Mycobacterium Tuberculosis H37Ra Research Articles

Critical view on antimicrobial, antibiofilm and cytotoxic activities of quinazolin-4(3H)-one derived schiff bases and their Cu(II) complexes

A series of nine 2,3-disubstituted-quinazolin-4(3H)-one derived Schiff bases and their three Cu(II) complexes was prepared and tested for their antimicrobial activities against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 and resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). All the substances were tested in vitro against Mycobacterium tuberculosis H37Ra ATCC 25177, M. kansasii DSM 44162 and M. smegmatis ATCC 700084. While anti-enterococcal and antimycobacterial activities were insignificant, 3-[(E)-(2-hydroxy-5-nitrobenzylidene)amino]-2-(2-hydroxy-5-nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (SB3) and its Cu(II) complex (SB3–Cu) demonstrated bacteriostatic antistaphylococcal activity. In addition, both compounds, as well as the other two prepared complexes, showed antibiofilm activity, which resulted in a reduction of biofilm formation and eradication of mature S. aureus biofilm by 80% even at concentrations lower than the values of their minimum inhibitory concentrations. In addition, the compounds were tested for their cytotoxic effect on the human monocytic leukemia cell line THP-1. The antileukemic efficiency was improved by the preparation of Cu(II) complexes from the corresponding non-chelated Schiff base ligands.

Fluvastatin Converts Human Macrophages into Foam Cells with Increased Inflammatory Response to Inactivated Mycobacterium tuberculosis H37Ra.

Cholesterol biosynthesis inhibitors (statins) protect hypercholesterolemic patients against developing active tuberculosis, suggesting that these drugs could help the host to control the pathogen at the initial stages of the disease. This work studies the effect of fluvastatin on the early response of healthy peripheral blood mononuclear cells (PBMCs) to inactivated Mycobacterium tuberculosis (Mtb) H37Ra. We found that in fluvastatin-treated PBMCs, most monocytes/macrophages became foamy cells that overproduced NLRP3 inflammasome components in the absence of immune stimulation, evidencing important cholesterol metabolism/immunity connections. When both fluvastatin-treated and untreated PBMCs were exposed to Mtb H37Ra, a small subset of macrophages captured large amounts of bacilli and died, concentrating the bacteria in necrotic areas. In fluvastatin-untreated cultures, most of the remaining macrophages became epithelioid cells that isolated these areas of cell death in granulomatous structures that barely produced IFNγ. By contrast, in fluvastatin-treated cultures, foamy macrophages surrounded the accumulated bacteria, degraded them, markedly activated caspase-1 and elicited a potent IFNγ/cytotoxic response. In rabbits immunized with the same bacteria, fluvastatin increased the tuberculin test response. We conclude that statins may enhance macrophage efficacy to control Mtb, with the help of adaptive immunity, offering a promising tool in the design of alternative therapies to fight tuberculosis.

Expanding the Chemical Diversity of Grisechelins via Heterologous Expression.

Thiazole scaffold-based small molecules exhibit a range of biological activities and play important roles in drug discovery. Based on bioinformatics analysis, a putative biosynthetic gene cluster (BGC) for thiazole-containing compounds was identified from Streptomyces sp. SCSIO 40020. Heterologous expression of this BGC led to the production of eight new thiazole-containing compounds, grisechelins E, F, and I-N (1, 2, 5-10), and two quinoline derivatives, grisechelins G and H (3 and 4). The structures of 1-10, including their absolute configurations, were elucidated by HRESIMS, NMR spectroscopic data, ECD calculations, and single-crystal X-ray diffraction analysis. Grisechelin F (2) is a unique derivative, distinguished by the presence of a salicylic acid moiety. The biosynthetic pathway for 2 was proposed based on bioinformatics analysis and in vivo gene knockout experiments. Grisechelin E (1) displayed moderate antimycobacterial activity against Mycobacterium tuberculosis H37Ra (MIC of 8 μg mL-1).

