Molecular Targets For Diagnosis Research Articles

Predominantly Orphan Secretome in the Lung Pathogen Mycobacterium abscessus Revealed by a Multipronged Growth-Phase-Driven Strategy.

The emerging lung pathogen Mycobacterium abscessus is understudied for its virulence determinants and molecular targets for diagnosis and therapeutics. Here, we report a comprehensive secretome (600 proteins) of this species, which was identified using a multipronged strategy based on genetic/genomic, proteomic, and bioinformatic approaches. In-solution digested bottom-up proteomics from various growth phases identified a total of 517 proteins, while 2D-GE proteomics identified 33 proteins. A reporter-gene-fusion-based genomic library that was custom-generated in this study enabled the detection of 23 secretory proteins. A genome-wide survey for N-terminal signal sequences using bioinformatic tools (Psortb 2.0 and SignalP 3.0) combined with a strategy of the subtraction of lipoproteins and proteins containing multiple transmembrane domains yielded 116 secretory proteins. A homology search against the M. tuberculosis database identified nine additional secretory protein homologs that lacked a secretory signal sequence. Considering the little overlap (80 proteins) among the different approaches used, this study emphasized the importance of using a multipronged strategy for a comprehensive understanding of the secretome. Notably, the majority of the secreted proteins identified (over 50%) turned out to be "orphans" (those with no known functional homologs). The revelation of these species-specific orphan proteins offers a hitherto unexplored repertoire of potential targets for diagnostic, therapeutic, and vaccine research in this emerging lung pathogen.

RBMX overexpression inhibits proliferation, migration, invasion and glycolysis of human bladder cancer cells by downregulating PKM2

To investigate the role of RNA-binding motif protein X-linked (RBMX) in regulating the proliferation, migration, invasion and glycolysis in human bladder cancer cells. A lentivirus vectors system and RNA interference technique were used to construct bladder cancer 1376 and UC-3 cell models with RBMX overexpression and knockdown, respectively, and successful cell modeling was verified using RT-qPCR and Western blotting. Proliferation and colony forming ability of the cells were evaluated using EdU assay and colony-forming assay, and cell migration and invasion abilities were determined using Transwell experiment. The expressions of glycolysis-related proteins M1 pyruvate kinase (PKM1) and M2 pyruvate kinase (PKM2) were detected using Western blotting. The effects of RBMX overexpression and knockdown on glycolysis in the bladder cancer cells were assessed using glucose and lactic acid detection kits. RT-qPCR and Western blotting confirmed successful construction of 1376 and UC-3 cell models with RBMX overexpression and knockdown. RBMX overexpression significantly inhibited the proliferation, clone formation, migration and invasion of bladder cancer cells, while RBMX knockdown produced the opposite effects. Western blotting results showed that RBMX overexpression increased the expression of PKM1 and decreased the expression of PKM2, while RBMX knockdown produced the opposite effects. Glucose consumption and lactate production levels were significantly lowered in the cells with RBMX overexpression (P < 0.05) but increased significantly following RBMX knockdown (P < 0.05). RBMX overexpression inhibits bladder cancer progression and lowers glycolysis level in bladder cancer cells by downregulating PKM2 expression, suggesting the potential of RBMX as a molecular target for diagnosis and treatment of bladder cancer.

Extracellular vesicles in the study of Alzheimer's and Parkinson's diseases: Methodologies applied from cells to biofluids.

Extracellular vesicles (EVs) are gaining increased importance in fundamental research as key players in disease pathogenic mechanisms, but also in translational and clinical research due to their value in biomarker discovery, either for diagnostics and/or therapeutics. In the first research scenario, the study of EVs isolated from neuronal models mimicking neurodegenerative diseases can open new avenues to better understand the pathological mechanisms underlying these conditions or to identify novel molecular targets for diagnosis and/or therapeutics. In the second research scenario, the easy availability of EVs in body fluids and the specificity of their cargo, which can reflect the cell of origin or disease profiles, turn these into attractive diagnostic tools. EVs with exosome-like characteristics, circulating in the bloodstream and other peripheral biofluids, constitute a non-invasive and rapid alternative to study several conditions, including brain-related disorders. In both cases, several EVs isolation methods are already available, but each neuronal model or biofluid presents its own challenges. Herein, a literature overview on EVs isolation methodologies from distinct neuronal models (cellular culture and brain tissue) and body fluids (serum, plasma, cerebrospinal fluid, urine and saliva) was carried out. Focus was given to approaches employed in the context of Alzheimer's and Parkinson's diseases, and the main research findings discussed. The topics here revised will facilitate the choice of EVs isolation methodologies and potentially prompt new discoveries in EVs research and in the neurodegenerative diseases field.

