In vitro susceptibility of nontuberculous mycobacteria in China.

OBJECTIVES: This study aimed to measure the prevalence of resistance to antimicrobial agents, and explore the risk factors associated with drug resistance by using nontuberculous Mycobacteria (NTM) isolates from China. METHODS: A total of 335 NTM isolates were included in our analysis. Broth dilution method was used to determine in vitro drug susceptibility of NTM isolates. RESULTS: Clarithromycin (CLA) was the most potent drug for Mycobacterium intracellulare (MI). The resistance rate of 244 MI isolates to CLA was 21%, yielding a minimum inhibitory concentrations (MIC)50 and MIC90 of 8 and 64 mg/L, respectively. 51% of 244 MI isolates exhibited resistance to amikacin (AMK). For 91 Mycobacterium abscessus complex (MABC) isolates, 6 (7%) and 49 (54%) isolates were categorized as resistant to CLA at day 3 and 14, respectively. The resistance rate to CLA for Mycobacterium abscessus subspecies abscessus (MAA) was dramatically higher than that for Mycobacterium abscessus subspecies massiliense (MAM). Additionally, the percentage of patients presenting fever in the CLA-susceptible group was significantly higher than that in the CLA-resistant group. CONCLUSIONS: Our data demonstrate that approximate one fifth of MI isolates are resistant to CLA. We have identified a higher proportion of CLA-resistant MAA isolates than MAM. The patients caused by CLA-resistant MI are at low risk for presenting with fever relative to CLA-susceptible group.

Real-time recombinase-aided amplification assay for rapid amplification of the IS1081 gene of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis (TB), is the leading cause of death due to a single infectious agent worldwide. Rapid and accurate diagnosis of MTB is critical for controlling TB especially in resource-limited countries, since any diagnosis delay increases the chances of transmission. Here, a real-time recombinase-aided amplification (RAA) assay targeting conserved positions in IS1081 gene of MTB, is successfully established to detect MTB. The intact workflow was completed within 30 min at 42 °C with no cross-reactivity observed for non-tuberculous mycobacteria and other clinical bacteria, and the detection limit for recombinant plasmid of MTB IS1081 was 163 copies/reaction at 95% probability, which was approximately 1.5-fold increase in analytical sensitivity for the detection of MTB, compared to conventional quantitative real-time PCR (qPCR; 244 copies/reaction). Furthermore, the result of clinical performance evaluation revealed an increased sensitivity of RAA assay relative to qPCR was majorly noted in the specimens with low bacteria loads. Our results demonstrate that the developed real-time RAA assay is a convenient, sensitive, and low-cost diagnostic tool for the rapid detection of MTB.

Iterative Methylation Leads to 3-Methylchuangxinmycin Production in Actinoplanes tsinanensis CPCC 200056.

A new congener of chuangxinmycin (CM) was identified from Actinoplanes tsinanensis CPCC 200056. Its structure was determined as 3-methylchuangxinmycin (MCM) by 1D and 2D NMR. MCM could be generated in vivo from CM by heterologous expression of the vitamin B12-dependent radical SAM enzyme CxnA/A1 responsible for methylation of 3-demethylchuangxinmycin (DCM) in CM biosynthesis, indicating that CxnA/A1 could perform iterative methylation for MCM production. In vitro assays revealed significant activities of CM, DCM, and MCM against Mycobacterium tuberculosis H37Rv and clinically isolated isoniazid/rifampin-resistant M. tuberculosis, suggesting that CM and its derivatives may have potential for antituberculosis drug development.

Rapid Mycobacterium abscessus antimicrobial susceptibility testing based on antibiotic treatment response mapping via Raman Microspectroscopy.

