Synthesis of narrow-spectrum anti-mycobacterial molecules without effect on the diversity of gut microbiota in mice based on the structure of rifampicin.

Rifampicin (RIF) is a broad-spectrum antimicrobial agent that is also a first-line drug for treating tuberculosis (TB). Based on the naphthyl ring structure of RIF this study synthesized 16 narrow-spectrum antimicrobial molecules that were specifically anti-Mycobacterium tuberculosis (Mtb). The most potent candidate was 2-((6-hydroxynaphthalen-2-yl) methylene) hydrazine-1-carbothioamide (compound 3c) with minimum inhibitory concentration (MIC) of 1 µg/mL against Mtb. Synergistic anti-Mtb test indicated that none of the combinations of 3c with the major anti-TB drugs are antagonistic. Consistent with RIF, compound 3c induced large amounts of reactive oxygen radicals (ROS) in the cells of Mtb. The killing kinetics of compound 3c and RIF are very similar. Furthermore, molecular docking showed that compound 3c was able to access the RIF binding pocket of the ß subunit of Mtb RNA polymerase (RNAP). Experiments in mice showed that compound 3c increased the variety of intestinal flora in mice, while RIF significantly decreased the diversity of intestinal flora in mice. In addition, compound 3c is non-toxic to animal cells with a selection index (SI) much more than 10. The evidence from this study suggests that the further development of 3c could contribute to the development of novel drug for TB treatment.

19 Schiff bases as antimycobacterial agents: synthesis, molecular docking and a plausible mechanism of action.

Aim: To discover novel anti-Mycobacterium tuberculosis (Mtb) drugs, 19 compounds were synthesized; their anti-Mtb effects were evaluated and mechanisms of action were preliminarily explored. Materials & methods: The compounds were synthesized and their anti-Mtb activity was elucidated using resazurin microtiter assays. The plausible target of the potential compound was investigated by microimaging techniques, gas chromatography-mass spectrometry analysis and molecular docking. Results: 19 compounds inhibited Mtb growth with minimum inhibitory concentrations ranging from 1 to 32 µg/ml. Compounds 1-17 showed inhibition of Mtb KatG enzyme. Compound 19, the most potent, might be an inhibitor of Pks13 polyketide synthase. Conclusion: This study suggests that 2-((6-fluoropyridin-3-yl)methylene) hydrazine-1-carbothioamide (19) is a potential anti-Mtb lead compound with a novel mechanism of action.

Globally, more than 1.6 million people die of tuberculosis (TB) and about 11 million new cases occur each year. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) has made it difficult to effectively treat TB. Therefore, 19 drugs were synthesized and assayed in the laboratory to verify whether they could inhibit the growth of Mtb. All compounds exhibit anti-Mtb effects at relatively low concentrations. Among them, compound 19 had a strong anti-Mtb effect, and its bactericidal effect on Mtb even exceeded that of isoniazid. In addition, it was preliminarily determined that compound 19 is a novel inhibitor of a key enzyme in the biosynthesis of Mtb cell walls. These findings demonstrate a potential new treatment option for TB but more research is needed to confirm the safety of these drugs.

Spatial and temporal heterogeneity of tumor immune microenvironment between primary tumor and brain metastases in NSCLC.

BACKGROUND: Brain metastasis is a common outcome in non-small cell lung cancer, and despite aggressive treatment, its clinical outcome is still frustrating. In recent years, immunotherapy has been developing rapidly, however, its therapeutic outcomes for primary lung cancer and brain metastases are not the same, suggesting that there may be differences in the immune microenvironment of primary lung cancer and brain metastases, however, we currently know little about these differences. METHODS: Seventeen paired samples of NSCLC and their brain metastases and 45 other unpaired brain metastases samples were collected for the current study. Immunohistochemical staining was performed on all samples for the following markers: immune checkpoints CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, IDO1, and EphA2; tumor-infiltrating lymphocytes (TILs) CD3, CD4, CD8, and CD20; tumor-associated microglia/macrophages (TAMs) CD68 and CD163; and tumor proliferation index Ki-67. The differences in expression of these markers were compared in 17 paired samples, and the effect of the expression level of these markers on the prognosis of patients was analyzed in lung adenocarcinoma brain metastases samples. Subsequently, multiplex immunofluorescence staining was performed in a typical lung-brain paired sample based on the aforementioned results. The multiplex immunofluorescence staining results revealed the difference in tumor immune microenvironment between primary NSCLC and brain metastases. RESULTS: In 17 paired lesions, the infiltration of CTLA-4+ (P = 0.461), PD-1+ (P = 0.106), CD3+ (P = 0.045), CD4+ (P = 0.037), CD8+ (P = 0.008), and CD20+ (P = 0.029) TILs in brain metastases were significantly decreased compared with primary tumors. No statistically significant difference was observed in the CD68 (P = 0.954) and CD163 (P = 0.654) TAM infiltration between primary NSCLC and paired brain metastases. In all the brain metastases lesions, the expression of PD-L1 is related to the time interval of brain metastases in NSCLC. In addition, the Cox proportional hazards regression models showed high expression of B7-H4 (hazard ratio [HR] = 3.276, 95% confidence interval [CI] 1.335-8.041, P = 0.010) and CD68 TAM infiltration (HR = 3.775, 95% CI 1.419-10.044, P = 0.008) were independent prognosis factors for lung adenocarcinoma brain metastases patients. CONCLUSIONS: Both temporal and spatial heterogeneity is present between the primary tumor and brain metastases of NCSLC. Brain metastases lesions exhibit a more immunosuppressive tumor immune microenvironment. B7-H4 and CD68+ TAMs may have potential therapeutic value for lung adenocarcinoma brain metastases patients.