Application of the Hollow-Fiber Infection Model to Personalized Precision Dosing of Isoniazid in a Clinical Setting.

The hollow-fiber infection model (HFIM) is a valuable tool for evaluating pharmacokinetics/pharmacodynamics relationships and determining the optimal antibiotic dose in monotherapy or combination therapy, but the application for personalized precision medicine in tuberculosis treatment remains limited. This study aimed to evaluate the efficacy of adjusted antibiotic doses for a tuberculosis patient using HFIM. Model-based Bayesian forecasting was utilized to assess the proposed reduction of the isoniazid dose from 300 mg daily to 150 mg daily in a patient with an ultra-slow-acetylation phenotype. The efficacy of the adjusted 150-mg dose was evaluated in a time-to-kill assay performed using the bacterial isolate Mycobacterium tuberculosis (Mtb) H37Ra in a HFIM that mimicked the individual pharmacokinetic profile of the patient. The isoniazid concentration observed in the HFIM adequately reflected the target drug exposures simulated by the model. After 7 days of repeated dose administration, isoniazid killed 4 log10 Mtb CFU/mL in the treatment arm, while the control arm without isoniazid increased 1.6 log10 CFU/mL. Our results provide an example of the utility of the HFIM for predicting the efficacy of specific recommended doses of anti-tuberculosis drugs in real clinical setting.

Synthesis and Structural Activity Relationship Study of Ursolic Acid Derivatives as Antitubercular Agent.

The chemical transformation of ursolic acid (UA) into novel C-3 aryl ester derivatives and in vitro and silico assessment of their antitubercular potential. UA is a natural pentacyclic triterpenoid with many pharmacological properties. Semisynthetic UA analogs have demonstrated enhanced anticancer, antimalarial, and antifilarial properties in our previous studies. The C-30 carboxylic group of previously isolated UA was protected, and various C-3 aryl ester derivatives were semi-synthesized. The agar dilution method was used to evaluate the in vitro antitubercular efficacy of Mycobacterium tuberculosis (Mtb) H37Ra. In silico docking studies of the active derivative were carried out against Mtb targets, catalase peroxidase (PDB: 1SJ2), dihydrofolate reductase (PDB: 4M2X), enoyl-ACP reductase (PDB: 4TRO), and cytochrome bc1 oxidase (PDB: 7E1V). The derivative 3-O-(2-amino,3-methyl benzoic acid)-ethyl ursolate (UA-1H) was the most active among the eight derivatives (MIC1 2.5 µg/mL) against Mtb H37Ra. Also, UA-1H demonstrated significant binding affinity in the range of 10.8-11.4 kcal/mol against the antiTb target proteins, which was far better than the positive control Isoniazid, Ethambutol, and co-crystallized ligand (HEM). Moreover, the predicted hit UA-1H showed no inhibition of Cytochrome P450 2D6 (CYP2D6), suggesting its potential for favorable metabolism in Phase I clinical studies. The ursolic acid derivative UA-1H possesses significant in vitro antitubercular potential with favorable in silico pharmacokinetics. Hence, further in vivo assessments are suggested for UA-1H for its possible development into a secure and efficient antitubercular drug.

Lanostane triterpenoids from artificially cultivated fruiting bodies of Ganoderma wiiroense

Seven undescribed lanostane triterpenoids (1–7) were isolated from artificially cultivated fruiting bodies of Ganoderma wiiroense, strain TBRC-BCC 60613. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. The C-23 absolute configuration of 1 was determined to be 23 S using the modified Mosher’s method. The isolated compounds were evaluated for their antitubercular and antimalarial activities. Compound 7 showed activity against Mycobacterium tuberculosis H37Ra (MIC 50 μg/ml).

Antimycobacterial Activities of Hydroxamic Acids and Their Iron(II/III), Nickel(II), Copper(II) and Zinc(II) Complexes.