OBJECTIVES: Antimicrobial susceptibility tests (ASTs) are pivotal tools for detecting and combating infections caused by multidrug-resistant rapidly growing mycobacteria (RGM) but are time-consuming and labor-intensive. DESIGN: We used a Mycobacterium abscessus-based RGM model to develop a rapid (24-h) AST from the beginning of the strain culture, the Clinical Antimicrobials Susceptibility Test Ramanometry for RGM (CAST-R-RGM). The ASTs obtained for 21 clarithromycin (CLA)-treated and 18 linezolid (LZD)-treated RGM isolates. RESULTS: CAST-R-RGM employs D2O-probed Raman microspectroscopy to monitor RGM metabolic activity, while also revealing bacterial antimicrobial drug resistance mechanisms. The results of clarithromycin (CLA)-treated and linezolid (LZD)-treated RGM isolates exhibited 90% and 83% categorical agreement, respectively, with conventional AST results of the same isolates. Furthermore, comparisons of time- and concentration-dependent Raman results between CLA- and LZD-treated RGM strains revealed distinct metabolic profiles after 48-h and 72-h drug treatments, despite similar profiles obtained for both drugs after 24-h treatments. CONCLUSIONS: Ultimately, the rapid, accurate, and low-cost CAST-R-RGM assay offers advantages over conventional culture-based ASTs that warrant its use as a tool for improving patient treatment outcomes and revealing bacterial drug resistance mechanisms.

Effects of short-term -30° HDT on contrast sensitivity.

Potential cognitive and physiological alterations due to space environments have been investigated in long-term space flight and various microgravity-like conditions, for example, head-down tilt (HDT), confinement, isolation, and immobilization. However, little is known about the influence of simulated microgravity environments on visual function. Contrast sensitivity (CS), which indicates how much contrast a person requires to see a target, is a fundamental feature of human vision. Here, we investigated how the CS changed by 1-h -30° HDT and determined the corresponding mechanisms with a perceptual template model. A quick contrast sensitivity function procedure was used to assess the CS at ten spatial frequencies and three external noise levels. We found that (1) relative to the + 30° head-up tilt (HUT) position, 1-h -30° HDT significantly deteriorated the CS at intermediate frequencies when external noise was present; (2) CS loss was not detected in zero- or high-noise conditions; (3) HDT-induced CS loss was characterized by impaired perceptual template; and (4) self-reported questionnaires indicated that subjects felt less pleasure and more excitement, less comfort and more fatigued by screen light, less comfort in the area around the eye, and serious symptoms such as piercing pain, blur acid, strain, eye burning, and dizziness after HDT. These findings improve our understanding of the negative effects of simulated microgravity on visual function and elucidate the potential risks of astronauts during space flight.

Alleviated Anxiety Boosts Memory Training Gain in Older Adults with Subjective Memory Complaints: A Randomized Controlled Trial.

OBJECTIVE: Older adults with subjective memory complaints (SMC) have a higher risk of dementia and commonly demonstrate symptoms of depression and anxiety. The study aimed to examine the effect of a memory training program for individuals with SMC, and whether additional group counseling aimed at alleviating depression and anxiety would boost memory training gains. DESIGN: A three-armed, double-blind, randomized controlled trial. SETTING AND PARTICIPANTS: Community-dwelling older adults with SMC, age ≥60. METHODS: Participants (n = 124) were randomly assigned to memory training (MT), group counseling (GC), or GC + MT intervention. The GT + MT group received 3 sessions of group counseling followed by a 4-week memory training, while the MT group attended reading and memory training, and the GC group received group counseling and health lectures. Cognitive function and symptoms of depression and anxiety were assessed at baseline, mid-, and post-intervention. RESULTS: After group counseling, the GC + MT and GC groups showed reduced symptoms of anxiety compared to the MT group. Memory training enhanced associative learning in both MT and GC + MT groups compare with the GC group, but the GC + MT group demonstrated a larger memory improvement (Cohen's d = 0.57) than the MT group (Cohen's d = 0.44). CONCLUSION AND IMPLICATIONS: Group counseling decreased symptoms of anxiety, memory training increased associative learning, and the combination of two intervention induced larger memory gain than memory training alone. The results suggest that it may need to integrate treatment for anxiety into memory training for older adults with SMC to achieve better intervention effect. TRIAL REGISTRATION: ChiCTR-IOR-15006165 in the Chinese Clinical Trial Registry.

Determination of critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against para-aminosalicylic acid with clinical isolates with thyA, folC and dfrA mutations.

