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Bacterial stress-signaling alarmones are important components of a protective network against diverse stresses such as nutrient starvation and antibiotic assault. pppGpp and ppGpp, collectively (p)ppGpp, have well-documented regulatory roles in gene expression and protein translation. Recent work has highlighted another key function of (p)ppGpp: inducing rapid and coordinated changes in cellular metabolism by regulating enzymatic activities, especially those involved in purine nucleotide synthesis. Failure of metabolic regulation by (p)ppGpp results in the loss of coordination between metabolic and macromolecular processes, leading to cellular toxicity. In this review, we document how (p)ppGpp and newly characterized nucleotides pGpp and (p)ppApp directly regulate these enzymatic targets for metabolic remodeling. We examine targets’ common determinants for alarmone interaction as well as their evolutionary diversification. We highlight classical and emerging themes in nucleotide signaling, including oligomerization and allostery along with metabolic interconversion and crosstalk, illustrating how they allow optimized bacterial adaptation to their environmental niches.

Chromosomes have their own territories and dynamically translocate in response to internal and external cues. However, whether and how territories and the relocation of chromosomes are controlled by other intracellular organelles remains unknown. Upon nutrient starvation and target of rapamycin complex 1 (TORC1) inactivation, micronucleophagy, which preferentially degrades nucleolar proteins, occurs at the nucleus-vacuole junction (NVJ) in budding yeast. Ribosomal DNA (rDNA) is condensed and relocated against the NVJ, whereas nucleolar proteins move towards the NVJ for micronucleophagic degradation, causing dissociation of nucleolar proteins from rDNA. These findings imply that the NVJ is the critical platform in the directional movements of rDNA and nucleolar proteins. Here, we show that cells lacking the NVJ (NVJΔ cells) largely lost rDNA condensation and rDNA-nucleolar protein separation after TORC1 inactivation. The macronucleophagy receptor Atg39, an outer nuclear membrane protein, accumulated at the NVJ and was degraded by micronucleophagy. These suggested that macronucleophagy is also dependent on the presence of the NVJ. However, micronucleophagy, but not macronucleophagy, was abolished in NVJΔ cells. This study clearly demonstrated that vacuoles controls intranuclear events, nucleolar dynamics, from outside of the nucleus via the NVJ under the control of TORC1.

In 2014, the coal cleaning chemical 4-methylcyclohexane methanol (MCHM) spilled into the water supply for 300,000 West Virginians. Initial toxicology tests showed relatively mild results, but the underlying effects on cellular biology were underexplored. Treated wildtype yeast cells grew poorly, but there was only a small decrease in cell viability. Cell cycle analysis revealed an absence of cells in S phase within thirty minutes of treatment. Cells accumulated in G1 over a six-hour time course, indicating arrest instead of death. A genetic screen of the haploid knockout collection revealed 329 high confidence genes required for optimal growth in MCHM. These genes encode three major cell processes: mitochondrial gene expression/translation, the vacuolar ATPase, and aromatic amino acid biosynthesis. The transcriptome showed an upregulation of pleiotropic drug response genes and amino acid biosynthetic genes and downregulation in ribosome biosynthesis. Analysis of these datasets pointed to environmental stress response activation upon treatment. Overlap in datasets included the aromatic amino acid genes ARO1, ARO3, and four of the five TRP genes. This implicated nutrient deprivation as the signal for stress response. Excess supplementation of nutrients and amino acids did not improve growth on MCHM, so the source of nutrient deprivation signal is still unclear. Reactive oxygen species and DNA damage were directly detected with MCHM treatment, but timepoints showed these accumulated slower than cells arrested. We propose that wildtype cells arrest from nutrient deprivation and survive, accumulating oxidative damage through the implementation of robust environmental stress responses.

Almost 140 years after the identification of Mycobacterium tuberculosis as the etiological agent of tuberculosis, important aspects of its biology remain poorly described. Little is known about the role of posttranscriptional control of gene expression and RNA biology, including the role of most of the small RNAs (sRNAs) identified to date. We have carried out a detailed investigation of the M. tuberculosis sRNA F6 and shown it to be dependent on SigF for expression and significantly induced in starvation conditions in vitro and in a mouse model of infection. Further exploration of F6 using an in vitro starvation model of infection indicates that F6 affects the expression of the essential chaperonins GroEL2 and GroES. Our results point toward a role for F6 during periods of low metabolic activity typically associated with long-term survival of M. tuberculosis in human granulomas. IMPORTANCE Control of gene expression via small regulatory RNAs (sRNAs) is poorly understood in one of the most successful pathogens, Mycobacterium tuberculosis. Here, we present an in-depth characterization of the sRNA F6, including its expression in different infection models and the differential gene expression observed upon deletion of the sRNA. Our results demonstrate that deletion of F6 leads to dysregulation of the two essential chaperonins GroEL2 and GroES and, moreover, indicate a role for F6 in the long-term survival and persistence of M. tuberculosis in the human host.

The stringent response is well conserved across bacterial species and is a key pathway involved both in bacterial survival and virulence and in the induction of antibiotic tolerance in Mycobacteria. It is mediated by the alarmone (p)ppGpp and the regulatory molecule inorganic polyphosphate in response to stress conditions such as nutrient starvation. Efforts to pharmacologically target various components of the stringent response have shown promise in modulating mycobacterial virulence and antibiotic tolerance. In this review, we summarize the current understanding of the stringent response and its role in virulence and tolerance in Mycobacteria, including evidence that targeting this pathway could have therapeutic benefit.