Hydroxamic acid (HA) derivatives display antibacterial and antifungal activities. HA with various numbers of carbon atoms (C2, C6, C8, C10, C12 and C17), complexed with different metal ions, including Fe(II/III), Ni(II), Cu(II) and Zn(II), were evaluated for their antimycobacterial activities and their anti-biofilm activities. Some derivatives showed antimycobacterial activities, especially in biofilm growth conditions. For example, 20-100 µM of HA10Fe2, HA10FeCl, HA10Fe3, HA10Ni2 or HA10Cu2 inhibited Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium marinum biofilm development. HA10Fe2, HA12Fe2 and HA12FeCl could even attack pre-formed Pseudomonas aeruginosa biofilms at higher concentrations (around 300 µM). The phthiocerol dimycocerosate (PDIM)-deficient Mycobacterium tuberculosis H37Ra was more sensitive to the ion complexes of HA compared to other mycobacterial strains. Furthermore, HA10FeCl could increase the susceptibility of Mycobacterium bovis BCG to vancomycin. Proteomic profiles showed that the potential targets of HA10FeCl were mainly related to mycobacterial stress adaptation, involving cell wall lipid biosynthesis, drug resistance and tolerance and siderophore metabolism. This study provides new insights regarding the antimycobacterial activities of HA and their complexes, especially about their potential anti-biofilm activities.

Herp regulates intracellular survival of Mycobacterium tuberculosis H37Ra in macrophages by regulating reactive oxygen species-mediated autophagy.

Several studies have suggested that endoplasmic reticulum (ER) stress is important in the pathogenesis of infectious diseases; however, the precise function of ER stress regulation and the role of Herp as a regulator in Mtb H37Ra-induced ER stress remain elusive. Therefore, our study investigated ER stress and autophagy associated with Herp expression in Mycobacterium tuberculosis-infected macrophages to determine the role of Herp in the pathogenesis of tuberculosis.

Malate:quinone oxidoreductase knockout makes Mycobacterium tuberculosis susceptible to stress and affects its in vivo survival

Mycobacterium tuberculosis H37Ra (Mtb-Ra) ORF MRA_2875, annotated as malate:quinone oxidoreductase (mqo), is thought to have a role in TCA cycle in converting malate to oxaloacetate. To study its physiological relevance, we developed mqo knockout (KO) in Mtb-Ra. A KO complemented (KOC) strain was also developed by complementing the KO with mqo over-expressing construct. Under normal in vitro conditions, KO does not show any growth defect but showed reduced CFU burden in macrophages and in mice lungs. In vitro studies with KO showed reduced fitness under oxidative and low pH stress, and also increased susceptibility to levofloxacin and D-cycloserine. Transcript analysis of mqo showed increased expression levels under oxidative and low pH stress. This is the first study to show physiological relevance of mqo encoded by MRA_2875 in Mtb-Ra under oxidative and low pH stress. In summary, the present study shows that MRA_2875 encoded malate:quinone oxidoreductase is a functional enzyme which contributes to oxidative stress and low pH tolerance, and survival in macrophages and in mice.

Design, Synthesis, and in vitro Biological Evaluation of ROS‐Generating Phenanthridin‐trione‐Epoxide Conjugates as Agents against Mycobacterium tuberculosis

AbstractTuberculosis (TB) is one of the devasting infectious diseases and continues to spread among people despite having several specific drugs. Total eradication of TB is one of the shared interests of both the World Health Organization (WHO) and India globally. A library of antitubercular 6‐((1‐(aryl/heteroaryl)‐1H‐1,2,3‐triazol‐4‐yl)methyl)oxireno[2,3‐b] phenanthridine‐5,7,9(6H,7aH,8aH)‐trione (6 a–e) has been prepared in five steps including click chemistry and tested against active and dormant strains of Mycobacterium tuberculosis H37Ra using XRMA protocol. The result showed the inhibitory potential of 6 d IC50 at 0.74 μg/mL concentration against active strain and at 0.9 μg/mL against the dormant strain of Mtb. ROS generating ability of the compounds has been confirmed by luminol, H2O2, and glutathione assays. The molecular docking with the thioredoxin protein of Mtb showed a docking score of −9.6 Kcal/mol. To understand the mechanism with the thioredoxin protein of Mtb, the adduct formation of compounds with cystine was confirmed with HPLC. The involvement of lead molecules with existing drugs can be helpful in the eradication of tuberculosis.

