Flavin-containing monooxygenase (FMO): Beyond xenobiotics.

Flavin-containing monooxygenases (FMOs), traditionally known for detoxifying xenobiotics, are now recognized for their involvement in endogenous metabolism. We recently discovered that an isoform of FMO, fmo-2 in Caenorhabditis elegans, alters endogenous metabolism to impact longevity and stress tolerance. Increased expression of fmo-2 in C. elegans modifies the flux through the key pathway known as One Carbon Metabolism (OCM). This modified flux results in a decrease in the ratio of S-adenosyl-methionine (SAM) to S-adenosyl-homocysteine (SAH), consequently diminishing methylation capacity. Here we discuss how FMO-2-mediated formate production during tryptophan metabolism may serve as a trigger for changing the flux in OCM. We suggest formate bridges tryptophan and OCM, altering metabolic flux away from methylation during fmo-2 overexpression. Additionally, we highlight how these metabolic results intersect with the mTOR and AMPK pathways, in addition to mitochondrial metabolism. In conclusion, the goal of this essay is to bring attention to the central role of FMO enzymes but lack of understanding of their mechanisms. We justify a call for a deeper understanding of FMO enzyme's role in metabolic rewiring through tryptophan/formate or other yet unidentified substrates. Additionally, we emphasize the identification of novel drugs and microbes to induce FMO activity and extend lifespan.

Fmo induction as a tool to screen for pro-longevity drugs.

Dietary restriction (DR) and hypoxia (low oxygen) extend lifespan in Caenorhabditis elegans through the induction of a convergent downstream longevity gene, fmo-2. Flavin-containing monooxygenases (FMOs) are highly conserved xenobiotic-metabolizing enzymes with a clear role in promoting longevity in nematodes and a plausible similar role in mammals. This makes them an attractive potential target of small molecule drugs to stimulate the health-promoting effects of longevity pathways. Here, we utilize an fmo-2 fluorescent transcriptional reporter in C. elegans to screen a set of 80 compounds previously shown to improve stress resistance in mouse fibroblasts. Our data show that 19 compounds significantly induce fmo-2, and 10 of the compounds induce fmo-2 more than twofold. Interestingly, 9 of the 10 high fmo-2 inducers also extend lifespan in C. elegans. Two of these drugs, mitochondrial respiration chain complex inhibitors, interact with the hypoxia pathway to induce fmo-2, whereas two dopamine receptor type 2 (DRD2) antagonists interact with the DR pathway to induce fmo-2, indicating that dopamine signaling is involved in DR-mediated fmo-2 induction. Together, our data identify nine drugs that each (1) increase stress resistance in mouse fibroblasts, (2) induce fmo-2 in C. elegans, and (3) extend nematode lifespan, some through known longevity pathways. These results define fmo-2 induction as a viable approach to identifying and understanding mechanisms of putative longevity compounds.

A diet of oxidative stress-adapted bacteria improves stress resistance and lifespan in C. elegans via p38-MAPK.

Organisms across taxa face stresses including variable temperature, redox imbalance, and xenobiotics. Successfully responding to stress and restoring homeostasis are crucial for survival. Aging is associated with a decreased stress response and alterations in the microbiome, which contribute to disease development. Animals and their microbiota share their environment; however, microbes have short generation time and can rapidly evolve and potentially affect host physiology during stress. Here, we leverage Caenorhabditis elegans and its simplified bacterial diet to demonstrate how microbial adaptation to oxidative stress affects the host's lifespan and stress response. We find that worms fed stress-evolved bacteria exhibit enhanced stress resistance and an extended lifespan. Through comprehensive genetic and metabolic analysis, we find that iron in stress-evolved bacteria enhances worm stress resistance and lifespan via activation of the mitogen-activated protein kinase pathway. In conclusion, our study provides evidence that understanding microbial stress-mediated adaptations could be used to slow aging and alleviate age-related health decline.

Defective quality control autophagy in Hyperhomocysteinemia promotes ER stress and consequent neuronal apoptosis through proteotoxicity.