Employing the optical redox imaging technique, we previously identified a significant redox shift of nicotinamide adenine dinucleotide (NAD and the reduced form NADH) in freshly isolated alveolar macrophages (AM) from ozone-exposed mice. The goal here was twofold: (a) to determine the NAD(H) redox shift in cryopreserved AM isolated from ozone-exposed mice and (b) to investigate whether there is a difference in the redox status between cryopreserved and freshly isolated AM. We found: (i) AM from ozone-exposed mice were in a more oxidized redox state compared to that from filtered air (FA)-exposed mice, consistent with the results obtained from freshly isolated mouse AM, (ii) under FA exposure, there was no significant NAD(H) redox difference between fresh AM that had been placed on ice for 2.5 h and cryopreserved AM, however, under ozone exposure, fresh AM were more oxidized than cryopreserved AM, (iii) via the use of nutrient starvation and replenishment and H2O2-induced oxidative stress of an AM cell line, we showed that this redox difference between cryopreserved and freshly isolated AM is likely the result of the double “hit”, i.e., the ozone-induced oxidative stress plus nutrient starvation that prevented freshly isolated AM from a full recovery after being on ice for a prolonged time period. The cryopreservation technique we developed eliminates/minimizes the effects of oxidative stress and nutrient starvation on cells. This method can be adopted to preserve lung macrophages from animal models or clinical patients for further investigations.

Mycobacterium tuberculosis (MTB) persists and survives antibiotic treatments by generating phenotypically heterogeneous drug-tolerant subpopulations. The surviving cells, persisters, are a major barrier to the relapse-free treatment of tuberculosis (TB), which is already killing >1.8 million people every year and becoming deadlier with the emergence of multidrug-resistant strains., Mycobacterium tuberculosis (MTB) generates phenotypic diversity to persist and survive the harsh conditions encountered during infection. MTB avoids immune effectors and antibacterial killing by entering into distinct physiological states. The surviving cells, persisters, are a major barrier to the timely and relapse-free treatment of tuberculosis (TB). We present for the first time, PerSort, a method to isolate and characterize persisters in the absence of antibiotic or other pressure. We demonstrate the value of PerSort to isolate translationally dormant cells that preexisted in small numbers within Mycobacterium species cultures growing under optimal conditions but that dramatically increased in proportion under stress conditions. The translationally dormant subpopulation exhibited multidrug tolerance and regrowth properties consistent with those of persister cells. Furthermore, PerSort enabled single-cell transcriptional profiling that provided evidence that the translationally dormant persisters were generated through a variety of mechanisms, including vapC30, mazF, and relA/spoT overexpression. Finally, we demonstrate that notwithstanding the varied mechanisms by which the persister cells were generated, they converge on a similar low-oxygen metabolic state that was reversed through activation of respiration to rapidly eliminate persisters fostered under host-relevant stress conditions. We conclude that PerSort provides a new tool to study MTB persisters, enabling targeted strategies to improve and shorten the treatment of TB. IMPORTANCE Mycobacterium tuberculosis (MTB) persists and survives antibiotic treatments by generating phenotypically heterogeneous drug-tolerant subpopulations. The surviving cells, persisters, are a major barrier to the relapse-free treatment of tuberculosis (TB), which is already killing >1.8 million people every year and becoming deadlier with the emergence of multidrug-resistant strains. This study describes PerSort, a cell sorting method to isolate and characterize, without antibiotic treatment, translationally dormant persisters that preexist in small numbers within Mycobacterium cultures. Characterization of this subpopulation has discovered multiple mechanisms by which mycobacterial persisters emerge and unveiled the physiological basis for their dormant and multidrug-tolerant physiological state. This analysis has discovered that activating oxygen respiratory physiology using l-cysteine eliminates preexisting persister subpopulations, potentiating rapid antibiotic killing of mycobacteria under host-relevant stress. PerSort serves as a new tool to study MTB persisters for enabling targeted strategies to improve and shorten the treatment of TB.

The tumor microenvironment is a nutrient-deficient region that alters the cancer cell phenotype to aggravate cancer pathology. The ability of cancer cells to tolerate nutrient starvation is referred to as austerity. Compounds that preferentially target cancer cells growing under nutrient-deficient conditions are being employed in anti-austerity approaches in anticancer drug discovery. Therefore, in this study, we investigated physcion (1) and 2-(2&prime, 3-epoxy-1&prime, 3&prime, 5&prime, heptatrienyl)-6-hydroxy-5-(3-methyl-2-butenyl) benzaldehyde (2) obtained from a culture extract of the marine-derived fungus Aspergillus species (sp.), which were isolated from an unidentified marine sponge, as anti-austerity agents. The chemical structures of 1 and 2 were determined via spectroscopic analysis and comparison with authentic spectral data. Compounds 1 and 2 exhibited selective cytotoxicity against human pancreatic carcinoma PANC-1 cells cultured under glucose-deficient conditions, with IC50 values of 6.0 and 1.7 &micro, M, respectively. Compound 2 showed higher selective growth-inhibitory activity (505-fold higher) under glucose-deficient conditions than under general culture conditions. Further analysis of the mechanism underlying the anti-austerity activity of compounds 1 and 2 against glucose-starved PANC-1 cells suggested that they inhibited the mitochondrial electron transport chain.

Many clinical trials are beginning to assess the effectiveness of compounds known to regulate autophagy in patients receiving anti-cancer chemotherapy. However, autophagy inhibition, through exogenous inhibitors, or activation, through starvation, has revealed conflicting roles in cancer management and chemotherapeutic outcome. This study aimed to assess the effect of amino acid starvation on doxorubicin-treated breast cancer cells by assessing the roles of autophagy and apoptosis. An in vitro breast cancer model consisting of the normal breast epithelial MCF12A and the metastatic breast cancer MDAMB231 cells was used. Apoptotic and autophagic parameters were assessed following doxorubicin treatments, alone or in combination with bafilomycin, ATG5 siRNA or amino acid starvation. Inhibition of autophagy, through ATG5 siRNA or bafilomycin treatment, increased caspase activity and intracellular doxorubicin concentrations in MCF12A and MDAMB231 cells during doxorubicin treatment. While amino acid starvation increased autophagic activity and decreased caspase activity and intracellular doxorubicin concentrations in MCF12A cells, no changes in autophagic parameters or caspase activity were observed in MDAMB231 cells. Our in vivo data showed that 24 h protein starvation during high dose doxorubicin treatment resulted in increased survival of tumor-bearing GFP-LC3 mice. Results from this study suggest that short term starvation during doxorubicin chemotherapy may be a realistic avenue for adjuvant therapy, especially with regards to the protection of non-cancerous cells. More research is however, needed to fully understand the regulation of autophagic flux during starvation.

Glioblastoma is a severe cancer with extremely poor survival. Its treatment typically involves a combination of surgery, chemotherapy, and radiation therapy. However, glioma stem-like cells (GSCs)-a subpopulation of tumor-propagating glioblastoma cells—cause post-treatment recurrence and are a major factor in the poor prognosis of the disease. GSCs have higher proliferation than non-GSCs and are more resistant to invasive chemotherapy and radiotherapy. In this study, we subjected GSCs to nutrient starvation (deprived of glucose, glutamine, and calcium) to determine whether cell death can be triggered as a potential strategy to improve treatment outcomes. Flow cytometry revealed that 35.1%, 96.1%, and 99.9% of starved GSCs underwent apoptosis on days 1, 3, and 5, respectively, along with nearly 100% autophagy on all three days. Western blots detected cleaved caspase-3 (an apoptosis marker) and phospho-beclin 1, LC 3B-I, LC 3B-II (autophagy markers) in C6 GSCs after nutrient starvation for 1, 3, 4, and 5 days. Transmission electron microscopic observation of GSC ultrastructure after starvation treatment revealed that compared with control GSCs, starved cells had more pyknotic nuclei, membrane bleb, swollen endoplasmic reticulum, degenerative mitochondria, lipid droplets, and microvilli loss. Thus, nutrient starvation stresses cells by increasing free radicals. Cell stress opens more channels between mitochondria and endoplasmic reticulum. This study demonstrated that nutrient starvation decreases proliferation by approximately 81%, while increasing apoptosis (99.9%) and autophagy (94.6%) in C6 GSCs by the fifth day. Nutrient starvation of GSCs may, therefore, be an effective therapeutic strategy that can trigger apoptotic and autophagic metabolic reprogramming in cancer cells., C6 glioma stem cells, Nutrient starvation, Apoptosis, Autophagy.

Persistence of infection despite extensive chemotherapy with antibiotics displaying low MICs is a hallmark of lung disease caused by Mycobacterium abscessus (Mab). Thus, the classical MIC assay is a poor predictor of clinical outcome. Discovery of more efficacious antibiotics requires more predictive in vitro potency assays. As a mycobacterium, Mab is an obligate aerobe and a chemo-organo-heterotroph – it requires oxygen and organic carbon sources for growth. However, bacteria growing in patients can encounter micro-environmental conditions that are different from aerated nutrient-rich broth used to grow planktonic cultures for MIC assays. These in vivo conditions may include oxygen and nutrient limitation which should arrest growth. Furthermore, Mab was shown to grow as biofilms in vivo. Here, we show Mab Bamboo, a clinical isolate we use for Mab drug discovery, can survive oxygen deprivation and nutrient starvation for extended periods of time in non-replicating states and developed an in vitro model where the bacterium grows as biofilm. Using these culture models, we show that non-replicating or biofilm-growing bacteria display tolerance to clinically used anti-Mab antibiotics, consistent with the observed persistence of infection in patients. To demonstrate the utility of the developed ‘persister’ assays for drug discovery, we determined the effect of novel agents targeting membrane functions against these physiological forms of the bacterium and find that these compounds show ‘anti-persister’ activity. In conclusion, we developed in vitro 'persister' assays to fill an assay gap in the Mab drug discovery compound progression and enable identification of novel lead compounds showing 'anti-persister' activity.

Upon activation, T-cells increase the uptake of glucose and glutamine to build the constituents of proliferating effectors. However, tumor and infected cells compete for the same nutrients. Several observations are consistently indicating that activated T-cells overcome this situation by engaging catabolic pathways. Here I discuss how these observations are reconciled with T-cells’ need of anabolic processes during activation.

In yeast, toxicity of acetaminophen (APAP), a frequently used analgesic and antipyretic drug, depends on ubiquitin-controlled processes. Previously, we showed a remarkable overlap in toxicity profiles between APAP and tyrosine, and a similarity with drugs like rapamycin and quinine, which induce degradation of the amino acid permease Tat2. Therefore, we investigated in yeast whether APAP reduced the expression levels of amino acid permeases. The protein levels of Tat2, Tat1, Mup1 and Hip1 were reduced, while the expression of the general permease Gap1 was increased, consistent with a nutrient starvation response. Overexpression of Tat1 and Tat2, but not Mup1, Hip1 and Gap1 conferred resistance to APAP. A tryptophan auxotrophic strain trp1Δ was more sensitive to APAP than wild-type and addition of tryptophan completely restored the growth restriction of trp1∆ upon APAP exposure, while tyrosine had an additive effect on APAP toxicity. Furthermore, intracellular aromatic amino acid concentrations were reduced upon APAP exposure. This effect was less prominent in ubiquitin-deficient yeast strains that were APAP resistant and showed a reduced degradation of high affinity amino acid permeases. APAP-induced changes in intracellular amino acid concentrations were also detected in hepatoma HepG2 cells indicating significance for humans. Electronic supplementary material The online version of this article (10.1007/s00726-018-2613-8) contains supplementary material, which is available to authorized users.

