Oncometabolites suppress DNA repair by disrupting local chromatin signalling.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

Role of mitochondrial and cytosolic folylpolyglutamate synthetase in one-carbon metabolism and antitumor efficacy of mitochondrial-targeted antifolates.

Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange between serine, glycine and formate. In both the cytosol and mitochondria, folates exist as polyglutamates with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets SHMT2 in the mitochondria, and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ~19-fold greater than AGF347 By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (~30-fold) more than cFPGS (~4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the non-classical SHMT2/SHMT1 inhibitor, SHIN1. These results suggest a critical role of mFPGS levels in determining anti-tumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer. Significance Statement AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT) 2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347 Mitochondrial FPGS levels play important roles in determining the anti-tumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.

Evaluation of a New DC-Ion Funnel Drift Tube for Use in Proton Transfer Reaction Mass Spectrometry.

We have developed and characterized a novel drift tube called the direct current-ion funnel (DC-ion funnel) drift tube, consisting of 20 traditional ring electrodes and 5 new DC-focusing electrodes (DC-FEs) for use in proton transfer reaction mass spectrometry (PTR-MS). Ion trajectory simulations demonstrate the ion focusing effect of the DC-FE and DC-ion funnel drift tube. Further comparative experiments show that the PTR-MS with the novel DC-ion funnel drift tube has a higher sensitivity (3.8-7.3 times for the volatile organic compounds considered in this work) than the PTR-MS with a traditional drift tube. Different from conventional radiofrequency (rf) focusing methods, the DC-ion funnel drift tube can realize ion focusing with only a DC electric field and no additional rf power supply, which makes it especially suitable for instruments requiring miniaturization and low power consumption to improve detection sensitivity. In addition, the DC-ion funnel drift tube can easily be coupled to other types of mass spectrometers to increase their detection sensitivity.

Increased Sensitivity in Proton Transfer Reaction Mass Spectrometry by Using a Novel Focusing Quadrupole Ion Funnel.

Sensitivity enhancement in proton transfer reaction mass spectrometry (PTR-MS) is an important development direction. We developed a novel drift tube called a focusing quadrupole ion funnel (FQ-IF) for use in PTR-MS to improve the sensitivity. The FQ-IF consists of 20 layers of stainless steel electrodes, and each layer has 4 quarter rings. The first 6 layers have a constant inner hole diameter of 22 mm; the latter 14 layers taper the inner diameter down to 8 mm. The FQ-IF drift tube can also operate in the direct current (DC) mode (similar to a conventional drift tube) and ion funnel (IF) mode (similar to a conventional ion funnel drift tube) by changing the voltage loading method. The simulation results show that the transmission efficiency of the FQ-IF is significantly improved compared to that of the other two modes. Further experiments show that the product ions of limonene tend to convert into smaller m/z fragment ions at higher voltages for the DC and IF modes. However, unlike the DC and IF modes, the distribution of product ions is stable at higher voltages for the FQ-IF. In other words, a higher RF voltage for the FQ-IF will not increase the collision energy of ions. In addition, the improvements in sensitivity for the FQ-IF range from 13.8 to 87.9 times compared to the DC mode and from 1.7 to 4.8 times compared to the IF mode for the 12 test compounds. The improvements in the limit of detection (LOD) for the FQ-IF range from 2.7 to 35.7 times compared to the DC mode. The FQ-IF provides a valuable reference for improving the sensitivity of PTR-MS and other mass spectrometers.

Inhibition of Drp1 ameliorates diabetic retinopathy by regulating mitochondrial homeostasis.

