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.

Correlation of serum 25-hydroxyvitamin D3 with chronic inflammation and insulin resistance in polycystic ovarian syndrome patients / 中国医师进修杂志

Objective:To discuss the correlation of serum 25-hydroxyvitamin D3[25(OH)D3] with chronic inflammation and insulin resistance (IR) in polycystic ovarian syndrome (PCOS) patients.Methods:One hundred and twenty-four PCOS patients registered from January 2018 to January 2020 in the Second Affiliated Hospital of Hebei North University were selected retrospectively. According to the difference of body mass index (BMI), the patients were divided into PCOS 1 group (BMI<25 kg/m 2, 64 cases) and PCOS 2 group (BMI≥25 kg/m 2, 60 cases). At the same time, 60 patients with simple obesity were selected as the obesity group and 58 healthy subjects were selected as the control group. The somatology indicators, gonadal hormone, serum 25(OH)D3, insulin resistance (IR) related index and chronic inflammation factors were measured, the correlations of serum 25(OH)D3 with relevant indicators were analyzed by Pearson correlation analysis. Results:The BMI, waist hip ratio, testosterone(T), luteinizing hormone (LH) / follicle-stimulating hormone (FSH), free androgen index(FAI), fasting insulin (FINS), fasting plasma glucose (FPG), insulin resistance index (HOMA-IR), insulin sensitivity index (ISI) in the four groups had significant differences ( P<0.05); the level of 25(OH)D3 in the PCOS 1 group was lower than that in the PCOS 2 group: (1.14 ± 0.36) nmol/L vs. (1.83 ± 0.25) nmol/L, P<0.05; the levels of FINS, HOMA-IR in the PCOS 2 group were higher than those in the PCOS 1 group, obesity group and control group: (13.26 ± 2.61) mg/L vs. (5.58 ± 1.03), (6.63 ± 1.42), (4.66 ± 0.85) mg/L, 1.49 ± 0.37 vs. 1.15 ± 0.20, 1.12 ± 0.22, 0.96 ± 0.11, P<0.05; the level of ISI in the PCOS 2 group was lower than that in the PCOS1 group, obesity group and control group: - 4.19 ± 0.78 vs. - 3.52 ± 0.74, - 3.23 ± 0.53, - 3.06 ± 0.54, P<0.05. The levels of interleukin-6(IL-6), transforming growth factor-β(TGF-β), tumor necrosis factor-α(TNF-α) in the four groups had significant differences ( P<0.05); the level of IL-6 in the PCOS 2 group was higher than that in the PCOS 1 group: (18.15 ± 4.93) ng/L vs. (14.77 ± 4.58) ng/L, P<0.05. The results of Pearson correlation analysis showed that the serum of 25(OH)D3 had negative correlation with IL-6, BMI, waist hip ratio, T, FINS, ISI, TGF-β and TNF-α( r = - 0.582, - 0.242, - 0.371, - 0.203, - 0.208, - 0.267, - 0.723, - 0.617, P<0.05). Conclusions:Serum 25(OH)D3 is correlated with chronic inflammation and IR, and involved into the genesis and progression of PCOS.

Ketogenic diet-mediated steroid metabolism reprogramming improves the immune microenvironment and myelin growth in spinal cord injury rats according to gene and co-expression network analyses.

The ketogenic diet has been widely used in the treatment of various nervous system and metabolic-related diseases. Our previous research found that a ketogenic diet exerts a protective effect and promotes functional recovery after spinal cord injury. However, the mechanism of action is still unclear. In this study, different dietary feeding methods were used, and myelin expression and gene level changes were detected among different groups. We established 15 RNA-seq cDNA libraries from among 4 different groups. First, KEGG pathway enrichment of upregulated differentially expressed genes and gene set enrichment analysis of the ketogenic diet and normal diet groups indicated that a ketogenic diet significantly improved the steroid anabolic pathway in rats with spinal cord injury. Through cluster analysis, protein-protein interaction analysis and visualization of iPath metabolic pathways, it was determined that Sqle, Sc5d, Cyp51, Dhcr24, Msmo1, Hsd17b7, and Fdft1 expression changed significantly. Second, through weighted gene co-expression network analysis showed that rats fed a ketogenic diet showed a significant reduction in the expression of genes involved in immune-related pathways, including those associated with immunity and infectious diseases. A ketogenic diet may improve the immune microenvironment and myelin growth in rats with spinal cord injury through reprogramming of steroid metabolism.

α-Synuclein in traumatic and vascular diseases of the central nervous system.

α-Synuclein (α-Syn) is a small, soluble, disordered protein that is widely expressed in the nervous system. Although its physiological functions are not yet fully understood, it is mainly involved in synaptic vesicle transport, neurotransmitter synthesis and release, cell membrane homeostasis, lipid synthesis, mitochondrial and lysosomal activities, and heavy metal removal. The complex and inconsistent pathological manifestations of α-Syn are attributed to its structural instability, mutational complexity, misfolding, and diverse posttranslational modifications. These effects trigger mitochondrial dysfunction, oxidative stress, and neuroinflammatory responses, resulting in neuronal death and neurodegeneration. Several recent studies have discovered the pathogenic roles of α-Syn in traumatic and vascular central nervous system diseases, such as traumatic spinal cord injury, brain injury, and stroke, and in aggravating the processes of neurodegeneration. This review aims to highlight the structural and pathophysiological changes in α-Syn and its mechanism of action in traumatic and vascular diseases of the central nervous system.

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.

Transcriptomic analysis of α-synuclein knockdown after T3 spinal cord injury in rats.

BACKGROUND: Endogenous α-synuclein (α-Syn) is involved in many pathophysiological processes in the secondary injury stage after acute spinal cord injury (SCI), and the mechanism governing these functions has not been thoroughly elucidated to date. This research aims to characterize the effect of α-Syn knockdown on transcriptional levels after SCI and to determine the mechanisms underlying α-Syn activity based on RNA-seq. RESULT: The establishment of a rat model of lentiviral vector-mediated knockdown of α-Syn in Sprague-Dawley rats with T3 spinal cord contusion (LV_SCI group). The results of the RNA-seq analysis showed that there were 337 differentially expressed genes (DEGs) between the SCI group and the LV_SCI group, and 153 DEGs specific to LV_SCI between the (SCI vs LV_SCI) and (SCI vs CON) comparisons. The top 20 biological transition terms were identified by Gene ontology (GO) analysis. The Kyoto Gene and Genomic Encyclopedia (KEGG) analysis showed that the LV_SCI group significantly upregulated the cholinergic synaptic & nicotine addiction and the neuroactive ligand receptor interaction signaling pathway. Enriched chord analysis analyzes key genes. Further cluster analysis, gene and protein interaction network analysis and RT-qPCR results showed that Chrm2 and Chrnb2 together significantly in both pathways. The proliferation of muscarinic cholinergic receptor subtype 2 (Chrm2) and nicotinic cholinergic receptor subtype ß2 (Chrnb2), and the neurogenesis were elevated in the injury site of LV_SCI group by immunofluorescence. Further by subcellular localization, the LV_SCI group enhanced the expression of Chrnb2 at the cell membrane. CONCLUSION: Knockdown of α-Syn after SCI enhance motor function and promote neurogenesis probably through enhancing cholinergic signaling pathways and neuroreceptor interactions. This study not only further clarifies the understanding of the mechanism of knockdown of α-Syn on SCI but also helps to guide the treatment strategy for SCI.

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.

MicroRNA-219 Inhibits Proliferation and Induces Differentiation of Oligodendrocyte Precursor Cells after Contusion Spinal Cord Injury in Rats.

MicroRNA-219 (miR-219) regulates the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) during central nervous system (CNS) development. OPCs only differentiate into oligodendrocytes (OLs) in the healthy CNS, but can generate astrocytes (As) after injury. We hypothesized that miR-219 may modulate OPC proliferation and differentiation in a cervical C5 contusion spinal cord injury (SCI) model. After injury, we observed a decrease in the miR-219 level and quantity of OLs and an increase in the number of OPCs and As. Silencing of miR-219 by its antagomir in vivo produced similar results, but of greater magnitude. Overexpression of miR-219 by its agomir in vivo increased the number of OLs and suppressed generation of OPCs and As. Luxol fast blue staining confirmed that SCI caused demyelination and that the extent of demyelination was attenuated by miR-219 overexpression, but aggravated by miR-219 reduction. Monocarboxylate transporter 1 (MCT-1) may be implicated in the regulation of OPC proliferation and differentiation mediated by miR-219 following contusion SCI. Collectively, our data suggest that miR-219 may mediate SCI-induced OPC proliferation and differentiation, and MCT-1 may participate in this process as a target of miR-219.

Lentivirus-mediated downregulation of α-synuclein reduces neuroinflammation and promotes functional recovery in rats with spinal cord injury.

BACKGROUND: The prognosis of spinal cord injury (SCI) is closely related to secondary injury, which is dominated by neuroinflammation. There is evidence that α-synuclein aggregates after SCI and that inhibition of α-synuclein aggregation can improve the survival of neurons after SCI, but the mechanism is still unclear. This study was designed to investigate the effects of α-synuclein on neuroinflammation after SCI and to determine the underlying mechanisms. METHOD: A T3 spinal cord contusion model was established in adult male Sprague-Dawley rats. An SNCA-shRNA-carrying lentivirus (LV-SNCA-shRNA) was injected into the injury site to block the expression of α-synuclein (forming the SCI+KD group), and the SCI and sham groups were injected with an empty vector. Basso-Beattie-Bresnahan (BBB) behavioural scores and footprint analysis were used to detect motor function. Inflammatory infiltration and myelin loss were measured in the spinal cord tissues of each group by haematoxylin-eosin (HE) and Luxol Fast Blue (LFB) staining, respectively. Immunohistochemistry, Western blot analysis, and RT-qPCR were used to analyse protein expression and transcription levels in the tissues. Immunofluorescence was used to determine the morphology and function of glial cells and the expression of matrix metalloproteinase-9 in the central canal of the spinal cord. Finally, peripheral serum cytokine levels were determined by enzyme-linked immunosorbent assay. RESULTS: Compared with the SCI group, the SCI+KD group exhibited reduced inflammatory infiltration, preserved myelin, and functional recovery. Specifically, the early arrest of α-synuclein inhibited the pro-inflammatory factors IL-1ß, TNF-α, and IL-2 and increased the expression of the anti-inflammatory factors IL-10, TGF-ß, and IL-4. The neuroinflammatory response was regulated by reduced proliferation of Iba1+ microglia/macrophages and promotion of the shift of M1-polarized Iba1+/iNOS+ microglia/macrophages to M2-polarized Iba1+/Arg1+ microglia/macrophages after injury. In addition, compared with the SCI group, the SCI+KD group also exhibited a smaller microglia/astrocyte (Iba1/GFAP) immunostaining area in the central canal, lower MMP-9 expression, and improved cerebrospinal barrier function. CONCLUSION: Lentivirus-mediated downregulation of α-synuclein reduces neuroinflammation, improves blood-cerebrospinal barrier function, promotes functional recovery, reduces microglial activation, and promotes the polarization of M1 microglia/macrophages to an M2 phenotype to confer a neuroprotective immune microenvironment in rats with SCI.

Ketogenic diet attenuates oxidative stress and inflammation after spinal cord injury by activating Nrf2 and suppressing the NF-κB signaling pathways.

Oxidative stress and inflammation are two key secondary pathological mechanisms following spinal cord injury (SCI). Ketogenic diet (KD) and its metabolite ß-hydroxybutyrate have been found to exhibit anti-oxidative and anti-inflammatory properties both in rats with SCI and in healthy rats; however, the underlying mechanisms are not yet fully understood. We investigated the effects of KD on the suppression of oxidative stress and inflammation, activation of nuclear factor-E2 related factor 2 (Nrf2), and inhibition of the nuclear factor-κB (NF-κB) signaling pathway in rats with SCI. We assessed functional recovery and evaluated the status of oxidative stress and inflammation using tests of superoxide dismutase and myeloperoxidase activity. We further assessed the presence of the proinflammatory cytokines tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interferon γ (IFN-γ) by ELISA. Western blotting was used to detect Nrf2 and NF-κB pathway-associated proteins in spinal cord tissue. Finally, we measured the levels of the NF-κB downstream genes TNF-α, IL-1ß, and IFN-γ by western blotting and real-time quantitative PCR. Following SCI, KD improved functional recovery, attenuated oxidative stress and inflammation, and induced Nrf2 activation. In addition, KD suppressed the NF-κB pathway and the expression of TNF-α, IL-1ß, and IFN-γ. Together, these findings provide new insight into the underlying regulatory mechanisms of KD.

[Effect of Interferon Combined with Thalidomid on HEL Cell Apoptosis and JAK2V617F Mutation Gene Expression].

OBJECTIVE: To investigate the effects of interferon-α2b combined with thalidomid(THD) on the proliferation and apoptosis of HEL cells, and expression of JAK2V617F-mutated gene. METHODS: The cell survival rates were assayed by CCK-8 after HEL cells were treated by interferon-α2b, thalidomid and thalidomid combined with interferon-α2b for 24, 48 and 72 hours, while the apoptosis and expression of JAK2V617F mutation gene were detected by flow cytometry and fluorescence quantitative PCR respectively after treatmemt for 48 hours. RESULTS: IFN-α2b combined with THD could more obviously inhibit the proliferation of HEL cells than IFN-α2b or THD alone for 24 and 48 and 72 hours, and the differences between groups were statistically significant(P<0.05). In addition, the apoptosis-inducing effect of IFN-α2b combined with THD on HEL cells was more obvious than that of IFN-α2b used alone, the differences was statistically significant(P<0.05). Although IFN-α2b combined with THD can decrease the JAK2V617F mutation gene expression more obviously than IFN-α2b alone,but the difference was no statistically significant (P>0.05). And the remaining groups showed statistically significance(P<0.05). CONCLUSION: IFN-α2b combined with THD can obviously inhibit the proliferation and induce apoptosis of HEL cells more than that of IFN-α2b alone, but the effect of reducing JAK2V617F mutation gene expression is not different.

Effect of Interferon Combined with Thalidomid on HEL Cell Apoptosis and JAK2V617F Mutation Gene Expression / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of interferon-α2b combined with thalidomid(THD) on the proliferation and apoptosis of HEL cells, and expression of JAK2V617F-mutated gene.</p><p><b>METHODS</b>The cell survival rates were assayed by CCK-8 after HEL cells were treated by interferon-α2b, thalidomid and thalidomid combined with interferon-α2b for 24, 48 and 72 hours, while the apoptosis and expression of JAK2V617F mutation gene were detected by flow cytometry and fluorescence quantitative PCR respectively after treatmemt for 48 hours.</p><p><b>RESULTS</b>IFN-α2b combined with THD could more obviously inhibit the proliferation of HEL cells than IFN-α2b or THD alone for 24 and 48 and 72 hours, and the differences between groups were statistically significant(P<0.05). In addition, the apoptosis-inducing effect of IFN-α2b combined with THD on HEL cells was more obvious than that of IFN-α2b used alone, the differences was statistically significant(P<0.05). Although IFN-α2b combined with THD can decrease the JAK2V617F mutation gene expression more obviously than IFN-α2b alone,but the difference was no statistically significant (P>0.05). And the remaining groups showed statistically significance(P<0.05).</p><p><b>CONCLUSION</b>IFN-α2b combined with THD can obviously inhibit the proliferation and induce apoptosis of HEL cells more than that of IFN-α2b alone, but the effect of reducing JAK2V617F mutation gene expression is not different.</p>

Volar locking plate versus external fixation for the treatment of unstable distal radial fractures: a meta-analysis of randomized controlled trials.

BACKGROUND: The choice between volar locking plates (VLP) and external fixation (EF) for unstable distal radius fractures have not reached a consensus. The meta-analysis of randomized controlled trials was performed to compare VLP with EF to determine the dominant strategy. MATERIALS AND METHODS: Meta-analysis was performed with a systematic search of studies conducted by using the PubMed, Embase, and Cochrane Central Register of Controlled Trials databases. The randomized controlled trials that compared VLP with EF was identified. Characteristics, functional outcomes, radiological results, and complications were manually extracted from all the selected studies. RESULTS: Six studies encompassing 445 patients met the inclusion criteria. There was significant difference between two procedures in disabilities of the arm shoulder and hand scores at 3,6, and 12 mo, visual analogue scale at 6 mo, grip strength at 3 mo, supination at 3 and 6 mo, extension at 3 mo, ulnar variance at 12 mo, and reoperation rate at 12 mo, postoperatively. However, there was no significant difference between flexion, pronation, radial deviation, and ulnar deviation at all follow-up points postoperatively and overall complications at 12 mo, postoperatively. CONCLUSIONS: EF had less reoperative rate due to complications, however, VLP had advantages in functional recovery in the early period after surgery, but two methods had similar functional recovery at 12 mo, postoperatively. Clinician should make the treatment decision with great caution for the patients who sustained unstable distal radial fractures.

LPS induces cardiomyocyte injury through calcium-sensing receptor.

Calcium-sensing receptor (CaSR) belongs to the family C of G-protein coupled receptors. We have previously demonstrated that CaSR could induce apoptosis of cultured neonatal rat ventricular cardiomyocytes in simulated ischemia/reperfusion. It remains unknown whether the CaSR has function in lipopolysaccharide (LPS)-induced myocardial injure. The aim of this study was to investigate whether the CaSR plays a role in LPS-induced myocardial injury. Cultured neonatal rat cardiomyocytes were treated with LPS, with or without pretreatment with the CaSR-specific agonist gadolinium chloride (GdCl3) or the CaSR-specific antagonist NPS2390. Release of TNF-α and IL-6 from cardiomyocytes was observed. Levels of malonaldehyde (MDA), lactate dehydrogenase (LDH), and activity of superoxide dismutase (SOD) were measured. In addition, apoptosis of the cardiomyocytes, [Ca(2+)]i and level of CaSR expression were determined. The results showed that LPS increased cardiomyocytes apoptosis, [Ca(2+)]i, MDA, LDH, TNF-α, IL-6 release, and CaSR protein expression. Compared with LPS treatment alone, pretreatment with GdCl3 further increased apoptosis of cardiomyocytes, MDA, LDH, TNF-α, IL-6 release, [Ca(2+)]i, and the expression of the CaSR protein. Conversely, pretreatment with NPS2390 decreased apoptosis of cardiomyocytes, MDA, LDH, TNF-α, IL-6 release, [Ca(2+)]i and the expression of the CaSR protein. These results demonstrate that LPS could induce cardiomyocyte injury. Moreover, LPS-induced cardiomyocyte injury was related to CaSR-mediated cardiomyocytes apoptosis, TNF-α, IL-6 release, and increase of intracellular calcium.