Heat Resistance and Salt Hypersensitivity in Lactococcus lactis Due to Spontaneous Mutation of llmg_1816 (gdpP) Induced by High-Temperature Growth

During construction of several gene deletion mutants in Lactococcus lactis MG1363 which involved a high-temperature (37.5°C) incubation step, additional spontaneous mutations were observed which resulted in stable heat resistance and in some cases salt-hypersensitive phenotypes. Whole-genome sequencing of one strain which was both heat resistant and salt hypersensitive, followed by PCR and sequencing of four other mutants which shared these phenotypes, revealed independent mutations in llmg_1816 in all cases. This gene encodes a membrane-bound stress signaling protein of the GdpP family, members of which exhibit cyclic dimeric AMP (c-di-AMP)-specific phosphodiesterase activity. Mutations were predicted to lead to single amino acid substitutions or protein truncations. An independent llmg_1816 mutant (Δ1816), created using a suicide vector, also displayed heat resistance and salt hypersensitivity phenotypes which could be restored to wild-type levels following plasmid excision. L. lactis Δ1816 also displayed improved growth in response to sublethal concentrations of penicillin G. High-temperature incubation of a wild-type industrial L. lactis strain also resulted in spontaneous mutation of llmg_1816 and heat-resistant and salt-hypersensitive phenotypes, suggesting that this is not a strain-specific phenomenon and that it is independent of a plasmid integration event. Acidification of milk by the llmg_1816-altered strain was inhibited by lower salt concentrations than the parent strain. This study demonstrates that spontaneous mutations can occur during high-temperature growth of L. lactis and that inactivation of llmg_1816 leads to temperature resistance and salt hypersensitivity.     

Glycolytic Flux Signals to mTOR through Glyceraldehyde-3-Phosphate Dehydrogenase-Mediated Regulation of Rheb

The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR''s access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.The mTOR complex 1 (mTORC1) signal transduction pathway acts as a central controller of cell growth in mammals (20, 23, 29). mTORC1 integrates a wide range of intracellular and extracellular signals, including insulin, availability of nutrients (glucose and amino acids), cellular energy status, and hypoxia, to regulate protein synthesis and cell growth (11, 12, 17, 36, 46). Many of these environmental cues are integrated into tuberous sclerosis complex (TSC1-TSC2), the major upstream regulator of mTORC1. In response to the absence of insulin and to the low-energy status of cells, the TSC1-TSC2 complex stimulates the GTPase function of Rheb, a small GTPase that acts as a proximal key activator of mTORC1, which leads to the inhibition of Rheb-mediated mTORC1 activation. In contrast, inactivation of the TSC1-TSC2 complex results in the accumulation of GTP-bound Rheb and thus activation of mTORC1 (3, 13, 21, 27, 32, 39). For this reason, both the loss of TSC proteins and the overexpression of Rheb cause hyperactivation of mTORC1 signaling, which is frequently observed in many common human cancers (2, 5, 19, 25, 33). Therefore, a tight regulation of Rheb activity is critical for the proper operation of the mTORC1 pathway in response to environmental cues.Rheb is an atypical member of the Ras superfamily of GTPases (1, 10, 47). As with other small GTPases, the activity of Rheb is regulated by its guanine nucleotide binding status. However, the negative control of GTP-bound Rheb by the TSC1-TSC2 complex has only recently been investigated, and the regulation of the nucleotide binding status of Rheb is not fully understood. A recent study proposed that translationally controlled tumor protein may function as a guanine nucleotide exchange factor for Rheb that causes the accumulation of GTP-bound Rheb (18). GTP-bound Rheb is essential for activating mTOR kinase (21, 28, 38). However, the interaction between Rheb and mTOR does not depend on the GTP binding status of Rheb (30), raising questions regarding the mechanism by which Rheb activates mTORC1. Recently, FKBP38 (immunophilin FK506-binding protein, 38 kDa) was found to be a direct binding partner of Rheb and an inhibitor of mTORC1 (4). GTP-bound Rheb binds FKBP38 and releases FKBP38 from mTORC1, resulting in activation of the mTORC1 pathway. However, there have been conflicting results regarding the effects of nutrient availability on Rheb activity (31, 37, 42, 50) and the effect of these newly identified regulators of Rheb function (44, 45). Thus, the precise molecular mechanisms underlying Rheb regulation and Rheb-mediated mTORC1 activation have remained unclear.In this study, we identified glyceraldehyde-3-phosphate (Gly-3-P) dehydrogenase (GAPDH) as a novel Rheb binding protein and a negative regulator of Rheb. We found that the interaction between GAPDH and Rheb is induced when the glycolytic flux is suppressed under low-glucose conditions to inhibit mTORC1. Here, we provide a molecular mechanism underlying the cross talk between the glycolytic flux and the mTORC1 signaling.     

