A new paradigm for generating high-quality cardiac pacemaker cells from mouse pluripotent stem cells.

Cardiac biological pacing (BP) is one of the future directions for bradyarrhythmias intervention. Currently, cardiac pacemaker cells (PCs) used for cardiac BP are mainly derived from pluripotent stem cells (PSCs). However, the production of high-quality cardiac PCs from PSCs remains a challenge. Here, we developed a cardiac PC differentiation strategy by adopting dual PC markers and simulating the developmental route of PCs. First, two PC markers, Shox2 and Hcn4, were selected to establish Shox2:EGFP; Hcn4:mCherry mouse PSC reporter line. Then, by stepwise guiding naïve PSCs to cardiac PCs following naïve to formative pluripotency transition and manipulating signaling pathways during cardiac PCs differentiation, we designed the FSK method that increased the yield of SHOX2+; HCN4+ cells with typical PC characteristics, which was 12 and 42 folds higher than that of the embryoid body (EB) and the monolayer M10 methods respectively. In addition, the in vitro cardiac PCs differentiation trajectory was mapped by single-cell RNA sequencing (scRNA-seq), which resembled in vivo PCs development, and ZFP503 was verified as a key regulator of cardiac PCs differentiation. These PSC-derived cardiac PCs have the potential to drive advances in cardiac BP technology, help with the understanding of PCs (patho)physiology, and benefit drug discovery for PC-related diseases as well.

SALL2 regulates neural differentiation of mouse embryonic stem cells through Tuba1a.

The spalt (Sal) gene family has four members (Sall1-4) in vertebrates, all of which play pivotal roles in various biological processes and diseases. However, the expression and function of SALL2 in development are still less clear. Here, we first charted SALL2 protein expression pattern during mouse embryo development by immunofluorescence, which revealed its dominant expression in the developing nervous system. With the establishment of Sall2 deficient mouse embryonic stem cells (ESCs), the in vitro neural differentiation system was leveraged to interrogate the function of SALL2, which showed impaired neural differentiation of Sall2 knockout (KO) ESCs. Furthermore, neural stem cells (NSCs) could not be derived from Sall2 KO ESCs and the generation of neural tube organoids (NTOs) was greatly inhibited in the absence of SALL2. Meanwhile, transgenic expression of E1 isoform of SALL2 restored the defects of neural differentiation in Sall2 KO ESCs. By chromatin immunoprecipitation sequencing (ChIP-seq), Tuba1a was identified as downstream target of SALL2, whose function in neural differentiation was confirmed by rescuing neural phenotypes of Sall2 KO ESCs when overexpressed. In sum, by elucidating SALL2 expression dynamics during early mouse development and mechanistically characterizing its indispensable role in neural differentiation, this study offers insights into SALL2's function in human nervous system development, associated pathologies stemming from its mutations and relevant therapeutic strategy.

A Profile of Drug-Resistant Mutations in Mycobacterium tuberculosis Isolates from Guangdong Province, China.

Guangdong Province, China's largest economy, has a high incidence of tuberculosis (TB). At present, there are few reports on the distribution, transmission and drug resistance of Mycobacterium tuberculosis (Mtb) strains in this region. In this study, we performed minimum inhibitory concentration testing for 14 anti-TB drugs and whole-genome sequencing of 713 clinical Mtb isolates from 20,662 sputum culture-positive tuberculosis patients registered at 31 tuberculosis drug resistance surveillance sites covering 20 cities in Guangdong Province from 2016 to 2018. Moreover, we evaluated genome-wide associations between mutations and drug resistance, and further investigated the differences in the MICs of mutations. The epidemiology, drug-resistant phenotypes and whole genome sequencing data of 713 clinical Mtb isolates were analyzed, revealing the lineage distribution and drug-resistant gene profiles in Guangdong Province. WGS combined with quantitative MIC measurements identified several novel loci associated with resistance, of which 16 loci were found to be related to resistance to more than one drug. This study analyzed the lineage distribution, prevalence characteristics and resistance-corresponding gene profiles of Mtb isolates in Guangdong province, and provided a theoretical basis for the formulation of tuberculosis prevention and control policy in the province. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01236-3.

Coarse particulate air pollution and mortality in a multidrug-resistant tuberculosis cohort.

