Exploring the regulatory mechanism of tRNA-derived fragments 36 in acute pancreatitis based on small RNA sequencing and experiments

BACKGROUND Acute pancreatitis(AP)is a disease featuring acute inflammation of the pancreas and histological destruction of acinar cells.Approximately 20%of AP patients progress to moderately severe or severe pancreatitis,with a case fatality rate of up to 30%.However,a single indicator that can serve as the gold standard for prognostic prediction has not been discovered.Therefore,gaining deeper insights into the underlying mechanism of AP progression and the evolution of the disease and exploring effective biomarkers are important for early diagnosis,progression evaluation,and precise treatment of AP.AIM To determine the regulatory mechanisms of tRNA-derived fragments(tRFs)in AP based on small RNA sequencing and experiments.METHODS Small RNA sequencing and functional enrichment analyses were performed to identify key tRFs and the potential mechanisms in AP.Reverse transcription quantitative polymerase chain reaction(RT-qPCR)was conducted to determine tRF expression.AP cell and mouse models were created to investigate the role of tRF36 in AP progression.Lipase,amylase,and cytokine levels were assayed to examine AP progression.Ferritin expression,reactive oxygen species,malondialdehyde,and ferric ion levels were assayed to evaluate cellular ferroptosis.RNA pull down assays and methylated RNA immunoprecipitation were performed to explore the molecular mechanisms.RESULTS RT-qPCR results showed that tRF36 was significantly upregulated in the serum of AP patients,compared to healthy controls.Functional enrichment analysis indicated that target genes of tRF36 were involved in ferroptosisrelated pathways,including the Hippo signaling pathway and ion transport.Moreover,the occurrence of pancreatic cell ferroptosis was detected in AP cells and mouse models.The results of interference experiments and AP cell models suggested that tRF-36 could promote AP progression through the regulation of ferroptosis.Furthermore,ferroptosis gene microarray,database prediction,and immunoprecipitation suggested that tRF-36 accelerated the progression of AP by recruiting insulin-like growth factor 2 mRNA binding protein 3(IGF2BP3)to the p53 mRNA m6A modification site by binding to IGF2BP3,which enhanced p53 mRNA stability and promoted the ferroptosis of pancreatic follicle cells.CONCLUSION In conclusion,regulation of nuclear pre-mRNA domain-containing protein 1B promoted AP development by regulating the ferroptosis of pancreatic cells,thereby acting as a prospective therapeutic target for AP.In addition,this study provided a basis for understanding the regulatory mechanisms of tRFs in AP.

Correlation of serum SARS-CoV-2 IgM and IgG serology and clinical outcomes in COVID-19 patients:Experience from a tertiary care centre

BACKGROUND The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus has become a pandemic for the last 2 years.Inflammatory response to the virus leads to organ dysfunction and death.Predicting the severity of inflammatory response helps in managing critical patients using serology tests IgG and IgM.AIM To investigate the correlation of the serology(IgM and IgG)with reverse transcriptase polymerase chain reaction(RT-PCR)status,disease severity[mild to critical],intensive care unit(ICU)admission,septic shock,acute kidney injury,and in-hospital mortality.METHODS We conducted a longitudinal study to correlate serum SARS-CoV-2 immunoglobulin M(IgM)and immunoglobulin G(IgG)serology with clinical outcomes in coronavirus disease 2019(COVID-19)patients.We analyzed patient data from March to December 2020 for those who were admitted at All India Institute of Medical Sciences Rishikesh.Clinical and laboratory data of these patients were collected from the e-hospital portal and analyzed.A correlation was seen with clinical outcomes and was assessed using MS Excel 2010 and SPSS software.RESULTS Out of 494 patients,the mean age of patients was 48.95±16.40 years and there were more male patients in the study(66.0%).The patients were classified as mild-moderate 328(67.1%),severe 131(26.8%),and critical 30(6.1%).The mean duration from symptom onset to serology testing was 19.87±30.53 d.In-hospital mortality was observed in 25.1%of patients.The seropositivity rate(i.e.,either IgG or IgM>10 AU)was 50%.IgM levels(AU/mL)(W=33428.000,P≤0.001)and IgG levels(AU/mL)(W=39256.500,P≤0.001),with the median IgM/IgG levels(AU/mL),were highest in the RT-PCR-Positive group compared to RT-PCR-Negative clinical COVID-19.There was no significant difference between the two groups in terms of all other clinical outcomes(disease severity,septic shock,ICU admission,mechanical ventilation,and mortality).CONCLUSION The study showed that serology levels are high in RT-PCR positive group compared to clinical COVID-19.However,serology cannot be useful for the prediction of disease outcomes.The study also highlights the importance of doing serology at a particular time as antibody titers vary with the duration of the disease.In week intervals there was a significant correlation between clinical outcomes and serology on week 3.

Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients

AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P< 0.001) and preoperative serum levels of CEA (P=0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented signif icant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RTPCR assay can provide useful information concerning disease stage and overall survival of CRC patients.

OVEREXPRESSION OF Akt-1 GENE IN HUMAN HEPATOCELLULAR CARCINOMA

Objective: To investigate the expression difference of protein kinase B/Akt (Akt-1) between hepatocellular carcinoma (HCC) and adjacent normal liver tissues through the use of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Northern blot. Methods: RT-PCR of 24 pairs of specimens and Northern blot of 4 pairs of specimens were performed to investigate the expression of Akt-1. Results: Akt-1 gene was overexpressed in 15 of 24 HCC (63.3%) by RT-PCR and in all HCC (4 paired tissues) by Northern blot. Conclusion: Akt-1 activation may play a role in the pathogenesis and progression of HCC. Akt-1 gene is reported to have changed in HCC for the first time. The precise relationship between Akt-1 and HCC is a matter of further investigation.