Diabetic retinopathy (DR) is a potentially blinding complication resulting from diabetes mellitus (DM). Retinal vascular endothelial cells (RMECs) dysfunction occupies an important position in the pathogenesis of DR, and mitochondrial disorders play a vital role in RMECs dysfunction. However, the detailed mechanisms underlying DR-induced mitochondrial disorders in RMECs remain elusive. In the present study, we used High glucose (HG)-induced RMECs in vitro and streptozotocin (STZ)-induced Sprague-Dawley rats in vivo to explore the related mechanisms. We found that HG-induced mitochondrial dysfunction via mitochondrial Dynamin-related protein 1(Drp1)-mediated mitochondrial fission. Drp1 inhibitor, Mdivi-1, rescued HG-induced mitochondrial dysfunction. Protein Kinase Cδ (PKCδ) could induce phosphorylation of Drp1, and we found that HG induced phosphorylation of PKCδ. PKCδ inhibitor (Go 6983) or PKCδ siRNA reversed HG-induced phosphorylation of Drp1 and further mitochondrial dysfunction. The above studies indicated that HG increases mitochondrial fission via promoting PKCδ/Drp1 signaling. Drp1 induces excessive mitochondrial fission and produces damaged mitochondrial, and mitophagy plays a key role in clearing damaged mitochondrial. Our study showed that HG suppressed mitophagy via inhibiting LC3B-II formation and p62 degradation. 3-MA (autophagy inhibitor) aggravated HG-induced RMECs damage, while rapamycin (autophagy agonist) rescued the above phenomenon. Further studies were identified that HG inhibited mitophagy by down-regulation of the PINK1/Parkin signaling pathway, and PINK1 siRNA aggravated HG-induced RMECs damage. Further in-depth study, we propose that Drp1 promotion of Hexokinase II (HK-II) separation from mitochondria, thus inhibiting HK-II-PINK1-mediated mitophagy. In vivo, we found that intraretinal microvascular abnormalities (IRMA), including retinal vascular leakage, acellular capillaries, and apoptosis were increased in STZ-induced DR rats, which were reversed by pretreatment with Mdivi-1 or Rapamycin. Altogether, our findings provide new insight into the mechanisms underlying the regulation of mitochondrial homeostasis and provide a potential treatment strategy for Diabetic retinopathy.

PGE2 promotes macrophage recruitment and neovascularization in murine wet-type AMD models.

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is a leading cause of blindness worldwide. Activated macrophages recruited to the injured eyes greatly contribute to the pathogenesis of choroidal neovascularization (CNV) in exudative AMD (wet AMD). This study describes the effects of cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) signalling on the macrophage activation and CNV formation of wet AMD. In a mouse model of laser-induced wet AMD, the mice received an intravitreal injection of celecoxib (a selective COX2 inhibitor). Optical coherence tomography (OCT), fundus fluorescein angiography (FFA), choroidal histology of the CNV lesions, and biochemical markers were assessed. The level of PGE2 expression was high in the laser-induced CNV lesions. Macrophage recruitment and CNV development were significantly less after celecoxib treatment. E-prostanoid1 receptor (EP1R)/protein kinase C (PKC) signalling was involved in M2 macrophage activation and interleukin-10 (IL-10) production of bone marrow-derived macrophages (BMDMs) in vitro. In addition, IL-10 was found to induce the proliferation and migration of human choroidal microvascular endothelial cells (HCECs). Thus, the PGE2/EP1R signalling network serves as a potential therapeutic target for CNV of the wet-type AMD. Video abstract.

Jeotgalibacillus aurantiacus sp. nov., a novel orange-pigmented species with a carotenoid biosynthetic gene cluster, isolated from wetland soil.