RATIONALE: The association between ambient coarse particulate matter (PM2.5-10) and mortality in multi-drug resistant tuberculosis (MDR-TB) patients has not yet been studied. The modifying effects of temperature and humidity on this association are completely unknown. OBJECTIVES: To evaluate the effects of long-term PM2.5-10 exposures, and their modifications by temperature and humidity on mortality among MDR-TB patients. METHODS: A Chinese cohort of 3469 MDR-TB patients was followed up from diagnosis until death, loss to follow-up, or the study's end, averaging 2567 days per patient. PM2.5-10 concentrations were derived from the difference between PM10 and PM2.5. Cox proportional hazard models estimated hazard ratios (HRs) per 3.74 µg/m3 (interquartile range, IQR) exposure to PM2.5-10 and all-cause mortality for the full cohort and individuals at distinct long-term and short-term temperature and humidity levels, adjusting for other air pollutants and potential covariates. Exposure-response relationships were quantified using smoothed splines. RESULTS: Hazard ratios of 1.733 (95% CI, 1.407, 2.135) and 1.427 (1.114, 1.827) were observed for mortality in association with PM2.5-10 exposures for the full cohort under both long-term and short-term exposures to temperature and humidity. Modifying effects by temperature and humidity were heterogenous across sexes, age, treatment history, and surrounding environment measured by greenness and nighttime light levels. Nonlinear exposure-response curves suggestes a cumulative risk of PM2.5-10-related mortality starting from a low exposure concentration around 15 µg/m3. CONCLUSION: Long-term exposure to PM2.5-10 poses significant harm among MDR-TB patients, with effects modified by temperature and humidity. Immediate surveillance of PM2.5-10 is crucial to mitigate the progression of MDR-TB severity, particularly due to co-exposures to air pollution and adverse weather conditions.

Biosafe cerium oxide nanozymes protect human pluripotent stem cells and cardiomyocytes from oxidative stress.

BACKGROUND: Cardiovascular diseases (CVDs) have the highest mortality worldwide. Human pluripotent stem cells (hPSCs) and their cardiomyocyte derivatives (hPSC-CMs) offer a valuable resource for disease modeling, pharmacological screening, and regenerative therapy. While most CVDs are linked to significant over-production of reactive oxygen species (ROS), the effects of current antioxidants targeting excessive ROS are limited. Nanotechnology is a powerful tool to develop antioxidants with improved selectivity, solubility, and bioavailability to prevent or treat various diseases related to oxidative stress. Cerium oxide nanozymes (CeONZs) can effectively scavenge excessive ROS by mimicking the activity of endogenous antioxidant enzymes. This study aimed to assess the nanotoxicity of CeONZs and their potential antioxidant benefits in stressed human embryonic stem cells (hESCs) and their derived cardiomyocytes (hESC-CMs). RESULTS: CeONZs demonstrated reliable nanosafety and biocompatibility in hESCs and hESC-CMs within a broad range of concentrations. CeONZs exhibited protective effects on the cell viability of hESCs and hESC-CMs by alleviating excessive ROS-induced oxidative stress. Moreover, CeONZs protected hESC-CMs from doxorubicin (DOX)-induced cardiotoxicity and partially ameliorated the insults from DOX in neonatal rat cardiomyocytes (NRCMs). Furthermore, during hESCs culture, CeONZs were found to reduce ROS, decrease apoptosis, and enhance cell survival without affecting their self-renewal and differentiation potential. CONCLUSIONS: CeONZs displayed good safety and biocompatibility, as well as enhanced the cell viability of hESCs and hESC-CMs by shielding them from oxidative damage. These promising results suggest that CeONZs may be crucial, as a safe nanoantioxidant, to potentially improve the therapeutic efficacy of CVDs and be incorporated into regenerative medicine.

HAND factors regulate cardiac lineage commitment and differentiation from human pluripotent stem cells.