Distinct miRNAs associated with various clinical presentations of SARS-CoV-2 infection

SummaryMicroRNAs (miRNAs) have been shown to play important roles in viral infections, but their associations with SARS-CoV-2 infection remain poorly understood. Here, we detected 85 differentially expressed miRNAs (DE-miRNAs) from 2,336 known and 361 novel miRNAs that were identified in 233 plasma samples from 61 healthy controls and 116 patients with COVID-19 using the high-throughput sequencing and computational analysis. These DE-miRNAs were associated with SASR-CoV-2 infection, disease severity, and viral persistence in the patients with COVID-19, respectively. Gene ontology and KEGG pathway analyses of the DE-miRNAs revealed their connections to viral infections, immune responses, and lung diseases. Finally, we established a machine learning model using the DE-miRNAs between various groups for classification of COVID-19 cases with different clinical presentations. Our findings may help understand the contribution of miRNAs to the pathogenesis of COVID-19 and identify potential biomarkers and molecular targets for diagnosis and treatment of SARS-CoV-2 infection.

Potential biomarkers that discriminate rheumatoid arthritis and osteoarthritis based on the analysis and validation of datasets

BackgroundRheumatoid arthritis (RA) and osteoarthritis (OA) share some similar arthritic symptoms, but different mechanisms underlie the pathogenesis of these two diseases. Analysis of differentially expressed molecules in rheumatoid arthritis and osteoarthritis may assist in improving diagnosis and treatment strategies in clinical practice.MethodsMicroarray and RNA-seq data were acquired from the gene expression omnibus database. Differentially expressed genes (DEGs) were identified using Bioconductor packages. Receiver operating characteristic curves were plotted to assess performance. Gene ontology enrichment analysis was conducted using the clusterProfiler application. During validation, synovial fluid was harvested from patients who had undergone in-hospital joint replacement, in which the expression of proteins was measured using enzyme-linked immunosorbent assays.ResultsCompared with OA samples, RA samples showed 14 genes to be upregulated and 3 to be downregulated. Gene ontology analysis indicated that DEGs principally included molecules responsible for the regulation of a synovial tissue inflammatory response. Seven genes displayed a good discriminatory power with an AUC higher than 0.90. ADAMDEC1 was the biomarker that most clearly discriminated RA from OA in the database, exhibiting an AUC of 0.999, a sensitivity of 100%, and a specificity of 97.8%. Following validation, the expression levels of ADAMDEC1 in the synovial fluid from RA patients were significantly higher than those in the synovial fluid from OA patients (P < 0.05). At the cut-off value of 1957 pg/mL, ADAMDEC1 expression in the synovial fluid discriminated RA from OA with an AUC of 0.951, a specificity of 88.6%, and a sensitivity of 92.9%.ConclusionThe differential expression of genes in RA compared with OA indicates potential targets for molecular diagnosis and treatment. The presence of ADAMDEC1 in synovial fluid is a good biomarker of RA.

Amplification of cestode DNA from the peri-anal region of naturally infected foxes by PCR and LAMP: proof of concept for a potential sampling strategy for diagnosing human taeniosis