Comparative interleukins and chemokines analysis of mice mesenchymal stromal cells infected with Mycobacterium tuberculosis H37Rv and H37Ra

Inflammatory pathways involving Mesenchymal stromal cells (MSCs) play an important role in Mycobacterium tuberculosis (Mtb) infection. H37Rv (Rv) is a standard virulent strain, however, H37Ra (Ra) is a strain with reduced virulence. Interleukins and chemokines production are known to promote inflammation resistance in mammalian cells and is recently reported to regulate mycobacterial immunopathogenesis via inflammatory responses. MSCs are very important cells during Mtb infection. However, the different expressions of interleukins and chemokines in the process of Mtb-infected MSCs between Ra and Rv remain unclear. We used the techniques of RNA-Seq, qRT-PCR, ELISA, and Western Blotting. We have shown that Rv infection significantly increased mRNA expressions of Mndal, Gdap10, Bmp2, and Lif, thereby increasing more differentiation of MSCs compared with Ra infection in MSCs. Further investigation into the possible mechanisms, we found that Rv infection enhanced more inflammatory response (Mmp10, Mmp3, and Ptgs2) through more activation of the TLR2-MAP3K1-JNK pathway than did Ra infection in MSCs. Further action showed that Rv infection enhanced more Il1α, Il6, Il33, Cxcl2, Ccl3, and Ackr3 production than did Ra infection. Rv infection showed more expressions of Mmp10, Mmp3, Ptgs2, Il1α, Il6, Il33, Cxcl2, Ccl3, and Ackr3 possibly through more active TLR2-MAP3K1-JNK pathway than did Ra infection in MSCs. MSCs may therefore be a new candidate for the prevention and treatment of tuberculosis.

Chemical analysis and antitubercular activity evaluation of the dried mycelial powders of the basidiomycete Ganoderma australe TBRC-BCC 22314

The isolation of lanostane triterpenoids possessing significant anti-tuberculosis (anti-TB) activity from mycelial cultures of the basidiomycete Ganoderma australe strain TBRC-BCC 22314 was previously reported. To demonstrate the potential of the dried mycelial powder for utilization in anti-TB medicinal products, its authentic chemical analysis was performed. Considering the possibility of the changes in the lanostane compositions and anti-TB activity by sterilization, both autoclave treated and non-autoclaved mycelial powder materials were chemically investigated. The study led to the identification of the lanostanes responsible for the activity of the mycelial extract against Mycobacterium tuberculosis H37Ra. The anti-TB activity of the extracts from autoclaved and non-autoclaved mycelial powders were the same (MIC 3.13 μg/mL). However, the analytical results revealed several unique chemical conversions of the lanostanes under the sterilization conditions. The most potent major lanostane, ganodermic acid S (1), was shown to be significantly active also against the extensively drug-resistant (XDR) strains of M. tuberculosis.

GlfT1 down-regulation affects Mycobacterium tuberculosis biofilm formation and its in-vitro and in-vivo survival