Association of pneumococcal carriage in infants with the risk of carriage among their contacts in Nha Trang,Vietnam: A nested cross-sectional survey

BackgroundInfants are at highest risk of pneumococcal disease. Their added protection through herd effects is a key part in the considerations on optimal pneumococcal vaccination strategies. Yet, little is currently known about the main transmission pathways to this vulnerable age group. Hence, this study investigates pneumococcal transmission routes to infants in the coastal city of Nha Trang, Vietnam.Methods and findingsIn October 2018, we conducted a nested cross-sectional contact and pneumococcal carriage survey in randomly selected 4- to 11-month-old infants across all 27 communes of Nha Trang. Bayesian logistic regression models were used to estimate age specific carriage prevalence in the population, a proxy for the probability that a contact of a given age could lead to pneumococcal exposure for the infant. We used another Bayesian logistic regression model to estimate the correlation between infant carriage and the probability that at least one of their reported contacts carried pneumococci, controlling for age and locality. In total, 1,583 infants between 4 and 13 months old participated, with 7,428 contacts reported. Few infants (5%, or 86 infants) attended day care, and carriage prevalence was 22% (353 infants). Most infants (61%, or 966 infants) had less than a 25% probability to have had close contact with a pneumococcal carrier on the surveyed day. Pneumococcal infection risk and contact behaviour were highly correlated: If adjusted for age and locality, the odds of an infant’s carriage increased by 22% (95% confidence interval (CI): 15 to 29) per 10 percentage points increase in the probability to have had close contact with at least 1 pneumococcal carrier. Moreover, 2- to 6-year-old children contributed 51% (95% CI: 39 to 63) to the total direct pneumococcal exposure risks to infants in this setting. The main limitation of this study is that exposure risk was assessed indirectly by the age-dependent propensity for carriage of a contact and not by assessing carriage of such contacts directly.ConclusionsIn this study, we observed that cross-sectional contact and infection studies could help identify pneumococcal transmission routes and that preschool-age children may be the largest reservoir for pneumococcal transmission to infants in Nha Trang, Vietnam.     

Phosphoinositide 3-kinase dependent regulation of Kv channels in dendritic cells.

The phosphoinositide 3 (PI3) kinase plays a pivotal role in the regulation of dendritic cells (DCs), antigen-presenting cells that are able to initiate primary immune responses and to establish immunological memory. PI3 kinase is an endogenous suppressor of interleukin 12 (IL-12) production in DCs that is triggered by Toll-like receptor signaling. Inhibition of IL-12 production limits T helper 1 (Th1) polarization. On the other hand, PI3 kinase is an important regulator of various ion channels. The present study aimed to explore whether ion channels in DCs are regulated by PI3 kinase and whether they are important for DC function. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by patch clamp. As a result, DCs express voltage-gated K(+) channels (Kv), which are blocked by Stichodactyla helianthus toxin (ShK, 2.5 nM). A significant upregulation of Kv currents was observed upon maturation of DCs as induced by stimulation of the cells with lipopolysaccharide (LPS, 0.1 microg/ml, 48 h). A dramatic increase of Kv current amplitude was observed following preincubation of the cells with LY294002 (100 nM), a specific inhibitor of PI3 kinase. PI3 kinase inhibitor wortmannin (100 nM) similarly increased Kv current. LY294002 treatment was further followed by a significant increase of IL-12 production. ShK (100 nM) significantly blunted the stimulation of IL-12 release by LPS but not when the cells were first pretreated with LY294002. The observations point to Kv channel sensitive and Kv channel insensitive regulation of DC function.     

A selective ‘Off–On’ fluorescent sensor for Zn2+ based on hydrazone–pyrene derivative and its application for imaging of intracellular Zn2+

A simple and effective fluorescent sensor based on hydrazone–pyrene has been synthesized. This probe displays a highly selective fluorescent enhancement with Zn²⁺, and application of this probe to detect the intrinsic Zn²⁺ ions present in pancreatic β-cells was successfully demonstrated.     

质粒pcDNA3—HGF的大规模纯经制备研究

质粒ｐｃＤＮＡ３－ＨＧＦ具有潜在的临床治疗缺血性疾病的应用前景。大规模纯化制备是质粒ＤＮＡ应用于基因治疗的关键步骤。质粒大规模纯化制备流程包括：发酵、离心收集细胞、碱性裂解、Ｑ－Ｓｅｐｈａｒｏｓｅ ＸＬ捕获质粒ＤＮＡ、Ｓｏｕｒｃｅ １５Ｑ精制质粒ＤＮＡ,所得纯超螺旋质粒ｐｃＤＮＡ３－ＨＧＦ符合质量标准。该纯化制备方法避免使用动物源性的酶及有毒试剂。     