The diagnosis of human taeniosis can be achieved through coproscopy, ELISA or PCR. An important limitation of these methods is the high turnaround time for stool sample collection and preparation, indicating the need for a straightforward sampling strategy. Due to the high metabolic activity and reproductive potential of Taenia spp., we hypothesise that parasite DNA (cells and eggs) present in the peri-anal region of the host can be exploited as a target for molecular diagnosis. We evaluated the feasibility of recovering parasite DNA from the peri-anal area of foxes naturally infected with Taenia spp. Before necropsy, cotton swabs were rubbed at the peri-anal region of foxes. DNA was extracted using alkaline lysis coupled with a commercial DNA isolation kit (method A) or alkaline lysis alone (method B). DNA was used in the multiplex-PCR assay (previously described and called here swab-PCR) and a novel LAMP assay detecting Taenia spp. commonly found in foxes (swab-LAMP). The results of these assays from 105 foxes were compared with the presence of intestinal helminths determined at necropsy and by the sedimentation and counting technique (SCT). The sensitivity of swab-PCR for detecting Taenia (n = 68) was 89.8% (95% CI, 77.7–96.6) and 89.5% (66.9–98.7) using methods A and B, respectively. The sensitivity of the swab-LAMP assay was 83.7% (70.3–92.7) using method A and 89.5% (66.9–98.7) with method B. We postulate that peri-anal swab sampling followed by simplified DNA extraction and LAMP might be a suitable strategy for surveillance of human taeniosis in resource-limited settings in the future.

The genome of the thin-necked bladder worm Taenia hydatigena reveals evolutionary strategies for helminth survival

Taenia hydatigena is a widespread gastrointestinal helminth that causes significant health problems in livestock industry. This parasite can survive in a remarkably wide range of intermediate hosts and affects the transmission dynamics of zoonotic parasites. T. hydatigena is therefore of particular interest to researchers interested in studying zoonotic diseases and the evolutionary strategies of parasites. Herein we report a high-quality draft genome for this tapeworm, characterized by some hallmarks (e.g., expanded genome size, wide integrations of viral-like sequences and extensive alternative splicing during development), and specialized adaptations related to its parasitic fitness (e.g., adaptive evolutions for teguments and lipid metabolism). Importantly, in contrast with the evolutionarily close trematodes, which achieve gene diversification associated with immunosuppression by gene family expansions, in T. hydatigena and other cestodes, this is accomplished by alternative splicing and gene loss. This indicates that these two classes have evolved different mechanisms for survival. In addition, molecular targets for diagnosis and intervention were identified to facilitate the development of control interventions. Overall, this work uncovers new strategies by which helminths evolved to interact with their hosts.

Molecular Detection Of Trypanosoma Evansi In Camel (Camelus Dromedarius) Using Internal Transcribed Spacer 1 of Ribosomal DNA

The purpose of this study was to determine clinical and subclinical prevalence of T. evansi among camels in Bikaner, India. In the present study, camels were examined parasitologically by blood smear examination and by Polymerase chain reaction (PCR) targeting ITS region of T. evansi. Blood samples of 74 camels were collected during the period from July 2019 to November 2019. The blood smear examination and molecular analysis showed 6.75 and 21.62%, respectively prevalence of T. evansi infection in camels, suggesting high sensitivity of diagnosis towards molecular tests. In comparison to parasitological methods, PCR proved fast, precise, sensitive detection diagnostic method for trypanosome infected camels. The adult camels possessed acute infection of 28.88% of T. evansi when compared to young camels with 10.34% infection, suggesting no significant difference (x2=1.141, P > 0.05). Hence, molecular diagnosis targeting ITS region among the infected trypanosome camels is more reliable and accurate for epidemiological survey and control programmes of trypanosomosis caused by T. evansi.

Typical numerical alterations in genome identified by array CGH analysis in neuroblastoma tumors

