Reverse Transcriptase-polymerase Chain Reaction Method Research Articles

MicroRNA and renal fibrosis in autosomal dominant polycystic kidney disease: a longitudinal study.

Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary kidney disorder that may progress to kidney failure, accounting for 5-10% of all patients with end-stage kidney disease (ESKD). Clinical data, as well as molecular genetics and advanced imaging techniques have provided surrogate prognostic biomarkers to predict rapid decline in kidney function, nonetheless enhanced tools for assessing prognosis for ADPKD are stillneeded. The aim of this study was to analyze specific microRNAs involved in the pathogenesis of ADPKD and in the development of renal fibrosis, evaluating their potential role as predictors of renal function loss. We evaluated kidney function by estimated glomerular filtration rate (eGFR) in 32 ADPKD patients in different stages of kidney disease at T0 and after a 24-month follow up (T1). Patients were divided into two groups: Rapid disease progression ([RP], n 15) and Non-rapid disease progression ([NRP], n 17), according to the Mayo Clinic classification criteria. At T0, ADPKD patients underwent plasma sampling for quantitative analysis of h-miR-17-5p, h-miR-21-5p and h-miR-199a-5p microRNA expression, using the quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) method and a 3T magnetic resonance imaging (MRI), using an advanced MRI imaging protocol, for the quantification of total kidney volume (TKV), total perfusion volume (TPV) and total fibrotic volume (TFV). The expression of h-miR17-5p was higher (p < 0.05) in ADPKD patients with rapid disease progression. h-miR-17-5p, h-miR-21-5p and h-mir-199-5p showed a positive and significant correlation with the eGFR slope (mL/min/1.73 m2/year) (p < 0.05) but not with the eGFR at both T0 and T1. Both total fibrotic volume (cm3) and height-adjusted total fibrotic volume (cm3/m) were positively and significantly correlated to h-miR 21-5p and h-miR 199-5p (p < 0.05), but not to total kidney volume (cm3) and height-adjusted total kidney volume (cm3/m). The microRNAs we studied were associated with fibrosis and renal damage, suggesting their possible role as biomarkers able to identify ADPKD patients at high risk of disease progression regardless of the degree of kidney function, and therefore suitable for medical therapy, and may help uncovering new molecular mechanisms underlying cystogenesis.

Diagnostic Performance of Saliva Samples in Asymtomatic COVID-19 Infected Patients in Kakamega Kenya

The pandemic coronavirus SARS‐CoV‐2 in the world has caused a large infected population suffering from COVID‐19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed which is non-invasive. Use of self-collected saliva can minimize healthcare worker exposure and expand testing capabilities for symptomatic and asymptomatic patients. The main aim of this study was to document the ability of patients to self-collect sufficient saliva specimens for SARS-CoV-2 in the quantitative detection by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) in asymptomatic patients by themselves under observation by a healthcare provider. The researcher recorded whether the patients were confident and the suitability of the specimen for laboratory testing that would inform clinical decision making. Seventy-one patients aged from 13 years and above were included between December 2020 and July, 2021. Saliva samples and Nasopharyngeal samples were taken from each patient. Quantitative PCR was performed to detect SARS-CoV-2 viral load in the nasopharyngeal samples and qualitative Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) was used to detect the presence of the virus in saliva samples. Results of saliva vs. nasopharyngeal samples testing using the two different methods were compared. Statistical analyses were performed. Out of the 350 samples tested, 314 samples were found to be Covid 19 positive. Result of the test was validated by the RT-PCR test. This showed that only 314 samples were tested both by saliva rapid test and PCR test while the rest 36 samples were not tested using RT-PCR method but were tested using saliva test. Thus, the salivary test based on pure oral saliva samples easily obtained by noninvasive techniques using RT-LAMP has the same agreement with the nasopharyngeal technique using RT-PCR one in asymptomatic COVID-19 patients.

Detection of Tilapia lake virus (TiLV) using semi-nested RT-PCR method in farmed Tilapia (Oreochromis niloticus) from ponds East Java, Indonesia

Tilapia lake virus disease is an emerging viral disease causing mortality up to 90% worldwide of aquaculture production. It has been recorded as a virus attacting wild and cultured tilapias in Asia, Africa, and Latin America. There is the urgent need for rapid Diagnostic techniques for viral diseases to identify infected tilapia to control the outspread in individual farms. This study aims to identify the presence of TiLV with the semi-nested Reverse Transcriptase-Polymerase Chain Reaction method allowing the rapid detection of TiLV in fish organs. A total of 4 samples of tilapia were isolated from different locations and then used a silica extraction kit to obtain RNA from the TiLV virus. Afterwards amplification step was carried out by semi-nested RT-PCR test. The primers used in PCR target the 250-bp fragment in the electrophoresis. The results showed that the 250 bp amplicon of TiLV RNA was detected at 1/4 tested Tilapia. PCR clearly identified infective TiLV viral RNA in tilapia that produced a cytopathic effect with protein similarity (weak homology) in the conserved Polymerase Sub Unit PB1 Influenza-C and the genome of Orthomyxoviruses causing brain, nervous system and liver damage in fish and may have applications as rapid species-specific virulence test.

A study to assess the impact of the cobas point-of-care RT-PCR assay (SARS-CoV-2 and Influenza A/B) on patient clinical management in the emergency department of the University of California at Davis Medical Center.

Rapid detection of SARS-CoV-2 is crucial for reduction of transmission and clinical decision-making. Several rapid (<30min) molecular point-of-care (POC) tests based on nucleic acid amplification exist for diagnosis of SARS-CoV-2 & Influenza A/B infections. This unblinded, pre-post study enrolled consecutive patients with symptoms/signs consistent with SARS-CoV-2 infection presenting to the University of California, Davis emergency department (ED). Outcomes following implementation of the cobas® SARS-CoV-2 & Influenza A/B test for use on the cobas Liat System (intervention: December 2020-May 2021) were compared with previous standard-of-care using centralized laboratory reverse transcriptase polymerase chain reaction (RT-PCR) methods (control: April 2020-October 2020). Electronic health records of 8879 symptomatic patient visits were analyzed, comprising 4339 and 4540 visits and 538 and 638 positive SARS-CoV-2 PCR test results in the control and intervention periods, respectively. Compared with the control period, turnaround time (TAT) was shorter in the intervention period (median 0.98vs 12.30h; p<0.0001). ED length of stay (LOS) was generally longer in the intervention period compared with the control period, but for those SARS-CoV-2-negative who were admitted, ED LOS was shorter (median 12.53vs 17.93h; p<0.0001). The rate of antibiotic prescribing was lower in the intervention than in the control period (42.86% vs 49.16%; p<0.0001) and antiviral prescribing was higher (7.64% vs 5.49%; p<0.0001). This real-world study confirms faster TAT with a POC RT-PCR method in an emergency care setting and highlights the importance of rapid SARS-CoV-2 detection to aid patient management and inform treatment decisions.

Comparative and Prospective Study on the Efficacy of RT-PCR and Rapid Antigen Test in Symptomatic COVID-19 Patients at Tertiary Care Hospital

The COVID-19 pandemic has primarily been controlled by testing for SARS-CoV-2 infections. Despite vaccines, testing will remain crucial for surveillance and screening, allowing for the detection of new variants in a timely manner and to isolate the infected people to lower the danger of the disease spreading further. The research study attempts to found out the efficiency of Reverse-transcriptase polymerase chain reaction (RT-PCR) and Rapid antigen tests in symptomatic COVID-19 patients at tertiary care hospitals. The research was performed on 1000 patients, both In-patients and Out-patients, who presented with COVID-19 symptoms. SARS-COV-2 nucleocapsid protein antigen was detected qualitatively with rapid antigen test in human nasal specimens through the immuno-chromatographic assay. The rapid test results were compared with a molecular test RT-PCR in which FAM, HEX, and ROX were the indicator dyes for the RdRp gene, E gene, and the internal control (RNAse P), respectively. Nearly 322 cases were positive with both RT-PCR and rapid antigen test methods. Fifty-nine samples yielded negative results with the rapid antigen test and positive with PCR. Three samples were negative with RT-PCR and positive with the rapid antigen test. The findings from our study show that the common symptoms are fever 92.2% and cough 74.1% in the reported test population. But in confirmed cases of RT-PCR showed cough at 74.1% was more prevalent, followed by fever at 41.3%. Rapid antigen test showed a overall sensitivity and specificity of 85.3% and 99.5%. According to World Health Organization, rapid antigen detection tests meet the minimum performance requirements of ≥80% sensitivity and ≥97% specificity. Hence, the present study meets this criterion and may perhaps be a probable tool for point-of-care in hospital settings.

Concomitant BCR-ABL and JAK2 V617F in a Patient with Myeloproliferative Neoplasm: A Case Report

Myeloproliferative neoplasm (MPN) is a clonal proliferation of the haematopoietic stem cells leading to activated tyrosine kinase signaling activity. Myeloproliferative neoplasm is classified into BCR-ABL positive chronic myeloid leukemia (CML) and BCR-ABL negative MPN which harbors JAK2 V617F mutation in most cases. The genetic combination of BCR-ABL and JAK2 V617F mutation is rare with estimated frequency of 0.4% based on recent study. Herein, we reported a case of a man diagnosed with CML detected by fluorescence in-situ hybdridisation (FISH) showing atypical BCR-ABL fusion pattern in 29% nucleated cells (cut-off levels ≥5% for positive signals) in the presence of JAK2 V617F mutation. However, the BCR-ABL transcript was not detected by the conventional reverse transcriptase-polymerase chain reaction (RT-PCR) method which was specific for major fragments. Interestingly, complete hematological remission was not achieved despite initiation of tyrosine kinase inhibitor (Imatinib). In conclusion, it is imperative to scrutinise CML cases for concomitant JAK2 V617F mutation especially patients with atypical clinical or laboratory findings. Therefore, close monitoring with clinical and ancillary technique especially FISH and molecular methods such as DNA sequencing were crucial to help achieve complete hematological, cytogenetic and deep molecular response alongside targeted therapy.

Bilirubin, an endogenous antioxidant that affects p53 protein and its downstream apoptosis/autophagy-related genes in LS180 and SW480 cell culture models of colorectal cancer

BackgroundColorectal cancer (CRC) is one of the commonest neoplasms worldwide, which its pathogenesis is strongly correlated with p53 mutations. Antioxidants are believed to decelerate the CRC progression, possibly through interfering with p53 and its downstream target genes and mechanisms. Regarding the potential antioxidant effects of bilirubin, as an incredible endogenous antioxidant, we sought to investigate how bilirubin affected the expression levels of p53 protein and its downstream target genes, including Mdm2, Bcl-2, BECN1 and LC3, in LS180 and SW480 cell culture models of CRC. Methods and resultsUsing the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide) assay, 50 and 100 μM concentrations of bilirubin were determined to be non-toxic for both LS180 and SW480 cell lines. Western blot analysis was employed to evaluate the protein expression levels of p53. The results revealed that p53 protein levels were higher in LS180 cells treated with bilirubin compared to the control group. Notwithstanding, in SW480 cells, no considerable changes were observed in p53 protein levels of treated cells compared to the control ones. The quantitative reverse transcriptase-polymerase chain reaction (q RT-PCR) method was used to measure the mRNA expression levels of the apoptosis/autophagy-related genes, Mdm2, Bcl-2, BECN1, and LC3 , as the p53's downstream target genes. Consequently, the expression of Bcl-2 and Mdm2 genes were affected by p53, while BECN1 and LC3 expression levels were decreased in both cell lines. ConclusionBilirubin is an endogenous antioxidant with significant anti-tumor effects in the studied CRC cell lines, probably through the regulation of p53 protein expression levels and subsequent control of apoptosis and autophagy, as two key processes involved in cell survival and progression of tumor cells.

Tracing COVID-19 Trails in Wastewater: A Systematic Review of SARS-CoV-2 Surveillance with Viral Variants

The emergence of new variants of SARS-CoV-2 associated with varying infectivity, pathogenicity, diagnosis, and effectiveness against treatments challenged the overall management of the COVID-19 pandemic. Wastewater surveillance (WWS), i.e., monitoring COVID-19 infections in communities through detecting viruses in wastewater, was applied to track the emergence and spread of SARS-CoV-2 variants globally. However, there is a lack of comprehensive understanding of the use and effectiveness of WWS for new SARS-CoV-2 variants. Here we systematically reviewed published articles reporting monitoring of different SARS-CoV-2 variants in wastewater by following the PRISMA guidelines and provided the current state of the art of this study area. A total of 80 WWS studies were found that reported different monitoring variants of SARS-CoV-2 until November 2022. Most of these studies (66 out of the total 80, 82.5%) were conducted in Europe and North America, i.e., resource-rich countries. There was a high variation in WWS sampling strategy around the world, with composite sampling (50/66 total studies, 76%) as the primary method in resource-rich countries. In contrast, grab sampling was more common (8/14 total studies, 57%) in resource-limited countries. Among detection methods, the reverse transcriptase polymerase chain reaction (RT-PCR)-based sequencing method and quantitative RT-PCR method were commonly used for monitoring SARS-CoV-2 variants in wastewater. Among different variants, the B1.1.7 (Alpha) variant that appeared earlier in the pandemic was the most reported (48/80 total studies), followed by B.1.617.2 (Delta), B.1.351 (Beta), P.1 (Gamma), and others in wastewater. All variants reported in WWS studies followed the same pattern as the clinical reporting within the same timeline, demonstrating that WWS tracked all variants in a timely way when the variants emerged. Thus, wastewater monitoring may be utilized to identify the presence or absence of SARS-CoV-2 and follow the development and transmission of existing and emerging variants. Routine wastewater monitoring is a powerful infectious disease surveillance tool when implemented globally.

A 30-year-old Woman with Multiple-Sites Metastasis Lung Adenocarcinoma and Coronavirus-19 Disease (COVID-19)

Background: Adenocarcinoma is the most common Non-Small Cell Lung Cancer (NSCLC) in non-smokers in Indonesia. It often metastasizes in multiple sites. Metastasis is a negative predictor for prognosis in lung adenocarcinoma. In this covid pandemic era we present a case of a 30-years-old woman with adenocarcinoma and multiple-sites metastasis, and Coronavirus-19 Disease (COVID-19).&#x0D; Case: A 30-year-old woman came to our hospital with multiple complaints of hearing loss, visual loss, and occasional shortness of breath. The patient had a history of wild-type EGFR adenocarcinoma and has been treated with 4 cycles of gemcitabine. She also presented with a lump in her neck. Further examination revealed multiple metastasis tumours in her brain, right eye, neck, and spine. Pleural fluid examination also showed a malignant pleural effusion. Nasopharyngeal swab using reverse transcriptase-polymerase chain reaction (rt-PCR) method revealed that she also suffers from COVID-19 with positive swab result. Despite adequate treatment, she rapidly deteriorates and passed away in hospital.&#x0D; Discussion: Management in this patient is complex due to multiple complications and limitation of management given in COVID-19 isolation ward. She presented with multiple equally complicated metastasis, in which treatment priority should be carefully assessed. Despite treatment by multiple specialists, her problems still persist and proved to be fatal.&#x0D; Conclusion: Although metastasis is common in adenocarcinoma, multiple metastasis is uncommon, and this case highlights the need of careful management priority in such patients in COVID-19 pandemic setting.&#x0D; Keywords: Adenocarcinoma, Multiple Metastasis, Lung Cancer, COVID-19

Urinary Mitochondrial Deoxyribonucleic Acid: A Novel Biomarker of Coronavirus Disease 2019-associated Acute Kidney Injury in Renal Transplant Recipients

Abstract Background: Acute kidney injury (AKI) is one of the common manifestations of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. An early identification of AKI is of paramount importance to modulate the management of AKI, limiting the severity, avoiding nephrotoxic medicines, and modifying the drug dose depending on renal function. Aim: To determine the utility of urinary mitochondrial deoxyribonucleic acid (umt-DNA) and neutrophil gelatinase-associated lipocalin (NGAL) in predicting coronavirus disease 2019 (COVID-19)-associated AKI and mitochondrial stress. Materials and Methods: Live-related renal transplant recipients (RTRs) (n = 66), who acquired SARS-CoV-2 infection and were admitted to a COVID hospital were included and subclassified into AKI (n = 19) with &gt; 1.5-fold rise in serum creatinine level from the pre-COVID-19 serum creatinine level, and non-AKI (n = 47) whose serum creatinine value remained stable, or a rise of &lt;1.5-fold of the baseline values of pre-COVID-19. A 50 mL urine sample was collected and urinary mt-DNA and NGAL was determined by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays methods. Results: Both the urinary mitochondrial gene NADH dehydrogenase (ND-1) and NGAL level were significantly higher in the AKI group compared to non-AKI. The mean ND-1 gene Ct in the AKI group was (19.44 ± 2.58 a.u) compared to non-AKI (21.77 ± 3.60; P = 0.013). The normalized ND-1 gene cycle of threshold (Ct) in AKI was 0.79 ± 0.11 a.u compared to non-AKI (0.89 ± 0.14; P = 0.007). The median urinary NGAL level in AKI group was (453.53; range, 320.22–725.02, 95% confidence interval [CI]) ng/mL compared to non-AKI (212.78; range, 219.80–383.06, 95% CI; P = 0.015). The median urine creatinine normalized uNGAL was 4.78 (0.58–70.39) ng/mg in the AKI group compared to 11.26 ng/mg (0.41–329.71) in the non-AKI group. The area under curve of ND-1 gene Ct was 0.725, normalized ND-1 Ct was 0.713, uNGAL was 0.663, and normalized uNGAL was 0.667 for detecting the AKI and mitochondrial stress. Conclusions: Urinary mt-DNA quantification can detect the post-COVID mitochondrial distress with higher sensitivity than uNGAL in RTRs.

Plant Virome Analysis by the Deep Sequencing of Small RNAs of Fritillaria thunbergii var. chekiangensis and the Rapid Identification of Viruses.

Thunberg fritillary (Fritillaria thunbergii), a perennial used in traditional Chinese herbal medicine, is a members of the family Liliaceae. The degeneration of germplasm is a severe problem in the production of Fritillaria thunbergii var. chekiangensis. However, no information about viral infections of F. thunbergii var. chekiangensis has been reported. In this study, we sequenced the small RNAs of F. thunbergii var. chekiangensis from leaves and bulbs, and viruses were identified using a phylogenetic analysis and BLAST search for sequence. In addition, multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was used to rapidly detect viruses in this variety. Our study first reported that five viruses infected F. thunbergii var. chekiangensis. Among them, fritillary virus Y (FVY), lily mottle virus (LMoV), Thunberg fritillary mosaic virus (TFMV), and hop yellow virus (HYV) had been reported in F. thunbergii, while apple stem grooving virus was first reported in the genus Fritillaria. A multiplex RT-PCR method was developed to rapidly test the four viruses FVY, LMoV, TFMV, and HYV in F. thunbergii var. chekiangensis. Our results provide a better understanding of the infection of F. thunbergii var. chekiangensis by viruses and a basic reference for the better design of suitable control measures.

Introducing a simple and cost-effective RT-PCR protocol for detection of DPYD*2A polymorphism: the first study in Kurdish population.

Fluoropyrimidines, the major chemotherapeutic agents in various malignancies treatment, are metabolized by dihydropyrimidine dehydrogenase (DPD). DPD deficiency can lead to severe and sometimes fatal toxicity. In the present study, we developed a simple protocol to detect the DPYD*2A variant. Common side effects in patients treated with these drugs were also evaluated in a Kurdish population. We established a reverse-transcriptase polymerase chain reaction (RT-PCR) technique for detection of DPYD*2A. Sanger sequencing was used to confirm the results. 121 Kurdish patients receiving fluoropyrimidine derivatives were enrolled, and clinical information regarding the dosage and toxicity was analyzed. Our RT-PCR method was able to detect one patient with heterozygous state for DPYD*2A (0.8%). The most observed adverse drug reactions were tingling, nausea, and hair loss. The frequency of patients with the toxicity of grade 3 or worse was 6.6%. This was the first study that detect DPYD*2A polymorphism in the Kurdish population. Our method was successfully able to detect the DPYD*2A variant and, due to its simplicity and cost-effectiveness, it may be considered as an alternative to the current methods, especially in developing countries. Our detected polymorphism rate at 0.8% is comparable with other studies. Despite the low rate of DPYD*2A polymorphism, pharmacogenetics assessment before beginning the treatment process is highly recommended due to its association with a high risk of severe toxicity.

Investigation of deformed wing virus, black queen cell virus, and acute bee paralysis virus infections in honey bees using reverse transcriptase-polymerase chain reaction (RT-PCR) method

Viruses are one of the most potential risk factors that negatively affect the different life stages of honey bees. This study was conducted to determine the presence of infections caused by the deformed wing virus (DWV), black queen cell virus (BQCV), and acute bee paralysis virus (ABPV) in honey bees in the beekeeping regions of Burdur, along with obtaining information about their prevalence in this particular region. In our study, the adult bees were taken from 31 different beehives and comb samples that had different honey bee breeds and were sampled randomly from 15 beekeeping areas within the region. The collected samples were analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR), and the prevalence of DWV, BQCV, and ABPV infections were determined to be 74.19% (23/31), 25.81%, (8/31) and 74.19% (23/31), respectively. In this study, the distribution of positive samples and the rates of multiple infections were determined in the colonies. Of the positive honey bee samples, 12 (%38.71) were detected only for one virus, 9 (%29.03) were positive for two viruses (DWV-ABPV) and 8 (%25.81) were positive for all three viruses. In the present study, the presence of the three bee viruses that caused significant damage to the colonies by multiple infections in the Burdur region was determined with RT-PCR. To our knowledge, this is the first report of three mentioned bee infections in honey bees in the Burdur region. The revealed epidemiological conditions lead to the conclusion that serious measures are needed to control these infections in this region.