Homocysteine (Hcy), produced physiologically in all cells, is an intermediate metabolite of methionine and cysteine metabolism. Hyperhomocysteinemia (HHcy) resulting from an in-born error of metabolism that leads to accumulation of high levels of Hcy, is associated with vascular damage, neurodegeneration and cognitive decline. Using a HHcy model in neuronal cells, primary cortical neurons and transgenic zebrafish, we demonstrate diminished autophagy and Hcy-induced neurotoxicity associated with mitochondrial dysfunction, fragmentation and apoptosis. We find this mitochondrial dysfunction is due to Hcy-induced proteotoxicity leading to ER stress. We show this sustained proteotoxicity originates from the perturbation of upstream autophagic pathways through an aberrant activation of mTOR and that protetoxic stress act as a feedforward cues to aggravate a sustained ER stress that culminate to mitochondrial apoptosis in HHcy model systems. Using chemical chaperones to mitigate sustained ER stress, Hcy-induced proteotoxicity and consequent neurotoxicity were rescued. We also rescue neuronal lethality by activation of autophagy and thereby reducing proteotoxicity and ER stress. Our findings pave the way to devise new strategies for the treatment of neural and cognitive pathologies reported in HHcy, by either activation of upstream autophagy or by suppression of downstream ER stress. Video Abstract.

Assessment of homozygosity in transgenic plants using selectable markers.

Production of homozygous transgenic plants is a prerequisite for the phenotypic analysis and/or for the commercial release of transgenic plants for cultivation. Here we present a simple protocol for the selection of homozygous transgenics using antibiotics as a selectable marker. The protocol has been used to select homozygous rice transgenic plants using hygromycin antibiotic. However, the described protocol can be used for selction of homozygous in any transgenic plants using a appropriate selectable marker. For complete details on the use and execution of this protocol, please refer to Passricha et al. (2016).1.

FMO rewires metabolism to promote longevity through tryptophan and one carbon metabolism in C. elegans.

Flavin containing monooxygenases (FMOs) are promiscuous enzymes known for metabolizing a wide range of exogenous compounds. In C. elegans, fmo-2 expression increases lifespan and healthspan downstream of multiple longevity-promoting pathways through an unknown mechanism. Here, we report that, beyond its classification as a xenobiotic enzyme, fmo-2 expression leads to rewiring of endogenous metabolism principally through changes in one carbon metabolism (OCM). These changes are likely relevant, as we find that genetically modifying OCM enzyme expression leads to alterations in longevity that interact with fmo-2 expression. Using computer modeling, we identify decreased methylation as the major OCM flux modified by FMO-2 that is sufficient to recapitulate its longevity benefits. We further find that tryptophan is decreased in multiple mammalian FMO overexpression models and is a validated substrate for FMO-2. Our resulting model connects a single enzyme to two previously unconnected key metabolic pathways and provides a framework for the metabolic interconnectivity of longevity-promoting pathways such as dietary restriction. FMOs are well-conserved enzymes that are also induced by lifespan-extending interventions in mice, supporting a conserved and important role in promoting health and longevity through metabolic remodeling.

Platelet Indices as Predictive Markers of Prognosis in Critically Ill Patients: A Prospective Study.