A Gram-stain-positive, orange-pigmented, rod-shaped and flagellated bacterial strain T12T was isolated from wetland soil in Kunyu Mountain Wetland in Yantai, China. The strain was able to grow at 15-40 °C (optimum 37 °C), at 0.0-9.0% NaCl (optimum 2%, w/v) and at pH 5.5-9.0 (optimum 8.5). A phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain T12T is a member of the family Planococcaceae, sharing 97.6% and 97.1% sequence similarity with the type strains of Jeotgalibacillus salarius and Jeotgalibacillus marinus, respectively. Genome-based analyses revealed a genome size of 3,506,682 bp and a DNA G + C content of 43.7%. Besides, the genome sequence led to 55.0-74.6% average amino acid identity values and 67.8-74.7% average nucleotide identity values between strain T12T and the current closest relatives. Digital DNA-DNA hybridization of strain T12T with the type strains of Jeotgalibacillus proteolyticus and J. marinus demonstrated 19.0% and 20.3% relatedness, respectively. The chemotaxonomic analysis showed that the sole quinone was MK-7. The predominant cellular fatty acids were iso-C15:0, anteiso-C15:0, C16:1ω7c alcohol and iso-C14:0. The polar lipids consisted of an unidentified aminolipid, phosphatidylglycerol, diphosphatidylglycerol and two unidentified phospholipids. Based on the polyphasic characterization, strain T12T is considered to represent a novel species, for which the name Jeotgalibacillus aurantiacus sp. nov. is proposed. The type strain is T12T (= KCTC 43296 T = MCCC 1K07171T).

Development and validation of a liquid chromatography coupled with tandem mass spectrometry method for determining total and unbound pamiparib in human plasma and brain tumors.

Pamiparib (BGB-290) is an orally bioavailable, small molecule inhibitor of poly (ADP-ribose) polymerase 1 (PARP1) and PARP2. A reversed-phase LC with tandem mass spectrometry method was developed and fully validated for determining total and unbound pamiparib concentrations in human plasma and brain tumor tissue. Plasma and tissue homogenate samples were prepared by methanol protein precipitation. Pamiparib and the internal standard [13 C2 ,15 N2 ]pamiparib were separated on a Waters BEH C18 (50 × 2.1 mm, 1.7 µm) column, with a gradient elution consisting of mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at a flow rate of 0.25 mL/min. The analytes were monitored with multiple reaction monitoring mode under positive electrospray ionization. The method was fully validated for specificity, linearity, accuracy and precision, matrix effect and recovery, and short- and long-term stability. The lower limit of quantitation was 0.5 nM of pamiparib in plasma or tissue homogenate. The calibration curve was linear over the pamiparib concentration range of 0.5-1000 nM in plasma. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method. Pamiparib was stable in plasma at -80°C for at least 6 months. The method was successfully applied to assess the plasma and tumor pharmacokinetics of total and unbound pamiparib in patients with glioma.

Discovery of 6-substituted thieno[2,3-d]pyrimidine analogs as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis in folate receptor expressing human tumors.

We discovered 6-substituted thieno[2,3-d]pyrimidine compounds (3-9) with 3-4 bridge carbons and side-chain thiophene or furan rings for dual targeting one-carbon (C1) metabolism in folate receptor- (FR) expressing cancers. Synthesis involved nine steps starting from the bromo-aryl carboxylate. From patterns of growth inhibition toward Chinese hamster ovary cells expressing FRα or FRß, the proton-coupled folate transporter or reduced folate carrier, specificity for uptake by FRs was confirmed. Anti-proliferative activities were demonstrated toward FRα-expressing KB tumor cells and NCI-IGROV1 ovarian cancer cells. Inhibition of de novo purine biosynthesis at both 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and glycinamide ribonucleotide formyltransferase (GARFTase) was confirmed by metabolite rescue, metabolomics and enzyme assays. X-ray crystallographic structures were obtained with compounds 3-5 and human GARFTase. Our studies identify first-in-class C1 inhibitors with selective uptake by FRs and dual inhibition of enzyme targets in de novo purine biosynthesis, resulting in anti-tumor activity. This series affords an exciting new platform for selective multi-targeted anti-tumor agents.

Characterization of a New Biofunctional, Exolytic Alginate Lyase from Tamlana sp. s12 with High Catalytic Activity and Cold-Adapted Features.

Alginate, a major acidic polysaccharide in brown algae, has attracted great attention as a promising carbon source for biorefinery systems. Alginate lyases, especially exo-type alginate lyase, play a critical role in the biorefinery process. Although a large number of alginate lyases have been characterized, few can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G) at low temperatures by means of an exolytic mode. In this study, the gene of a new exo-alginate lyase-Alys1-with high activity (1350 U/mg) was cloned from a marine strain, Tamlana sp. s12. When sodium alginate was used as a substrate, the recombinant enzyme showed optimal activity at 35 °C and pH 7.0-8.0. Noticeably, recombinant Alys1 was unstable at temperatures above 30 °C and had a low melting temperature of 56.0 °C. SDS and EDTA significantly inhibit its activity. These data indicate that Alys1 is a cold-adapted enzyme. Moreover, the enzyme can depolymerize alginates polyM and polyG, and produce a monosaccharide as the minimal alginate oligosaccharide. Primary substrate preference tests and identification of the final oligosaccharide products demonstrated that Alys1 is a bifunctional alginate lyase and prefers M to G. These properties make Alys1 a valuable candidate in both basic research and industrial applications.

Placing covered self-expanding metal stents by suspension laryngoscope in benign tracheal stenosis.

BACKGROUND: Tracheobronchial stent placement for malignant airway strictures has been proved to improve respiratory function, but experience for benign tracheobronchial stenoses is limited. The purpose of our study is to investigate the efficacy of covered expandable metallic stents, inserted through a suspension laryngoscope, treating tracheal stenosis following intubation or tracheostomy. METHODS: From 2010 to 2018, 67 adult patients with the benign tracheal stenosis, underwent stent placement, using a suspension laryngoscope. According to the date of stent placement and stent caliber, these patients have been subdivided into two groups: Group 1 (from 2010 to 2013, stent caliber ranging from 16 to 20 mm) and Group 2 (from 2014 to 2018, stent caliber ranging from 18 to 22 mm). Complications, related reinterventions, and long-term prognosis were retrospectively evaluated. RESULTS: Primary successful stent placement was achieved and symptoms were improved in all patients. Complications occurred in 27 (40.3%) cases. Among these, there were 14 (20.9%) cases with stent migration, 10 (14.93%) with granulation tissue formation and 3 (4.48%) with pneumonia. Stent migration in Group 1 was nearly 30% higher than that in Group 2 (P = 0.002). Five of the 8 patients who had placement of 16 mm stents had stent migration, more often than with 20 mm stents (P = 0.002). Ten patients' trachea had slight narrowing but without any symptoms. Six patients still had granulation tissue but without any growth at least two-year follow-up (2 patients whose stents were removed more than 1 year after placement). Even without tracheal narrowing and granulation tissue, 5 patients felt persistent shortness of breath. 92.5% of the patients reported to be satisfied with significant improvement in symptoms. CONCLUSIONS: Patients with tracheal obstruction secondary to intubation or tracheostomy can benefit from tracheal stents. Placing 16 mm stents might lead to stent migration more easily than 20 mm stents. Tracheal stents placed by a suspension laryngoscope provide a reasonable alternative to open surgery for patients with benign tracheal stenosis or obstruction.

An Extra-Large-Pore Pure Silica Zeolite with 16×8×8-Membered Ring Pore Channels Synthesized using an Aromatic Organic Directing Agent.

Extra-large-pore zeolites for processing large molecules have long been sought after by both the academia and industry. However, the synthesis of these materials, particularly extra-large-pore pure silica zeolites, remains a big challenge. Herein we report the synthesis of a new extra-large-pore silica zeolite, designated NUD-6, by using an easily synthesized aromatic organic cation as structure-directing agent. NUD-6 possesses an intersecting 16×8×8-membered ring pore channel system constructed by four-connected (Q4 ) and unusual three-connected (Q3 ) silicon species. The organic cations in NUD-6 can be removed in nitric acid to yield a porous material with high surface area and pore volume. The synthesis of NUD-6 presents a feasible means to prepare extra-large pore silica zeolites by using assembled aromatic organic cations as structure-directing agents.