Tumours evolve to cope with environmental stresses or challenges such as nutrient starvation, depletion of survival factors, and unbalanced mechanical forces. The uncontrolled growth and aberrant deregulation of core cell homeostatic pathways induced by genetic mutations create an environment of stress. Here, we explore how the adaptations of tumours to the changing environment can drive changes in the motility machinery of cells, affecting migration, invasion, and metastasis. Tumour cells can invade individually or collectively, or they can be extruded out of the surrounding epithelium. These mechanisms are thought to be modifications of normal processes occurring during development or tissue repair. Therefore, tumours may activate these pathways in response to environmental stresses, enabling them to survive in hostile environments and spread to distant sites. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P(2), a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P(2)-TRPML1-ALG-2-dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation.

The aging at cellular level manifests itself in the form of uncontrolled formation of ROS, chronic inflammation, and increased susceptibility to cellular stress. Aging is often regarded as a risk factor for several diseases due to several age-associated pathological changes in cells. Sestrin (Sesn) is an important molecule for controlling normal cellular physiology and play a significant role in the progression of certain age-associated cellular pathologies. This review deals with the structure, function, regulation, signaling network, and the potential role of Sesn in age-associated cellular pathophysiology. The cellular response mediated by Sesn under stressful conditions and rescue mechanism is discussed. It would be interesting to find out the precise physiological role of Sesn in the regulation of cellular aging. The anti-aging activity of Sesn may benefit to prevent various age-associated diseases and have clinical utility in diagnostic and therapeutic intervention.

The mechanisms of cancer cell adaptation to tumor microenvironmental conditions, such as hypoxia and nutrient starvation, are currently receiving much attention as possible therapeutic targets. In an attempt to identify selectively cytotoxic substances against cancer cells adapted to nutrient starvation, 4 abietane-type diterpenes, sugiol (1), 6-α-hydroxysugiol (2), cryptojaponol (3), and 6-hydroxy-5,6-dehydrosugiol (4), were isolated from the bark of Taxodium distichum L. Rich var. distichum (bald cypress). Compounds 1, 2, and 4 showed potent cytotoxic activity against PANC-1 cells adapted to nutrient-starved conditions with half-maximal effective concentration (EC50) values of 6.4-9.2 µM, whereas the EC50 values of these compounds against PANC-1 cells under general culture conditions were more than 100 µM. Alternatively, compound 3, which we report for the first time in the genus Taxodium, showed moderate cytotoxicity against PANC-1 cells under nutrient-starved conditions with an EC50 of 37.9 µM. The selective index (S.I.), which compared the activity under nutrient-starved conditions with that under general culture conditions, was low (7.9). Further investigation revealed that the selective cytotoxic activity of compound 2 might be affecting the mitochondria.

Summary Mycobacterium tuberculosis (MTB) is able to persist in the host for long periods of time, even during antibiotic treatment. Eliminating persister cells, which are implicated as the primary reason for treatment failure, is essential for shortening TB treatment regimen. Here, we report a novel methodology, Per-Sort, to identify and sort miniscule numbers of translationally dormant mycobacterial cells within an isogenic mycobacterium population. Using Per-Sort we have discovered that translationally dormant cells pre-exist (under optimal growth conditions) as a fraction of a percent of isogenic mycobacterial cultures, suggesting they are generated stochastically as a bet hedging strategy. We show that this pre-existing translationally dormant subpopulation of cells are tolerant to antibiotics, small in size, low in oxidative metabolism, and expand in number upon nutrient starvation. Finally, through transcriptional profiling at single cell resolution, we’ve determined that the pre-existing persisters are a heterogeneous mix of vapC30, mazF, and relA/spoT overexpressing cells that are eliminated and sensitized to antibiotic killing through induction of respiration. Highlights We have developed a novel method, Per-Sort, to isolate a small proportion of translationally dormant cells that pre-exist in Mycobacterium spp . cultures growing under optimal conditions but dramatically increase in proportion under stressful conditions. The pre-existing translationally dormant cells have lower oxygen consumption, significantly longer lag phase to initiate growth in nutrient rich medium, and high tolerance to >10x MIC concentrations of isoniazid (INH) and rifampicin (RIF), indicating they are a subpopulation of persister cells. Single-cell expression profiling demonstrated that the persisters are a heterogenous mixture of toxin (VapC30 and MazF) and alarmone response (RelA/SpoT) expressing cells. A shared outcome of high toxin and alarmone response is reduced cellular oxidative metabolism, which we demonstrate is reversed upon addition of L-cysteine to reduce the proportion of pre-existing persister cells and increase killing by INH and RIF.

We present a method capable of detecting single slow-growing and growth-arrested cells in a bacterial culture composed of physiologically and phenotypically different cells. Unlike the use of transcriptional reporters to gauge the metabolic activities in cells, here, we fuse two different fluorescent proteins with distinctive maturation rates to construct a timer to directly determine the growth rate of single Pseudomonas aeruginosa cells. We demonstrate that the dual-color fluorescent timer can indicate the slow-growing and growth-arrested cells from bacterial cultures in the presence of various environmental stresses, including nutrient starvation or antibiotic treatments, which greatly expand the methods for detecting and isolating persister cells.

Cytoplasmic lipid droplets are important organelles in nearly every eukaryotic and some prokaryotic cells. Storing and providing energy is their main function, but they do not work in isolation. They respond to stimuli initiated either on the cell surface or in the cytoplasm as conditions change. Cellular stresses such as starvation and invasion are internal insults that evoke changes in droplet metabolism and dynamics. This review will first outline lipid droplet assembly and then discuss how droplets respond to stress and in particular nutrient starvation. Finally, the role of droplets in viral and microbial invasion will be presented, where an unresolved issue is whether changes in droplet abundance promote the invader, defend the host, to try to do both. The challenges of stress and infection are often accompanied by changes in physical contacts between droplets and other organelles. How these changes may result in improving cellular physiology, an ongoing focus in the field, is discussed.

The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48 h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis. The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis. The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could contribute to the delayed apoptosis of hypertrophic chondrocytes in the growth plate, and emphasize the antiapoptotic properties of HAS2., Graphical abstract Nutrient starvation triggers apoptosis in wt fibroblasts, but not in fro/fro fibroblasts (in which nSMase2 is inactive). The resistance to nutrient deprivation-induced cell death in fro/fro cells is mediated by an increased expression of HAS2 and HA secretion and from the subsequent expression of the heat-shock protein Hsp72 that acts as a chaperone and anti-apoptotic protein. HAS2 is not protective against apoptosis evoked by oxLDL and H2O2, and fro/fro fibroblasts are not resistant to these agents., Highlights • Apoptosis evoked by oxidized LDL and H2O2 is comparable in fro/fro and wt fibroblasts. • fro/fro fibroblasts resist to apoptosis evoked by nutrient starvation. • HAS2 increased expression protects fro/fro fibroblasts against apoptosis. • HAS2 regulates the expression of the antiapoptotic heat-shock protein HsP72.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Legionella is an opportunistic waterborne pathogen of increasing public health significance. Pasteurization, otherwise known as super-heat and flush (increasing water temperature to above 70°C and flushing all outlets), has been identified as an important mechanism for the disinfection of Legionella in manufactured water systems. However, several studies have reported that this procedure was ineffective at remediating water distribution systems as Legionella was able to maintain long term persistent contamination. Up to 25% of L. pneumophila cells survived heat treatment of 70°C, but all of these were in a viable but non-culturable state. This demonstrates the limitations of the culture method of Legionella detection currently used to evaluate disinfection protocols. In addition, it has been demonstrated that pasteurization and nutrient starvation can select for thermal tolerant strains, where L. pneumophila was consistently identified as having greater thermal tolerance compared to other Legionella species. This review demonstrates that further research is needed to investigate the effectiveness of pasteurization as a disinfection method. In particular, it focuses on the potential for pasteurization to select for thermal tolerant L. pneumophila strains which, as the primary causative agent of Legionnaires disease, have greater public health significance compared to other Legionella species.

Long noncoding RNAs play a pivotal role in tumor progression, but their role in cancer cells in the nutrient-starved tumor microenvironment remains unknown. Here, we show that a nutrient starvation-responsive long noncoding RNA, JHDM1D antisense 1 (JHDM1D-AS1), promotes tumorigenesis by regulating angiogenesis in response to nutrient starvation. Expression of JHDM1D-AS1 was increased in cancer cells. In addition, expression of JHDM1D-AS1 was increased in clinical tumor samples compared to that in normal tissue. Stable expression of JHDM1D-AS1 in human pancreatic cancer (PANC-1 and AsPC-1) cells promoted cell growth in vitro. Remarkably, these JHDM1D-AS1-expressing cells showed a significant increase in tumor growth in vivo that was associated with increased formation of CD31+ blood vessels and elevated infiltration of CD11b+ macrophage lineage cells into tumor tissues. Genome-wide analysis of tumor xenografts revealed that expression of genes for tumor-derived angiogenic factors such as hHGF and hFGF1 concomitant with host-derived inflammation-responsive genes such as mMmp3, mMmp9, mS100a8, and mS100a9 was increased in tumor xenografts of JHDM1D-AS1-expressing pancreatic cancer cells, leading to a poor prognosis. Our results provide evidence that increased JHDM1D-AS1 expression under nutrient starvation accelerates tumor growth by upregulating angiogenesis, thus laying the foundation for improved therapeutic strategies.

In the course of searching for selective growth inhibitors of the cancer cells adapted to nutrient starvation, a new 3-alkylpyridine alkaloid named N-methylniphatyne A (1) was isolated from an Indonesian marine sponge of Xestospongia sp. The chemical structure of 1 was determined on the basis of the spectroscopic analysis and comparison with the synthesized 1 and its analogues. Compound 1 showed the cytotoxic activity against PANC-1 cells under the condition of glucose starvation with IC50 value of 16 µM, whereas no growth-inhibition was observed up to 100 µM under the general culture conditions.

Published version. Source at https://doi.org/10.3389/fmars.2016.00068 Marine microalgae are considered a potentially new and valuable source of biologically active molecules for applications in the food industry as well as in the pharmaceutical, nutraceutical, and cosmetic sectors. They can be easily cultured, have short generation times and enable an environmentally-friendly approach to drug discovery by overcoming problems associated with the over-utilization of marine resources and the use of destructive collection practices. In this study, 21 diatoms, 7 dinoflagellates, and 4 flagellate species were grown in three different culturing conditions and the corresponding extracts were tested for possible antioxidant, anti-inflammatory, anticancer, anti-diabetes, antibacterial, and anti-biofilm activities. In addition, for three diatoms we also tested two different clones to disclose diversity in clone bioactivity. Six diatom species displayed specific anti-inflammatory, anticancer (blocking human melanoma cell proliferation), and anti-biofilm (against the bacteria Staphylococcus epidermidis) activities whereas, none of the other microalgae were bioactive against the conditions tested for. Furthermore, none of the 6 diatom species tested were toxic on normal human cells. Culturing conditions (i.e., nutrient starvation conditions) greatly influenced bioactivity of the majority of the clones/species tested. This study denotes the potential of diatoms as sources of promising bioactives for the treatment of human pathologies.

Proximal tubular epithelial cells are particularly sensitive to damage. In search of a biomarker, this study evaluated the potential of different cell activation models (hypoxia/replenishment and protein overload) to lead to a release of TFF3 (Trefoil factor 3). Surprisingly, we found disparity in the ability of the different stimuli to enhance the intracellular abundance of TFF3 and its release: While conditions of nutrient starvation and damage associated with replenishment lead to intracellular abundance of TFF3 in the absence of TFF3 release, stimulation with an excess amount of albumin did not yield accumulation of TFF3. By contrast, incubation of cells with a purified lambda light chain preparation from a patient with multiple myeloma provoked the presence of TFF3 in the cell supernatant. We therefore propose that elevations of TFF3 in renal disease might be more revelatory for the cause of restitution than previously thought.