BACKGROUND & OBJECTIVES: Accurate determination of antimicrobial resistance profiles is of great importance to formulate optimal regimens against multidrug-resistant tuberculosis (MDR-TB). Although para-aminosalicylic acid (PAS) has been widely used clinically, the reliable testing methods for PAS susceptibility were not established. Herein, we aimed to establish critical test concentration for PAS on the Mycobacterial Growth Indicator Tube (MGIT) 960 in our laboratory settings. METHODS: A total of 102 clinical isolates were included in this study, including 82 wild-type and 20 resistotype isolates. Minimum inhibitory concentration (MIC) was determined by MGIT 960. Whole-genome sequencing was used to identify the mutation patterns potentially conferring PAS resistance. Sequence alignment and structure modelling were carried out to analyze potential drug-resistant mechanism of folC mutant. RESULTS: Overall, the Minimum inhibitory concentration (MIC) distribution demonstrated excellent separation between wild-type and resistotype isolates. The wild-type population were all at least 1 dilution below 4 µg/ml, and the resistotype population were no lower than 4 µg/ml, indicating that 4 µg/ml was appropriate critical concentration to separate these two populations. Of 20 mutant isolates, 12 (60.0%) harbored thyA mutations, 2 (10%) had a mutation on upstream of dfrA, and the remaining isolates had folC mutations. Overall, thyA and folC mutations were scattered throughout the whole gene without any one mutation predominating. All mutations within thyA resulted in high-level resistance to PAS (MIC > 32 µg/ml); whereas the MICs of isolates with folC mutations exhibited great diversity, ranged from 4 to > 32 µg/ml, and sequence and structure analysis partially provided the possible reasons for this diversity. CONCLUSIONS: We propose 4 µg/ml as tentative critical concentration for MGIT 960. The major mechanism of PAS resistance is mutations within thyA and folC in MTB isolations. The whole-gene deletion of thyA locus confers high-level resistance to PAS. The diversity of many distinct mutations scattered throughout the full-length folC gene challenges the PCR-based mutation analysis for PAS susceptibility.

Expression profiling of TRIM gene family reveals potential diagnostic biomarkers for rifampicin-resistant tuberculosis.

The epidemic of pulmonary tuberculosis (TB), especially rifampin-resistant tuberculosis (RR-TB) presents a major challenge for TB control today. However, there is a lack of reliable and specific biomarkers for the early diagnosis of RR-TB. We utilized reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to profile the transcript levels of 72 tripartite motif (TRIM) genes from a discovery cohort of 10 drug-sensitive tuberculosis (DS-TB) patients, 10 RR-TB patients, and 10 healthy controls (HCs). A total of 35 differentially expressed genes (DEGs) were screened out, all of which were down-regulated. The bio functions and pathways of these DEGs were enriched in protein ubiquitination, regulation of the viral process, Interferon signaling, and innate immune response, etc. A protein-protein interaction network (PPI) was constructed and analyzed using STRING and Cytoscape. Twelve TRIM genes were identified as hub genes, and seven (TRIM1, 9, 21, 32, 33, 56, 66) of them were verified by RT-qPCR in a validation cohort of 95 subjects. Moreover, we established the RR-TB decision tree models based on the 7 biomarkers. The receiver operating characteristic (ROC) analyses showed that the models exhibited the areas under the curve (AUC) values of 0.878 and 0.868 in discriminating RR-TB from HCs and DS-TB, respectively. Our study proposes potential biomarkers for RR-TB diagnosis, and also provides a new experimental basis to understand the pathogenesis of RR-TB.

Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking.

BACKGROUND: Herbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC-MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development. RESULTS: The genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by 1H NMR spectrum. CONCLUSIONS: A herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the "cryptic" herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.

The fronto-insular cortex causally mediates the default-mode and central-executive networks to contribute to individual cognitive performance in healthy elderly.

The triple network model that consists of the default-mode network (DMN), central-executive network (CEN), and salience network (SN) has been suggested as a powerful paradigm for investigation of network mechanisms underlying various cognitive functions and brain disorders. A crucial hypothesis in this model is that the fronto-insular cortex (FIC) in the SN plays centrally in mediating interactions between the networks. Using a machine learning approach based on independent component analysis and Bayesian network (BN), this study characterizes the directed connectivity architecture of the triple network and examines the role of FIC in connectivity of the model. Data-driven exploration shows that the FIC initiates influential connections to all other regions to globally control the functional dynamics of the triple network. Moreover, stronger BN connectivity between the FIC and regions of the DMN and the CEN, as well as the increased outflow connections from the FIC are found to predict individual performance in memory and executive tasks. In addition, the posterior cingulate cortex in the DMN was also confirmed as an inflow hub that integrates information converging from other areas. Collectively, the results highlight the central role of FIC in mediating the activity of large-scale networks, which is crucial for individual cognitive function.