BACKGROUND: Transcription factors HAND1 and HAND2 (HAND1/2) play significant roles in cardiac organogenesis. Abnormal expression and deficiency of HAND1/2 result in severe cardiac defects. However, the function and mechanism of HAND1/2 in regulating human early cardiac lineage commitment and differentiation are still unclear. METHODS: With NKX2.5eGFP H9 human embryonic stem cells (hESCs), we established single and double knockout cell lines for HAND1 and HAND2, respectively, whose cardiomyocyte differentiation efficiency could be monitored by assessing NKX2.5-eGFP+ cells with flow cytometry. The expression of specific markers for heart fields and cardiomyocyte subtypes was examined by quantitative PCR, western blot and immunofluorescence staining. Microelectrode array and whole-cell patch clamp were performed to determine the electrophysiological characteristics of differentiated cardiomyocytes. The transcriptomic changes of HAND knockout cells were revealed by RNA sequencing. The HAND1/2 target genes were identified and validated experimentally by integrating with HAND1/2 chromatin immunoprecipitation sequencing data. RESULTS: Either HAND1 or HAND2 knockout did not affect the cardiomyocyte differentiation kinetics, whereas depletion of HAND1/2 resulted in delayed differentiation onset. HAND1 knockout biased cardiac mesoderm toward second heart field progenitors at the expense of first heart field progenitors, leading to increased expression of atrial and outflow tract cardiomyocyte markers, which was further confirmed by the appearance of atrial-like action potentials. By contrast, HAND2 knockout cardiomyocytes had reduced expression of atrial cardiomyocyte markers and displayed ventricular-like action potentials. HAND1/2-deficient hESCs were more inclined to second heart field lineage and its derived cardiomyocytes with atrial-like action potentials than HAND1 single knockout during differentiation. Further mechanistic investigations suggested TBX5 as one of the downstream targets of HAND1/2, whose overexpression partially restored the abnormal cardiomyocyte differentiation in HAND1/2-deficient hESCs. CONCLUSIONS: HAND1/2 have specific and redundant roles in cardiac lineage commitment and differentiation. These findings not only reveal the essential function of HAND1/2 in cardiac organogenesis, but also provide important information on the pathogenesis of HAND1/2 deficiency-related congenital heart diseases, which could potentially lead to new therapeutic strategies.

The profile of genome-wide DNA methylation, transcriptome, and proteome in streptomycin-resistant Mycobacterium tuberculosis.

Streptomycin-resistant (SM-resistant) Mycobacterium tuberculosis (M. tuberculosis) is a major concern in tuberculosis (TB) treatment. However, the mechanisms underlying streptomycin resistance remain unclear. This study primarily aimed to perform preliminary screening of genes associated with streptomycin resistance through conjoint analysis of multiple genomics. Genome-wide methylation, transcriptome, and proteome analyses were used to elucidate the associations between specific genes and streptomycin resistance in M. tuberculosis H37Rv. Methylation analysis revealed that 188 genes were differentially methylated between the SM-resistant and normal groups, with 89 and 99 genes being hypermethylated and hypomethylated, respectively. Furthermore, functional analysis revealed that these 188 differentially methylated genes were enriched in 74 pathways, with most of them being enriched in metabolic pathways. Transcriptome analysis revealed that 516 genes were differentially expressed between the drug-resistant and normal groups, with 263 and 253 genes being significantly upregulated and downregulated, respectively. KEGG analysis indicated that these 516 genes were enriched in 79 pathways, with most of them being enriched in histidine metabolism. The methylation level was negatively related to mRNA abundance. Proteome analysis revealed 56 differentially expressed proteins, including 14 upregulated and 42 downregulated proteins. Moreover, three hub genes (coaE, fadE5, and mprA) were obtained using synthetic analysis. The findings of this study suggest that an integrated DNA methylation, transcriptome, and proteome analysis can provide important resources for epigenetic studies in SM-resistant M. tuberculosis H37Rv.

Realization and Control of Bulk and Surface Modes in 3D Nanomagnonic Networks by Additive Manufacturing of Ferromagnets.

The high-density integration in information technology fuels the research on functional 3D nanodevices. Particularly ferromagnets promise multifunctional 3D devices for nonvolatile data storage, high-speed data processing, and non-charge-based logic operations via spintronics and magnonics concepts. However, 3D nanofabrication of ferromagnets is extremely challenging. In this work, an additive manufacturing methodology is reported, and unprecedented 3D ferromagnetic nanonetworks with a woodpile-structure unit cell are fabricated. The collective spin dynamics (magnons) at frequencies up to 25 GHz are investigated by Brillouin Light Scattering (BLS) microscopy and micromagnetic simulations. A clear discrepancy of about 10 GHz is found between the bulk and surface modes, which are engineered by different unit cell sizes in the Ni-based nanonetworks. The angle- and spatially-dependent modes demonstrate opportunities for multi-frequency signal processing in 3D circuits via magnons. The developed synthesis route will allow one to create 3D magnonic crystals with chiral unit cells, which are a prerequisite toward surface modes with topologically protected properties.

