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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.

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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.

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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.

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Autoimmune lymphoproliferative syndrome (ALPS) is an incurable disease mainly caused by the defect of Fas-mediated apoptosis and characterized by nonmalignant autoimmune lymphoproliferation. Stabilized ß-catenin could not only potentiate Fas-mediated T cell apoptosis via upregulating the expression of Fas on activated T cells, but also potentiate T cell apoptosis via intrinsic apoptotic pathway. In the present study, we introduced ß-catTg into lpr/lpr mice and aimed to explore the potential role of stabilized ß-catenin (ß-catTg) in the development of ALPS-like phenotypes of lpr/lpr mice. We found that the total splenocyte cells and some compositions were slightly downregulated in ß-catTglpr/lpr mice, especially the CD4 and CD8 TEM cells were significantly reduced. Meanwhile, stabilized ß-catenin obviously decreased the numbers of spleen TCRß+CD4-CD8- T (DNT) cells, and the levels of some serum proinflammatory factors also were lowered in ß-catTglpr/lpr mice. Beyond that, stabilized ß-catenin slightly lowered the levels of the serum autoantibodies and the scores of kidney histopathology of ß-catTglpr/lpr mice compared with lpr/lpr mice. Our study suggested that stabilized ß-catenin ameliorated some ALPS-like symptoms of lpr/lpr mice by potentiating Fas-independent signal-mediated T cell apoptosis, which might uncover a potential novel therapeutic direction for ALPS.

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Lupus nephritis (LN) is the major clinical manifestation of systemic lupus erythematosus. LN is promoted by T helper 17 (Th17) cells, which are the major pro-inflammatory T cell subset contributing to autoimmunity regulation. Nuclear factor erythroid 2-related factor 2 (NRF2) is critical for suppressing reactive oxygen species (ROS) and relieving oxidant stress by regulating antioxidant gene expression. Previous studies have demonstrated that Nrf2 deficiency promotes drug-induced or spontaneous LN. However, whether NRF2 regulates Th17 function during LN development is still unclear. In this study, we introduced Nrf2 deficiency into a well-known LN model, the B6/lpr mouse strain, and found that it promoted early-stage LN with altered Th17 activation. Th17 cells and their relevant cytokines were dramatically increased in these double-mutant mice. We also demonstrated that naïve T cells from the double-mutant mice showed significantly increased differentiation into Th17 cells in vitro, with decreased expression of the Th17 differentiation suppressor Socs3 and increased phosphorylation of STAT3. Our results demonstrated that Nrf2 deficiency promoted Th17 differentiation and function during LN development. Moreover, our results suggested that the regulation of Th17 differentiation via NRF2 could be a therapeutic target for the treatment of subclinical LN patients.

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OBJECTIVE: To investigate the dynamic variation of subsets of myeloid derived suppressor cells (MDSC) and their ratio in septic mice, and to discuss their role in the development of sepsis. METHODS: Male C57BL/6 mice were randomly divided into sepsis model group and sham group according to random number table. Polymicrobial sepsis was induced by using cecal ligation and puncture (CLP), while mice in sham group only underwent laparotomy and laparorrhaphy without CLP. Thirty mice in each group were used to observe living condition, and the 20-day survival rate was compared between the two groups. In addition, subsets of MDSC in peripheral blood, spleen and bone marrow were analyzed with flow cytometry for other 60 mice (12 mice at each time point, as 0, 3, 7, 12 and 20 days). Spleens were harvested at 7 days for weighing, and single cell suspension of spleen tissue was prepared for splenocyte counting. Histopathologic changes in spleen tissue and liver tissue were observed under light microscope after hematoxylin and eosin (HE) stain. RESULTS: (1) No mice died in sham group within 20 days after the operation. On the other hand, 10 mice in model group died within 20 days, and the difference in survival rate between the two groups was statistically significant (100.0% vs. 66.7%, χ(2) = 11.861, P = 0.001). (2) The spleens in model group showed obvious enlargement and significantly outweighed as compared with those in sham group (mg: 413.33±41.63 vs. 111.67±17.56, t = 11.564, P = 0.000), and the total count of splenocytes was significantly higher than that in sham group (×10(9)/L: 21.20±2.43 vs. 1.87±0.06, t = 13.578, P = 0.005). (3) Pathological sections with HE staining showed that the liver tissue and spleen tissue remained normal in sham group. In model group, the hepatic tissue showed acute inflammatory reaction, including tissue disruption, capillary congestion, infiltration of neutrophils, marked edema of hepatocytes and focal hepatocellular necrosis. Abnormalities were also found in the spleen tissue: the red pulp and white pulp were disordered, splenic sinus was congested with numerous red cells, the splenic capsule thickened, immature myeloid cells with circular nuclei proliferated in the subcapsular region and perivascular region, splenic cord and splenic sinus were infiltrated with a large number of hematopoietic cells. (4) No significant changes in the monocytic MDSC (M-MDSC) and granulocytic MDSC (G-MDSC), and their ratio were found in peripheral blood, spleen and bone marrow at every time point in sham group. On the other hand, in model group, the ratio of M-MDSC and G-MDSC was continuously increased in peripheral blood, spleen and bone marrow, and M-MDSC only slightly decreased at 20 days. On the other hand, the ratio of M-MDSC/G-MDSC rose at first followed by a decrease. The ratio of M-MDSC/G-MDSC in peripheral blood was higher than 1 from 3 days after the operation, reaching the peak at 12 days (compared with 0 day: 4.16±0.53 vs. 0.79±0.11, P < 0.05), while the ratio of M-MDSC/G-MDSC in spleen and bone marrow after CLP were lower than 1 at all time points, reaching the peak on 7 days after the operation (compared with 0 day: 0.70±0.06 vs. 0.25±0.02 in spleen, 0.39±0.06 vs. 0.11±0.01 in bone marrow, both P < 0.05), and then gradually decreased afterwards. CONCLUSIONS: Subgroups of MDSCs were continuously aggregated in the peripheral blood, spleen and bone marrow, and their ratio rose first and decreased afterwards along with the development of sepsis, and the changes may reflect the change of immune status at different stages of sepsis.

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Epidemiological evidence indicates that assisted reproductive technologies (ART) may be associated with several epigenetic diseases such as Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS). Selection of sperm by density-gradients in ART has improved DNA integrity and sperm quality; however, epigenetic alterations associated with this approach are largely unknown. In the present study, we investigated DNA methylation and histone retention profiles in raw sperm and selected sperm derived from the same individual and separated by using density-gradients. Results from a study group consisting of 93 males demonstrated that both global DNA methylation and histone retention levels decreased in density selected sperm. Compared to unselected raw sperm, histone transition rates decreased by an average of 27.2% in selected sperm, and the global methylation rate was 3.8% in unselected sperm and 3.3% in the selected sperm. DNA methylation and histone retention location profiling analyses suggested that these alterations displayed specific location patterns in the human genome. Changes in the pattern of hypomethylation largely occurred in transcriptional factor gene families such as HOX, FOX, and GATA. Histone retention increased in 67 genes, whereas it was significantly clustered in neural development-related gene families, particularly the olfactory sensor gene family. Although a causative relationship could not be established, the results of the present study suggest the possibility that sperm with good density also possess unique epigenetic profiles, particularly for genes involved in neural and olfactory development. As increasing evidence demonstrates that epigenetics plays a key role in embryonic development and offspring growth characteristics, the specific epigenetic alterations we observed in selected sperm may influence the transcriptional process and neural development in embryos.

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