Identification of urine biomarkers predictive of prolonged QTc interval in multidrug-resistant tuberculosis patients treated with bedaquiline.

The most frequent adverse event associated with bedaquiline (BDQ) is the QTc interval prolongation; however, there was no biomarkers that could be used to predict the occurrence of QTc prolongation in BDQ-treated patients. In this study, we employed the ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) to generate metabolic profiling for the discovery of potential predictive urine biomarkers of QTc prolongation in these patients. Untargeted metabolomic technique was used to concentrate the differential metabolic pathway, and targeted metabolomic technique was subsequently performed to identify predictive biomarkers for QTc prolongation. A total of 45 rifampicin-resistant TB (RR-TB) and multidrug-resistant TB (MDR-TB) patients were enrolled in our study, including 15 RR/MDR-TB patients with QTc interval prolongation (QIP) and 30 RR/MDR-TB patients with QTc interval un-prolongations (QIU). Untargeted technique revealed that the lipid metabolism was the most differential metabolic pathway between two groups. Further targeted technique identified four differential metabolites, including betaine, LPE (18:2), LPE (20:3), and LPE (20:4). The combined analysis of metabolisms revealed that the combined use of LPE (20:3) and LPE (20:4) had the best performance for predicting the occurrence of QTc prolongation in TB patients, yielding a sensitivity of 87.4% and a specificity of 78.5%. In addition, with the progression of BDQ treatment, the LPEs exhibited persistent difference in the BDQ-treated TB patients experiencing QTc interval prolongation. In conclusion, our data demonstrate that the combined use of LPE (20:3) and LPE (20:4) yields promising performance for predicting the occurrence of QTc interval prolongation in BDQ-treated patients.

Discovering common pathogenetic processes between tuberculosis and COVID-19 by bioinformatics and system biology approach.

Background: SARS-CoV-2, the cause of the COVID-19 pandemic, poses a significant threat to humanity. Individuals with pulmonary tuberculosis (PTB) are at increased risk of developing severe COVID-19, due to long-term lung damage that heightens their susceptibility to full-blown disease. Methods: Three COVID-19 datasets (GSE157103, GSE166253, and GSE171110) and one PTB dataset (GSE83456) were obtained from the Gene Expression Omnibus databases. Subsequently, data were subjected to weighted gene co-expression network analysis(WGCNA)followed by functional enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. These analyses revealed two overlapping disease-specific modules, each comprising co-regulated genes with potentially related biological functions. Using Cytoscape, we visualised the interaction network containing common disease-related genes found within the intersection between modules and predicted transcription factors (TFs). Real-time qPCR was conducted to quantify expression levels of these genes in blood samples from COVID-19 and PTB patients. Finally, DisGeNET and the Drug Signatures database were employed to analyze these common genes, unveiling their connections to clinical disease features and potential drug treatments. Results: Examination of the overlap between COVID-19 and PTB gene modules unveiled 11 common genes. Functional enrichment analyses using KEGG and GO shed light on potential functional relationships among these genes, providing insights into their potential roles in the heightened mortality of PTB patients due to SARS-CoV-2 infection. Furthermore, results of various bioinformatics-based analyses of common TFs and target genes led to identification of shared pathways and therapeutic targets for PTB patients with COVID-19, along with potential drug treatments for these patients. Conclusion: Our results unveiled a potential biological connection between COVID-19 and PTB, as supported by results of functional enrichment analysis that highlighted potential biological processes and signaling pathways shared by both diseases. Building on these findings, we propose potential drug treatments for PTB patients with COVID-19, pending verification of drug safety and efficacy through laboratory and multicentre studies before clinical use.

Antigen-specific chemokine profiles as biomarkers for detecting Mycobacterium tuberculosis infection.