Gibberellin biosynthesis is required for CPPU-induced parthenocarpy in melon.

Spraying N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU), an exogenous cytokinin (CK) growth regulator, is the conventional method for inducing fruit set during melon (Cucumis melo L.) production; however, the mechanism by which CPPU induces fruit set is unclear. Through histological and morphological observations, fruit size was comparable between CPPU-induced fruits and normal pollinated fruits because CPPU-induced fruits had higher cell density but smaller cell size compared with normal pollinated fruits. CPPU promotes the accumulation of gibberellin (GA) and auxin and decreases the level of abscisic acid (ABA) during fruit set. Moreover, application of the GA inhibitor paclobutrazol (PAC) partially inhibits CPPU-induced fruit set. Transcriptome analysis revealed that CPPU-induced fruit set specifically induced the GA-related pathway, in which the key synthase encoding gibberellin 20-oxidase 1 (CmGA20ox1) was specifically upregulated. Further study indicated that the two-component response regulator 2 (CmRR2) of the cytokinin signaling pathway, which is highly expressed at fruit setting, positively regulates the expression of CmGA20ox1. Collectively, our study determined that CPPU-induced melon fruit set is dependent on GA biosynthesis, providing a theoretical basis for the creation of parthenocarpic melon germplasm.

The gut microbiota mediates protective immunity against tuberculosis via modulation of lncRNA.

The gut-lung axis has been implicated as a potential therapeutic target in lung disorders. While increasing evidence suggests that gut microbiota plays a critical role in regulating host immunity and contributing to tuberculosis (TB) development and progression, the underlying mechanisms whereby gut microbiota may impact TB outcomes are not fully understood. Here, we found that broad-spectrum antibiotics treatment increased susceptibility to Mycobacterium tuberculosis (M. tuberculosis) infection and modulated pulmonary inflammatory responses in mouse M. tuberculosis infection model. We then identified a commensal gut bacteria-regulated lncRNA, termed lncRNA-CGB, which was down-regulated by dysbiosis of gut microbiota during TB infection. Furthermore, we found that Bacteroides fragilis (B. fragilis) was a direct regulator of lncRNA-CGB, and oral administration of B. fragilis enhanced expression of lncRNA-CGB and promoted anti-TB immunity. Genomic knock-out of lncRNA-CGB led to reduced IFN-γ expression and impaired anti-TB immunity, therefore leading to detrimental effects on M. tuberculosis infection. Mechanistically, lncRNA-CGB interacted with EZH2 and negatively regulated H3K27 tri-methylation (H3K27Me3) epigenetic programming, leading to enhanced IFN-γ expression. Thus, this work not only uncovered previously unrecognized importance of gut bacteria-lncRNA-EZH2-H3K27Me3 axis in conferring immune protection against TB but also identified a potential new paradigm to develop a microbiota-based treatment against TB and potentially other diseases.

Allogeneic Vγ9Vδ2 T-Cell Therapy Promotes Pulmonary Lesion Repair: An Open-Label, Single-Arm Pilot Study in Patients With Multidrug-Resistant Tuberculosis.

The WHO's "Global tuberculosis report 2020" lists tuberculosis (TB) as one of the leading causes of death globally. Existing anti-TB therapy strategies are far from adequate to meet the End TB Strategy goals set for 2035. Therefore, novel anti-TB therapy protocols are urgently needed. Here, we proposed an allogeneic Vγ9Vδ2 T-cell-based immunotherapy strategy and clinically evaluated its safety and efficacy in patients with multidrug-resistant TB (MDR-TB). Eight patients with MDR-TB were recruited in this open-label, single-arm pilot clinical study. Seven of these patients received allogeneic Vγ9Vδ2 T-cell therapy adjunct with anti-TB drugs in all therapy courses. Cells (1 × 108) were infused per treatment every 2 weeks, with 12 courses of cell therapy conducted for each patient, who were then followed up for 6 months to evaluate the safety and efficacy of cell therapy. The eighth patient initially received four courses of cell infusions, followed by eight courses of cell therapy plus anti-MDR-TB drugs. Clinical examinations, including clinical response, routine blood tests and biochemical indicators, chest CT imaging, immune cell surface markers, body weight, and sputum Mycobacterium tuberculosis testing, were conducted. Our study revealed that allogeneic Vγ9Vδ2 T cells are clinically safe for TB therapy. These cells exhibited clinical efficacy in multiple aspects, including promoting the repair of pulmonary lesions, partially improving host immunity, and alleviating M. tuberculosis load in vivo, regardless of their application in the presence or absence of anti-TB drugs. This pilot study opens a new avenue for anti-TB treatment and exhibits allogeneic Vγ9Vδ2 T cells as promising candidates for developing a novel cell drug for TB immunotherapy. Clinical Trial Registration: (https://clinicaltrials.gov/ct2/results?cond=&term=NCT03575299&cntry=&state=&city=&dist=) ( NCT03575299).

Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of Mycobacterium smegmatis Leading to Increased Vancomycin Resistance.

In recent years, the treatment of tuberculosis is once again facing a severe situation because the existing antituberculosis drugs have become weaker and weaker with the emergence of drug-resistant Mycobacterium tuberculosis (Mtb). The studies of cell division and cell cycle-related factors in Mtb are particularly important for the development of new drugs with broad-spectrum effects. Mycobacterium smegmatis (Msm) has been used as a model organism to study the molecular, physiological, and drug-resistant mechanisms of Mtb. Bioinformatics analysis has predicted that MSMEG_6171 is a MinD-like protein of the septum site-determining protein family associated with cell division in Mycobacterium smegmatis. In our study, we use ultrastructural analysis, proteomics, metabolomics, and molecular biology techniques to comprehensively investigate the function of MSMEG_6171. Overexpression of MSMEG_6171 in Msm resulted in elongated cells, suggesting an important role of MSMEG_6171 in regulating cell wall morphology. The MSMEG_6171 overexpression could enhance the bacterial resistance to vancomycin, ethionamide, meropenem, and cefamandole. The MSMEG_6171 overexpression could alter the lipid metabolism of Msm to cause the changes on cellular biofilm property and function, which enhances bacterial resistance to antibiotics targeting cell wall synthesis. MSMEG_6171 could also induce the glyceride and phospholipid alteration in vivo to exhibit the pleiotropic phenotypes and various cellular responses. The results showed that amino acid R249 in MSMEG_6171 was a key site that can affect the level of bacterial drug resistance, suggesting that ATPase activity is required for function.

Giant Piezospintronic Effect in a Noncollinear Antiferromagnetic Metal.

One of the main bottleneck issues for room-temperature antiferromagnetic spintronic devices is the small signal read-out owing to the limited anisotropic magnetoresistance in antiferromagnets. However, this could be overcome by either utilizing the Berry-curvature-induced anomalous Hall resistance in noncollinear antiferromagnets or establishing tunnel-junction devices based on effective manipulation of antiferromagnetic spins. In this work, the giant piezoelectric strain modulation of the spin structure and the anomalous Hall resistance in a noncollinear antiferromagnetic metal-D019 hexagonal Mn3 Ga-is demonstrated. Furthermore, tunnel-junction devices are built with a diameter of 200 nm to amplify the maximum tunneling resistance ratio to more than 10% at room-temperature, which thus implies significant potential of noncollinear antiferromagnets for large signal-output and high-density antiferromagnetic spintronic device applications.

Anomalous Hall Effect, Robust Negative Magnetoresistance, and Memory Devices Based on a Noncollinear Antiferromagnetic Metal.

We report the successful fabrication of noncollinear antiferromagnetic D019 Mn3Ge thin films on insulating oxide substrates. The anomalous Hall effect and the large parallel negative magnetoresistance that is robust up to 53 T are observed in the thin films, which may provide evidence for the recent theoretical prediction of the existence of Weyl fermions in antiferromagnetic Mn3Ge. More importantly, we integrate the Mn3Ge thin films onto ferroelectric PMN-PT substrates and manipulate the longitudinal resistance reversibly by electric fields at room temperature, demonstrating the anisotropic magnetoresistance effect in noncollinear antiferromagnets, which thus illustrates the potential of antiferromagnetic Mn3Ge for information storage applications.