Amino acid-linked platinum(II) compounds: non-canonical nucleoside preferences and influence on glycosidic bond stabilities.

Nucleobases serve as ideal targets where drugs bind and exert their anticancer activities. Cisplatin (cisPt) preferentially coordinates to 2'-deoxyguanosine (dGuo) residues within DNA. The dGuo adducts that are formed alter the DNA structure, contributing to inhibition of function and ultimately cancer cell death. Despite its success as an anticancer drug, cisPt has a number of drawbacks that reduce its efficacy, including repair of adducts and drug resistance. Some approaches to overcome this problem involve development of compounds that coordinate to other purine nucleobases, including those found in RNA. In this work, amino acid-linked platinum(II) (AAPt) compounds of alanine and ornithine (AlaPt and OrnPt, respectively) were studied. Their reactivity preferences for DNA and RNA purine nucleosides (i.e., 2'-deoxyadenosine (dAdo), adenosine (Ado), dGuo, and guanosine (Guo)) were determined. The chosen compounds form predominantly monofunctional adducts by reacting at the N1, N3, or N7 positions of purine nucleobases. In addition, features of AAPt compounds that impact the glycosidic bond stability of Ado residues were explored. The glycosidic bond cleavage is activated differentially for AlaPt-Ado and OrnPt-Ado isomers. Formation of unique adducts at non-canonical residues and subsequent destabilization of the glycosidic bonds are important features that could circumvent platinum-based drug resistance.

Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression.

Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells.

Gold nanoparticle conjugated Rad6 inhibitor induces cell death in triple negative breast cancer cells by inducing mitochondrial dysfunction and PARP-1 hyperactivation: Synthesis and characterization.

We recently developed a small molecule inhibitor SMI#9 for Rad6, a protein overexpressed in aggressive breast cancers and involved in DNA damage tolerance. SMI#9 induces cytotoxicity in cancerous cells but spares normal breast cells; however, its therapeutic efficacy is limited by poor solubility. Here we chemically modified SMI#9 to enable its conjugation and hydrolysis from gold nanoparticle (GNP). SMI#9-GNP and parent SMI#9 activities were compared in mesenchymal and basal triple negative breast cancer (TNBC) subtype cells. Whereas SMI#9 is cytotoxic to all TNBC cells, SMI#9-GNP is endocytosed and cytotoxic only in mesenchymal TNBC cells. SMI#9-GNP endocytosis in basal TNBCs is compromised by aggregation. However, when combined with cisplatin, SMI#9-GNP is imported and synergistically increases cisplatin sensitivity. Like SMI#9, SMI#9-GNP spares normal breast cells. The released SMI#9 is active and induces cell death via mitochondrial dysfunction and PARP-1 stabilization/hyperactivation. This work signifies the development of a nanotechnology-based Rad6-targeting therapy for TNBCs. FROM THE CLINICAL EDITOR: Protein Rad6 is overexpressed in breast cancer cells and its blockade may provide a new treatment against 3N breast cancer. The authors conjugated a small molecule inhibitor SMI#9 for Rad6 to gold nanoparticles in this study and showed that this new formulation specifically targeted chemo-resistant breast cancer cells and highlighted the importance of nanotechnology in drug carrier development.

Comparison of Two Molecular Assays For Detecting Smear Negative Pulmonary Tuberculosis.