Background: Latent tuberculosis (TB) infection can progress to active TB, which perpetuates community transmission that undermines global TB control efforts. Clinically, interferon-γ release assays (IGRAs) are commonly used for active TB case detection. However, low IGRA sensitivity rates lead to false-negative results for a high proportion of active TB cases, thus highlighting IGRA ineffectiveness in differentiating MTB-infected individuals from healthy individuals. Methods: Participants enrolled at Beijing Chest Hospital from May 2020-April 2022 were assigned to healthy control (HC), LTBI, IGRA-positive TB, and IGRA-negative TB groups. Screening cohort MTB antigen-specific blood plasma chemokine concentrations were measured using Luminex xMAP assays then were verified via testing of validation cohort samples. Results: A total of 302 individuals meeting study inclusion criteria were assigned to screening and validation cohorts. Testing revealed significant differences in blood plasma levels of CXCL9, CXCL10, CXCL16, CXCL21, CCL1, CCL19, CCL27, TNF-α, and IL-4 between IGRA-negative TB and HC groups. Levels of CXCL9, CXCL10, IL-2, and CCL8 biomarkers were predictive for active TB, as reflected by AUC values of ≥0.9. CXCL9-based enzyme-linked immunosorbent assay sensitivity and specificity rates were 95.9% (95%CI: 91.7-98.3) and 100.0% (92.7-100.0), respectively. Statistically similar AUC values were obtained for CXCL9 and CXCL9-CXCL10 assays, thus demonstrating that combined analysis of CXCL10 and CXCL9 levels did not improve active TB diagnostic performance. Conclusion: The MTB antigen stimulation-based CXCL9 assay may compensate for low IGRA diagnostic accuracy when used to diagnose IGRA-negative active TB cases and thus is an accurate and sensitive alternative to IGRAs for detecting MTB infection.

Joint spatial-temporal association of response codes (STEARC) effect: Mental timelines embodied interpersonally.

A co-action task was used to explore the effect of social interactions on temporal judgements, in comparison with an individual-task condition. In Experiment 1, the co-actors sat either individually (individual condition) or alongside a partner (joint condition) in front of a monitor and then responded to time-related words (e.g. yesterday, tomorrow). In Experiment 2, co-actors sat separately in front of two monitors and categorized the words either individually or jointly. Participants' response times to past- and future-related words in the individual conditions of both experiments had no significant difference. However, in the joint conditions, the responses were faster when the past-time words were mapped toward the participants on the left than when future-time words were mapped toward them. Our data support the existence of a specific mapping between past-time-left space and future-time-right space. This suggests that the two cooperators probably shared a similar mental timeline.

Serum Cytokine Biomarkers for Use in Diagnosing Pulmonary Tuberculosis versus Chronic Pulmonary Aspergillosis.

Background: Aspergillus fumigatus-induced chronic pulmonary aspergillosis (CPA), the most common pulmonary tuberculosis (TB) sequela, tends to occur after pulmonary infection with the intracellular pathogen Mycobacterium tuberculosis (Mtb). Timely and accurate detection of A. fumigatus infection of pulmonary TB patients would undoubtedly greatly improve patient prognosis. Currently, the galactomannan (GM) antigen test is commonly used to detect A. fumigatus infection but has poor sensitivity that renders this assay inadequate for use in clinical practice. Design or Methods: Given the fact CPA and TB induce different host immune responses, we evaluated serum cytokine level profiles of CPA, TB patients and patients with both diseases (CPA-TB) for multiple cytokines and cytokine combinations. Results: The results revealed significantly higher serum levels of numerous proinflammatory cytokines, including IL-1ß, IL-6, IL-8, IL-12p70, IFN-α, IFN-γ and TNF-α, in peripheral blood of CPA-TB patients versus that of TB patients. IL-8 levels alone provided the best discriminatory performance for distinguishing between TB and either CPA-TB patients (AUC = 0.949) or CPA patients (AUC = 0.964). Moreover, both IL-8 and TNF-α (AUC = 0.996) levels could be used to distinguish between TB and CPA-TB patients. Likewise, IL-8, TNF-α and IL-6 levels together could be used to distinguish between CPA-TB and TB patients. Conclusion: In this study, multiple cytokines were identified that may serve as potential biomarkers for use in detecting TB patients with CPA. Furthermore, our results should enhance understanding of how immune system dysfunctions influence susceptibility to Mtb and/or A. fumigatus infections.

Integrated bioinformatics analysis of dendritic cells hub genes reveal potential early tuberculosis diagnostic markers.

BACKGROUND: Dendritic cells (DCs) are most potent antigen-processing cells and play key roles in host defense against Mycobacterium tuberculosis (MTB) infection. In this study, hub genes in DCs during MTB infection were first investigated using bioinformatics approaches and further validated in Monocyte-derived DCs. METHODS: Microarray datasets were obtained from Gene Expression Omnibus (GEO) database. Principal component analysis (PCA) and immune infiltration analysis were performed to select suitable samples for further analysis. Differential analysis and functional enrichment analysis were conducted on DC samples, comparing live MTB-infected and non-infected (NI) groups. The CytoHubba plugin in Cytoscape was used to identify hub genes from the differentially expressed genes (DEGs). The expression of the hub genes was validated using two datasets and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in human monocyte-derived DCs. Enzyme-linked immunosorbent assay (ELISA) was used to validate interferon (IFN) secretion. Transcription factors (TFs) and microRNAs (miRNAs) that interact with the hub genes were predicted using prediction databases. The diagnostic value of the hub genes was evaluated using receiver operating characteristic (ROC) curves and area under the curve (AUC) values. RESULTS: A total of 1835 common DEGs among three comparison groups (18 h, 48 h, 72 h after MTB infection) were identified. Six DEGs (IFIT1, IFIT2, IFIT3, ISG15, MX1, and RSAD2) were determined as hub genes. Functions enrichment analysis revealed that all hub genes all related to IFN response. RT-qPCR showed that the expression levels of six hub genes were significantly increased after DC stimulated by live MTB. According to the results of ELISA, the secretion of IFN-γ, but not IFN-α/ß, was upregulated in MTB-stimulated DCs. AUC values of six hub genes ranged from 84 to 94% and AUC values of 5 joint indicators of two hub genes were higher than the two hub genes alone. CONCLUSION: The study identified 6 hub genes associated with IFN response pathway. These genes may serve as potential diagnostic biomarkers in tuberculosis (TB). The findings provide insights into the molecular mechanisms involved in the host immune response to MTB infection and highlight the diagnostic potential of these hub genes in TB.

Antimicrobial Effect of Oxazolidinones and Its Synergistic Effect with Bedaquiline Against Mycobacterium abscessus Complex.

Purpose: Unsatisfactory efficacies of currently recommended anti-Mycobacterium abscessus complex (MABC) treatment regimens have led to development of novel drugs to combat MABC infections. In this study, we evaluated in vitro antimicrobial activities of bedaquiline (BDQ) and four oxazolidinones against MABC isolates. Methods: The resazurin microplate assay was performed to determine minimum inhibitory concentrations (MICs) of BDQ and four oxazolidinones, including tedizolid (TZD), sutezolid (SZD), delpazolid (DZD), and linezolid (LZD), against 65 MABC isolates. A checkerboard method was used to investigate efficacies of various antimicrobial drug combinations. Results: BDQ MICs for MABC isolates ranged from <0.031 to 1 µg/mL, while MIC50 and MIC90 values were 0.125 µg/mL and 0.25 µg/mL, respectively. TZD MIC50 and MIC90 values for MABC isolates were 1 µg/mL and 4 µg/mL, respectively, which were fourfold lower than corresponding LZD values (P < 0.001). DZD MIC90 values for MABC isolates was 8 µg/mL, which were 0.5-fold lower than corresponding LZD values (P < 0.01). MICs of BDQ, SZD, and LZD for M. abscessus subspecies massiliense isolates were significantly lower than corresponding MICs for M. abscessus subspecies abscessus isolates (P < 0.05). Notably, use of oxazolidinones (DZD, SZD, LZD, or TZD) with BDQ against MABC isolates led to reduction of the oxazolidinone median MIC range from 4 to 0.125 µg/mL to 1-0.031 µg/mL. Conclusion: These results demonstrated excellent BDQ inhibitory activity against MABC isolates. TZD exhibited stronger antimicrobial efficacy against MABC isolates as compared to efficacies of DZD, SZD, and LZD. Importantly, MICs of oxazolidinones were markedly decreased when they were combined with BDQ, thus suggesting that combinations of BDQ and oxazolidinones may be effective treatments for MABC infections.

Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens.