Electric-Field-Controlled Antiferromagnetic Spintronic Devices.

In recent years, the field of antiferromagnetic spintronics has been substantially advanced. Electric-field control is a promising approach for achieving ultralow power spintronic devices via suppressing Joule heating. Here, cutting-edge research, including electric-field modulation of antiferromagnetic spintronic devices using strain, ionic liquids, dielectric materials, and electrochemical ionic migration, is comprehensively reviewed. Various emergent topics such as the Néel spin-orbit torque, chiral spintronics, topological antiferromagnetic spintronics, anisotropic magnetoresistance, memory devices, 2D magnetism, and magneto-ionic modulation with respect to antiferromagnets are examined. In conclusion, the possibility of realizing high-quality room-temperature antiferromagnetic tunnel junctions, antiferromagnetic spin logic devices, and artificial antiferromagnetic neurons is highlighted. It is expected that this work provides an appropriate and forward-looking perspective that will promote the rapid development of this field.

Potential Genes Related to Levofloxacin Resistance in Mycobacterium tuberculosis Based on Transcriptome and Methylome Overlap Analysis.

Drug-resistant Mycobacterium tuberculosis (M. tuberculosis) has become an increasingly serious public health problem and has complicated tuberculosis (TB) treatment. Levofloxacin (LOF) is an ideal anti-tuberculosis drug in clinical applications. However, the detailed molecular mechanisms of LOF-resistant M. tuberculosis in TB treatment have not been revealed. Our study performed transcriptome and methylome sequencing to investigate the potential biological characteristics of LOF resistance in M. tuberculosis H37Rv. In the transcriptome analysis, 953 differentially expressed genes (DEGs) were identified; 514 and 439 DEGs were significantly downregulated and upregulated in the LOF-resistant group and control group, respectively. The KEGG pathway analysis revealed that 97 pathways were enriched in this study. In the methylome analysis, 239 differentially methylated genes (DMGs) were identified; 150 and 89 DMGs were hypomethylated and hypermethylated in the LOF-resistant group and control group, respectively. The KEGG pathway analysis revealed that 74 pathways were enriched in this study. The overlap study suggested that 25 genes were obtained. It was notable that nine genes expressed downregulated mRNA and upregulated methylated levels, including pgi, fadE4, php, cyp132, pckA, rpmB1, pfkB, acg, and ctpF, especially cyp132, pckA, and pfkB, which were vital in LOF-resistant M. tuberculosis H37Rv. The overlapping genes between transcriptome and methylome could be essential for studying the molecular mechanisms of LOF-resistant M. tuberculosis H37Rv. These results may provide informative evidence for TB treatment with LOF.

CRISPR/Cas12a-based biosensing platform for precise and efficient screening of CRISPR/Cas9-induced biallelic mutants.

CRISPR/Cas9 is a robust tool to manipulate genes in a wide range of species. Although several methods are introduced to identify the CRISPR/Cas9-induced mutations, they are labor-intensive, costly, and not easy to use or were sequence-limited. Moreover, few of them could identify the biallelic mutants that are the desired outcomes of targeted mutagenesis. Recently, a CRISPR/Cas12a-mediated biosensing platform was developed to detect nucleic acids based on the collateral DNA cleavage activity of Cas12a; it was highly sensitive, specific, rapid, and cost-efficient for genotyping, mutation detection, and single nucleotide polymorphism (SNP) identification, thereby deeming it as an innovative method for screening the CRISPR/Cas9-induced biallelic mutants. Thus, the CRISPR/Cas12a-based biosensing platform has been successfully utilized for screening 23 CRISPR/Cas9-induced biallelic mutants in Thp-1 cells, which were also confirmed by direct sequencing and ELISA. The precision and efficiency of CRISPR/Cas12a-based biosensing platform make it a promising tool for screening of CRISPR/Cas9-induced biallelic mutants in the future.

Anti-tuberculosis (TB) chemotherapy dynamically rescues Th1 and CD8+ T effector levels in Han Chinese pulmonary TB patients.

