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.

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.

Effect of physical exercise on sleep quality in college students: Mediating role of smartphone use.

OBJECTIVE: To investigate the effect of physical exercise on sleep quality and the mediating effect of smartphone use behavior in college students. METHODS: A cross-sectional study design was adopted. An online survey of 5,075 college students was conducted using the Physical Activity Rating Scale-3, the Pittsburgh Sleep Quality Index, and the Mobile Phone Addiction Tendency Scale. RESULTS: The sleep quality of college students was poor, and the proportion of college students with good sleep quality was 23.567%. A significant correlation existed between sleep quality and physical exercise (r = -0.159, P < 0.001) and mobile phone addiction (r = 0.355, P < 0.001). Physical exercise can predict sleep quality in college students (ß = -0.011, P < 0.001). Smartphone use plays a part in mediating the process by which physical exercise affects sleep quality. CONCLUSION: Chinese college students have poor sleep quality. Physical exercise and smartphone use behavior are important factors affecting the sleep quality of college students. Physical exercise can directly predict the sleep quality of college students and can predict the sleep quality of college students through the mediating effect of smartphone use behavior.

Icariin as a potential anticancer agent: a review of its biological effects on various cancers.

Numerous chemical compounds used in cancer treatment have been isolated from natural herbs to address the ever-increasing cancer incidence worldwide. Therein is icariin, which has been extensively studied for its therapeutic potential due to its anti-inflammatory, antioxidant, antidepressant, and aphrodisiac properties. However, there is a lack of comprehensive and detailed review of studies on icariin in cancer treatment. Given this, this study reviews and examines the relevant literature on the chemopreventive and therapeutic potentials of icariin in cancer treatment and describes its mechanism of action. The review shows that icariin has the property of inhibiting cancer progression and reversing drug resistance. Therefore, icariin may be a valuable potential agent for the prevention and treatment of various cancers due to its natural origin, safety, and low cost compared to conventional anticancer drugs, while further research on this natural agent is needed.

Effects of phosphorus application on yield, quality and phosphorus use efficiency of edible sweetpotato: A case study of Xushu 32.

To explore the appropriate amount of phosphorus (P) fertilizer and improve economic yield and P use efficiency of edible sweetpotato, we took Xushu 32 as an example and compared the effects of different P application rates on yield, quality, P accumulation and P use efficiency of edible sweetpotato based on a two-year field experiment (soil available P content was 31.70 mg·kg-1) from 2018 to 2019. There were five P application levels (P2O5), including 0 (P0), 25 (P25), 50 (P50), 75 (P75) and 100 kg·hm-2(P100). The results showed that, 1) compared with P0, P application significantly increased the yield of fresh sweetpotao and commodity potato, with the effects being the stongest under P75 treatment, followed by P50 treatment. However, there was no significant difference between the two treatments. 2) P application significantly increased the contents of starch and reducing sugar in storage root. The contents of soluble sugar and protein increased significantly under P50 treatment. 3) Du-ring the growth period of 90 to 120 d, P fertilizer supply significantly increased P accumulation and dry matter accumulation of sweetpotato. 4) The apparent P use efficiency (APUE) decreased with increasing P application rates, while P agronomic efficiency (PAE) increased first and then decreased with the increases of P application rates, which was significantly higher under P50 than other treatments. Taking into account the yield, quality, economic yield and P utilization rate of edible sweetpotato, the optimal dosage of P2O5 is 50 kg·hm-2 under the experimental conditions.

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.

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.