Objective: Tuberculosis diagnosis requires rapid, simple and highly sensitive methods. Clustered regularly interspaced short palindromic repeats (CRISPRs) and associated protein (Cas) systems are increasingly being used for clinical diagnostic applications, due to their high flexibility, sensitivity and specificity. We developed a sensitive Mycobacterium tuberculosis (MTB) complex polymerase chain reaction (PCR)-CRISPR/Cas13a detection method (CRISPR-MTB) and then evaluated its performance in detecting MTB in clinical specimens. Methods: The conserved MTB IS1081 sequence was used to design CRISPR-derived RNAs (crRNAs) and T7 promoter sequencing-containing PCR primers for use in the CRISPR-MTB assay, then assay performance was evaluated using 401 clinical specimens. Results: The CRISPR-MTB assay provided a low limit of detection of 1 target sequence copy/µL and excellent specificity. Furthermore, use of the assay to detect MTB in bronchoalveolar lavage fluid (BALF), sputum and pus samples provided superior sensitivity (261/268, 97.4%) as compared to sensitivities of acid-fast bacilli (130/268, 48.5%) and mycobacterial culture (192/268, 71.6%) assays, and comparable or greater sensitivity to that of GeneXpert MTB/RIF (260/268, 97.0%). Conclusion: The CRISPR-MTB assay, which provides excellent sensitivity and specificity for MTB detection in sputum, BALF and pus samples, is a viable alternative to conventional tests used to diagnose TB in resource-limited settings.

Attenuated Cytokine-Induced Memory-Like Natural Killer Cell Responses to Mycobacterium tuberculosis in Tuberculosis Patients.

Purpose: This study aimed to investigate the phenotype, proliferation and functional alterations of cytokine-induced memory-like natural killer (CIML NK) cells from healthy subjects and TB patients, and assessed the efficacy of CIML NK cells in response to H37Rv-infected U937 cells in vitro. Methods: Fresh peripheral blood mononuclear cells (PBMCs) were isolated from healthy people and tuberculosis patients and activated for 16h using low-dose IL-15, or IL-12, IL-15, IL-18 combination or IL-12, IL-15, IL-18 and MTB H37Rv lysates, respectively, followed by low-dose IL-15 maintenance for another 7 days. Then, the PBMCs were co-cultured with K562 and H37Rv-infected U937, and the purified NK cells were co-cultured with H37Rv infected U937. The phenotype, proliferation and response function of CIML NK cells were assessed using flow cytometry. Finally, colony forming units were enumerated to confirm the survival of intracellular MTB. Results: CIML NK phenotypes from TB patients were similar to healthy controls. CIML NK cells undergo higher rates of proliferation after IL-12/15/18 pre-activation. Moreover, the poor expansion potential of CIML NK cells co-stimulated with MTB lysates. CIML NK cells from healthy individuals showed enhanced IFN-γ functional to H37Rv infected U937 cells, along with significantly enhanced killing of H37Rv. However, the CIML NK cells from TB patients show attenuated IFN-γ production and now enhanced the ability of killing intracellular MTB compared to those from healthy donors after co-cultured with H37Rv infected U937. Conclusion: CIML NK cells from healthy individuals exist the increased ability of IFN-γ secretion and boosted anti-MTB activity in vitro, which from TB patients show impaired IFN-γ production and no enhanced anti-MTB activity compared to those from healthy donors. Additionally, we observe the poor expansion potential of CIML NK cells co-stimulated with antigens from MTB. These results open up new possibilities for NK cell-based anti-tuberculosis immunotherapeutic strategies.

An interaction effect of external noise and exposure duration on the spatial contrast sensitivity function.

It has been demonstrated that contrast sensitivity is sensitive to stimulus exposure duration. Here, we investigated how the duration effect on contrast sensitivity was modulated by the spatial frequency and intensity of external noise. Through a contrast detection task, the contrast sensitivity function under 10 spatial frequencies, three external noise, and two exposure duration conditions was measured. The temporal integration effect was defined by the difference in contrast sensitivity or the area under the log contrast sensitivity function between short and long exposure durations. We found that (1) the temporal integration effect was less pronounced in the zero-noise condition than in the low- or high-noise condition; (2) in the zero-noise condition, a stronger temporal integration effect was observed at high spatial frequencies; (3) in the high-noise condition, a stronger temporal integration effect was observed at low spatial frequencies; (4) the spatial-frequency-dependent transient or sustained mechanism is also sensitive to external noise level; and (5) perceptual template model analysis revealed that both decreased additive internal noise and an improved perceptual template accounted for the temporal integration effect, and these two factors were tuned to spatial frequency.