Role of serological rapid antibody test in the management of possible COVID-19 cases

Although the detection of viral particles by reverse transcription polymerase chain reaction(RT-PCR)is the gold standard diagnostic test for coronavirus disease 2019(COVID-19),the false-negative results constitute a big challenge.AIM To examine a group of patients diagnosed and treated as possible COVID-19 pneumonia whose multiple nasopharyngeal swab samples were negative for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)by RT-PCR but then serological immunoglobulin M/immunoglobulin G(IgM/IgG)antibody against SARS-CoV-2 were detected by rapid antibody test.METHODS Eighty possible COVID-19 patients who had at least two negative consecutive COVID-19 RT-PCR test and were subjected to serological rapid antibody test were evaluated in this study.RESULTS The specific serological total IgM/IgG antibody against SARS-CoV-2 was detected in twenty-two patients.The mean age of this patient group was 63.2±13.1-yearsold with a male/female ratio of 11/11.Cough was the most common symptom(90.9%).The most common presenting chest computed tomography findings were bilateral ground glass opacities(77.2%)and alveolar consolidations(50.1%).The mean duration of time from appearance of first symptoms to hospital admission,to hospital admission,to treatment duration and to serological positivity were 8.6 d,11.2 d,7.9 d,and 24 d,respectively.Compared with reference laboratory values,serologically positive patients have shown increased levels of acute phase reactants,such as C-reactive protein,ferritin,and procalcitonin and higher inflammatory markers,such as erythrocyte sedimentation rate,lactate dehydrogenase enzyme,and fibrin end-products,such as D-dimer.A left shift on white blood cell differential was observed with increased neutrophil counts and decreased lymphocytes.CONCLUSION Our study demonstrated the feasibility of a COVID-19 diagnosis based on rapid antibody test in the cases of patients whose RT-PCR samples were negative.Detection of antibodies against SARS-CoV-2 with rapid antibody test should be included in the diagnostic algorithm in patients with possible COVID-19 pneumonia.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Quantitative detection of Cymbidium mosaic virus by real time PCR

The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(cp)gene of Cymbidium mosaic virus(CyMV).The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity.The standard curve was drawn based on the linear relationship between the logarithm(base 10)of the quantity of target sequence and cycle threshold[C(T)].While the concentration of plasmid DNA falling within the range of 2.6×10^(7)to 2.6×10^(2)copies per tube established a regression equation,y=-0.3583x+10.32,and related coefficient:r^(2)=0.995.The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube.The naturally infected samples of Phalaenopsis sp.and the artificially inoculated samples of Arachnis sp.with trace CyMV were quantitatively detected using this method.CyMVin the positive samples of Phalaenopsis sp.and Arachnis sp.was confirmed by DNA sequencing and cp gene homeology blast.The results showed that CyMV extracted from the leaves of orchid in Hangzhou,Zhejiang Province,China,could be derived from Kunming city(KM),Yunnan Province,China.This method characterized by high sensitivity,specificity,and precision is suitable for early diagnosis and quantitative detection of CyMV.

Expression and clinical implication of PRL-1 and PRL-3 in transitional cell carcinoma of bladder

The mRNA and protein expression of phos-phatase of regenerating liver 1(PRL-1)and phosphatase of regenerating liver 3(PRL-3)in transitional cell carcinoma of bladder(BTCC)and normal epithelia of bladder was investigated,and the relationship between the BTCC and pathological changes was clarified.The expression of PRL-1 and PRL-3 mRNA was detected by using reverse transcription polymerase chain reaction(RT-PCR)in 30 cases of BTCC and 10 cases of normal bladder,and the expression of PRL-1 and PRL-3 protein was checked by using immunohistochemistry in 30 cases of BTCC and 15 cases of normal bladder.The expression levels of PRL-1 and PRL-3 mRNA and protein were higher in BTCC than those in normal bladder epithelia(P<0.05).The increased expression of PRL-1 and PRL-3 mRNA and protein was detectable in deep invasion and metastasis of BTCC(P<0.05).There was no correlation between the expres-sion of PRL-1 and PRL-3 and gender,age or recurrence of BTCC(all P>0.05).A significantly positive correlation was found between PRL-1 and PRL-3 in BTCC(P<0.05).PRL-1 and PRL-3 are expressed consistently and may contribute to the growth,differentiation,invasion and metastasis of BTCC.

A versatile method for enumeration and characterization of circulating tumour cells from patients with breast cancer

Aim:To establish a standardized protocol for the isolation and enumeration of circulating tumor cells(CTCs)from peripheral blood of patients with metastatic breast cancer.Methods:The protocol used tumor cells spiked in a lymphoid cell line with detection by flow cytometry and quantitative reverse transcription polymerase chain reaction(QRT-PCR).Cells of the human mammary cancer subtypes were spiked into Jurkat cells,which served as the lymphocyte designate in numbers from 10 to 500 per 105 Jurkat cells.This mixed population was probed for CD45,EpCAM,and pancytokeratin acquired from flow cytometry and characterized by microscopy.QRT-PCR was done for CK-19,MUC-1,EpCAM,and GAPDH.Validation was attained with blood samples from 22 patients with metastatic breast cancer and 20 healthy individuals.Results:Flow cytometry could detect 1 breast cancer cell per 100,000 Jurkat cells,with similar detection levels in the breast cancer subtypes.Samples from patients with breast cancer showed a range of CTCs from 1-85 per 10 mL of blood.Quantitation of expression for EpCAM,CK-19,Muc-1,and Her2neu confirmed the presence of CTCs in 76%of samples.Conclusion:Density gradient and immunomagnetic enrichment accomplished isolation of CTCs and quantitation was achieved using flow cytometry.Combined QRT-PCR and imaging further validated these findings,rendering a robust methodology.