Primary intracranial sarcomas: a clinicopathological investigation.

Background: The purpose of this study is to present a series of primary intracranial sarcomas (PIS), a rare type of tumor of the central nervous system, in order to improve our understanding of the disease. These tumors are heterogeneous and prone to recurrence after resection, exhibiting a high mortality rate. As PIS has yet to be understood and studied on a large scale, it is vital for further evaluation and research. Methods: Our study included 14 cases of PIS. The patients' clinical, pathological, and imaging features were retrospectively analyzed. Additionally, targeted DNA next-generation sequencing (NGS) was applied for the 481-gene panel to detect gene mutations. Results: The average age for PIS patients was 31.4 years. Headache (7, 50.0%) was the most common symptom leading to the hospital visit. Twelve cases had PIS located in the supratentorial area and two in the cerebellopontine angle region. The maximum tumor diameter ranged from 19.0 mm to 130.0 mm, with an average diameter of 50.3 mm. Pathological types of tumors were heterogeneous, with chondrosarcoma being the most common, followed by fibrosarcoma. Eight of the 10 PIS cases that underwent MRI scanning showed gadolinium enhancement; 7 of these cases were heterogeneous, and 1 of them was garland-like. Targeted sequencing was performed in two cases and identified mutations in genes such as NRAS, PIK3CA, BAP1, KDR, BLM, PBRM1, TOP2A, DUSP2, and CNV deletions of SMARCB1. Additionally, the SH3BP5::RAF1 fusion gene was also detected. Of the 14 patients, 9 underwent a gross total resection (GTR), and 5 chose subtotal resection. Patients who underwent GTR displayed a trend toward superior survival. Among the 11 patients with available follow-up information, one had developed lung metastases, three had died, and eight were alive. Conclusion: PIS is extremely rare compared to extracranial soft sarcomas. The most common histological type of intracranial sarcoma (IS) is chondrosarcoma. Patients who underwent GTR of these lesions showed improved survival rates. Recent advancements in NGS aided in the identification of diagnostic and therapeutic PIS-relevant targets.

Synthesis, antimycobacterial evaluation, and molecular docking study of 1,2,4-triazole derivatives.

Fifteen 1,2,4-triazole derivatives were synthesised in this study and their MIC values against Mycobacterium tuberculosis (Mtb) ranged from 2 to 32 µg/mL. Furthermore, their antimycobacterial activity was positively correlated with the KatG enzyme docking score. Among the 15 compounds, compound 4 showed the strongest bactericidal activity with an MIC of 2 µg/mL. The selectivity index of compound 4 is more than 10, indicating that the compound has low toxicity to animal cells and has the potential to become a drug. Molecular docking indicates that compound 4 can bind firmly to the Mtb KatG active site. The experimental results showed that compound 4 inhibited Mtb KatG and caused the accumulation of ROS in Mtb cells. We speculate that compound 4 causes the accumulation of ROS by inhibiting KatG, and ROS produces oxidative destruction, leading to the death of Mtb. This study provides a new idea for the development of novel anti-Mtb drugs.

Clinical significance and molecular annotation of cellular morphometric subtypes in lower-grade gliomas discovered by machine learning.

BACKGROUND: Lower-grade gliomas (LGG) are heterogeneous diseases by clinical, histological, and molecular criteria. We aimed to personalize the diagnosis and therapy of LGG patients by developing and validating robust cellular morphometric subtypes (CMS) and to uncover the molecular signatures underlying these subtypes. METHODS: Cellular morphometric biomarkers (CMBs) were identified with artificial intelligence technique from TCGA-LGG cohort. Consensus clustering was used to define CMS. Survival analysis was performed to assess the clinical impact of CMBs and CMS. A nomogram was constructed to predict 3- and 5-year overall survival (OS) of LGG patients. Tumor mutational burden (TMB) and immune cell infiltration between subtypes were analyzed using the Mann-Whitney U test. The double-blinded validation for important immunotherapy-related biomarkers was executed using immunohistochemistry (IHC). RESULTS: We developed a machine learning (ML) pipeline to extract CMBs from whole-slide images of tissue histology; identifying and externally validating robust CMS of LGGs in multicenter cohorts. The subtypes had independent predicted OS across all three independent cohorts. In the TCGA-LGG cohort, patients within the poor-prognosis subtype responded poorly to primary and follow-up therapies. LGGs within the poor-prognosis subtype were characterized by high mutational burden, high frequencies of copy number alterations, and high levels of tumor-infiltrating lymphocytes and immune checkpoint genes. Higher levels of PD-1/PD-L1/CTLA-4 were confirmed by IHC staining. In addition, the subtypes learned from LGG demonstrate translational impact on glioblastoma (GBM). CONCLUSIONS: We developed and validated a framework (CMS-ML) for CMS discovery in LGG associated with specific molecular alterations, immune microenvironment, prognosis, and treatment response.