Mycobacterial galactan biosynthesis is critical for cell viability and growth, therefore an effort was made to study galactofuranosyl transferase 1, encoded by MRA_3822 in Mycobacterium tuberculosis H37Ra (Mtb-Ra). Galactofuranosyl transferases are involved in the biosynthesis of mycobacterial cell wall galactan chain and have been shown to be essential for in-vitro growth of Mycobacterium tuberculosis. In Mtb-Ra and Mycobacterium tuberculosis H37Rv (Mtb-Rv), two galactofuranosyl transferases are present, GlfT1 acts as initiator of galactan biosynthesis and GlfT2 continues with the subsequent polymerization events. GlfT2 has been well studied however GlfT1 inhibition/down-regulation and its effect on mycobacterial survival fitness has not been evaluated. To study the Mtb-Ra survival after GlfT1 silencing, Mtb-Ra knockdown and complemented strains were developed. In this study we show that GlfT1 down-regulation leads to increased susceptibility to ethambutol. Expression of glfT1 was up-regulated in the presence of ethambutol, and also in the presence of oxidative and nitrosative stress and upon exposure to low pH. Also, reduced biofilm formation, increased accumulation of ethidium bromide, and reduced tolerance to peroxide, nitric oxide and acid stress, were observed. The present study also demonstrates that GlfT1 down-regulation leads to reduced survival of Mtb-Ra in macrophages and in mice.

Synthesis of New 4′-(Substituted phenyl)spiro[indoline-3,3′-[1,2,4]triazolidine]-2,5′-diones as Antimicrobial, Antitubercular, and Antifungal Agents: An Insight into the ADME and Toxicity Prediction as well as in-silico Molecular Docking Studies

Spiro-compounds are a significant class of naturally occurring materials distinguished by their prominent biological characteristics. Spiro[indole-thiazolidines] analogues find an extensive application in the development of biologically active compounds. The synthesis of the 4′-(substitutedphenyl)spiro[indoline-3,3′-[1,2,4]triazolidine]-2,5′-diones (3a-d) described here was quick and efficient, and it produced excellent yields. The synthesis was carried out in a hydro-alcoholic (EtOH:H2O; 1:2 v/v) system with an addition of 30% mol of boric acid in glycerol. The structure of the prepared compounds was validated using infrared (IR), nuclear magnetic resonance (1H & 13C NMR) and mass spectral (GC-MS) data, followed by their antibacterial, antitubercular and antifungal activities. Among the series, 4́-(3-chloro-4-fluorophenyl)spiro[indoline-3,3́-[1,2,4]triazolidine]-2,5́-dione (3d) was found to be the most potent, with minimum inhibitory concentrations (MICs) values ranging from 4 to 8 µg/mL against both the gram positive (Bacillus subtilis and Staphylococcus aureus) as well as gram negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains. However, the compound 3d was found to have inconsequential activity against the mycobacterium strains (Mycobacterium tuberculosis H37Ra and M. bovis) and less activity against fungal strains (Aspergillus niger and Candida albicans). On the other hand 4́-(2-methoxyphenyl)spiro[indoline-3,3́-[1,2,4]triazolidine]-2,5́-dione (3c) displayed moderate but significant antifungal activity. The title compounds (3a-d) were further subjected to ADME and toxicity prediction as well as in-silico molecular docking studies against bacterial DNA gyrase (PDB ID: 6KZV). All the compounds (3a-d) followed the Lipinski's rule of five and were predicted as free from toxicities like immunotoxicity, mutagenicity and cytotoxicity and placed in Class IV category (300 < LD50 ≤ 2000). The docking score of the compound 3d was found to be −6.689 kcal/mol and demonstrated H-bond interactions with the residue Arg76 and Asp73 via water molecule.

LncRNA DANCR restrained the survival of mycobacterium tuberculosis H37Ra by sponging miR-1301-3p/miR-5194.

Tuberculosis is a worldwide contagion caused by Mycobacterium tuberculosis (MTB). MTB is characterized by intracellular parasitism and is semi-dormant inside host cells. The persistent inflammation caused by MTB can form a granuloma in lesion regions and intensify the latency of bacteria. In recent years, several studies have proven that long non-coding RNAs (lncRNAs) play critical roles in modulating autophagy. In our study, the Gene Expression Omnibus (GEO) databases were searched for lncRNAs that are associated with tuberculosis. We found that lncRNA differentiation antagonizing non-protein coding RNA (DANCR) increased in the peripheral blood samples collected from 54 pulmonary tuberculosis patients compared to 23 healthy donors. By constructing DANCR overexpression cells, we analyzed the possible cellular function of DANCR. After analyzing our experiments, it was found that the data revealed that upregulation of DANCR facilitated the expression of signal transducer and activator of transcription 3, autophagy-related 4D cysteine peptides, autophagy-related 5, Ras homolog enriched in the brain, and microtubule-associated protein 1A/1B light chain 3 (STAT3, ATG4D, ATG5, RHEB, and LC3, respectively) by sponging miR-1301-3p and miR-5194. Immunofluorescence analysis indicated that DANCR played a positive role in both autophagosome formation and fusion of autolysosomes in macrophages. The colony-forming unit (CFU) assay data also showed that the cells overexpressing DANCR were more efficient in eliminating the intracellular H37Ra strain. Consequently, these data suggest that DANCR restrained intracellular survival of M. tuberculosis by promoting autophagy via miR-1301-3p and miR-5194.

In Vitro and In Silico Studies of Glycyrrhetinic Acid Derivatives as Antitubercular Agents

Background: Glycyrrhetinic acid (GA) is a biologically active triterpenoid acid, isolated from the root of the Glycyrrhiza plant species. In our earlier studies, the semisynthetic analogs of GA have been reported to possess improved anticancer activities against various cell lines, antimalarial, and antifilarial activities. Method: GA was isolated and characterized from roots of Glycyrrhiza glabra and converted to its various C-3 aryl ester derivatives via the protection of C-30 carboxylic group. Antitubercular activity was determined against Mycobacterium tuberculosis H37Ra by Agar dilution assay. The in-silico docking was performed for the most active analogue against three antitubercular targets, catalase peroxidase, dihydrofolate reductase and enoyl-ACP reductase. Results: The derivatives, Methyl glycyrrhetinate (GA-1), 3-O-(4-methyl-phenyl)-ethanoyl methyl glycyrrhetinate (GA-1a), 3-O-(4-fluoro phenyl)-ethanoyl methyl glycyrrhetinate (GA-1c), 3-O-(4-methoxy trans cinnamyl)-ethanoyl methyl glycyrrhetinate (GA-1e) and 3-O-{(4-chlorophenyl)-ethanoyl methyl glycyrrhetinate (GA-1g) showed improved antitubercular activity (in the range 38.76 to 51.546 mM) over the parent molecule (MIC &gt;106.157 mM). The derivative, 3-O-(4-aminobenzoyl)- methyl glycyrrhetinate (GA-1h) was found most active (MIC 20.695 mM) which was further supported by high binding affinity with the selected antitubercular target proteins in in silico docking studies. Conclusion: Synthetic modifications on GA led to C-3 aryl ester derivatives with improved antitubercular activities. Further studies for the development of GA-1h as potential antitubercular lead is therefore warranted.

Toll-like Receptor 2 Mediates VEGF Overexpression and Mesothelial Hyperpermeability in Tuberculous Pleural Effusion.

Toll-like receptor (TLR) is essential for the immune response to Mycobacterium tuberculosis (MTB) infection. However, the mechanism whereby TLR mediates the MTB-induced pleural mesothelial hyperpermeability in tuberculous pleural effusion (TBPE) remains unclear. Pleural effusion size and pleural fluid levels of vascular endothelial growth factor (VEGF) and soluble TLR2 (sTLR2) in patients with TBPE (n = 36) or transudative pleural effusion (TPE, n = 16) were measured. The effects of MTB H37Ra (MTBRa) on pleural mesothelial permeability and the expression of VEGF and zonula occludens (ZO)-1 in human pleural mesothelial cells (PMCs) were assessed. Levels of VEGF and sTLR2 were significantly elevated in TBPE compared to TPE. Moreover, effusion VEGF levels correlated positively, while sTLR2 values correlated negatively, with pleural effusion size in TBPE. In human PMCs, MTBRa substantially activated JNK/AP-1 signaling and upregulated VEGF expression, whereas knockdown of TLR2 remarkably inhibited MTBRa-induced JNK phosphorylation and VEGF overexpression. Additionally, both MTBRa and VEGF markedly reduced ZO-1 expression and induced pleural mesothelial permeability, while TLR2 silencing or pretreatment with anti-VEGF antibody significantly attenuated the MTBRa-triggered effects. Collectively, TLR2 mediates VEGF overproduction and downregulates ZO-1 expression in human PMCs, leading to mesothelial hyperpermeability in TBPE. Targeting TLR2/VEGF pathway may confer a potential treatment strategy for TBPE.