Environmental enrichment compensates for the effects of stress on disease progression in Tg2576 mice, an Alzheimer's disease model

Various environmental factors are known to influence the onset and progression of Alzheimer's disease (AD). Environmental enrichment was reported to improve cognitive performance in various Alzheimer's transgenic mice via an amyloid-related or unrelated mechanism. However, stress has been found to accelerate amyloid deposition and cognitive deficits in many AD models. The aim of this study was to determine whether environmental enrichment compensates for the effects of stress on disease progression in the Tg2576 mice, an established AD model. We housed Tg2576 mice under environmental enrichment, enrichment plus stress, stress, or control conditions at 3 months of age. In this study, we first report that environmental enrichment counteracts the effects of stress in terms of cognitive deficits, tau phosphorylation, neurogenesis, and neuronal proliferation during AD-like disease progression. These results strongly implicate the importance of environmental factors as a major modulator for the disease progression of AD.     

Protective effects of Danshensu from the aqueous extract of Salvia miltiorrhiza (Danshen) against homocysteine-induced endothelial dysfunction

Homocysteine (Hcy) is a by-product of methionine metabolism. An imbalance of Hcy in the body may lead to hyperhomocysteinemia, a condition with elevated Hcy concentration in blood that may be one of the risk factors responsible for the development of several vascular diseases (thromboembolism, atherosclerosis, stroke, vascular diseases and dementia). Radix Salvia miltiorrhiza (Danshen), a well-known Chinese medicinal herb that can activate and improve blood microcirculation, is noticeable for its beneficial effect in treating cardiovascular diseases. The present study is to demonstrate the protective effect of Danshen extract against the homocysteine-induced adverse effect on human umbilical vein endothelial cell (HUVEC). Homocysteine (5 mM) not only decreased the cell viability but also caused the disruption of capillary-like structure formation in vitro. The protective effect of Danshen aqueous extract and its active compounds on endothelial cell function were demonstrated through an in vitro tube formation assay, which mimics the new blood vessel formation. To identify the active components in the aqueous extract of Danshen, the content was characterized by instrumental analysis using high performance liquid chromatography with diode array detector (DAD) and electrospray tandem mass spectrometry (ESI-MS/MS). Interestingly, Danshen extract and its pure compounds showed different effectiveness in protecting HUVEC against Hcy-induced injury according to the following descending order: Danshen aqueous extract, 3-(3,4-dihydroxy-phenyl)-2-hydroxy-propionic acid (Danshensu), protocatechuic acid, catechin and protocatechualdehyde. We believed that such findings might provide evidence in understanding the beneficial effects of Danshen on the cardiovascular system.     

Risk factors of Streptococcus suis infection in Vietnam. A case-control study

Background

Streptococcus suis infection, an emerging zoonosis, is an increasing public health problem across South East Asia and the most common cause of acute bacterial meningitis in adults in Vietnam. Little is known of the risk factors underlying the disease.

Methods and Findings

A case-control study with appropriate hospital and matched community controls for each patient was conducted between May 2006 and June 2009. Potential risk factors were assessed using a standardized questionnaire and investigation of throat and rectal S. suis carriage in cases, controls and their pigs, using real-time PCR and culture of swab samples. We recruited 101 cases of S. suis meningitis, 303 hospital controls and 300 community controls. By multivariate analysis, risk factors identified for S. suis infection as compared to either control group included eating “high risk” dishes, including such dishes as undercooked pig blood and pig intestine (OR₁ = 2.22; 95%CI = [1.15–4.28] and OR₂ = 4.44; 95%CI = [2.15–9.15]), occupations related to pigs (OR₁ = 3.84; 95%CI = [1.32–11.11] and OR₂ = 5.52; 95%CI = [1.49–20.39]), and exposures to pigs or pork in the presence of skin injuries (OR₁ = 7.48; 95%CI = [1.97–28.44] and OR₂ = 15.96; 95%CI = [2.97–85.72]). S. suis specific DNA was detected in rectal and throat swabs of 6 patients and was cultured from 2 rectal samples, but was not detected in such samples of 1522 healthy individuals or patients without S. suis infection.

Conclusions

This case control study, the largest prospective epidemiological assessment of this disease, has identified the most important risk factors associated with S. suis bacterial meningitis to be eating ‘high risk’ dishes popular in parts of Asia, occupational exposure to pigs and pig products, and preparation of pork in the presence of skin lesions. These risk factors can be addressed in public health campaigns aimed at preventing S. suis infection.     

Nitric Oxide Induction of Parkin Translocation in PTEN-induced Putative Kinase 1 (PINK1) Deficiency: FUNCTIONAL ROLE OF NEURONAL NITRIC OXIDE SYNTHASE DURING MITOPHAGY*

The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser¹⁴¹²) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease.