Comparison of High-resolution Melting Curve Analysis with Specific Target Gene Sequencing to Identify the Most Common Species of Aspergillus and Fusarium

Background: Currently, it appears that new molecular-based methods could substitute microscopic and culture assessment for the first-line detection of microorganisms isolated from clinical specimens. However, it will remain the "continual strategy" until this technology is attuned to identifying all fungi that can be isolated from biological specimens. Objectives: The present study aimed to validate a high-resolution melting (HRM) technique to identify clinical filamentous fungi. Moreover, it was attempted to compare the results with those of the target gene’s polymerase chain reaction (PCR) sequencing. Methods: A total of 54 specimens of bronchoalveolar lavage (BAL), nail, ear discharge, blood culture, and cornea were collected from patients suspected of fungal infection. All Fusarium spp. and Aspergillus spp. were recognized based on Tef-α and beta-tubulin region sequencing, as well as PCR-HRM analysis. Results: The Tef-α sequence analysis revealed the most frequent spp. to be Fusarium solani followed by F. oxysporum (n = 3), F. caucasicum (n = 3), F. coeruleum (n = 3), F. falciforme (n = 1), F. proliferatum (n = 1), F. brevicatenulatum (n = 1), F. globosom (n = 1), and F. verticillioides (n = 1). Based on the beta-tubulin sequences, Aspergillus flavus (n = 10), A. fumigatus (n = 7), A. niger (n = 2), A. terreus (n = 1), and A. orezea (n = 1) were identified in this study. Furthermore, the dataset analysis of PCR-HRM revealed that 33 isolates belonging to Fusarium spp. were F. solani (n = 24), F. oxysporum (n = 3), F. proliferatum (n = 3), F. falciforme (n = 1), F. verticillioides (n = 1), and F. brevicatenulatum (n = 1). Moreover, isolates (n = 21) belonging to Aspergillus spp. included A. flavus (n = 11), A. fumigatus (n = 7), A. niger (n = 2), and A. terreus (n = 1). Conclusions: The sequencing method has a time-consuming and costly nature, and there exists conformity between the sequence results of the Tef-α/beta-tubulin regions and PCR-HRM. The PCR-HRM method is a reliable approach in the clinical laboratory to identify Aspergillus and Fusarium spp. However, some closely related spp. show no curve algorithm differences in PCR-HRM.

Exosomal miRNA-328-3p Targets ZO-3 and Inhibits Porcine Epidemic Diarrhea Virus Proliferation

As essential transfer carriers for cell-to-cell communication and genetic material, exosomes carry microRNAs that participate in the regulation of various biological processes. MicroRNA is a type of single-stranded noncoding RNA that can bind to specific target gene mRNA to degrade or inhibit its translation, thereby regulating the expression of related target genes. At present, it is known that a variety of microRNAs are involved in the viral infection process, but there are few reports on research into the specific microRNAs involved in PEDV infection. In this study, we isolated and identified exosomes in PEDV-infected Vero E6 cells. Using transcriptomics technology, we found that miRNA-328-3p was significantly downregulated in exosomes following PEDV infection. Moreover, exosomal miRNA-328-3p inhibits cell infection by PEDV through targeting and inhibition of tight junction protein-3 (TJP-3/ZO-3) in recipient cells. Our findings provide evidence that after infecting cells, PEDV downregulates the expression of miRNA-328-3p, and the resulting reduced inhibition of the target ZO-3 protein helps to enhance PEDV infection. These results provide new insights for understanding the regulatory mechanism of PEDV infection.

Inflammatory Pathways and In Vivo Studies of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a physically incapacitating disorder that significantly disturbs patients' quality of life. IBD is classified into two main pathophysiological forms, ulcerative colitis and Crohn's disease. Literature studies indicate that chronic colitis may contribute to the development of up to 25% of all diagnosed colorectal tumors. The complexity and development of IBD onset is intermediated by a variety of inflammatory mediators and pathways that are intrinsically linked and are summarized in this chapter. This complexity resulted in the development of various in vivo models to surpass the enormous challenges in the finding of new drugs for IBD treatment. These models are mostly based on rodents and on three types of inflammatory activation: chemical induction, transfer of naïve CD4+ T cells, and generation of engineered mouse strains, with specific target gene manipulations. The most broadly described models in literature are here presented and discussed.

Isolation, identification and molecular detection of selected probiotic bacteria from broiler chickens and their related environment

This study was aimed to isolate and identify the potential probiotic bacteria from broiler chickens and their related environment of the two districts in Bangladesh by using cultural and biochemical techniques and detection of the selected isolated probiotic potential bacterial species by using polymerase chain reaction (PCR). In this context five probiotic bacteria species of five bacteria genera were screened out from 750 samples in where there were 186 Lactobacillus genus isolates, 102 Bacillus genus isolates, 112 Streptococcus genus isolates, 220 Enterococcus genus isolates and 33 Bifidobacteriumgenus isolates on the basis of biochemical properties like 10% solution of total 22 different sugars. Other biochemical tests like MR-VP, Oxidase, Catalase, Citrate utilization, MIU and Indole test were major. Genus was detected through specific genus target gene and 16S r DNA based PCR. Selected probiotic bacteria species were detected through species specific target gene PCR. We confirmed 23 Lactobacillus acidophilus species isolates, 14 Bacillus subtilisisolates, 19 Streptococcus thermophilusisolates, 31 Enterococcus faeciumisolates and 09 Bifidobacteriumbifidumisolates at species level with ddlgene, apr-E gene, 16S-23S ITS gene, BIBI gene, Stp-TH gene primers. The novelty of this study is at first time in Bangladesh, we isolated and identified five selected potential probiotic species from the gastro-intestinal tract of broiler chickens and their related environmental sources by not only using traditional cultural, biochemical techniques but also genus and species specific target gene based PCR. Asian J. Med. Biol. Res. September 2020, 6(3): 383-399

miR-766-3p suppressed tumorigenesis, epithelial-mesenchymal transition, and metastasis by targeting BCL9L via β -catenin signaling pathway in osteosarcoma cells

Background Emerging evidence has indicated that abnormal microRNAs (miRNAs) play critical roles in carcinogenesis and progression of osteosarcoma (OS). The aim of this study was to clarify the relationship between miR-766-3p expression and osteosarcoma development and to explore its potential mechanism. Methods miR-766-3p was the most downregulated miRNA by analyzing GSE65071 from the GEO database. RT-PCR and western blot was performed to determine miR-766-3p expression and its specific target gene in human OS samples and cell lines. CCK-8 proliferation, colony formation, EdU, wound-healing, and transwell assays were used respectively to evaluate the influences of miR-766-3p depletion or ectopic expression on OS proliferation, migration and invasion in vitro. And a mouse tumorigenicity model was conducted to investigate effects of miR-766-3p in vivo. Moreover, we identified directly interactions between miR-766-3p and its specific target gene using luciferase reporter assays. Results miR-766-3p expression was overexpressed in OS tissues and cell lines, and ectopic miR-766-3p expression repressed the malignant level of OS, including cell proliferation, migration, invasion and epithelial to mesenchymal transition (EMT) in vitro and in vivo. B-Cell Lymphoma 9-Like Protein (BCL9L) was negatively correlated with miR-766-3p expression in human OS tissue, and was validated as a downstream target of miR-766-3p by the luciferase reporter assay and Western blotting. Rescue experiment indicated that BCL9L could restore the effects of miR-766-3p on OS migration and invasion. The β-Catenin signaling pathway was demonstrated as being implicated in the miR-766-3p/BCL9L axis. Conclusions In conclusion, miR-766-3p is a negative regulator of BCL9L and a risk factor for tumor metastasis in OS progression.

Detection of SARS-CoV-2 from raw patient samples by coupled high temperature reverse transcription and amplification

The SARS-CoV-2 beta coronavirus is spreading globally with unprecedented consequences for modern societies. The early detection of infected individuals is a pre-requisite for all strategies aiming to contain the virus. Currently, purification of RNA from patient samples followed by RT-PCR is the gold standard to assess the presence of this single-strand RNA virus. However, these procedures are time consuming, require continuous supply of specialized reagents, and are prohibitively expensive in resource-poor settings. Here, we report an improved nucleic-acid-based approach to detect SARS-CoV-2, which alleviates the need to purify RNA, reduces handling steps, minimizes costs, and allows evaluation by non-specialized equipment. The use of unprocessed swap samples and the ability to detect as little as three viral genome equivalents is enabled by employing a heat-stable RNA- and DNA-dependent DNA polymerase, which performs the double task of stringent reverse transcription of RNA at elevated temperatures as well as PCR amplification of a SARS-CoV-2 specific target gene. As results are obtained within 2 hours and can be read-out by a hand-held LED-screen, this novel protocol will be of particular importance for large-scale virus surveillance in economically constrained settings.

Investigation of the Causes of Shigatoxigenic Escherichia coli PCR Positive and Culture Negative Samples

Molecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon.

Detection Method for Salmonella Typhimurium and Salmonella Enteritidis using Real-Time Polymerase Chain Reaction

Salmonella is a pathogenic bacterium that can cause serious harm to humans. Chicken carcasses have been reported contaminated by Salmonella, especially S. Typhimurium and S. Enteritidis. These two serovars are very difficult to be confirmed and distinguished using biochemical analysis, therefore a rapid method for detection and differentiation of both is required. The objective of this study was to evaluate designed primer for detection and differentiation of S. Typhimurium and S. Enteritidis on chicken carcasses using real time Polymerase Chain Reaction (rt-PCR). Detection of Salmonella spp. was conducted using primer sequence from invA gene. Differentiation of both Salmonella serovars was conducted using specific target gene from S. Typhimurium (STM) and specific virulence plasmid of S. Enteritidis (Prot6E). The result showed that invA primer effective to detect all species Salmonella tested and has good specificity that could not detect Escherichia coli and Shigella dysenteriae in the similar melting temperature. Two specific primers STM and prot6E have distinguished between S. Typhimurium and S. Enteritidis. Sensitivity of method showed very good with 0.5 μM primer concentration of invA, STM and prot6E that were 0.2 pg, 22 pg and 28 pg respectively. Initial trial showed that this method can be applied for detection of Salmonella spp. and two serovars in chicken carcasses.

miR-624-5p promoted tumorigenesis and metastasis by suppressing hippo signaling through targeting PTPRB in osteosarcoma cells

Background Accumulating evidence indicates that aberrant microRNA (miRNA) expression contributes to osteosarcoma progression. This study aimed to elucidate the association between miR-624-5p expression and osteosarcoma (OS) development and to investigate its underlying mechanism. Methods We analyzed GSE65071 from the GEO database and found miR-624-5p was the most upregulated miRNA. The expression of miR-624-5p and its specific target gene were determined in human OS specimens and cell lines by RT-PCR and western blot. The effects of miR-624-5p depletion or ectopic expression on OS proliferation, migration and invasion were evaluated in vitro using CCK-8 proliferation assay, colony formation assay, transwell assay, would-healing assay and 3D spheroid BME cell invasion assay respectively. We investigated in vivo effects of miR-624-5p using a mouse tumorigenicity model. Besides, luciferase reporter assays were employed to identify interactions between miR-624-5p and its specific target gene. Results miR-624-5p expression was upregulated in OS cells and tissues, and overexpressing miR-624-5p led to a higher malignant level of OS, including cell proliferation, migration and invasion in vitro and in vivo. Protein tyrosine phosphatase receptor type B (PTPRB) was negatively correlated with miR-624-5p expression in OS tissues. Using the luciferase reporter assay and Western blotting, PTPRB was confirmed as a downstream target of miR-624-5p. PTPRB restored the effects of miR-624-5p on OS migration and invasion. The Hippo signaling pathway was identified as being involved in the miR-624-5p/PTPRB axis. Conclusions In conclusion, our results suggest that miR-624-5p is a negative regulator of PTPRB and a risk factor for tumor metastasis in OS progression.

KARAKTERISASI MICRORNA TRIPLE HELIX MELALUI METODE HYDROGEL SCAFFOLD SPESIFIK TERHADAP MICRORNA-26A DAN HSA-MICRORNA-15A: MODALITAS KURATIF MUTAKHIR PADA PENYAKIT KATARAK

Cataract is an eye disease that is marked by it decrease in lens transparency. Cataract is a major cause of blindness. Data The World Health Organization (WHO) in 2010 showed that there were 285 million people have visual impairments worldwide, 39 million of them blindness where 33% of them are caused by cataracts. Governance cataracts are currently limited to surgical efforts and administration of eye drops as antioxidant. But both have flaws because of the risk of complications limited ocular and lens penetrating ability. Therefore, the author reviewed the potential for triple helix microRNA characterization through specific Hydrogel Scaffold methods against microRNA-26a and hsa-microRNA-15a as the latest curative modalities on cataract disease. microRNA is a non coding RNA molecule consisting of 22 nucleotide. microRNA is known to have a specific target gene so it will increase the effectiveness of therapy in cataracts. The method used in this writing is a study of literature. The author reviews various sources, the majority come from a global journal. The author enters several keywords such as cataract, microRNA, triplex nanoparticle, and Hydrogel Scaffold. The website is used including nature, sciencedirect, pubmed, and ebsco. After extraction based on the inclusion and exclusion criteria that have been determined, the journal is then analyzed and synthesized whose results are written in this review literature. based on literature obtained, microRNA-26a has decreased expression in cataract cases. So that when microRNA-26a is transfected, there will be an increase in regulation which can increase the positive effect on inhibiting the process of fibrosis oncataract pathomechanism mediated by Notch / Jagged-1. Meanwhile, microRNA- 15a overexpresses, so that when transfected antagomir microRNA-15a, then this microRNA will decrease and give negative feedback so the expression of bcl-2 and mcl-1 as antiapoptotic and antioxidant proteins will be increased. To increase its stability, both microRNA-26a and antagomir microRNA-15a will be formed into a triple helix RNA structure through addition dendrimer with the Hydogel Scaffold method. This allows the microRNA become more stable in circulation and can be released in specific genes. By therefore, through the triple helix RNA characterization specific to microRNA-15a and microRNA-26a, cataract therapy is expected to be more effective and efficient. Keywords : cataracts, triple helix microRNA, microRNA-15a, microRNA-2