Universal stress proteins (Usps) are among the most highly induced genes when bacteria are subjected to several stress conditions such as heat shock, nutrient starvation or the presence of oxidants or other stress agents.Escherichia colihas five small Usps and one tandem-type Usp. UspE (or YdaA) is the tandem-type Usp and consists of two Usp domains arranged in tandem. To date, the structure of UspE remains to be elucidated. To contribute to the molecular understanding of the function of the tandem-type UspE, UspE fromE. coliwas overexpressed and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. Crystals of UspE were obtained by sitting-drop vapour diffusion. A diffraction data set was collected to a resolution of 3.2 Å from flash-cooled crystals. The crystals belonged to the tetragonal space groupI4122 orI4322, with unit-cell parametersa=b= 121.1,c = 241.7 Å.

An intermittent feeding strategy in fed-batch cultivation has been widely applied to control the accumulation of overflow toxic metabolites and oxygen limitation. However, successive depletions of carbon source by carbon-limited feeding might induce considerable stress to host cells, such as nutrient starvation, which may evoke the enhanced degradation of secreted recombinant proteins by proteolysis. To prevent this degradation, we evaluated novel nutrient feeding strategies for the production of MOX promoter-driven recombinant human serum albumin in the fed-batch cultivation of Hansenula polymorpha. Compared to conventional feeding strategies such as exponential or intermittent feeding strategy, feeding strategies developed in the present study significantly reduced the degradation of recombinant products and yielded high levels of intact recombinant human serum albumin in fed-batch cultivation.

Summary Interruption of the (p)ppGpp synthetase gene (rel) of Sorangium cellulosum So ce56 resulted in loss of ppGpp accumulation after norvaline treatment during exponential growth phase. The rel mutant failed to produce wild-type levels of the polyketides chivosazol and etnangien in production media. In wild-type cells expression of the chivosazol biosynthetic operon can be significantly increased by norvaline or α-methylglucoside. This induction does not occur in the rel mutant. The rel mutant also lost the capability to form multicellular fruiting bodies under nutrient starvation.

Reprogramming of cellular metabolism is crucial for T cells responding to environmental cues. Blagih et al. (2015) reveal that the kinase AMPK directs metabolic adaption of effector T cells upon nutrient limitation and contributes to immune responses in vivo.

Nutrient starvation occurs in plants either by the insufficiency of nutrients in the soil or by their unavailability in plant absorbable form. Nutrient malnutrition is an age-old problem, aggravated by the human demand for more food which had led to the development of nutrient-hungry crop varieties. Ironically, what once lauded as a boon to mankind, the intensive agriculture, is turning to be a multifaceted bane in the form of depletion of natural reserves of inorganic fertilisers, price rise of farm inputs, environmental degradation due to nutrient residues and socio-economic and political divide among farming communities and nations. With the low availability of nutrients, plants are subjected to tremendous stress that jeopardises their normal physiology and survival itself. Rice, the major staple crop on earth is set to suffer any or all of the above problems in the near future. Immediate reduction of fertiliser input is the only viable solution to this problem, but it is going to trigger low production from farmlands. Therefore, nutrient input reduction should be done in conjunction with the development of low nutrient happy rice varieties. There is enough variability for nutrient response within the rice gene pool including low nutrient tolerance, which is to be tapped for the development of new varieties. In addition, low nutrient tolerant varieties can help in producing the best out of marginal lands that are rendered unsuitable for high-yielding varieties due to low nutrient status. This chapter overviews the developments in breeding towards nutrient deficiency tolerant rice varieties as a sustainable solution for future agriculture.

A combination of experimental and theoretical approaches was used to investigate the role of nutrient starvation as a potential trigger for biofilm detachment. Experimental observations of detachment in a variety of biofilm systems were made with pure cultures of Pseudomonas aeruginosa . These observations indicated that biofilms grown under continuous-flow conditions detached after flow was stopped, that hollow cell clusters were sometimes observed in biofilms grown in flow cells, and that lysed cells were apparent in the internal strata of colony biofilms. When biofilms were nutrient starved under continuous-flow conditions, detachment still occurred, suggesting that starvation and not the accumulation of a metabolic product was responsible for triggering detachment in this particular system. A cellular automata computer model of biofilm dynamics was used to explore the starvation-dependent detachment mechanism. The model predicted biofilm structures and dynamics that were qualitatively similar to those observed experimentally. The predicted features included centrally located voids appearing in sufficiently large cell clusters, gradients in growth rate within these clusters, and the release of most of the biofilm with simulated stopped-flow conditions. The model was also able to predict biofilm sloughing resulting solely from this detachment mechanism. These results support the conjecture that nutrient starvation is an environmental cue for the release of microbes from a biofilm.

A range of pathophysiological factors can result in a perturbation or restriction of fetal growth, and the cardiovascular, neuroendocrine and metabolic adaptations of the fetus to these stimuli will depend on their nature, timing and intensity. The critical importance of these physiological adaptations for both immediate survival and long-term health outcomes has provided an impetus for experimental studies of the nature and consequences of specific fetal adaptations to a poor intrauterine environment. This review summarizes data from recent studies that have focused on the responses of the fetal cardiovascular, sympathoadrenal, hypothalamo-pituitary-adrenal and renin-angiotensin systems to experimental restriction of placental function in the sheep and discusses the consequences of these adaptations for fetal, neonatal and adult health.

Research marches on the feet of methodology. Advances are made when we have acquired the means to utilize the accrued information. In this way, investigation into the influence of energy restriction in cancer has gone through three distinct periods. After the initial observation by Moreschi in 1909, there was about a decade of active research in this area. Then interest waned, possibly because the field had gone as far as it could considering the knowledge and methodology available at the time. Interest was rekindled in 1940 due, principally, to the work coming from the laboratories of Tannenbaum at the Michael Reese Hospital in Chicago and Baumann at the University of Wisconsin. Another decade of active research followed. In this period we learned how to design experimental diets and interest was expressed in dietary constituents. By 1950 publications on this type of research had dwindled and the field lay virtually dormant for 30 years. Since the early 1980s research on this topic has blossomed and we now know enough about physiology and molecular biology to probe the mechanisms underlying the phenomenon. Energy flux, as in exercise, also inhibits carcinogenesis. Energy restriction modulates oxidative DNA damage and enhances DNA repair. It is now apparent that energy restriction affects adrenal metabolism, insulin metabolism, and various aspects of gene expression. Understanding the basic mechanisms should provide important insights into control of tumor proliferation.

The CH(2)Cl(2)-soluble extract of Angelica pubescens was found to kill PANC-1 cancer cells preferentially under nutrition starvation at a concentration of 50 microg/ml, with virtually no cytotoxicity under nutrient-rich conditions. Further bioassay-guided fractionation and isolation led to the isolation of a novel compound named angelmarin as the primary compound responsible for the preferential cytotoxicity; the compound exhibited 100% preferential cytotoxicity against PANC-1 cells at a concentration of 0.01 microg/ml.

Tumour progression is characterized by massive cellular proliferation associated with alterations of the tumour microenvironment. Hence, the tumour microenvironment is considered to be of great importance for tumourigenesis and, as a consequence, might influence the response to antitumour therapy. The microenvironmental alterations comprise hypoxia, acidosis, nutrient starvation, as well as increased interstitial fluid pressure (Denko 2008; Milosevic et al. 2004; Pouyssegur et al. 2006; Vaupel et al. 1989) and are largely the result of a defective and/or inadequate tumour vasculature which develops during rapid tumour growth (Bertout et al. 2008; Brown and Giaccia 1998; Vaupel et al. 1989; Vaupel 2004). Hypoxia is present in virtually every solid tumour and probably represents the most persistent of the microenvironmental hallmarks that sway tumour progression.

15 páginas, 2 figuras -- PAGS nros. 704-718, Development in the ascomycete A. nidulans is principally determined by environmental signals. Adaptability to oxidative stimuli can derive in changes of growth patterns and/or the activation of sexual or asexual reproductive cycles but this model fungus might also respond to high osmotic or salt concentrations, the redox state, the availability and quantity of carbon or nitrogen sources and the degree or quality of illumination. Since each cell within the colony follows a single morphogenetic program at a time, all these environmental cues might be sensed and integrated into a limited number of intracellular signals which, finally, would activate the required morphogenetic program and repress the others. This signaling mainly occurs through stress response pathways. The present review aims to summarize the available knowledge on how these pathways transduce environmental stimuli to mediate morphological changes in Aspergillus nidulans., This work was supported by the Spanish Ministerio de Educación y Ciencia through grant BFU2009-08701 to E.A.E, and by the Basque Government through grant IT393- 10 to U.U. O.E. is a contract researcher of The University of The Basque Country

Glucose-starved cells of Vibrio parahaemolyticus were compared with non-starved counterparts with respects to heat, osmotic, and oxidative challenges. The starved cells demonstrated greater thermal and oxidative resistance than did the non-starved cells. The starved cells also showed greater resistance against low osmotic challenge than did the non-starved cells although both cells showed a comparable resistance against high osmotic challenge.

The IκB kinase (IKK) pathway is an essential mediator of inflammatory, oncogenic, and cell stress pathways. Recently IKK was shown to be essential for autophagy induction in mammalian cells independent of its ability regulate NF-κB, but the mechanism by which this occurs is unclear. Here we demonstrate that the p85 regulatory subunit of PI3K is an IKK substrate, phosphorylated at S690 in vitro and in vivo in response to cellular starvation. Cells expressing p85 S690A or inhibited for IKK activity exhibit increased Akt activity following cell starvation, demonstrating that p85 phosphorylation is required for starvation-induced PI3K feedback inhibition. S690 is in a conserved region of the p85 cSH2 domain, and IKK-mediated phosphorylation of this site results in decreased affinity for tyrosine-phosphorylated proteins and decreased PI3K membrane localization. Finally, leucine deprivation is shown to be necessary and sufficient for starvation-induced, IKK-mediated p85 phosphorylation and PI3K feedback inhibition.

Latent tuberculosis (TB) infects one-third of the world. We present evidence for the existence of a latent state of TB in humans, cite new approaches to diagnosis and treatment, and identify several models that attempt to mimic the latent state. Persistent infection in mice and in vitro systems of microaerophilic and/or anaerobic growth and nutrient starvation have been the most productive models in yielding insights into the host and mycobacterial pathways involved in the latent state. These pathways may serve as targets for better diagnosis, treatment, and prevention of latent TB in man. Copyright 2002, Elsevier Science (USA). All rights reserved.

Pseudomonas aeruginosa cells were inoculated into different waters and sampled after different periods of starvation in order to evaluate the influences of storage under daylight or dark conditions, the presence or absence of the autochthonous flora, the chemical composition of the water and the storage temperature, on survival. Survival was investigated by plate counts on selective and nonselective agar media. Light, low temperature (4C) and presence of the autochthonous flora negatively influenced the survival of P. aeruginosa during starvation in water. Higher survival rates were observed in waters with high mineral content. During starvation, cells developed sensitivity to the selective medium demonstrating that research is needed in the development of new media, or improvement in the existing ones, for the enumeration of P. aeruginosa in water. Current selective media/methodologies for detecting P. aeruginosa in mineral waters may seriously underestimate the levels of or presence of this organism which might represent, in some cases, a hazard to the public health. Selon une directive europeenne, le traitement des eaux minerales destinees a l'embouteillage est limite et, en particulier, aucun traitement susceptible de modifier le taux de colonies viables de l'eau minerale naturelle n'est permis. Le sujet de cette etude est de determiner le comportement de Pseudomonas aeruginosa, en tant qu'indicateur d'hygiene dans les procedes d'embouteillage et de modele d'organisme pathogene pendant le jeune prolonge dans des bouteilles d'eaux de differentes compositions et stockees dans des conditions variees. Les influences d'un stockage a la lumiere ou l'obscurite, la temperature, la presence d'une flore autochtone et la composition chimique de l'eau sont recherchees. La capacite de l'organisme a croitre sur un milieu selectif apres le jeune en bouteille est aussi etudiee.

Jun-ya Ueda,1,2 Sirivan Athikomkulchai,3 Ryuta Miyatake,4 Ikuo Saiki,2 Hiroyasu Esumi,5,6 Suresh Awale1,21Frontier Research Core for Life Sciences, 2Institute of Natural Medicine, University of Toyama, Toyama, Japan; 3Faculty of Pharmacy, Srinakharinwirot University, Nakhon Nayok, Thailand; 4Graduate School of Science and Engineering, University of Toyama, Toyama, Japan; 5Research Institute for Biomedical Sciences, Tokyo University of Science, 6National Cancer Center Hospital East, Chiba, JapanAbstract: Human pancreatic tumors are known to be highly resistant to nutrient starvation, and this prolongs their survival in the hypovascular (austere) tumor microenvironment. Agents that retard this tolerance to nutrient starvation represent a novel antiausterity strategy in anticancer drug discovery. (+)-Grandifloracin (GF), isolated from Uvaria dac, has shown preferential toxicity to PANC-1 human pancreatic cancer cells under nutrient starvation, with a PC50 value of 14.5 µM. However, the underlying mechanism is not clear. In this study, GF was found to preferentially induce PANC-1 cell death in a nutrient-deprived medium via hyperactivation of autophagy, as evidenced by a dramatic upregulation of microtubule-associated protein 1 light chain 3. No change was observed in expression of the caspase-3 and Bcl-2 apoptosis marker proteins. GF was also found to strongly inhibit the activation of Akt, a key regulator of cancer cell survival and proliferation. Because pancreatic tumors are highly resistant to current therapies that induce apoptosis, the alternative cell death mechanism exhibited by GF provides a novel therapeutic insight into antiausterity drug candidates.Keywords: (+)-grandifloracin, antiausterity strategy, PANC-1, nutrient starvation

The effects of potentially perturbating influences on the respiration of glucose-grown Candida utilis were studied using an open oxygen electrode system. Periods of anaerobiosis as short as 2 min produced an oscillation in respiration after the air supply was restored. Longer exposure to anoxia was followed by an overshoot in dissolved oxygen after switching back to a gas phase of air. Centrifugation, cold shock or nutrient starvation caused less disturbance to respiration rates than did anaerobiosis. The high frequency oscillations (period about 5 min) resulting from anaerobic-aerobic transitions are contrasted with the slow cell cycle-dependent oscillations previously observed in synchronous cultures.

Amoebae of Dictyostelium discoideum, treated with 0.2 mM 5-bromodeoxyuridine (5-BUdR) for one generation, display an altered differentiation pattern. In this work we analysed the consequence of this treatment on the RNA synthesis during growth and during the first 7 h of starvation. The results show that 1. The rate of ribosomal RNA (rRNA) synthesis of exponentially growing treated cells decreases by 25%. 2. Nutrient starvation by itself induces an immediate 70% decrease in the rate of rRNA synthesis. When amoebae are pretreated with 5-BUdR, the inhibition of rRNA synthesis observed during the growth phase occurs also during starvation and therefore lowers the residual activity normally observed during that period.

We used the technique of Mu d-directed lac operon fusion formation in an effort to identify loci in Salmonella typhimurium which are transcriptionally regulated by nutrient starvation conditions. We identified lacZ operon fusions in eight genetic loci, all of which exhibited increased transcription when starved for two or more of the following nutrients: nicotinate, phosphate, ammonium, glucose, and sulfate. The loci have been designated stiA to stiH for starvation-inducible loci. Mutations in two sti loci (stiC and stiD) significantly decreased cell viability during prolonged periods of nicotinate starvation, stiA and stiD are linked and map at 30 min. The stiC, stiE, stiG, and stiH loci mapped at approximately 77, 43, 88, and 56 min, respectively, on the S. typhimurium linkage map.

Legionellae are among the most important waterborne pathogens in industrialized countries. Monitoring and surveillance of Legionella in engineered water systems is usually performed with culture-based methods. Since the advent of culture-independent techniques, it has become clear that Legionella concentrations are often several orders of magnitude higher than those measured by culture-based techniques and that a variable proportion of these non-culturable cells are viable. In engineered water systems, the formation of these viable but non-culturable (VBNC) cells can be caused by different kinds of stress, such as, and most importantly, nutrient starvation, oxidative stress and heat. In this study, the formation of VBNC cells of six Legionella strains under conditions of starvation was monitored in mono-species microcosms for up to one year using a combination of different viability indicators. Depending on the strain, complete loss of culturability was observed from 11 days to 8 weeks. During the starvation process, three distinct phases and different sub-populations of VBNC cells were identified. Until complete loss of culturability, the number of membrane-intact cells decreased rapidly to 5.5-69% of the initial cell concentration. The concentration of the sub-population with low esterase activity dropped to 0.03-55%, and the concentration of the highly esterase-active sub-population dropped to 0.01-1.2% of the initial concentration; these sub-populations remained stable for several weeks to months. Only after approximately 200 days of starvation, the number of VBNC cells started to decrease below detection limits. The most abundant VBNC sub-populations were characterized by partially damaged membranes and low esterase-activity. With this study, we showed that upon starvation, a stable VBNC Legionella community may be present over several months in a strain-dependent manner even under harsh conditions. Even after one year of starvation, a small proportion of L. pneumophila cells with high esterase-activity was detected. We speculate that this highly active VBNC subpopulation is able to infect amoebae and human macrophages.