Detection of Bean Common Mosaic Virus in Bean Seeds by Immunocapture RT-PCR and DAS-ELISA Methods

This study was carried out in bean seeds in Isparta and Antalya provinces to determine Bean common mosaic virus (BCMV) in 2016. For this purpose; 106 seed samples were collected from the research area. Seed samples were used for biological, serological, and molecular methods. The virus incidence, as a percentage of seeds samples for BCMV, was found 87.73% by DAS-ELISA and 90.56% by Immunocapture Reverse Transcriptase-Polymerase Chain Reaction (IC-RT-PCR) methods. As a result of the study, BCMV was detected in a total of 96 (90.56%) seed samples. 93 samples were detected positive with the DAS-ELISA method, while 96 samples were found to be infected with BCMV using the IC-RT-PCR method. In the IC-RT-PCR method, approximately 850 bp of the coat protein gene was amplified with specific primers and BCMV specific bands were obtained at the expected levels.

Role of Zinc and Clinicopathological Factors for COVID-19-Associated Mucormycosis (CAM) in a Rural Hospital of Central India: A Case-Control Study.

IntroductionCoronavirus disease 2019 (COVID-19)has been a difficult enemy to beat for healthcare professionals around the world. However, even before the end of the COVID-19 pandemic, there has been an emergence of a new combatant in the form of opportunistic fungal infections with a high rate of morbidity and mortality, creating havoc throughout the globe.MethodsA case-control single-center study was conducted in Datta Meghe Institute of Medical Sciences, Wardha, Maharashtra. All the subjects who were included in the study were tested positive for COVID-19 through the reverse transcriptase-polymerase chain reaction (RT-PCR) method and the cases were defined as patients with biopsy-proven mucormycosis, whereas control were subjects who did not develop mucormycosis. The duration of the study was three months, from June 2021 to August 2021.ResultA total of 55 cases and 50 controls were enrolled in the study. The use of zinc was found to be significantly associated with COVID-19-associated mucormycosis, with 89.1% of the cases having a history of zinc intake and only 52% of controls having a history of zinc intake( p-value <0.001). Diabetes mellitus was found to be significantly associated with COVID-19-associated mucormycosis with 83.6% of the cases and 16% of the controls having diabetes mellitus (p-value <0.001). Although the use of steroids in cases was more with 98.2% of the cases and 54% of the control receiving steroids; this difference was not significant statistically (p-value of 1.00).ConclusionWe conclude that apart from diabetes mellitus and other immunosuppressive states, zinc might be the hidden culprit behind the sudden surge of COVID-19-associated mucormycosis worldwide owing to the self-administration of zinc by the patients to acquire innate immunity and over-prescription of multivitamins by the treating clinicians. However, this association required further studies in order to be proved.

Abstract P2-08-22: Correlation of ER (ESR1), PR (PGR), and HER2 (ERBB2) at protein and mRNA levels in operable breast cancer

Abstract Background:. We aimed to analyze the concordance between ER (ESR1), PR (PGR), and HER2 (ERBB2) levels and breast cancer subtyping results obtained by using the immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR) methods and to assess recurrence-free survival (RFS) between subtypes as determined by the two methods. Patients and Methods:. We included a total of 441 operable breast cancer patients, consisting of the training (n = 116), testing (n = 56) and validation (n = 269) cohorts in this study. Protein levels of estrogen receptor (ER) and progesterone receptor (PR) were obtained by using the IHC and that of human epidermal growth factor receptor 2 (HER2) by the IHC/FISH methods. Expression (mRNA) levels of the corresponding genes, ESR1, PGR, and ERBB2, respectively, were determined by the RT-PCR method. We then compared the status of ER (ESR1), PR (PGR), and HER2 (ERBB2) in the training, testing and validation cohorts. We categorized all validation cases into luminal (ER/PR-positive and HER2-negative), HER2 (HER2-positive regardless of ER/PR status), and triple negative (TN: ER-, PR-, and HER2-negative) subtypes accordingly. The concordance of the two methods and RFS estimated by the Kaplan-Meier methods of the three different subtypes were revealed to evaluate the clinical performance. Results:. The concordance rates between the two methods in the validation cohort were 95.1% (Kappa = 0.74) for ER (ESR1), 91.1% (Kappa = 0.67) for PR (PGR), and 93.6% (Kappa = 0.59) for HER2 (ERBB2). Kappa statistic was 0.66 for the resulting luminal, HER2 and TN subtypes. The 5-year RFS was 83.3% for luminal vs. 73.9% for HER2 vs. 48.4% for TN subtypes as determined by the IHC (p = 0.019), and 84.3% vs. 66.3% vs. 54.4% by the RT-PCR method (p = 0.016). Conclusions:. Whereas discordance existed for some cases between the IHC and RT-PCR methods for determination of the ER (ESR1), PR (PGR), and HER2 (ERBB2) status and the resulting subtypes; both the methods showed values in RFS prognosis of the population studied. It is arguable that supplementing the conventional IHC with the molecular method for subtyping of breast cancer may have beneficial clinical implications. Citation Format: Lei Lei, Xiaojia Wang, Skye Hung-Chun Cheng. Correlation of ER (ESR1), PR (PGR), and HER2 (ERBB2) at protein and mRNA levels in operable breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-22.

Can the 21-gene recurrence score (RS) assay spare some patients from unnecessary adjuvant chemotherapy in hormone–receptor-positive, HER2 negative early breast cancer with low burden axillary disease

Background: Breast cancer is regarded as the most common malignancy in females worldwide,it is representing 25% of all cancer cases diagnosed in women.The Oncotype DX recurrence score (RS) assay analyzes gene expression using a reverse transcriptase-polymerasechain reaction method,the tested panel includes 21 genes(16cancer-related genes and 5 reference genes).Its introduction resulted in significant changes in the adjuvant treatment decisions.It provides prognostic information for distant disease recurrence in with low burden axillary disease,ER +ve,HER2-ve early breast cancer that is independent of clinico-pathologic characteristics.

Development, Optimization, and In Vitro Evaluation of Novel Oral Long-Acting Resveratrol Nanocomposite In-Situ Gelling Film in the Treatment of Colorectal Cancer.

This study aimed to develop and evaluate sustained-release (SR) long-acting oral nanocomposites in-situ gelling films of resveratrol (Rv) to treat colorectal cancer. In these formulations, Rv-Soy protein (Rv-Sp) wet granules were prepared by the kneading method and then encapsulated in the sodium alginate (NA) dry films. The prepared nanocomposite in-situ gels films were characterized using dynamic light scattering, Fourier-transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy. The optimized formulations were further evaluated based on drug encapsulation efficiency, pH-drug release profile, swelling study, and storage time effects. The optimized formulation was tested for its anticancer activity against colorectal cancer cells using the cytotoxicity assessment, apoptosis testing, cell cycle analysis, gene expression analysis, and protein estimation by the reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay methods, respectively. The optimum film showed encapsulation efficiency of 97.87% ± 0.51 and drug release of 14.45% ± 0.043 after 8 h. All physiochemical characterizations confirmed, reasoned, and supported the drug release experiment’s findings and the encapsulation assay. The Rv nanocomposite formulation showed concentration-dependent cytotoxicity enhanced apoptotic activity as compared to free Rv (p < 0.05). In addition, Rv nanocomposite formulation caused a significant increase in Bcl-2-associated protein X (Bax) and a decrease in expression of B-cell lymphoma 2, interleukin 1 beta, IL-6, and tumor necrosis factor-alpha (Bcl2, IL-1β, IL-6, and TNF-α respectively) compared to that of free Rv in HCT-116 cells. These results suggest that long-acting Rv nanocomposite gels could be a promising agent for colorectal cancer treatment.

Serum levels of inflammatory biomarkers, glycaemic control indices and leptin receptors expression in adult male Wistar rats exposed to Pyrethroids

BACKGROUND: Due to widespread use of pesticides, an individual’s lifetime exposure to pesticide mixtures is unavoidable, resulting in potentiation of toxic effects such as changing metabolic signaling temporarily or permanently due to the formation of free radicals among exposed individuals. OBJECTIVE: This study then looked at the expression of leptin and insulin receptors in Wistar rats exposed to pyrethroid and its effects on adipokines and glycemic indices. METHODS: 84 Wistar rats were randomly selected and divided into two groups. 51 rats were exposed to 1.2 % w/v pyrethroid insecticides, while the remaining 33 rats were grouped as non-exposed. The groups were divided into three different groups, each with 7, 21, and 41 days. The animals were anesthetically sacrificed and samples of blood were collected after days of exposure. The levels of total antioxidant status (TAS), malondialdehyde (MDA), glutathione (GSH), hydrogen peroxide (H2O2), nitric oxide (NO), glucose and lipids were determined. Blood activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) were also determined. ELISA was used to assess leptin, adiponectin, insulin, CRP, IL-6, and TNFα. Glycaemic indices were calculated appropriately. Leptin and Insulin expression was determined using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Data obtained were statistically compared, and P≤0.05 was considered significant. RESULTS: The mean TChol, TAG, and HDL-C were significantly different (p &lt; 0.05) among the exposed groups. The serum mean levels of SOD, GPx, CAT, GSH, TAS, QUICKI, TNFα, IL6, and CRP were significantly reduced (p &lt; 0.05), while mean levels of MDA, H2O2, NO, insulin, HOMA-IR, FIRI, leptin, and adiponectin were significantly elevated (p &lt; 0.05) in exposed groups. The relative expression of insulin and leptin genes in exposed and non-exposed groups was also shown by column. CONCLUSION: It is self-evident that pyrethroid chemicals exposure may disrupt carbohydrate and lipid metabolism by raising the risk of obesity among the exposed groups.

Abstract 468: Feasibility of digital PCR for the detection of ALK rearrangement in the plasma of non-small cell lung cancer patients

Abstract Introduction: Currently, the three primary methods of detecting ALK rearrangement are fluorescent in situ hybridization, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR). The former two methods use tissue as the laboratory materials, but some patients do not have sufficient tissue to perform the tests. The RT-PCR method can be conducted by both tissue and blood, but its detection of ALK rearrangement seems not sensitive enough. Digital PCR (dPCR) is a biotechnology refinement of conventional PCR methods that may be a more precise method than RT-PCR. Materials and Methods: An EML4-ALK variant 1 (v1) synthetic sequence, a variant 2 (v2) synthetic sequence, and a variant 3 (v3) non-small cell lung cancer (NSCLC) cell line (NCI-H2228) were used to evaluate the performance of our dPCR assay. To examine the performance PCR on clinical samples, we used both dPCR and RT-PCR assays to quantify the expression levels of EML4-ALK variants (v1, v2, and v3) in the circulating tumor RNAs derived from 414 plasma samples of 30 NSCLC patients during their ALK inhibitor treatment. Results: We have designed a multiplex dPCR assay for the detection of EML4-ALK in a v1 synthetic sequence (with 8,000 copies), a v2 synthetic sequence (with 10,000 copies), and a v3 cell line (with 100 copies). Our results showed high specificities and precisions (8,757 ± 592 copies, 11,070 ± 7,791 copies, and 93 ± 14 copies, respectively) for detecting all three variants simultaneously. On the other hand, our results observed good linearities (R2 = 0.9996, 0.9985, and 0.9991, respectively) in all three variants. To determine our dPCR assay's limit of detection for v1 and v2 synthetic sequences (each with four copies), our assay successfully measured the copy numbers of both v1 and v2 (4.1 ± 1.6 and 3.9 ± 1.8 copies, respectively). Comparing the sensitivities of our dPCR assay with RT-PCR assay, dPCR was able to detect down to 2 copies (for v1 and v2) and 20 pg of cDNA (for v3) while RT-PCR could only detect 200 copies (for v1 and v2) and 200 pg of cDNA (for v3). After confirming the promising performance of our dPCR assay, we tried to detect EML4-ALK variants by both dPCR and RT-PCR assays in the plasma samples of our recruited patients but mostly negative. Only one sample detected 39.2 copies of EML4-ALK v3 using dPCR assay. Conclusion: dPCR assay is a precise method with good performance at detecting EML4-ALK variants in synthetic sequences and cell line, which is more sensitive than RT-PCR. However, EML4-ALK variants could not be detected in the majority of our plasma samples. This may be due to insufficient circulating RNAs for detection. In addition, RNA may be degraded by RNase in plasma and that platelet may be a better alternative than plasma for the detection of RNAs in patient samples. Additional testing is warranted to detect EML4-ALK variants in the platelet samples of another NSCLC patient cohort. Citation Format: William C. Cho, Alvin H. Fong, King Y. Fung, Jeannie Y. Chik, Jacky Y. Li. Feasibility of digital PCR for the detection of ALK rearrangement in the plasma of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 468.