Dynamic interactions between Candida albicans and different streptococcal species in a multispecies oral biofilm

The oral cavity is colonized by a plethora of bacteria, fungi, and archaea, including streptococci of the mitis group (MSG) and the yeast Candida albicans. This study aims to investigate the role of streptococcal species in the development of oral biofilm and the cross-kingdom interactions between some of the members of the commensal MSG and the pathogen yeast C. albicans using a multispecies supragingival biofilm model. A total of nine different in vitro biofilms were grown, quantified with culture analyses, and visually examined with confocal laser scanning microscopy (CLSM). A four-species biofilm without any streptococcal species was used as a basic biofilm. In each subsequent inoculum, one species of MSG was added and afterward combined with Streptococcus mutans. The eight-species biofilm contained all eight strains used in this study. Culture analyses showed that the presence of S. mutans in a four-species biofilm with Streptococcus oralis or S. oralis subsp. tigurinus did not differ significantly in C. albicans colony-forming unit (CFU) counts compared to biofilms without S. mutans. However, compared to other mitis species, Streptococcus gordonii combined with S. mutans resulted in the lowest CFUs of C. albicans. Visual observation by CLSM showed that biofilms containing both S. mutans and one species of MSG seemed to induce the formation of filamentous form of C. albicans. However, when several species of MSG were combined with S. mutans, C. albicans was again found in its yeast form

Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability

Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand tumor metabolism. To measure the extracellular nutrient levels available to tumors, we utilized quantitative metabolomics methods to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors

Risk of developing active tuberculosis following tuberculosis screening and preventive therapy for Tibetan refugee children and adolescents in India: An impact assessment.

BackgroundTuberculosis (TB) rates among Tibetan refugee children and adolescents attending boarding schools in India are extremely high. We undertook a comprehensive case finding and TB preventive treatment (TPT) program in 7 schools in the Zero TB Kids project. We aimed to measure the TB infection and disease burden and investigate the risk of TB disease in children and adults who did and did not receive TPT in the schools.Methods and findingsA mobile team annually screened children and staff for TB at the 7 boarding schools in Himachal Pradesh, India, using symptom criteria, radiography, molecular diagnostics, and tuberculin skin tests. TB infection (TBI) was treated with short-course regimens of isoniazid and rifampin or rifampin. TB disease was treated according to Tibetan and Indian guidelines. Between April 2017 and December 2019, 6,582 schoolchildren (median age 14 [IQR 11-16] years) and 807 staff (median age 40 [IQR 33-48] years) were enrolled. Fifty-one percent of the students and 58% of the staff were females. Over 13,161 person-years of follow-up in schoolchildren (median follow-up 2.3 years) and 1,800 person-years of follow-up in staff (median follow-up 2.5 years), 69 TB episodes occurred in schoolchildren and 4 TB episodes occurred in staff, yielding annual incidence rates of 524/100,000 (95% CI 414-663/100,000) person-years and 256/100,000 (95% CI 96-683/100,000) person-years, respectively. Of 1,412 schoolchildren diagnosed with TBI, 1,192 received TPT. Schoolchildren who received TPT had 79% lower risk of TB disease (adjusted hazard ratio [aHR] 0.21; 95% CI 0.07-0.69; p = 0.010) compared to non-recipients, the primary study outcome. Protection was greater in recent contacts (aHR 0.07; 95% CI 0.01-0.42; p = 0.004), the secondary study outcome. The prevalence of recent contacts was 28% (1,843/6,582). Two different TPT regimens were used (3HR and 4R), and both were apparently effective. No staff receiving TPT developed TB. Overall, between 2017 and 2019, TB disease incidence decreased by 87%, from 837/100,000 (95% CI 604-1,129/100,000) person-years to 110/100,000 (95% CI 36-255/100,000) person-years (p ConclusionsIn this study, following implementation of a school-wide TB screening and preventive treatment program, we observed a significant reduction in the burden of TB disease and TBI in children and adolescents. The benefit of TPT was particularly marked for recent TB contacts. This initiative may serve as a model for TB detection and prevention in children and adolescents in other communities affected by TB

MFSD12 mediates the import of cysteine into melanosomes and lysosomes

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. Dozens of genes contribute to the wide variation in human pigmentation. Many of these genes encode proteins that localize to the melanosome—the organelle, related to the lysosome, that synthesizes pigment—but have unclear functions1,2. Here we describe MelanoIP, a method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use this method to study MFSD12, a transmembrane protein of unknown molecular function that, when suppressed, causes darker pigmentation in mice and humans3,4. We find that MFSD12 is required to maintain normal levels of cystine—the oxidized dimer of cysteine—in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation5,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal-storage disease caused by inactivation of the lysosomal cystine exporter cystinosin7–9. Thus, MFSD12 is an essential component of the cysteine importer for melanosomes and lysosomes

Dihydroxyacetone phosphate signals glucose availability to mTORC1

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1

Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction

© 2021 National Academy of Sciences. All rights reserved. In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10. Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction

Histidine catabolism is a major determinant of methotrexate sensitivity

The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase1, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis2. Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production3. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials4, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR–Cas9-based screen6,7. This screen yielded FTCD, which encodes an enzyme—formimidoyltransferase cyclodeaminase—that is required for the catabolism of the amino acid histidine8, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.National Cancer Institute (U.S.) (Grant R01 CA129105)United States. Department of Defense (Grant W81XWH-15-1-0337)EMBO Long-Term Fellowship (ALTF 350-2012)American Association for Cancer Research (Grant 16-40-38-KANA)American Cancer Society (Grant PF-12-099-01-TBG)EMBO Long-Term Fellowship (ALTF 1-2014

Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability

Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand tumor metabolism. To measure the extracellular nutrient levels available to tumors, we utilized quantitative metabolomics methods to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors.NIH (Grants R01CA168653, R01CA201276, P30CA1405141, T32GM007287, T32GM007753, F32CA213810 and F32CA210421

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.NIH (Grant R01CA103866)NIH (Grant R01CA129105)NIH (Grant R37AI047389)Department of Defense (Grant W81XWH-15-1-0230

Methionine synthase is essential for cancer cell proliferation in physiological folate environments

Folate metabolism can be an effective target for cancer treatment. However, standard cell culture conditions utilize folic acid, a non-physiological folate source for most tissues. We find that the enzyme that couples folate and methionine metabolic cycles, methionine synthase, is required for cancer cell proliferation and tumour growth when 5-methyl tetrahydrofolate (THF), the major folate found in circulation, is the extracellular folate source. In such physiological conditions, methionine synthase incorporates 5-methyl THF into the folate cycle to maintain intracellular levels of the folates needed for nucleotide production. 5-methyl THF can sustain intracellular folate metabolism in the absence of folic acid. Therefore, cells exposed to 5-methyl THF are more resistant to methotrexate, an antifolate drug that specifically blocks folic acid incorporation into the folate cycle. Together, these data argue that the environmental folate source has a profound effect on folate metabolism, determining how both folate cycle enzymes and antifolate drugs impact proliferation