Monitoring Bevacizumab-Induced Tumor Vascular Normalization by Intravoxel Incoherent Motion Diffusion-Weighted MRI.

BACKGROUND: Accurate monitoring of tumor blood vessel normalization progression is beneficial to accurate treatment of patients. At present, there is a lack of safe and noninvasive monitoring methods. PURPOSE: To serial monitor the vascular normalization time window of tumor antiangiogenesis treatment through intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) and histopathological methods. STUDY TYPE: Exploratory animal study. POPULATION: Sixty rat C6 glioma models were randomly and equally divided into the control groups (N = 30) and bevacizumab treatment groups (N = 30). Twenty-five for magnetic resonance imaging (MRI) and five for electron microscope testing in each group. FIELD STRENGTH/SEQUENCE: T1-weighted imaging (T1WI), T2WI with a fast spin echo sequence and IVIM-DWI with a spin-echo echo-planar imaging sequence at 3 T. ASSESSMENT: IVIM-DWI quantitative parameters (f, D, D*, and fD*) were obtained on days 0, 2, 4, 6, and 8 after bevacizumab treatment. After MRI, the microvessel density (MVD), pericyte coverage, and hypoxia-inducible factor-1α (HIF-1α) were assessed. Electron microscope observation was performed at each time point. STATISTICAL TESTS: One-way analysis of variance and Student's t-tests were used to compare differences within and between groups. Spearman's correlation coefficient (r) assess the correlation between IVIM and pathological parameters. The intragroup correlation coefficient was determined to assess the repeatability of each IVIM parameter. RESULTS: The IVIM-DWI perfusion parameters (f and fD*) of the treated group were higher than the control group on days 2 and 4. Compared to the control group, MVD decreased on days 2 and pericyte coverage increased on days 4 in the treatment group. Electron microscopy showed that the tight junctions of the treatment group were prolonged on days 2-4. In the control group, f had the highest correlation with MVD (r = 0.689). In the treated group, f had a good correlation with pericyte coverage (r = 0.557), HIF-1α had a moderately positive correlation with f (r = 0.480) and fD*(r = 0.447). DATA CONCLUSION: The vascular normalization time window of bevacizumab treatment of glioma was days 2-4 after antiangiogenesis treatment, which could be monitored noninvasively by IVIM-DWI. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 3.

Genetic alteration and clonal evolution of primary glioblastoma into secondary gliosarcoma.

AIMS: Secondary gliosarcoma (SGS) rarely arises post treatment of primary glioblastoma multiforme (GBM), and contains gliomatous and sarcomatous components. The origin and clonal evolution of SGS sarcomatous components remain uncharacterized. Therapeutic radiation is mutagenic and can induce sarcomas in patients with other tumor phenotypes, but possible causal relationships between radiotherapy and induction of SGS sarcomatous components remain unexplored. Herein, we investigated the clonal origin of SGS in a patient with primary GBM progressing into SGS post-radiochemotherapy. METHODS: Somatic mutation profile in GBM and SGS was examined using whole-genome sequencing and deep-whole-exome sequencing. Mutation signatures were characterized to investigate relationships between radiochemotherapy and SGS pathogenesis. RESULTS: A mutation cluster containing two founding mutations in tumor-suppressor genes NF1 (variant allele frequency [VAF]: 50.0% in GBM and 51.1% in SGS) and TP53 (VAF: 26.7% in GBM and 50.8% in SGS) was shared in GBM and SGS. SGS exhibited an overpresented C>A (G>T) transversion (oxidative DNA damage signature) but no signature 11 mutations (alkylating-agents - exposure signature). Since radiation induces DNA lesions by generating reactive oxygen species, the mutations observed in this case of SGS were likely the result of radiotherapy rather than chemotherapy. CONCLUSIONS: Secondary gliosarcoma components likely have a monoclonal origin, and the clone possessing mutations in NF1 and TP53 was likely the founding clone in this case of SGS.

Correction: Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mulberry.

[This corrects the article DOI: 10.1371/journal.pone.0155814.].

Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mul-berry.

A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol.