Glycolytic ATP fuels phosphoinositide 3-kinase signaling to support effector T helper 17 cell responses.

Aerobic glycolysis-the Warburg effect-converts glucose to lactate via the enzyme lactate dehydrogenase A (LDHA) and is a metabolic feature of effector T cells. Cells generate ATP through various mechanisms and Warburg metabolism is comparatively an energy-inefficient glucose catabolism pathway. Here, we examined the effect of ATP generated via aerobic glycolysis in antigen-driven T cell responses. Cd4CreLdhafl/fl mice were resistant to Th17-cell-mediated experimental autoimmune encephalomyelitis and exhibited defective T cell activation, migration, proliferation, and differentiation. LDHA deficiency crippled cellular redox balance and inhibited ATP production, diminishing PI3K-dependent activation of Akt kinase and thereby phosphorylation-mediated inhibition of Foxo1, a transcriptional repressor of T cell activation programs. Th17-cell-specific expression of an Akt-insensitive Foxo1 recapitulated the defects seen in Cd4CreLdhafl/fl mice. Induction of LDHA required PI3K signaling and LDHA deficiency impaired PI3K-catalyzed PIP3 generation. Thus, Warburg metabolism augments glycolytic ATP production, fueling a PI3K-centered positive feedback regulatory circuit that drives effector T cell responses.

Lactate dehydrogenase activity drives hair follicle stem cell activation.

Although normally dormant, hair follicle stem cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier 1 (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allows them to remain dormant and yet quickly respond to appropriate proliferative stimuli.

Deletion of Lactate Dehydrogenase-A in Myeloid Cells Triggers Antitumor Immunity.

Immunometabolism is emerging as a critical determinant of cancer pathophysiology. In this study, we explored the contributions of macrophage-expressed lactate dehydrogenase-A (LDH-A) to tumor formation in a K-Ras murine model of lung carcinoma. Myeloid-specific deletion of LDH-A promoted accumulation of macrophages with a CD86high and MCP-1high M1-like phenotype that suppressed tumor growth. This phenotypic effect was accompanied by reduced VEGF expression and angiogenesis, diminished numbers of PD-L1+ cancer cells, increased numbers of CD3+ T cells, and activation status of CD8+ T cells. Furthermore, it was associated with more pronounced antitumor T-cell immunity via induction of IL17 and IFNγ-producing CD8+ T (Tc17 and Tc1) cells, likely via suppression of lactate-driven PD-L1 expression. Our results suggest that expressions of LDH-A and lactate by macrophage in the tumor microenvironment are major drivers of T-cell immunosuppression, strongly supporting the concept of targeting stromal LDH-A as an effective strategy to blunt tumoral immune escape. Cancer Res; 77(13); 3632-43. ©2017 AACR.

Hereditary lactate dehydrogenase M-subunit deficiency with late-developing pustular psoriasis-like lesions.

Hereditary lactate dehydrogenase (LDH) M-subunit deficiency is very rare and we have found reports of close to a dozen cases in the published work, two of which were associated with pustular psoriasis-like lesions. We report a third case of pustular psoriasis-like eruptions associated with LDH M-subunit deficiency, which occurred 24 years after the diagnosis of LDH M-subunit deficiency. These cases indicate that abnormal activity of LDH can induce pustular psoriatic lesions in the long term. Some patients with symptoms of hereditary LDH M-subunit deficiency have antecedent annular scaly plaque lesions, that resemble psoriatic lesions. We discuss a hypothesis to explain this scenario.

Engineering Corynebacterium glutamicum for the production of 2,3-butanediol.

BACKGROUND: 2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions. RESULTS: A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol(-1) yield on glucose, an overall productivity of 0.2 g L(-1) h(-1) and a titer of 6.3 g L(-1). CONCLUSIONS: We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.

Lactate dehydrogenase B is associated with the response to neoadjuvant chemotherapy in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) comprises a subset of head and neck squamous cell carcinoma (HNSCC) with poor therapeutic outcomes and high glycolytic dependency. Neoadjuvant chemotherapy regimens of docetaxel, cisplatin and 5-fluorouracil (TPF) are currently accepted as standard regimens for HNSCC patients with a high risk of distant metastatic spread. However, the antitumor outcomes of TPF neoadjuvant chemotherapy in HNSCC remain controversial. This study investigated the role of lactate dehydrogenase B (LDHB), a key glycolytic enzyme catalyzing the inter-conversion between pyruvate and lactate, in determining chemotherapy response and prognosis in OSCC patients. We discovered that a high protein level of LDHB in OSCC patients was associated with a poor response to TPF regimen chemotherapy as well as poor overall survival and disease-free survival. Our in-depth study revealed that high LDHB expression conferred resistance to taxol but not 5-fluorouracil or cisplatin. LDHB deletion sensitized OSCC cell lines to taxol, whereas the introduction of LDHB decreased sensitivity to taxol treatment. Taxol induced a pronounced impact on LDHB-down-regulated OSCC cells in terms of apoptosis, G2/M phase cell cycle arrest and energy metabolism. In conclusion, our study highlighted the critical role of LDHB in OSCC and proposed that LDHB could be used as a biomarker for the stratification of patients for TPF neoadjuvant chemotherapy and the determination of prognosis in OSCC patients.

Adaptation and validation of the Inventory of Family Protective Factors for the Portuguese culture / Adaptação e Validação do Instrumento Inventory of Family Protective Factors para a cultura portuguesa / Adaptación y Validación del Instrumento Inventory of Family Protective Factors para la cultura portuguesa

OBJECTIVES: to adapt and validate the Inventory of Family Protective Factors (IFPF) for the Portuguese culture. This instrument assesses protective factors that contribute to family resilience. Studies addressing resilience are embedded within the salutogenic paradigm, i.e. it addresses protective factors of individuals or groups without underestimating risk factors or vulnerability. METHOD: in order to assess the IFPF's linguistic and conceptual equivalence, the instrument was translated, retro-translated and the think-aloud protocol was used. We then verified the instrument's sensitiveness, reliability and validity of results to assess its psychometric characteristics. A factor analysis was performed of the principal components with varimax rotation of the scale's items and Cronbach's alpha coefficient was calculated for each dimension. A total of 85 families with disabled children, selected through simple random sampling, self-administered the instrument. RESULTS: the IFPF presents psychometric characteristics that are appropriate for the Portuguese population (Cronbach's alpha = .90). CONCLUSION: the IFPF was adapted and validated for the Portuguese culture and is an instrument to be used in studies intended to assess protective factors of family resilience. .

OBJETIVOS: adaptar e validar o Inventory of Family Protective Factors para a cultura portuguesa. Esse instrumento avalia os fatores protetores que contribuem para a resiliência familiar. Os estudos sobre resiliência inserem-se no paradigma salutogênico, abordando os fatores protetores dos indivíduos, ou grupos, sem subestimar os fatores de risco ou vulnerabilidade. MÉTODO: para avaliar a equivalência linguística e conceitual do Inventory of Family Protective Factors realizou-se a tradução, retroversão e reflexão falada; para aferir as características psicométricas do instrumento, verificou-se a sensibilidade, confiabilidade e a validade dos resultados. Foi realizada uma análise fatorial de componentes principais com rotação Varimax dos itens da escala e calculou-se o coeficiente alfa de Cronbach para cada dimensão. Através de uma amostragem aleatória simples, aplicou-se esse instrumento a 85 famílias de crianças com necessidades especiais que o autopreencheram. RESULTADOS: o Inventory of Family Protective Factors apresenta características psicométricas adequadas para a população portuguesa (alfa de Cronbach de .90). CONCLUSÃO: o Inventory of Family Protective Factors foi adaptado e validado para a cultura portuguesa. Considera-se que se trata de um instrumento útil para estudos nos quais há a proposta de avaliar os fatores protetores da resiliência familiar. .

OBJETIVOS: adaptar y validar el Inventory of Family Protective Factors (IFPF) para la cultura portuguesa. Este instrumento evalúa los factores protectores que contribuyen para la resiliencia familiar. Los estudios sobre resiliencia se insieren en el paradigma salutogénico, abordando los factores protectores de los individuos o grupos, sin subestimar los factores de riesgo o vulnerabilidad. MÉTODO: para evaluar la equivalencia lingüística y conceptual del IFPF realizamos la traducción, retrotraducción y reflexión hablada; para evaluar las características psicométricas del instrumento verificamos la sensibilidad, confiabilidad y la validez de los resultados. Realizamos un análisis factorial de los componentes principales con rotación varimax de los ítems de la escala y calculamos el coeficiente Alpha de Cronbach para cada dimensión. A través de un muestreo aleatorio simple, aplicamos este instrumento a 85 familias de niños con necesidades especiales que lo autollenaron. RESULTADOS: el IFPF presenta características psicométricas adecuadas para la población portuguesa (alfa de Cronbach de 0,90). CONCLUSIÓN: el IFPF fue adaptado y validado para la cultura portuguesa. Consideramos que se trata de un instrumento útil para estudios que se propongan evaluar los factores protectores de la resiliencia familiar. .

Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth.

UNLABELLED: L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD+-dependent L-lactate dehydrogenase. Naturally, Escherichia coli does not produce L-lactate in noticeable amounts, but can catabolize it via a dehydrogenation reaction mediated by an FMN-dependent L-lactate dehydrogenase. In aims to make the E. coli strain to produce L-lactate, three L-lactate dehydrogenase genes from different bacteria were cloned and expressed. The L-lactate producing strains, 090B1 (B0013-070, ΔldhA::diflldD::Pldh-ldhLca), 090B2 (B0013-070, ΔldhA::diflldD::Pldh-ldhStrb) and 090B3 (B0013-070, ΔldhA::diflldD::Pldh-ldhBcoa) were developed from a previously developed D-lactate over-producing strain, E. coli strain B0013-070 (ack-ptappspflBdldpoxBadhEfrdA) by: (1) deleting ldhA to block D-lactate formation, (2) deleting lldD to block the conversion of L-lactate to pyruvate, and (3) expressing an L-lactate dehydrogenase (L-LDH) to convert pyruvate to L-lactate under the control of the ldhA promoter. Fermentation tests were carried out in a shaking flask and in a 25-l bioreactor. Strains 090B1, 090B2 or 090B3 were shown to metabolize glucose to L-lactate instead of D-lactate. However, L-lactate yield and cell growth rates were significantly different among the metabolically engineered strains which can be attributed to a variation between temperature optimum for cell growth and temperature optimum for enzymatic activity of individual L-LDH. In a temperature-shifting fermentation process (cells grown at 37°C and L-lactate formed at 42°C), E. coli 090B3 was able to produce 142.2 g/l of L-lactate with no more than 1.2 g/l of by-products (mainly acetate, pyruvate and succinate) accumulated. In conclusion, the production of lactate by E. coli is limited by the competition relationship between cell growth and lactate synthesis. Enzymatic properties, especially the thermodynamics of an L-LDH can be effectively used as a factor to regulate a metabolic pathway and its metabolic flux for efficient L-lactate production. HIGHLIGHTS: The enzymatic thermodynamics was used as a tool for metabolic regulation. Minimizing the activity of L-lactate dehydrogenase in growth phase improved biomass accumulation. Maximizing the activity of L-lactate dehydrogenase improved lactate productivity in production phase.

Bacterial pyruvate production from alginate, a promising carbon source from marine brown macroalgae.

Marine brown macroalgae is a promising source of material for biorefining, and alginate is one of the major components of brown algae. Despite the huge potential availability of alginate, no system has been reported for the production of valuable compounds other than ethanol from alginate, hindering its further utilization. Here we report that a bacterium, Sphingomonas sp. strain A1, produces pyruvate from alginate and secretes it into the medium. High aeration and deletion of the gene for d-lactate dehydrogenase are critical for the production of high concentrations of pyruvate. Pyruvate concentration and productivity were at their maxima (4.56 g/l and 95.0 mg/l/h, respectively) in the presence of 5% (w/v) initial alginate, whereas pyruvate produced per alginate consumed and % of theoretical yield (0.19 g/g and 18.6%, respectively) were at their maxima at 4% (w/v) initial alginate. Concentration of pyruvate decreased after it reached its maximum after cultivations for 2 or 3 days at 145 strokes per minute. Our study is the first report to demonstrate the production of other valuable compounds than ethanol from alginate, a promising marine macroalgae carbon source.

2,3-Butanediol recovery from fermentation broth by alcohol precipitation and vacuum distillation.

This study presents a new and effective downstream process to recover 2,3-butanediol (2,3-BD) from fermentation broth which is produced by a recombinant Klebsiella pneumoniae strain. The ldhA-deficient K. pneumoniae strain yielded about 90 g/L of 2,3-BD, along with a number of by-products, such as organic acids and alcohols, in a 65 h fed-batch fermentation. The pH-adjusted cell-free fermentation broth was firstly concentrated until 2,3-BD reached around 500 g/L by vacuum evaporation at 50°C and 50 mbar vacuum pressure. The concentrated solution was further treated using light alcohols, including methanol, ethanol, and isopropanol, for the precipitation of organic acids and inorganic salts. Isopropanol showed the highest removal efficiency, in which 92.5% and 99.8% of organic acids and inorganic salts were precipitated, respectively. At a final step, a vacuum distillation process enabled the recovery of 76.2% of the treated 2,3-BD, with 96.1% purity, indicating that fermentatively produced 2,3-BD is effectively recovered by a simple alcohol precipitation and vacuum distillation.

Lactato en sangre de cordón umbilical y estado metabólico en recién nacidos con sufrimiento fetal intraparto / Umbilical cord lactate and metabolic status in newborns with intrapatum fetal distress

Objetivo. Relacionar las concentraciones de lactato en sangre de cordón umbilical y el estado metabólico en recién nacidos con sufrimiento fetal intraparto. Materiales y métodos. Se obtuvo un segmento de cordón umbilical de recién nacidos a término para medir de las concentraciones de lactato de la arteria umbilical, el pH y el déficit de base, tanto de la arteria como de la vena umbilical. Resultados. De los 200 cordones umbilicales analizados, 19 (9,5%) pertenecían a recién nacidos con diagnóstico de estado fetal no reactivo debido a cambios en la cardiotocografía (grupo A). En el grupo B se ubicaron los recién nacidos, sin alteraciones. Los valores promedio de pH y déficit de base de la arteria y vena umbilical fueron significativamente más altos en los recién nacidos del grupo A que en los recién nacidos del grupo B (p < 0,05). Las concentraciones de lactato en los recién nacidos con sufrimiento fetal (grupo A) fueron de 5,37 ± 0,99 nmol/L y en los recién nacidos sin sufrimiento fetal fue de (grupo B) fue de 4,21 ± 0,88 nmol/L (p < 0,05). En los recién nacidos del grupo A, se encontró una correlación modera, negativa y significativa entre las concentraciones de lactato y el déficit de base de la vena umbilical (r = –0,554; p < 0,05). Conclusión. Los recién nacidos con sufrimiento fetal intraparto tienen concentraciones más elevadas de lactato y que existe una correlación entre las concentraciones y el déficit de base en la vena umbilical solo en este grupo de recién nacidos (AU)

Objective: To relate lactate concentrations in umbilical cord blood and metabolic status in newborns with intrapartum fetal distress. Materials and methods: A portion of umbilical cord was obtained from term newborns to measure lactate concentrations in the umbilical artery, and pH and base deficit in theumbilical artery and vein. Results: Of 200 umbilical cords analyzed, 19 (9.5%) were from newborns with a diagnosis of nonreactive fetal status based on changes in cardiotocography (group A). Newborns without alterations were assigned to group B. Mean values of pH and base deficit of the umbilical artery and vein were significantly higher in group A than in group B (P<.05). Lactate concentrations were 5.37 +/- 0.99 nmol/L in group A and 4.21 +/- 0.88 nmol/L in group B (P<.05). In group A, a moderate, negative and significant association was found between lactate concentrations and base deficit in the umbilical vein (r = —0,554; P>,05). Conclusion: Lactate concentrations were higher in newborns with intrapartum fetal distress. These concentrations were correlated with base deficit in the umbilical vein in group A but not in group B (AU)

In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production.

Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising bulk chemical with wide industrial applications, along with various byproducts. In this study, the in silico gene knockout simulation of K. oxytoca was carried out for 2,3-BD overproduction by inhibiting the formation of byproducts. The knockouts of ldhA and pflB genes were targeted with the criteria of maximization of 2,3-BD production and minimization of byproducts formation. The constructed K. oxytoca ΔldhA ΔpflB strain showed higher 2,3-BD yields and higher final concentrations than those obtained from the wild-type and ΔldhA strains. However, the simultaneous deletion of both genes caused about a 50 % reduction in 2,3-BD productivity compared with K. oxytoca ΔldhA strain. Based on previous studies and in silico investigation that the agitation speed during 2,3-BD fermentation strongly affected cell growth and 2,3-BD synthesis, the effect of agitation speed on 2,3-BD production was investigated from 150 to 450 rpm in 5-L bioreactors containing 3-L culture media. The highest 2,3-BD productivity (2.7 g/L/h) was obtained at 450 rpm in batch fermentation. Considering the inhibition of acetoin for 2,3-BD production, fed-batch fermentations were performed using K. oxytoca ΔldhA ΔpflB strain to enhance 2,3-BD production. Altering the agitation speed from 450 to 350 rpm at nearly 10 g/L of acetoin during the fed-batch fermentation allowed for the production of 113 g/L 2,3-BD, with a yield of 0.45 g/g, and for the production of 2.1 g/L/h of 2,3-BD.

Attenuation of LDHA expression in cancer cells leads to redox-dependent alterations in cytoskeletal structure and cell migration.

Aerobic glycolysis, the preferential use of glycolysis even in the presence of oxygen to meet cellular metabolic demands, is a near universal feature of cancer. This unique type of metabolism is thought to protect cancer cells from damaging reactive oxygen species (ROS) produced in the mitochondria. Using the cancer cell line MDA-MB-435 it is shown that shRNA mediated knockdown of lactate dehydrogenase A (LDHA), a key mediator of aerobic glycolysis, results in elevated mitochondrial ROS production and a concomitant decrease in cell proliferation and motility. Redox-sensitive proteins affected by oxidative stress associated with LDHA knockdown were identified by Redox 2D-PAGE and mass spectrometry. In particular, tropomyosin (Tm) isoforms Tm4, Tm5NM1 and Tm5NM5, proteins involved in cell migration and cytoskeletal dynamics, exhibited changes in disulfide bonding and co-localized with peri-nuclear actin aggregates in LDHA knockdown cells. In contrast, treatment with the thiol-based antioxidant N-acetylcysteine promoted the relocalization of Tms to cortical actin microfilaments and partially rescued the migration defects associated with attenuated LDHA expression. These results suggest that aerobic glycolysis and reduced mitochondrial ROS production create an environment conducive to cytoskeletal remodeling; key events linked to the high cell motility associated with cancer.

Engineered Bacillus subtilis 168 produces L-malate by heterologous biosynthesis pathway construction and lactate dehydrogenase deletion.

In the present work, Bacillus subtilis was engineered to produce L-malate. Initially, the study revealed that the slight fumarase activity under anaerobic conditions is extremely favourable for L-malate one-step fermentation accumulation. Subsequently, an efficient heterologous biosynthesis pathway formed by Escherichia coli phosphoenolpyruvate carboxylase and Saccharomyces cerevisiae malate dehydrogenase was introduced into B. subtilis, which led to 6.04 ± 0.19 mM L-malate production. Finally, the L-malate production was increased 1.5-fold to 9.18 ± 0.22 mM by the deletion of lactate dehydrogenase. Under two-stage fermentation conditions, the engineered B. subtilis produced up to 15.65 ± 0.13 mM L-malate, which was 86.3 % higher than that under anaerobic fermentation conditions. Though the L-malate production by the recombinant was low, this is the first attempt to produce L-malate in engineered B. subtilis and paves the way for further improving L-malate production in B. subtilis.

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of the lactate dehydrogenase-deficient recombinant Klebsiella pneumoniae overexpressing an aldehyde dehydrogenase.

In the present study, the lactate dehydrogenase-deficient (ldhA(-)) recombinant Klebsiella pneumoniae overexpressing an ALDH (KGSADH) was developed and the co-production of 3-HP and PDO from glycerol by this recombinant under resting cell conditions was examined. The new recombinant did not produce any appreciable lactate, which seriously inhibits the production of 3-HP and PDO. The final titers of 3-HP and PDO by the ldhA(-) recombinant strain at 60 h were 252.2 mM and 308.7 mM, respectively, which were improved by approximately 30% and 50%, respectively, compared to those by the counterpart recombinant strain, which was the wild type for ldhA. In addition, after deleting ldhA, the cumulative yield on glycerol and specific production rate of these two metabolites (3-HP and PDO) were enhanced by 41.4% and 52%, respectively.

LDH-A influences hypoxia-inducible factor 1α (HIF1 α) and is critical for growth of HT29 colon carcinoma cells in vivo.

Serum lactate dehydrogenase (LDH) is a well-known clinical surrogate parameter. A high activity of LDH is associated with a poor prognosis in different tumor types. Here we demonstrate by a gene silencing approach that LDH-A is critical for in vivo but not in vitro growth of HT29 colon carcinoma cells. We provide evidence that the suppression of the LDH-A gene leads to an increased level of hypoxia inducible factor 1α (HIF1α) but in consequence not to an increase of HIF1 regulated proteins such as carbonic anhydrase IX (CAIX), vascular endothelial growth factor (VEGF), prolyl-hydroxylase 2 (PHD2), and factor-inhibiting HIF (FIH) in cell cultures and tumor lysates. This effect is independent of LDH activity in vivo. We conclude that LDH-A has an influence on the activity of HIF1α and thus on the adaptation of cells to a hypoxic tumor microenvironment in HT29 colon cells. We suggest the use of LDH-M as a potential therapeutic target for anticancer treatment.