CD4+/CD8+ T cells play a major role in conferring immune protection against tuberculosis (TB), but it remains unknown how the immune responses of CD4+/CD8+ T cells exactly correlate with the clinical variables and disease statuses during anti-TB chemotherapy. To address this, several major immune parameters of CD4+/CD8+ T cells in peripheral blood derived from pulmonary TB patients and healthy volunteers were evaluated. We observed that active TB infection induced lower CD3+ T cell and CD4+ T cell levels but higher CD8+T cell levels, while anti-TB chemotherapy reversed these effects. Also, anti-TB treatment induced enhanced production of IL-2 and IFN-γ but reduced expression of IL-10 and IL-6. Moreover, the dynamic changes of CD3, CD4, and CD8 levels did not show a significant association with sputum smear positivity. However, the frequencies of IL-2+CD4+ or IL-10 + CD4+ T effector subpopulation or IL-1ß production in peripheral blood showed significant difference between patients positive for sputum smear and patients negative for sputum smear after anti-TB treatment. These findings implicated that recovery of Th1/CD8+T cell effector levels might be critical immunological events in pulmonary TB patients after treatment and further suggested the importance of these immunological parameters as potential biomarkers for prediction of TB progress and prognosis.

Genome-wide DNA methylation and transcriptome and proteome changes in Mycobacterium tuberculosis with para-aminosalicylic acid resistance.

Previous studies have reported that genome-wide DNA methylation and differentially expressed genes and proteins are closely associated with drug resistance in Mycobacterium tuberculosis (M. tuberculosis). However, no reports have explored such associations in para-aminosalicylic acid (PAS)-resistant M. tuberculosis H37Rv. Here, we investigated genome-wide methylation and transcriptome and proteome changes to explore the associations between specific genes and PAS resistance in M. tuberculosis H37Rv. The results revealed that 1,388 differentially methylated (1,161 hypermethylated and 227 hypomethylated) genes, 214 significantly differentially expressed (103 up- and 111 down-regulated) genes and 137 differentially expressed (48 up- and 89 down-regulated) proteins were regulated by PAS in M. tuberculosis H37Rv. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that metabolic pathways and ABC transporters were closely associated with differentially methylated and expressed genes, respectively. In addition, correlation analysis revealed that differentially methylated genes were negatively correlated with their transcriptional levels in PAS-resistant M. tuberculosis H37Rv. Furthermore, the existence of five hypermethylated candidate genes (esxC, fabG3, fbpB, papA1 and pks2) in PAS-resistant M. tuberculosis H37Rv was verified using protein-protein interaction analysis in the STRING database. The integrated DNA methylation and transcriptome and proteome analysis could provide valuable resources for epigenetics studies in PAS-resistant M. tuberculosis H37Rv.

Multi-omics comparisons of p-aminosalicylic acid (PAS) resistance in folC mutated and un-mutated Mycobacterium tuberculosis strains.

p-Aminosalicylic acid (PAS) is an important second-line antibiotic for treating multidrug-resistant tuberculosis (MDR-TB). Due to gastrointestinal disturbance and intolerance, its potent and efficacy in the treatment of extensively drug-resistant (XDR)-TB commonly are poor. Thus, it is important to reveal the mechanism of susceptibility and resistance of Mycobacterium tuberculosis (Mtb) to this drug. Herein, we screened and established PAS-resistant (PASr) folC mutated and un-mutated Mtb strains, then utilized a multi-omics (genome, proteome, and metabolome) analysis to better characterize the mechanisms of PAS resistance in Mtb. Interestingly, we found that promotion of SAM-dependent methyltransferases and suppression of PAS uptake via inhibiting some drug transport associated membrane proteins were two key pathways for the folC mutated strain evolving into the PASr Mtb strain. However, the folC un-mutated strain was resistant to PAS via uptake of exogenous methionine, mitigating the role of inhibitors, and promoting DfrA, ThyA and FolC expression. Beyond these findings, we also found PAS resistance in Mtb might be associated with the increasing phenylalanine metabolism pathway. Collectively, our findings uncovered the differences of resistant mechanism between folC mutated and un-mutated Mtb strains resistant to PAS using multi-omics analysis and targeting modulators to these pathways may be effective for treatment of PASr Mtb strains.