&lt;abstract&gt;&lt;sec&gt; &lt;title&gt;Introduction&lt;/title&gt; &lt;p&gt;The clinical variability in the course of neuroblastoma (NB) is closely linked to diverse genetic changes acquired by tumor cells. Rapid NB progression is associated with oncogene MYCN amplification (MNA) and segmental chromosomal aberrations (SCA). Alternatively, numerical chromosomal alterations (NCA) have positive impact on treatment. So far, no studies have been undertaken to identify NCA that may group NB patients. Therefore, the aim of the study was to identify NCA typical for NB.&lt;/p&gt; &lt;/sec&gt;&lt;sec&gt; &lt;title&gt;Materials and methods&lt;/title&gt; &lt;p&gt;Copy number alterations in NB tumor genome (fresh samples N = 94; formalin-fixed paraffin-embedded specimens N = 66) were analyzed with a pangenomic array CGH technique.&lt;/p&gt; &lt;/sec&gt;&lt;sec&gt; &lt;title&gt;Results&lt;/title&gt; &lt;p&gt;The profile with NCA was observed in 72 (45%) cases, NCA+SCA in 37 (23%), normal in 35 (22%) and MNA in 16 (10%). Samples with NCA were characterized by whole chromosome gains: 17, 7, 6 (78%, 65%, 51%, respectively) and copy loss of chromosome 14 (57%). Similarly to NCA, patients with a combined NCA and SCA profile were also characterized by gain of whole chromosome 17 and 7 (35% both) and loss of chromosome 14 (38%), but with lower frequency. In the combined NCA and SCA profiles, typical NB changes such as deletion 1p36 (27%) and gain 17q (41%) were observed, as well as deletion 11q (24%). The same alterations were detected in MNA samples (44%, 44%, 19%, respectively). A difference was found in spanning 11q deletion between MNA and NCA+SCA subgroup, which may suggest new prognostic markers in NB. In MNA subgroup specific NCA was not indicated.&lt;/p&gt; &lt;/sec&gt;&lt;sec&gt; &lt;title&gt;Conclusions&lt;/title&gt; &lt;p&gt;The hypothesis that NCA in NB tumors are more frequent in younger children with good prognosis was confirmed. To gain new insights into the pathogenesis of NB and to establish molecular targets for diagnosis and therapy, candidate genes in the altered chromosomal regions must be investigated.&lt;/p&gt; &lt;/sec&gt;&lt;/abstract&gt;

Expression of Somatostatin Receptor Subtypes (SSTR-1-SSTR-5) in Pediatric Hematological and Oncological Disorders.

Hematological and oncological disorders represent leading causes of childhood mortality. Neuropeptide somatostatin (SST) has been previously demonstrated in various pediatric tumors, but limited information exists on the expression and characteristics of SST receptors (SSTR) in hematological and oncological disorders of children. We aimed to investigate the expression of mRNA for SSTR subtypes (SSTR-1–5) in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of SSTRs were further studies by ligand competition assay. Our results show that the pediatric tumor samples highly expressed mRNA for the five SSTR subtypes with various patterns. The mRNA for SSTR-2 was detected in all specimens independently of their histological type. A Hodgkin lymphoma sample co-expressed mRNA for all five SSTR subtypes. SSTR-3 and SSTR-5 were detected only in malignant specimens, such as rhabdomyosarcoma, Hodgkin lymphoma, acute lymphoblastic leukemia, and a single nonmalignant condition, hereditary spherocytosis. The incidence of SSTR-1 and SSTR-4 was similar (60%) in the 15 specimens investigated. Radioligand binding studies demonstrated the presence of specific SSTRs and high affinity binding of SST analogs in pediatric solid tumors investigated. The high incidence of SSTRs in hematological and oncological disorders in children supports the merit of further investigation of SSTRs as molecular targets for diagnosis and therapy.

Abstract 1981: Cell free DNA promotes malignant transformation in non-tumor cells

Abstract Cell free DNA (cfDNA) are present at various biological fluids and when released by tumor cells it can contribute with the induction of pro-tumorigenic events, such as malignant transformations and metastasis. In this work we evaluated the effects of tumor derived cfDNA, isolated from blood of prostate cancer patients, in the non-tumoral prostate cellular linages RWPE-1 and PNT-2. We quantified, characterized and evaluated the function of the isolated cfDNA by qPCR of tumor progression related genes and MS-metabolomics. The results showed that the isolated cfDNA was able to induce the increase expression of MMP9 and CD44 in the normal prostate cellular linages, as well as to alter the consumption of tryptophan by the cells, these changes can indicate the sifting of the cells metabolism in favor of a tumoral profile. Those results demonstrate the potential of tumor derived cfDNA to alter receptor cells profiles, triggering malignant transformation events, making them a good target for new therapies and molecular targets for diagnosis. Citation Format: Aline Gomes Souza, Mario Machado Martins, Victor Alexandre Bastos, Natassia Caroline Resende, Paula Sousa Santos, Karina Marangoni, Luiz Ricardo Goulart, Vivian Alonso Goulart. Cell free DNA promotes malignant transformation in non-tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1981.

Progress in the correlation between PTPN12 gene expression and human tumors.

Background:The global morbidity of cancer is rising rapidly. Despite advances in molecular biology, immunology, and cytotoxic and immune-anticancer therapies, cancer remains a major cause of death worldwide. Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a new member of the cytoplasmic protein tyrosine phosphatase family, isolated from a cDNA library of adult colon tissue. Thus far, no studies have reviewed the correlation between PTPN12 gene expression and human tumors.Methods:This article summarizes the latest domestic and international research developments on how the expression of PTPN12 relates to human tumors. The extensive search in Web of Science and PubMed with the keywords including PTPN12, tumor, renal cell carcinoma, proto-oncogenes, tumor suppressor genes was undertaken.Results:More and more studies have shown that a tumor is essentially a genetic disease, arising from a broken antagonistic function between proto-oncogenes and tumor suppressor genes. When their antagonistic effect is out of balance, it may cause uncontrolled growth of cells and lead to the occurrence of tumors. PTPN12 is a tumor suppressor gene, so inhibiting its activity will lead directly or indirectly to the occurrence of tumors.Conclusion:The etiology, prevention, and treatment of tumors have become the focus of research around the world. PTPN12 is a tumor suppressor gene. In the future, PTPN12 might serve as a novel molecular marker to benefit patients, and even the development of tumor suppressor gene activation agents can form a practical research direction.

Pondicherry declaration on the identification and detection of Entamoeba histolytica.

Stake holders meet on “Identification and Detection of Entamoeba histolytica” was conducted on July 21, 2019 at Sri Balaji Vidyapeeth Deemed-to-be-University, Pondicherry. This programme was of national importance, since the amoebiasis is being increasingly reported from different parts of India because of poor socioeconomic conditions and sanitation levels. Experts in amoebiasis research across India attended this meeting. This meeting was conducted with an objective to frame the guidelines on the identification and detection of E. histolytica with reference to conventional diagnostic methods and molecular diagnosis targeting appropriate genes of E. histolytica. The recommendations of the panel were released as declaration on the diagnosis of amoebiasis and were circulated to various administrative and scientific bodies in India as reference policy document on the diagnosis of amoebiasis.

MEX3C regulates lipid metabolism to promote bladder tumorigenesis through JNK pathway.

Purpose: Bladder cancer (BC) is the most common urinary cancer among men with a high rate of deaths despite the improved medical technology and treatment. Recent evidence demonstrated that Mex-3 RNA-Binding Family Member C (MEX3C) plays various roles in different biological activities, but its molecular mechanisms underlying the pathogenesis of BC remain unclear yet. The aim of this research was to explore the expression patterns of MEX3C and its biological functions in human BC.Materials and methods: The Cancer Genome Atlas (TCGA) and Oncomine databases were jointly used to analyze the expression of MEX3C in BC and its correlation with the clinicopathological features, while real-time PCR and immunohistochemistry analysis were used to verify the predicted results. Wound-healing assay, Matrigel invasion assay, BODIPY staining and Western blot analysis were used in a cell model to assess the effect of MEX3C on the lipid metabolism, invasion and migration of BC and its mechanisms.Results: MEX3C was highly expressed in BC tissues and cells compared with their normal counterparts, and its expression was positively correlated with the clinicopathological features, especially the invasiveness phenotype. Overexpression of MEX3C accumulated lipid droplets and promoted cell adhesion, invasion and migration. We further demonstrated that MEX3C regulated lipid metabolism and promoted tumor development and progression through activation of JNK signaling and upregulating the JNK downstream protein levels of sterol regulatory element-binding proteins-1, fatty acid synthase and acetyl-CoA carboxylase-1.Conclusion: Here we identified MEX3C as a new oncogene to promote bladder tumorigenesis by regulating lipid metabolism through Mitogen-activated protein kinase/c-Jun N-terminal kinase (MAPK/JNK) pathway. These findings suggest a new role of MEX3C in promoting BC tumorigenesis and provide a novel biomarker or molecular target for diagnosis or treating BC.

PET Imaging Biomarkers of Anti-EGFR Immunotherapy in Esophageal Squamous Cell Carcinoma Models.

Epidermal growth factor receptor (EGFR) is overexpressed and considered as a proper molecular target for diagnosis and targeted therapy of esophageal squamous cell carcinoma (ESCC). This study evaluated the usefulness of PET imaging biomarkers with 64Cu-PCTA-cetuximab and 18F-FDG-PET for anti-EGFR immunotherapy in ESCC models. In vivo EGFR status and glucose metabolism by cetuximab treatment were evaluated using 64Cu-PCTA-cetuximab and 18F-FDG-PET, respectively. Therapeutic responses with imaging biomarkers were confirmed by western blot and immunohistochemistry. TE-4 and TE-8 tumors were clearly visualized by 64Cu-PCTA-cetuximab, and EGFR expression on TE-8 tumors showed 2.6-fold higher uptake than TE-4. Tumor volumes were markedly reduced by cetuximab in TE-8 tumor (92.5 ± 5.9%), but TE-4 tumors were refractory to cetuximab treatment. The SUVs in 64Cu-PCTA-cetuximab and 18F-FDG-PET images were statistically significantly reduced by cetuximab treatment in TE-8 but not in TE-4. 64Cu-PCTA-cetuximab and 18F-FDG-PET images were well correlated with EGFR and pAkt levels. 64Cu-PCTA-cetuximab immuno-PET had a potential for determining EGFR level and monitoring therapeutic response by anti-EGFR therapy. 18F-FDG-PET was also attractive for monitoring efficacy of anti-EGFR therapy. In conclusion, PET imaging biomarkers may be useful for selecting patients that express target molecules and for monitoring therapeutic efficacy of EGFR-targeted therapy in ESCC patients.

Immunohistochemical expression analysis of leucine-rich PPR-motif-containing protein (LRPPRC), a candidate colorectal cancer biomarker identified by shotgun proteomics using iTRAQ

BackgroundColorectal cancer (CRC) is the fourth most frequent cause of cancer deaths in the world. Novel biomarkers for the diagnosis, prognosis, and treatment of CRC are required to improve the clinical strategy. MethodsWe applied shotgun proteomics using isobaric tags for relative and absolute quantitation (iTRAQ) to identify novel biomarkers of CRC, and then we detected leucine-rich PPR-motif-containing protein (LRPPRC) expression in 83 normal colorectal tissues and 133 CRC tissues by immunohistochemistry. ResultsA total of 570 proteins were identified using iTRAQ. We validated the expression of LRPPRC protein by immunohistochemical analysis of the 77 proteins that showed expression changes in the cancer tissues >1.5-fold the levels in the normal tissues. The expression levels of LRPPRC were significantly higher in CRC tissues than those in normal colorectal tissues, and the expression levels were related with tumor differentiation and especially high in moderately differentiated CRC tissues. ConclusionWe identified a novel, differentially expressed protein, LRPPRC, which has the potential to serve as a molecular target for diagnosis and/or prognosis of CRC.

The protein arginine methyltransferase 5 promotes malignant phenotype of hepatocellular carcinoma cells and is associated with adverse patient outcomes after curative hepatectomy.

The prognosis of advanced hepatocellular carcinoma (HCC) is dismal. Novel molecular targets for diagnosis and therapy is urgently required. This study evaluated expression and functions of the protein arginine methyltransferase 5 (PRMT5) in HCC. Using HCC cell lines, the expression levels of PRMT5 mRNA were determined using the quantitative real-time reverse-transcription polymerase chain reaction, and the effect of a small interfering PRMT5-siRNA on cell phenotype was evaluated. Further, PRMT5 expression was determined in 144 pairs of resected liver tissues to evaluate its clinical significance. Regardless of their differentiated phenotypes, nine HCC cell lines expressed different levels of PRMT5 mRNA. Inhibition of PRMT5 expression significantly decreased the proliferation, invasion, and migration of HCC cell lines. Although the level of PRMT5 mRNA was not influenced by patient's background liver status, it was significantly higher in HCC tissues than in the corresponding noncancerous tissues. High levels of PRMT5 mRNA in HCC tissues were significantly associated with advanced disease stage and adverse prognosis. In conclusion, our results indicate that PRMT5 may act as a putative oncogene in HCC and that the levels of PRMT5 mRNA represent a promising prognostic marker and a potential target of molecular therapy for HCC.

Identification of New PCR Targets and its Validation for Development of Nucleic Acid-based Detection Assay for Melioidosis

Burkholderia pseudomallei the gram negative, soil saprophyte is the causative agent of melioidosis in human and animals. Development of rapid, sensitive, species specific and cost effective molecular assays are needed for detection of B. pseudomallei from clinical and environmental samples and to differentiate it from other closely related bacterial species. In this study, insilico approach was used to identify new species specific gene targets for molecular diagnosis of B. pseudomallei. The identified targets were then analyzed by SYBR Green real time PCR assay for their specificity, sensitivity and presence across different Indian clinical and soil isolates of B. pseudomallei. Out of the three targets studied SYBR Green real time PCR assay targeting bpss0091 gene of B. pseudomallei was found 100% specific, having detection limit of 12.3fg/µl DNA. The bpss0091 gene target was present in all clinical and soil isolates of B. pseudomallei tested thus suggesting bpss0091 gene based SYBR Green real time PCR assay will be useful for detection of B. pseudomallei in different geographical regions.

MiR-135b, a key regulator of malignancy, is linked to poor prognosis in human myxoid liposarcoma.

Myxoid/round cell (RC) liposarcomas (MLS) were originally classified into two distinct populations based on histological differences; a myxoid component and a RC component. It is notable that, depending on an increase of the RC component, the prognosis significantly differs. Hence, the RC component is associated with metastasis and poor prognosis. However, the molecular mechanisms that contribute to the malignancy of the RC component still remain largely unknown. Here, we report microRNA-135b (miR-135b), a key regulator of the malignancy, highly expressed in the RC component and promoting MLS cell invasion in vitro and metastasis in vivo through the direct suppression of thrombospondin 2 (THBS2). Decreased THBS2 expression by miR-135b increases the total amount of matrix metalloproteinase 2 (MMP2) and influences cellular density and an extracellular matrix structure, thereby resulting in morphological change in tumor. The expression levels of miR-135b and THBS2 significantly correlated with a poor prognosis in MLS patients. Overall, our study reveals that the miR-135b/THBS2/MMP2 axis is tightly related to MLS pathophysiology and has an important clinical implication. This work provides noteworthy evidence for overcoming metastasis and improving patient outcomes, and sheds light on miR-135b and THBS2 as novel molecular targets for diagnosis and therapy in MLS.

Restoration of miR-20a expression suppresses cell proliferation, migration, and invasion in HepG2 cells.

ObjectiveTo study microRNA (miR)-20a expression in hepatocellular carcinoma (HCC) and its effects on the proliferation, migration, and invasion of HepG2.MethodsThe real-time polymerase chain reaction was used to detect the expression of miR-20a in HCC tissue and normal tissue, as well as in HCC cell lines and normal liver cells. miR-20a mimic and miR negative control (NC) were transfected into HepG2 cells. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay was used to detect cell proliferation. Annexin fluorescein isothiocyanate/propidium iodide assay was run to examine the early apoptosis of cells. Transwell chamber assay was carried out to investigate the cell invasion and migration abilities.ResultsmiR-20a was lowly expressed both in HCC tissues and HCC cell lines. After transfection of exogenous miR-20 mimics, miR-20a expression in HepG2 cells was significantly increased by 61.29% compared to the blank group (P<0.01). MTT assay showed that the growth of HepG2 cells in the miR-20a mimics group was significantly inhibited, and optical density values during the 36–96 hour time period were dramatically decreased compared to the blank group (P<0.01). Apoptosis rates of the miR-20a mimics group were higher than those of the blank and NC groups (both P<0.01). The number of HCC cells after transfection by miR-20a mimics in the G1 and S phases were 15.88% and 7.89%, respectively, which were lower than in the blank and NC groups (both P<0.05). Transwell assay showed that in the miR-20a mimics group the number of cell migration and invasion were 0.459 and 0.501 times that of the blank group (both P<0.01), and the migration and inhibition rates were 54.1% and 51.4%, respectively. After closing target gene CCND1 in HepG2 cells, the number of cell migration and invasion in the small interfering (si)-CCND1 group were 0.444 and 0.435 times that of the si-NC group (P<0.05); and compared to the si-NC group, the migration and inhibition rates were 55.6% and 56.5%, respectively.ConclusionmiR-20a can inhibit the growth, invasion, and migration of HepG2 cells, and is therefore promising as a new molecular target for diagnosis and therapy of HCC.