Study on the effect of γδ T cells expanded in vitro to kill hepatocellular carcinoma cells.

γδ T cells for tumor cell immunotherapy has recently become a hot topic. To investigate the stimulation of expanded γδ T cells in vitro to kill liver cancer cells and its mechanism, and in vivo validation. Peripheral blood mononuclear cells (PBMCs) were isolated and amplified. The proportion of γδ T cells in T cells was determined using flow cytometry. γδ T cells were selected as effector cells, and HepG2 cells as target cells in the cytotoxicity experiment. NKG2D blocker was used to block effector cells from identifying target cells, and PD98059 was used to block intracellular signaling pathways. The nude mice tumor model was established in two batches, the tumor growth curve was drawn, and the tumor formation effect was tested using small animal imager to verify the killing effect of γδ T cells. The γδ T cells in the three experimental groups exhibited a large amount of amplification (P < 0.01). In the killing experiment, the killing rate of γδ T cells stimulated by zoledronate (ZOL) in the experimental group was significantly higher than that in the HDMAPP group and the Mycobacterium tuberculosis H37Ra strain (Mtb-Hag) group (P < 0.05). The blocking effect of PD98059 is stronger than that of the NKG2D blocker (P < 0.05). Among them, in the HDMAPP group, when the target ratio was 40:1, the NKG2D blocker exhibited a significant blocking effect (P < 0.05). Alternatively, in the ZOL group, when the effect ratio was 10:1, the effector cells were blocked significantly after treatment using PD98059 (P < 0.05). In vivo experiments verified the killing effect of γδ T cells. According to the tumor growth curve, there was a difference between the experimental and control groups after cell treatment (P < 0.05). ZOL has high amplification efficiency and a positive effect on killing tumor cells.

Mycobacterium tuberculosis retains viability in RNAlater buffer but not in GTC-TCEP and DNA/RNA Shield

Efficient inactivation of clinical samples containing mycobacteria is crucial for biosafety during shipment and handling. When stored in RNAlater, Mycobacterium tuberculosis H37Ra remains viable, and our results suggest that at -20 °C and 4 °C changes in the mycobacterial transcriptome are possible. Only GTC-TCEP and DNA/RNA Shield inactivate sufficiently for shipment.

Targeted isolation of antitubercular cycloheptapeptides and an unusual pyrroloindoline-containing new analog, asperpyrroindotide A, using LC–MS/MS-based molecular networking

Further insights on the secondary metabolites of a soft coral-derived fungus Aspergillus versicolor under the guidance of MS/MS-based molecular networking led to the isolation of seven known cycloheptapeptides, namely, asperversiamides A–C (1–3) and asperheptatides A–D (4–7) and an unusual pyrroloindoline-containing new cycloheptapeptide, asperpyrroindotide A (8). The structure of 8 was elucidated by comprehensive spectroscopic data analysis, and its absolute configuration was determined by advanced Marfey’s method. The semisynthetic transformation of 1 into 8 was successfully achieved and the reaction conditions were optimized. Additionally, a series of new derivatives (10−19) of asperversiamide A (1) was semi-synthesized and their anti-tubercular activities were evaluated against Mycobacterium tuberculosis H37Ra. The preliminary structure−activity relationships revealed that the serine hydroxy groups and the tryptophan residue are important to the activity.