behaviours over women/girls, attitudes about men's perpetration of violence on a woman/girl and perceptions about peer reactions to young men acting in gender-equitable ways. Effects were particularly significant among young men who attended regularly, underscoring the importance of regular attendance in such programmes.

The role of squash cytology in rapid on-site adequacy checking and rapid diagnosis in image-guided gun biopsy at a tertiary cancer center.

OBJECTIVE: To evaluate the role of squash cytology in rapid on-site adequacy checking (ROSAC) of image-guided gun biopsy and to determine its diagnostic accuracy at a tertiary cancer center. STUDY DESIGN: This was a prospective study on 183 patients undergoing image-guided biopsy. Squash smears were prepared from biopsy cores and checked for adequacy by cytotechnologists. When adequate, more cores were sampled from the same area for histopathology. If inadequate, the procedure was repeated at the same sitting on a different area. The squash smears were reported by cytopathologists within 4 h after staining with conventional Papanicoloau stain. The results were compared with the final histopathology report. RESULTS: The sampling was representative in 95.6% cases with concordance for adequacy in 97.3% cases. The sensitivity, specificity, positive predictive value and negative predictive value of squash cytology for diagnosis of the lesion were 99.4, 92.7, 97.7 and 97.4%, respectively. CONCLUSIONS: Squash cytology is an ideal and cost-effective technique for ROSAC of image-guided biopsies, which ensures adequacy, avoids repeat procedures and prevents delay in diagnosis. It can be effectively performed by trained cytotechnologists in radiology clinics. Squash cytology is also a cost-effective tool offering rapid diagnosis which expedites planning of treatment.

Safety and Effectiveness of Liv.52 DS in Patients With Varied Hepatic Disorders: An Open-Label, Multi-centre, Phase IV Study.

Background The hepatoprotective function of polyherbal formulation Liv.52 in chronic liver diseases is well recognized in published literature. The objective of this open-label, phase IV study was to further strengthen and validate its safety and effectiveness using a large patient pool in a real-world scenario and provide scientific data on symptomatic improvement and supportive treatment in liver function with improvement in quality of life. Methods Adult patients of either sex with one or more clinical symptoms like fatigue, nausea, anorexia, abdominal pain or discomfort, muscle cramps, jaundice, or any other signs and symptoms with a history suggestive of mild-to-moderate hepatic disorders like alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), drug-induced hepatotoxicity, or hepatitis were treated with two Liv.52 DS tablets (oral) twice daily for 12 weeks. Results Out of the 1000 enrolled patients, 962 (96%) completed the study with the following subgroups ALD: 375 (38.9%), NAFLD: 379 (39.3%), drug-induced hepatotoxicity: 78 (8.1%), hepatitis: 130 (13.5%). The mean age of enrolled patients was 37.7 years, and the majority of them, 785 (78.5%) were men. The common adverse events observed (with >1.5% incidence) in the study were abdominal pain: 26 (2.6%) and headache: 17 (1.7%). Liv.52 showed statistically significant improvement (P<0.0001) in various clinical signs and symptoms in the majority of patients namely, fatigue: 357/723 (49%), anorexia: 485/620 (78.2%), jaundice: 48/52 (92%). Majority of the patients showed significant improvements from baseline to end of 12 weeks in the liver function test parameters namely, aspartate aminotransferase: 633/840 (75.36%), alanine aminotransferase: 592/729 (81.21%), serum bilirubin: 244/347 (70.32%), alkaline phosphatase: 279/355 (78.59%) with P<0.0001 for all parameters. Statistically significant improvement (P<0.005) was also seen in all the components of the chronic liver disease questionnaire (CLDQ) scores from baseline to 12 weeks. Conclusions The study demonstrated that Liv.52 was hepatoprotective and well tolerated in the study population after treatment for 12 weeks. Significant improvements were seen in clinical signs and symptoms, laboratory parameters of liver function, and CLDQ scores from baseline to 12 weeks. No significant or new safety signals emerged from this study.

Fine-needle aspiration cytology of breast lesions with spontaneous infarction: a five-year study.

OBJECTIVE: To put forth the cytological features and diagnostic pitfalls of spontaneously infarcted breast lesions on fine-needle aspiration cytology (FNAC). STUDY DESIGN: We present 19 cases of spontaneously infarcted breast lesions encountered on FNAC over a 5-year period. Histological follow-up was correlated wherever available. RESULTS: The majority of cases were seen in the second decade of life. The smears in all 19 cases were cellular. The cytomorphologic findings were scattered dyscohesive cells (n = 16), ghost cells (n = 11) and necrosis (n = 10). The dyscohesive cells were small and had a normal nuclear cytoplasmic ratio with pyknotic nuclei. The presence of viable epithelial cells or stromal fragments helped in the diagnosis of the primary breast lesion and was seen in all 19 cases. Cytology diagnoses were infarcted fibroadenoma (n = 11), infarcted breast lesion (n = 3), Phyllodes tumor (n = 2), papillary lesion (n = 2) and infarcted benign breast lesion (n = 1). Histopathology was available in 13 cases, 12 were concordant and 1 was inadequate for primary diagnosis. CONCLUSION: An infarcted breast lesion poses diagnostic difficulties on cytology. It needs to be differentiated from inflammatory lesions and malignancy. A cytopathologist should be aware of the entity and recognize its cytomorphologic features.

Dysplastic megakaryocytes and eosinophilic precursors in the diagnosis of myeloid sarcoma on lymph node fine-needle aspiration cytology: a case series.

OBJECTIVE: To analyze the cases diagnosed as myeloid sarcoma on fine-needle aspiration cytology (FNAC) of lymph nodes. STUDY DESIGN: Ten cases of lymph node aspirate diagnosed as myeloid sarcoma were analyzed. FNAC was performed as a routine outpatient procedure in all cases. Correlation with peripheral smear, bone marrow examination, flow cytometry and cytogenetics was done wherever possible. RESULTS: Diagnosis of a hematologic malignancy, before fine-needle aspiration, was available in only 2 cases. All 10 cases showed eosinophilic precursors while five aspirates showed megakaryocytes with dysplastic forms. Of the 10 cases, 3 were diagnosed as acute myeloid leukemia, 3 as chronic myeloid leukemia, 1 case as juvenile myelomonocytic leukemia, 1 case proved to be precursor T acute lymphoblastic leukemia/lymphoma and 2 patients did not show blasts on peripheral blood smear but showed blasts, dysplastic megakaryocytes and eosinophilic precursors in the aspirate. CONCLUSION: Thorough workup to search for an underlying hematological malignancy should be done whenever dysplastic megakaryocytes and/or eosinophilic precursors are seen in lymph node aspirate.

Key cytological findings in FNA from infantile digital fibromatosis.

BACKGROUND: Infantile digital fibromatosis (IDF) or inclusion body fibromatosis is a benign proliferation of fibroblastic and myofibroblastic cells. Its most common site is the digits of young children and it is named for the intracytoplasmic inclusions that are detected in lesional cells. CASE: A two and a half-year-old male child presented with a single flesh-colored nodule on the dorsal aspect of his right little toe since the sixth month of life. FNAC from the lesion showed characteristic cytomorphological features of IDF. CONCLUSION: IDF is a rare lesion occurring in children or infants. There are many case reports describing histopathological features of IDF. To our knowledge, the typical inclusion bodies as cytomorphological findings in IDF have not been described in the literature. This is a rare case diagnosed on cytology and confirmed on histopathology.

snoRNPs: Functions in Ribosome Biogenesis.

Ribosomes are perhaps the most critical macromolecular machine as they are tasked with carrying out protein synthesis in cells. They are incredibly complex structures composed of protein components and heavily chemically modified RNAs. The task of assembling mature ribosomes from their component parts consumes a massive amount of energy and requires greater than 200 assembly factors. Among the most critical of these are small nucleolar ribonucleoproteins (snoRNPs). These are small RNAs complexed with diverse sets of proteins. As suggested by their name, they localize to the nucleolus, the site of ribosome biogenesis. There, they facilitate multiple roles in ribosomes biogenesis, such as pseudouridylation and 2'-O-methylation of ribosomal (r)RNA, guiding pre-rRNA processing, and acting as molecular chaperones. Here, we reviewed their activity in promoting the assembly of ribosomes in eukaryotes with regards to chemical modification and pre-rRNA processing.

Adenocarcinoma in a tailgut cyst: A rare case report.

Tailgut cysts (TGCs) are rare congenital lesions derived from the remnants of primitive hindgut and are usually lined by squamous, transitional, or glandular epithelium. Malignant transformation in TGC may occur which is still rarer. Most common malignancies that arise from these cysts are adenocarcinomas. Preoperative diagnosis is difficult as high degree of suspicion is required for the diagnosis. We report here a case of adenocarcinoma arising in a tale gut cyst diagnosed preoperatively and till date very few cases have been reported in literature.

Role of fine needle aspiration cytology in diagnosis of brown tumor secondary to parathyroid adenoma.

Brown tumor (BT) is caused by altered metabolism of calcium resulting from hyperparathyroidism (primary or secondary). The most common cause of hyperparathyroidism is isolated parathyroid adenoma (PA), and the most common symptoms are hypercalcemia related. BT is considered as a late manifestation of PA and usually diagnosed after surgical treatment of the bony lesion. Fine-needle aspiration cytology (FNAC) is a cheap, easy, and less traumatic procedure and should be performed in all lesions wherever possible as unnecessary surgeries may be avoided. We here report a rare case of PA presenting primarily as BT and diagnosed on FNAC.

Cytomorphological Findings of Follicular Dendritic Cell Sarcoma on Fine-Needle Aspiration Cytology.

BACKGROUND: Follicular dendritic cell sarcoma (FDCS) is a rare neoplasm arising from follicular dendritic cells of germinal centers. The most common site of origin is lymph nodes and it may mimic a variety of tumors at that location, including carcinomas and sarcomas. Diagnosis is frequently missed on cytology as there are very few case reports describing the cytological characteristics of the lesion. Even on histology, a high degree of suspicion is required for an appropriate diagnosis. CASE: A 60-year-old male presented with a gradually increasing left submandibular mass that had been present for 3 months. Fine-needle aspiration cytology (FNAC) was performed, showing many clusters as well as scattered epithelioid cells with spindled to oval nuclei, nuclear pleomorphism, grooves, inclusions, and uniformly dispersed mature lymphocytes throughout the smears. The diagnosis of FDCS was suspected and was confirmed on histopathology and immunohistochemistry. CONCLUSION: FNAC can be a cheap, easy, and helpful tool in obtaining a diagnosis of FDCS as there are few characteristic cytological features that are better recognized than histology.

Role of Field Staining in the Cytological Assessment of Intraoperative Surgical Specimens.

OBJECTIVE: To study the role of Field staining in scrape smears for intraoperative cytological (IOC) diagnosis. Specimens were assessed for categorizing among benign and malignant lesions, lymph node status, and adequacy of surgical cut margins as per specimen. Technique, adequacy, and quality were assessed along with comparison of cytological diagnosis with final histopathological diagnosis obtained on routine hematoxylin and eosin-stained slides. STUDY DESIGN: A prospective observational study was conducted over a period of 1 year from November 2016 to October 2017 in the Department of Pathology of our Institute. RESULTS: 50 cases were studied, and scrape smears were stained with Field stain. Results were satisfactory in terms of adequacy and attaining the objectives of the study. A diagnostic accuracy of 98% was observed with an average turnaround time of 5 min. A single case of low-grade glioma was found to be discordant. CONCLUSIONS: Use of Field staining for intraoper-ative cytological assessment of surgical specimens has 98% concordance with the final histopathological diagnosis and achieved the aim of the study. With its low costs, easy availability, short turnaround time, and simple technique, it will be helpful in IOC as an alternative to present techniques especially in financially constrained settings.

Leiomyosarcoma of Renal Vein - A Rare Case Report.

Leiomyosarcomas (LMS) arising from vascular channel are rare and more often arise from inferior vena cava and pulmonary arteries. Primary renal vein LMS are even rarer and occur predominantly in females with peak in fifth and sixth decade. Preoperative diagnosis is difficult because these are rare tumours and present with symptoms and radiological findings similar to Renal Cell Carcinoma (RCC). We report a case of 55-year-old female who presented with abdominal discomfort with radiology showing a renal mass with features of RCC, radical nephrectomy was done and resected tumour showed an attachment to the wall of renal vein with morphology resembling LMS.

Post-transcriptional regulation of ribosomal protein genes during serum starvation in Entamoeba histolytica.

Ribosome synthesis involves all three RNA polymerases which are co-ordinately regulated to produce equimolar amounts of rRNAs and ribosomal proteins (RPs). Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in E. histolytica rDNA transcription continues but pre-rRNA processing slows down and unprocessed pre-rRNA accumulates during serum starvation. To investigate the regulation of RP genes under stress we measured transcription of six selected RP genes from the small- and large-ribosomal subunits (RPS6, RPS3, RPS19, RPL5, RPL26, RPL30) representing the early-, mid-, and late-stages of ribosomal assembly. Transcripts of these genes persisted in growth-stressed cells. Expression of luciferase reporter under the control of two RP genes (RPS19 and RPL30) was studied during serum starvation and upon serum replenishment. Although luciferase transcript levels remained unchanged during starvation, luciferase activity steadily declined to 7.8% and 15% of control cells, respectively. After serum replenishment the activity increased to normal levels, suggesting post-transcriptional regulation of these genes. Mutations in the sequence -2 to -9 upstream of AUG in the RPL30 gene resulted in the phenotype expected of post-transcriptional regulation. Transcription of luciferase reporter was unaffected in this mutant, and luciferase activity did not decline during serum starvation, showing that this sequence is required to repress translation of RPL30 mRNA, and mutations in this region relieve repression. Our data show that during serum starvation E. histolytica blocks ribosome biogenesis post-transcriptionally by inhibiting pre-rRNA processing on the one hand, and the translation of RP mRNAs on the other.

A host factor supports retrotransposition of the TRE5-A population in Dictyostelium cells by suppressing an Argonaute protein.

BACKGROUND: In the compact and haploid genome of Dictyostelium discoideum control of transposon activity is of particular importance to maintain viability. The non-long terminal repeat retrotransposon TRE5-A amplifies continuously in D. discoideum cells even though it produces considerable amounts of minus-strand (antisense) RNA in the presence of an active RNA interference machinery. Removal of the host-encoded C-module-binding factor (CbfA) from D. discoideum cells resulted in a more than 90 % reduction of both plus- and minus-strand RNA of TRE5-A and a strong decrease of the retrotransposition activity of the cellular TRE5-A population. Transcriptome analysis revealed an approximately 230-fold overexpression of the gene coding for the Argonaute-like protein AgnC in a CbfA-depleted mutant. RESULTS: The D. discoideum genome contains orthologs of RNA-dependent RNA polymerases, Dicer-like proteins, and Argonaute proteins that are supposed to represent RNA interference pathways. We analyzed available mutants in these genes for altered expression of TRE5-A. We found that the retrotransposon was overexpressed in mutants lacking the Argonaute proteins AgnC and AgnE. Because the agnC gene is barely expressed in wild-type cells, probably due to repression by CbfA, we employed a new method of promoter-swapping to overexpress agnC in a CbfA-independent manner. In these strains we established an in vivo retrotransposition assay that determines the retrotransposition frequency of the cellular TRE5-A population. We observed that both the TRE5-A steady-state RNA level and retrotransposition rate dropped to less than 10 % of wild-type in the agnC overexpressor strains. CONCLUSIONS: The data suggest that TRE5-A amplification is controlled by a distinct pathway of the Dictyostelium RNA interference machinery that does not require RNA-dependent RNA polymerases but involves AgnC. This control is at least partially overcome by the activity of CbfA, a factor derived from the retrotransposon's host. This unusual regulation of mobile element activity most likely had a profound effect on genome evolution in D. discoideum.