RhoC in association with TET2/WDR5 regulates cancer stem cells by epigenetically modifying the expression of pluripotency genes.

Emerging evidence illustrates that RhoC has divergent roles in cervical cancer progression where it controls epithelial to mesenchymal transition (EMT), migration, angiogenesis, invasion, tumor growth, and radiation response. Cancer stem cells (CSCs) are the primary cause of recurrence and metastasis and exhibit all of the above phenotypes. It, therefore, becomes imperative to understand if RhoC regulates CSCs in cervical cancer. In this study, cell lines and clinical specimen-based findings demonstrate that RhoC regulates tumor phenotypes such as clonogenicity and anoikis resistance. Accordingly, inhibition of RhoC abrogated these phenotypes. RNA-seq analysis revealed that RhoC over-expression resulted in up-regulation of 27% of the transcriptome. Further, the Infinium MethylationEPIC array showed that RhoC over-expressing cells had a demethylated genome. Studies divulged that RhoC via TET2 signaling regulated the demethylation of the genome. Further investigations comprising ChIP-seq, reporter assays, and mass spectrometry revealed that RhoC associates with WDR5 in the nucleus and regulates the expression of pluripotency genes such as Nanog. Interestingly, clinical specimen-based investigations revealed the existence of a subset of tumor cells marked by RhoC+/Nanog+ expression. Finally, combinatorial inhibition (in vitro) of RhoC and its partners (WDR5 and TET2) resulted in increased sensitization of clinical specimen-derived cells to radiation. These findings collectively reveal a novel role for nuclear RhoC in the epigenetic regulation of Nanog and identify RhoC as a regulator of CSCs. The study nominates RhoC and associated signaling pathways as therapeutic targets.

Yoga and music intervention to reduce depression, anxiety, and stress during COVID-19 outbreak on healthcare workers.

AIM: To investigate impact of Yoga and Music Intervention on anxiety, stress, and depression levels of health care workers during the COVID-19 outbreak. METHODS: This study was conducted to assess psychological responses of 240 healthcare workers during COVID-19 outbreak. We used Yoga and Music Intervention in normal and abnormal subjects based on Depression Anxiety and Stress Scale-42 (DASS-42). RESULTS: Of all 209 participants, 105 (50.23%) had symptoms of depression (35.88%), anxiety (40.19), and stress (34.92%) alone or in combination. The data suggest that there is significant improvement in test scores after intervention. Majority of persons with abnormal score exhibited improved DASS-42 score on combined interventions of Yoga and music compared to control group. Even subjects without abnormalities on DASS-42 score also showed improved DASS-42 scores in intervention (n = 52) group compared to nonintervention (n = 52) group. CONCLUSIONS: Our findings highlighted the significance of easily available, simple, inexpensive, safe nonpharmacological interventions like Yoga and Music therapy to overcome stress, anxiety, and depression in present times.

Metagenomics Analysis of Thrombus Samples Retrieved from Mechanical Thrombectomy.

PURPOSE: The purpose of this study was to assess the microbiota in middle cerebral artery thrombi retrieved in mechanical thrombectomy arising out of symptomatic carotid plaque within 6 hours of acute ischemic stroke. Thrombi were subjected to next-generation sequencing for a bacterial signature to determine their role in atherosclerosis. MATERIALS AND METHODS: We included 4 human middle cerebral artery thrombus samples (all patients were male). The median age for the patients was 51±13.6 years. Patients enrolled in the study from Pacific Medical University and Hospital underwent mechanical thrombectomy in the stroke window period. All patients underwent brain magnetic resonance angiography (MRA) and circle of Willis and neck vessel MRA along with the standard stroke workup to establish stroke etiology. Only patients with symptomatic carotid stenosis and tandem lesions with ipsilateral middle cerebral artery occlusion were included in the study. Thrombus samples were collected, stored at -80 degrees, and subjected to metagenomics analysis. RESULTS: Of the 4 patients undergoing thrombectomy for diagnosis with ischemic stroke, all thrombi recovered for bacterial DNA in qPCR were positive. More than 27 bacteria were present in the 4 thrombus samples. The majority of bacteria were Lactobacillus, Stenotrophomonas, Pseudomonas, Staphylococcus, and Finegoldia. CONCLUSION: Genesis of symptomatic atherosclerotic carotid plaque leading to thromboembolism could be either due to direct mechanisms like acidification and local inflammation of plaque milieu with lactobacillus, biofilm dispersion leading to inflammation like with pseudomonas fluorescence, or enterococci or indirect mechanisms like Toll 2 like signaling by gut microbiota.

Gene expression profiling of CD34(+) cells from patients with myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the increased proliferation of hematopoietic stem cell precursors and mature blood cells. Mutations of Janus kinase 2 (JAK2), Calreticulin (CALR) and MPL (myeloproliferative leukemia virus) are key driver mutations in MPN. However, the molecular profile of triple negative MPN has been a subject of ambiguity over the past few years. Mutations of, methylcytosine dioxygenase TET2, polycomb group protein ASXL1 and histone-lysine N-methyltransferase EZH2 genes have accounted for certain subsets of triple negative MPNs but the driving cause for majority of cases is still unexplored. The present study performed a microarray-based transcriptomic profile analysis of bone marrow-derived CD34(+) cells from seven MPN samples. A total of 21,448 gene signatures were obtained, which were further filtered into 472 upregulated and 202 downregulated genes. Gene ontology and protein-protein interaction (PPI) network analysis highlighted an upregulation of genes involved in cell cycle and chromatin modification in JAK2V617F negative vs. positive MPN samples. Out of the upregulated genes, seven were associated with the hematopoietic stem cell signature, while forty-seven were associated with the embryonic stem cell signature. The majority of the genes identified were under the control of NANOG and E2F4 transcription factors. The PPI network indicated a strong interaction between chromatin modifiers and cell cycle genes, such as histone-lysine N-methyltransferase SUV39H1, SWI/SNF complex subunit SMARCC2, SMARCE2, chromatin remodeling complex subunit SS18, tubulin ß (TUBB) and cyclin dependent kinase CDK1. Among the upregulated epigenetic markers, there was a ~10-fold increase in MYB expression in JAK2V617F negative samples. A significant increase in total CD34 counts in JAK2V617F negative vs. positive samples (P<0.05) was also observed. Overall, the present data showed a distinct pattern of expression in JAK2V617F negative vs. positive samples with upregulated genes involved in epigenetic modification.

High frequency of exon 20 S768I EGFR mutation detected in malignant pleural effusions: A poor prognosticator of NSCLC.

BACKGROUND: Lung cancer is the cause of a fourth of all cancer-related deaths. About a third of all lung adenocarcinoma tumours harbour mutations on exons 18 to 21 of the epidermal growth factor receptor (EGFR) gene. Detection of these mutations allows for targeted therapies in the form of EGFR Tyrosine kinase inhibitors. Recently, "liquid biopsies" have emerged as an alternative to conventional tissue mutation detection. AIM: In this pilot study, we attempted to optimize EGFR mutation detection from malignant pleural effusions (MPEs) as "liquid biopsies" when tissue biopsies were unavailable. Resulting mutations were then to be mapped on the EGFR gene and explored using cBioPortal, a public cancer genomic database. METHODS AND RESULTS: We first attempted a direct sequencing approach and showed that single nucleotide variants (SNVs) were likely to be missed in MPEs. We then switched to and optimized an EGFR mutant-specific quantitative polymerase chain reaction-based assay. This assay was piloted on n = 10 pleural effusion samples (one non-malignant pleural effusion as a negative control). 5/9 (55.55%) samples harboured EGFR mutations with 2/9 (22.22%) being exon 19 deletions and 3/9 (33.33%) the S768I mutation. The frequency of the S768I SNV in our study was significantly higher than that observed in other studies (~0.2%). Utilizing cBioPortal data, we report that patients with S768I have a shorter median survival time (6 months vs 38 months), progression-free survival time (8 months vs 44 months) and lower tumor mutation count compared to patients with other EGFR mutations. CONCLUSIONS: The shorter survival of patients with the S768I SNV predicts aggressive disease and poor prognosis as a result of this mutation. Studies in larger cohorts and/or animal models are necessary to confirm these findings.

Role of synaptophysin in the intraoperative assessment of quadrantic innervation of the proximal doughnut in Hirschsprung disease.

BACKGROUND: Symptoms may persist in a retained aganglionic segment of the colon after corrective (pull-through) surgery in Hirschsprung disease (HD). Thus, it is important to assess the proximal doughnut for innervation abnormalities intraoperatively by frozen sections stained with conventional haematoxylin and eosin stain and supported by rapid acetylcholinesterase (AChE) histochemistry. When the doughnut is proximal to the sigmoid colon, AChE is not useful and requires ratification by yet another rapid technique and hence this study. METHODS: Two pathologists independently evaluated fresh doughnuts from the proximal bowel clinically assumed to be of normal innervation intraoperatively and chosen for anastomosis in patients with HD along with controls using AChE and synaptophysin (SY) immunohistochemistry. RESULTS: From 38 patients with HD, 28 doughnuts (63.7%) showed normal innervation with intense SY activity in the mucosa, the muscularis and the ganglion cells. The circumferential aganglionic doughnuts (abnormal innervation) (n= 6, 13.6%) showed neither SY-positive fibres in the mucosa nor in the muscularis. The abnormal transition zone doughnuts (n=10, 22.7%) showed involvement of three quadrants of the doughnut in one, two quadrants in three and one quadrant in six with decreased SY-positive fibres in the muscularis and scattered ganglion cells with a statistically significant measure of agreement of (κ=0.973) between the two. CONCLUSION: The pattern, intensity and distribution of SY-positive fibres in the muscularis propria of the doughnut of the proximal bowel chosen intraoperatively for anastomosis in HD can identify sectors with abnormal innervation allowing the surgeon to seek normal innervation status more proximally to avoid complications.

Appendicular Biopsy in Total Colonic Aganglionosis: A Histologically Challenging and Inadvisable Practice.