OBJECTIVE: To compare the performance of MTBDRplus V2 and Xpert MTB/RIF for detecting smear negative pulmonary tuberculosis (PTB). METHODS: Clinical PTB suspects were enrolled consecutively in Anhui Chest Hospital and Xi'an Chest Hospital from January to December in 2014. The sputum samples of smear negative PTB suspects were collected and decontaminated. The sediment was used to conduct MTBDRplus V2, Xpert MTB/RIF and drug susceptibility test (DST). All the samples with discrepant drug susceptibility result between molecular methods and phenotypic method were confirmed by DNA sequencing. RESULTS: A total of 1973 cases were enrolled in this study. The detection rates of Mycobacterium tuberculosis complex (MTBC) by MTBDRplus V2 and Xpert MTB/RIF were 27.67% and 27.98%, respectively. When setting MGIT culture result as a gold standard, the sensitivity and specificity of MTBDRplus V2 were 86.74% and 93.84%, and the sensitivity and specificity of Xpert MTB/RIF were 86.55% and 93.43%, respectively. For the detection of the resistance to rifampin, the sensitivity and specificity of MTBDRplus V2 were 94.34% and 96.62%, and the sensitivity and specificity of Xpert MTB/RIF were 88.68% and 95.96%, respectively. For the detection of the resistance to isoniazid, the sensitivity and specificity of MTBDRplus V2 were 77.38% and 98.02%, respectively. CONCLUSION: MTBDRplus V2 and Xpert MTB/RIF can be used to detect MTBC in smear negative samples with satisfactory performance.

SHMT2 reduces fatty liver but is necessary for liver inflammation and fibrosis in mice.

Non-alcoholic fatty liver disease is associated with an irregular serine metabolism. Serine hydroxymethyltransferase 2 (SHMT2) is a liver enzyme that breaks down serine into glycine and one-carbon (1C) units critical for liver methylation reactions and overall health. However, the contribution of SHMT2 to hepatic 1C homeostasis and biological functions has yet to be defined in genetically modified animal models. We created a mouse strain with targeted SHMT2 knockout in hepatocytes to investigate this. The absence of SHMT2 increased serine and glycine levels in circulation, decreased liver methylation potential, and increased susceptibility to fatty liver disease. Interestingly, SHMT2-deficient mice developed simultaneous fatty liver, but when fed a diet high in fat, fructose, and cholesterol, they had significantly less inflammation and fibrosis. This study highlights the critical role of SHMT2 in maintaining hepatic 1C homeostasis and its stage-specific functions in the pathogenesis of NAFLD.

Screening, epidemic trends and drug sensitivity analysis of nontuberculous mycobacteria in a local area of China.

OBJECTIVE: To analyze the isolation rate, prevalence trends, species distribution, and drug sensitivity of non-tuberculous mycobacteria (NTM) in Anhui Province, providing a reference for diagnosis and treatment strategies. METHODS: Specimens from suspected mycobacterial infection patients at Anhui Chest Hospital (including outpatients and inpatients) from January 2021 to December 2023 were cultured. Identified NTM strains were analyzed for species distribution and drug sensitivity. RESULTS: Of 10,519 mycobacteria strains cultured, 1,589 were NTM (15.11%). The top four species were Mycobacterium intracellulare (75.36%), Mycobacterium abscessus (11.78%), Mycobacterium kansasii (7.09%), and Mycobacterium avium (2.85%). NTM strains showed high sensitivity to amikacin and clarithromycin (≥90%) and significant sensitivity to rifabutin, moxifloxacin, and rifampicin (89.03%-79.61%). They exhibited high resistance to imipenem/cilastatin, sulfamethoxazole, minocycline, and doxycycline (≥95%). CONCLUSION: NTM isolation rates in Anhui have remained stable, with the predominant species being M. intracellulare, M. kansasii, M. abscessus, and M. avium. NTM strains are highly sensitive to amikacin, clarithromycin, rifabutin, moxifloxacin, and rifampicin. These findings can guide diagnosis, treatment strategies, and drug selection for NTM disease in Anhui Province.

Mitochondrial and Cytosolic One-Carbon Metabolism Is a Targetable Metabolic Vulnerability in Cisplatin-Resistant Ovarian Cancer.

One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared with normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin-sensitive (A2780, CaOV3, IGROV1) and cisplatin-resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359, and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism, which offers a promising new platform for therapy.