Accurate and affordable detection of rifampicin and isoniazid resistance in Tuberculosis sputum specimens by multiplex PCR-multiple probes melting analysis.

BACKGROUND: Escalating cases of multidrug-resistant tuberculosis (MDR-TB) pose a major challenge to global TB control efforts, necessitating innovative diagnostics to empower decentralized detection of gene mutations associated with resistance to rifampicin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis (M. tuberculosis) in resource-constrained settings. METHODS: Combining multiplex fluorescent PCR and Multiple Probes Melting Analysis, we identified mutations in the rpoB, katG, ahpC and inhA genes from sputum specimens. We first constructed a reference plasmid library comprising 40 prevalent mutations in the target genes' resistance determining regions and promoters, serving as positive controls. Our assay utilizes a four-tube asymmetric PCR method with specifically designed molecular beacon probes, enabling simultaneous detection of all 40 mutations. We evaluated the assay's effectiveness using DNA isolated from 50 clinically confirmed M. tuberculosis sputum specimens, comparing our results with those obtained from Sanger sequencing and retrospective validation involving bacteriological culture and phenotypic drug susceptibility testing (pDST). We also included the commercial Xpert MTB/RIF assay for accuracy comparison. RESULTS: Our data demonstrated remarkable sensitivity in detecting resistance to RIF and INH, achieving values of 93.33% and 95.24%, respectively, with a specificity of 100%. The concordance between our assay and pDST was 98.00%. Furthermore, the accuracy of our assay was comparable to both Sanger sequencing and the Xpert assay. Importantly, our assay boasts a 4.2-h turnaround time and costs only $10 per test, making it an optimal choice for peripheral healthcare settings. CONCLUSION: These findings highlight our assay's potential as a promising tool for rapidly, accurately, and affordably detecting MDR-TB.

Development and validation of a droplet digital PCR assay for the evaluation of PML-RARα fusion transcripts in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is an aggressive disease that requires prompt treatment. Promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion genes resulting from reciprocal translocation are considered a molecular basis for diagnosing APL. Moreover, PML-RARα fusion gene testing is an essential tool for monitoring the response to therapy via minimal residual disease and providing a diagnosis before rapid disease progression in APL. The present study developed a novel droplet digital PCR (ddPCR) assay to rapidly detect two PML-RARα variants (bcr1 and bcr3) and compared its limit of detection (LOD) with quantitative PCR (qPCR). It was demonstrated that the LOD of ddPCR for PML-RARα reached 0.001%, and the evaluation of high copy number samples of PML-RARα by ddPCR correlated well with qPCR. Furthermore, clinical sample testing with ddPCR found that 34 and 24% samples were bcr-1-positive and bcr3-positive, respectively. However, according to qPCR, 30% of the samples were bcr1-positive and 20% were bcr3-positive. In addition, the concordance rate between ddPCR and qPCR reaction was 86%. While monitoring minimal residual disease, the PML-RARα mutation rate of three patients who recovered well decreased to 0.34%. However, one patient who was bcr3-positive and relapsed had a mutation rate of 13% while in remission, indicating that the bcr3 isoform may be an adverse prognostic factor affecting recovery. Therefore, the present results suggested that this novel ddPCR assay may be useful for monitoring and evaluating the treatment effects and prognosis of APL.

Ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry analysis of the impact of processing on toxic components of Kansui Radix.

BACKGROUND: Kansui Radix (Gansui in Chinese), the dried tuber of Euphorbia kansui, is a Chinese medicinal herb commonly used for the treatment of oedema and ascites with dyspnea. Because of its toxic nature, the herb is usually processed with vinegar to reduce the toxicity. A report has shown that the contents of toxic terpenoids in Gansui decreased after processing with vinegar. However, comprehensive comparison of the chemical profiles between vinegar-processed and raw Gansui has not yet been conducted. METHODS: An ultra-high-performance liquid chromatography in conjunction with ultra-high resolution quadrupole time-of-flight mass spectrometry (UHPLC UHD Q-TOF MS/MS) method was developed for the analysis of chemical profiles of vinegar-processed and raw Gansui in this study. RESULTS: Results showed that processing with vinegar caused conspicuous chemical changes. Among the altered components, 11 toxic terpenoids, 3-O-benzoyl-13-O- dodecanoylingenol/20-O-benzoyl-13-O-dodecanoylingenol, kansuinine D, kansuinine A, 3-O-benzoyl-13-O-dodecanoylingenol/20-O-benzoyl-13-O-dodecanoylingenol, 3-O- benzoylingenol/20-O-benzoylingenol, 20-O-(2'E,4'Z-decadienoyl)ingenol/20-O-(2'E,4'E- decadienoyl)ingenol/3-O-(2'E,4'Z-decadienoyl)ingenol/3-O-(2'E,4'E-decadienoyl)ingenol, 3-O-(2'E,4'Z-decadienoyl)-20-deoxyingenol,3-O-(2'E,4'Z-,ecadienoyl)-5-O-acetylingenol,3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol,3-O-(2,3-dimethylbutanoyl)-13-O-dodecanoylingenol, were tentatively identified. The contents of most of these terpenoids were obviously decreased after processing with reductions of 6.66-95.25%. CONCLUSION: Our findings could help us understand the chemical basis for the toxicity reduction of Gansui afforded by processing with vinegar. Further investigations are warranted to establish the relationship between processing-induced chemical changes and the reduction of toxicity of Gansui.

Relationship of proteomic variation and toxin synthesis in the dinoflagellate Alexandrium tamarense CI01 under phosphorus and inorganic nitrogen limitation.

Paralytic shellfish toxins (PSTs) are originated from cyanobacteria and dinoflagellates, including Alexandrium tamarense, the common dinoflagellate species. In this study, a toxic dinoflagellate strain of A. tamarense CI01 was selected for studying the PSTs' concentration and the related protein variation during the whole cell cycle under different nutrient conditions. High-performance liquid chromatography, 2-D DIGE and Western blotting were used collectively for protein profiling and identification. Results showed that the toxin content was suppressed under nitrogen limiting condition, but enhanced in phosphorous limiting medium. Based on the results of proteomics analysis, 7 proteins were discovered to be related to the PSTs biosynthesis of A. tamarense CI01, including S-adenosylhomocysteine hydrolase, ornithine cyclodeaminase, argininosuccinate synthase, methyluridine methyltransferase cystine ABC transporter, phosphoserine phosphatase, argininosuccinate synthase and acyl-CoA dehydrogenase, which corresponds to the metabolism of the methionine, cysteine, ornithine, arginine and proline. Moreover, some photosynthesis relating proteins also increased their expression during PST synthesis period in A. tamarense CI01, such as phosphoenolpyruvate carboxylase, chloroplast phosphoglycerate kinase, peridinin-chlorophyll α-binding protein, Mg(2+) transporter protein and chloroplast phosphoglycerate kinase. The above findings are in support of our hypothesis that these proteins are involved in toxin biosynthesis of A. tamarense CI01, but cause-and-effect mechanisms need to be investigated in further studies.

Comparison of three protein extraction procedures from toxic and non-toxic dinoflagellates for proteomics analysis.

Three methods for extraction and preparation of high-quality proteins from both toxic and non-toxic dinoflagellates for proteomics analysis, including Trizol method, Lysis method and Tris method, were compared with the subsequent protein separation profiles using 2-D differential gel electrophoresis (2-D DIGE), Coomassie Blue and silver staining. These methods showed suitability for proteins with different pIs and molecular weights. Tris method was better for low molecular weight and low pI protein isolation; whereas both Lysis and Trizol method were better for high-molecular weight and high pI protein purification. Trizol method showed good results with Alexandrium species and Gynodinium species, and the background in gel was much clearer than the other two methods. At the same time, only Lysis method caused breaking down of the target proteins. On the other hand, Trizol method obtained higher concentration of ribulose-1,5-bisphosphate carboxylase/oxygenase proteins by Western-blotting, while Tris method was the best for peridinin-chlorophyll-protein complexes protein and T1 protein preparation. DIGE was better than Coomassie Blue and silver staining, except for some limitations, such as the high cost of the dyes, relatively short shelf life and the requirements for extensive and special image capturing equipment. Some proteins related to PSTs synthesis in dinoflagellates are hydrophobic with high molecular weight or binding on membranes and Trizol method performed better than Tris method for these proteins. The Trizol method and 2-D DIGE were effective combination for proteomics investigations of dinoflagellates. This procedure allows reliable and high recovery efficiency of proteins from dinoflagellates for better understanding on their occurrence and toxin-production for physiological and biochemical information.

Silencing LINC00987 ameliorates adriamycin resistance of acute myeloid leukemia via miR-4458/HMGA2 axis.

BACKGROUND: Most patients with acute myeloid leukemia (AML) eventually develop drug resistance, leading to a poor prognosis. Dysregulated long gene non coding RNAs (lincRNAs) have been implicated in chemoresistance in AML. Unfortunately, the effects of lincRNAs which participate in regulating the Adriamycin (ADR) resistance in AML cells remain unclear. Thus, the purpose of this study is to determine LINC00987 function in ADR-resistant AML. METHODS: In this study, ADR-resistant cells were constructed. LINC00987, miRNAs, and HMGA2 mRNA expression were measured by qRT-PCR. P-GP, BCRP, and HMGA2 protein were measured by Western blot. The proliferation was analyzed by MTS and calculated IC50. Soft agar colony formation assay and TUNEL staining were used to analyze cell colony formation and apoptosis. Xenograft tumor experiment was used to analyze the xenograft tumor growth of ADR-resistant AML. RESULTS: We found that higher expression of LINC00987 was observed in AML patients and associated with poor overall survival in AML patients. LINC00987 expression was increased in ADR-resistant AML cells, including ADR/MOLM13 and ADR/HL-60 cells. LINC00987 downregulation reduces ADR resistance in ADR/MOLM13 and ADR/HL-60 cells in vitro and in vivo, while LINC00987 overexpression enhanced ADR resistance in MOLM13 and HL-60 cells. Additionally, LINC00987 functions as a competing endogenous RNA for miR-4458 to affect ADR resistance in ADR/MOLM13 and ADR/HL-60 cells. HMGA2 is a target of miR-4458. LINC00987 knockdown and miR-4458 overexpression reduced HMGA2 expression. HMGA2 overexpression enhanced ADR resistance, which reversed the function of LINC00987 silencing in suppressing ADR resistance of ADR/MOLM13 and ADR/HL-60 cells. CONCLUSIONS: Downregulation of LINC00987 weakens ADR resistance by releasing miR-4458 to deplete HMGA2 in ADR/MOLM13 and ADR/HL-60. Therefore, LINC00987 may act as the therapeutic target for treating chemoresistant AML.

Differentiation between glioma recurrence and treatment effects using amide proton transfer imaging: A mini-Bayesian bivariate meta-analysis.

Background: Amide proton transfer (APT) imaging as an emerging MRI approach has been used for distinguishing tumor recurrence (TR) and treatment effects (TEs) in glioma patients, but the initial results from recent studies are different. Aim: The aim of this study is to systematically review and quantify the diagnostic performance of APT in assessing treatment response in patients with post-treatment gliomas. Methods: A systematic search in PubMed, EMBASE, and the Web of Science was performed to retrieve related original studies. For the single and added value of APT imaging in distinguishing TR from TEs, we calculated pooled sensitivity and specificity by using Bayesian bivariate meta-analyses. Results: Six studies were included, five of which reported on single APT imaging parameters and four of which reported on multiparametric MRI combined with APT imaging parameters. For single APT imaging parameters, the pooled sensitivity and specificity were 0.85 (95% CI: 0.75-0.92) and 0.88 (95% CI: 0.74-0.97). For multiparametric MRI including APT, the pooled sensitivity and specificity were 0.92 (95% CI: 0.85-0.97) and 0.83 (95% CI: 0.55-0.97), respectively. In addition, in the three studies reported on both single and added value of APT imaging parameters, the combined imaging parameters further improved diagnostic performance, yielding pooled sensitivity and specificity of 0.91 (95% CI: 0.80-0.97) and 0.92 (95% CI: 0.79-0.98), respectively, but the pooled sensitivity was 0.81 (95% CI: 0.65-0.93) and specificity was 0.82 (95% CI: 0.61-0.94) for single APT imaging parameters. Conclusion: APT imaging showed high diagnostic performance in assessing treatment response in patients with post-treatment gliomas, and the addition of APT imaging to other advanced MRI techniques can improve the diagnostic accuracy for distinguishing TR from TE.

Development of a diagnostic assay by three-tube multiplex real-time PCR for simultaneous detection of nine microorganisms causing acute respiratory infections.

Acute respiratory infections are widespread in vulnerable populations of all ages and are characterized by a variety of symptoms. The underlying infection can be caused by a multitude of microorganisms, including viruses and bacteria. Early detection of respiratory infections through rapid pathogen screening is vital in averting infectious respiratory disease epidemics. This study utilized a multiplex real-time PCR system to develop a three-tube reverse transcription-PCR (RT-PCR) assay, enabling simultaneously detect nine respiratory pathogens, including: influenza A and B, adenovirus, respiratory syncytial virus (RSV), Streptococcus pneumoniae, Legionella pneumophila, Haemophilus influenzae, Chlamydia pneumoniae, and Mycoplasma pneumoniae. This technique utilizes a one-step assay, with specifically designed TaqMan primer-probe sets combined in the same tube. This assay provided rapid and simplified detection of the nine prevalent pathogens, as well as increased sensitivity and reduced cross-contamination. This assay was evaluated using 25 related viral/bacterial strains as positive references, the other 25 irrelevant strains as negative controls, and clinical specimens from 179 patients. All positive strains were detected with no amplification of the non-target microorganism mixtures and the assay's detection limits ranged between 250-500 copies/ml (1.25-2.5 copies/reaction). A total of 167 (93.3%) samples tested positive for at least one of the pathogens identified; 109 of these samples were from patients confirmed to have RSV infections. The diagnostic accuracy of our assay was further confirmed by matching results from classical direct immunofluorescence assay and nucleotide sequencing. These data demonstrate the innovative multiplex real-time PCR assay as a promising alternative to the current approaches used for early screening of acute respiratory infections.

Identification of SARS-CoV-2 Variants and Their Clinical Significance in Hefei, China.

The ongoing coronavirus disease 2019 (COVID-19) pandemic represents one of the most exigent threats of our lifetime to global public health and economy. As part of the pandemic, from January 10 to March 10, 2020, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) began to spread in Hefei (Anhui Province, China) with a total of 174 confirmed cases of COVID-19. During this period, we were able to gather critical information on the transmission and evolution of pathogens through genomic surveillance. Particularly, the objective of our study was to track putative variants of SARS-CoV-2 circulating in Hefei for the first time and contribute to the global effort toward elucidating the molecular epidemic profile of the virus. Patients who showed symptoms of COVID-19 were routinely tested for SARS-CoV-2 infections via RT-PCR at the First Affiliated Hospital of Anhui Medical University. Whole-genome sequencing was performed on 97 clinical samples collected from 29 confirmed COVID-19 patients. As a result, we identified a local novel single-nucleotide polymorphism site (10,380) harboring a G → T mutation (Gly → Val) in Hefei. Further phylogenetic network analysis with all the sequences of SARS-CoV-2 deposited in GenBank collected in East and Southeast Asia revealed a local subtype of S-type SARS-CoV-2 (a1) harboring a C → T synonymous mutation (Leu) at position 18,060 of ORF1b, likely representing a local SARS-CoV-2 mutation site that is obviously concentrated in Hefei and the Yangtze River Delta region. Moreover, clinical investigation on the inflammatory cytokine profile of the patients suggested that mutations at positions 18,060 (the shared variable site of subtype a1) and 28,253(harboring a C → T synonymous mutation, Phe) were associated with milder immune responses in the patients.

Genetic and biochemical analyses of chlorobenzene degradation gene clusters in Pandoraea sp. strain MCB032.

Pandoraea sp. strain MCB032 was isolated as an emerging chlorobenzene degrader from a functionally stable bioreactor where species succession had occurred. In this study, two gene clusters encoding chlorobenzene metabolic functions have been cloned. Within the cbs gene cluster, CbsA and CbsB are similar to the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase in Ralstonia sp. JS705 and shown to transform chlorobenzene to 3-chlorocatechol. The clc gene cluster shows strong similarity to the clc genes of Ralstonia sp. JS705 and encodes chlorocatechol 1,2-dioxygenase (ClcA) and other enzymes, which catalyze the conversion of chlorocatechol to 3-oxoadipate. The Michaelis constants (K (m)) values of ClcA for catechol, 3-methylcatechol and 3-chlorocatechol were determined as 10.0, 8.9 and 3.4 muM, respectively. CbsX, a putative transport protein present in the cbs cluster of strain MCB032 but not in those of other chlorobenzene degraders, shows 76 and 53% identities to two previously identified transport proteins involved in toluene degradation, TbuX from Ralstonia pickettii PKO1 and TodX from Pseudomonas putida F1. The presence of the transport protein in strain MCB032 likely provides a mechanistic explanation for its higher chlorobenzene affinity and may well be the basis for the competitive advantage of this strain in the bioreactor.

Evaluation of EGFR mutations in NSCLC with highly sensitive droplet digital PCR assays.

Targeted drugs have been widely used in the treatment of patients with lung cancer, particularly for those with non­small cell lung cancer (NSCLC). Plasma cell­free DNA is an emerging clinical tool for the detection of epidermal growth factor receptor (EGFR) gene mutation in patients with lung cancer. Detection of circulating tumor (ct) DNA by droplet digital PCR (ddPCR) is a highly sensitive and minimally invasive alternative for the assessment and management of cancer. In the present study, four ddPCR systems were developed to detect the 19DELs, L858R, T790M and C797S mutations of the EGFR gene in plasma ctDNA samples, and all exhibited higher sensitivity compared with the amplification refractory mutation system (ARMS)­PCR assays. The results revealed that the sensitivity of the ddPCR assays for the four major types of EGFR mutant reached 0.04%. In total, 50 plasma ctDNA samples were collected from patients with NSCLC to detect the 19DELs, L858R, T790M and C797S mutations by ddPCR and ARMS­PCR. All the mutations except for C797S were detected and the concordance rates between ddPCR and ARMS­PCR were 96% (19DELs), 98% (L858R) and 100% (T790M). The fraction of EGFR mutation ranged from 0.43 to 68.07% using the ddPCR method. Therefore, the present study suggests that the four ddPCR testing systems could be used for early detection of EGFR mutations in plasma samples, so that patients can better select the targeted drugs according to the EGFR mutation.