Background The reliability of intraoperative evaluation of ganglion cells in the appendix as a guide to a diagnosis of total colonic aganglionosis is unclear. Objective To evaluate the diagnostic utility of appendicular innervation in colonic Hirschsprung disease (HD) and TCA. Methods Prospective, systematic study of ganglion cells and the neural plexii in appendices from cases (HD and TCA) and age matched controls with frozen and paraffin sections, rapid acetylcholinesterase (AChE) and immunohistochemistry. Results A total of 48 appendices (28 controls, 20 cases; 19 frozen) were evaluated. Of these 48, 30 were neonates. Ganglion cell clusters were smaller in controls (28) and HD (6) than those in the rectum, distorted at places and mimicked lymphocytes and endothelial cells, especially in neonates. Complete study of 13 appendices in TCA showed absence of ganglion cells, hypertrophic nerves, AChE activity, and calretinin staining. In 2/13 TCA, an erroneous frozen section identification of ganglia was later corrected based on AChE histochemistry and a panel of IHC stains. Ileal biopsies guided the placement of a ganglionic ileostomy in all. One case each of skip segment aganglionosis in a TCA and variable hypoganglionosis in long segment colonic HD is reported. Conclusion Intraoperative characterization of appendicular innervation as a guide to the diagnosis of TCA is unreliable, in part because of the possibility of skip segment disease/variable hypoganglionosis. We propose terminal ileal biopsies for diagnosis and leveling of aganglionosis. AChE on frozen/calretinin on paraffin tissue is the best approach to avoid diagnostic errors.

Improvised double-embedding technique of minute biopsies: a mega boon to histopathology laboratory.

INTRODUCTION: Optimal orientation of minute mucosal biopsies is essential for a definite diagnosis in gastrointestinal pathology or to visualize neural plexuses in Hirschsprung disease. The problem of minute size of the biopsy and its orientation gets compounded when they are from neonates and mandates exhaustive strip cuts, thus delaying reporting. AIM: A modified agar-paraffin technique is aimed to make tissue embedding efficient and user-friendly by inking mapping biopsies (one or more) either fresh or fixed with surgical coloring inks followed by embedding first in agar after orientation and followed thereafter by processing, re-embedding in paraffin wax, sectioning and staining. RESULTS: The tissues in agar paraffin block were found to be well processed, firm, held secure and well preserved. The blocks were easy to cut, with serial sections of thickness 2-3 µ and easy to spread. The colored inks remained permanently on the tissues both in the block as well as on the sections which helped in easy identification of tissues. Agar did not interfere with any stain such as Hematoxylin and Eosin or with histochemical stains, enzyme histochemistry or immunohistochemistry. Inking biopsies and pooling them in a block when obtained from the same patient reduced the number of tissue blocks. CONCLUSION: The modified agar-paraffin embedding technique is a simple reliable user friendly method that can greatly improve the quality of diagnostic information from minute biopsies by optimal orientation, better quality of sections, faster turnaround time and cost-effectiveness by economizing on the number of paraffin blocks, manpower, chemical reagents and laboratory infrastructure.

Calretinin immunohistochemistry versus improvised rapid Acetylcholinesterase histochemistry in the evaluation of colorectal biopsies for Hirschsprung disease.

BACKGROUND: Acetylcholinesterase (AChE) histochemistry on rectal mucosal biopsies accurately diagnoses Hirschsprung disease (HD), but is not widely employed as it requires special tissue handling and pathologist expertise. Calretinin immunohistochemistry (IHC) has been reported to be comparable to AChE staining with the loss of expression correlating with aganglionosis. AIM: The aim was to evaluate calretinin IHC as a primary diagnostic tool in comparison to the improvised rapid AChE technique in the diagnosis of HD. MATERIALS AND METHODS: A total of 74 rectal biopsies (18 fresh frozen - 18 cases, 56 formalin fixed - 33 cases) from 51 cases of suspect HD were evaluated with hematoxylin and eosin/AChE/Calretinin. Ten biopsies each from ganglionated and aganglionated segments served as positive and negative controls. Ileal (3), appendiceal (3) and ring bowel (2) biopsies were also included. Two pathologists blinded to the clinical details evaluated the histomorphology with AChE and calretinin. Observations were statistically analyzed and Cohen's k coefficient employed to assess agreement between two pathologists and calretinin and the AChE. RESULTS: The study confirmed HD in 26 and non-HD in 25 cases. There were 7 neonates, 5 low level biopsies and 14 "inadequate" biopsies. The results of calretinin were comparable with AChE with a statistically significant measure of agreement of k = 0.973 between the two. One false-positive case of HD was noted with calretinin. The advantages and disadvantages of calretinin versus AChE are discussed. CONCLUSION: Calretinin is a reliable single immune marker for ruling out HD by its specific positive mucosal staining of formalin fixed rectal biopsy. The improvised AChE staining remains indispensable to confirm HD on fresh biopsies and thus, along with calretinin IHC maximizes the diagnostic accuracy of HD in difficult cases.