INTRODUCTION: Platelets (PLTs) are dynamic blood molecules which perform multiple physiological functions. Platelet derangements are commonly encountered in intensive care units (ICUs). The relationship of PLT indices with all-cause mortality, acute physiology and chronic health evaluation IV (APACHE IV), diabetes mellitus (DM), and length of stay in ICU is debatable and hence this study was undertaken to bridge this gap of knowledge. MATERIALS AND METHODS: Prospective data were collected for 20 months in the ICU of our hospital. Platelet indices were analyzed among survivors and non-survivors. Acute physiology and chronic health evaluation IV scores were used to study the relationship between PLT indices and illness severity. Receiver operating characteristic curves were constructed to compare the performances of PLT indices in predicting mortality, while the effect of DM on PLT indices was evaluated using regression analysis. RESULTS: A total of 170 out of 345 patients (119 survivors, 51 non-survivors) met the study criteria. Patients with decreased PLT count and plateletcrit (PCT) (p < 0.001 and 0.001, respectively), increased mean platelet volume (MPV) and platelet distribution width (PDW) (p = 0.014 and 0.004, respectively) had a significant correlation with increased risk of mortality than those with normal PLT indices. These patients also had a higher APACHE IV and acute physiology score (p < 0.001). No significant relationship was found between the PLT indices and the length of ICU stay. The influence of each PLT index adjusted to DM was significant in univariate regression analysis, whereas in multivariate only PDW had a significant influence. CONCLUSION: Patients with low PLT, PCT and high MPV, PDW were associated with more severe illness, poor prognosis, and a higher risk of mortality. Platelet distribution width is the preferred PLT index in a diabetic patient to predict clinical status. CLINICAL SIGNIFICANCE: Platelet indices which are routinely available can be effectively used as a morbidity and mortality indicator in critically ill patients. HOW TO CITE THIS ARTICLE: Samuel D, Bhat AN, Prabhu VM. Platelet Indices as Predictive Markers of Prognosis in Critically Ill Patients: A Prospective Study. Indian J Crit Care Med 2020;24(9):817-822.

Ncl1-mediated metabolic rewiring critical during metabolic stress.

Nutritional limitation has been vastly studied; however, there is limited knowledge of how cells maintain homeostasis in excess nutrients. In this study, using yeast as a model system, we show that some amino acids are toxic at higher concentrations. With cysteine as a physiologically relevant example, we delineated the pathways/processes that are altered and those that are involved in survival in the presence of elevated levels of this amino acid. Using proteomics and metabolomics approach, we found that cysteine up-regulates proteins involved in amino acid metabolism, alters amino acid levels, and inhibits protein translation-events that are rescued by leucine supplementation. Through a comprehensive genetic screen, we show that leucine-mediated effect depends on a transfer RNA methyltransferase (NCL1), absence of which decouples transcription and translation in the cell, inhibits the conversion of leucine to ketoisocaproate, and leads to tricarboxylic acid cycle block. We therefore propose a role of NCL1 in regulating metabolic homeostasis through translational control.

Game change in Indian Health Care System through reforms in medical education curriculum focusing on primary care- Recommendations of a joint working group.

Despite the stated aim of Medical Council of India (body regulating medical education in India) to produce an Indian Medical Graduate with requisite knowledge, skills, attitudes, values and responsiveness, so that he or she may function appropriately and effectively as a doctor of first contact of the community while being globally relevant, it appears that we failed. The joint working group extensively consisting of medical teachers have come up with suggestions which may work as the game changer in Indian Health care system. The key is to dedicate medical education towards primary care.

Proteomic profile of 4-PBA treated human neuronal cells during ER stress.

Perturbations affecting the homoeostasis of endoplasmic reticulum (ER) activate an adaptive signaling known as the unfolded protein response or UPR. Many studies have reported the association between neurological disorders and ER stress. Decreasing ER stress may therefore aid in therapeutic control of neuronal diseases. Sodium 4-phenylbutyrate (4-PBA), a small molecule, has been shown to alleviate ER stress and various neurological diseases, but the mechanistic basis of its action is not well understood. Using an iTRAQ based LC-MS technique we have delineated the effect of 4-PBA on the proteome of human neuroblastoma cells (SK-N-SH) during Tunicamycin-induced ER stress. The proteomic profile of 4-PBA-treated cells revealed that 4-PBA does not alter the cellular proteome to adapt towards ER stress. However, it can alleviate both the toxicity and proteomic alterations, induced by an ER stress inducer. Hence, the therapeutic effect of 4-PBA is primarily due to its ability to resolve ER stress rather than its ability to alter the expression of proteins required for maintaining ER proteostasis. Thus, we posit here that 4-PBA acts as an authentic chemical chaperone by aiding protein folding in the ER.

A Clinical Study of Acute Kidney Injury in Tropical Acute Febrile Illness.

INTRODUCTION: Tropical Acute Febrile Illness (TAFI) is one of the most common causes of morbidity within the community. Acute Kidney Injury (AKI) due to infective and non infective causes is a major complication. Presence of AKI is a major cause of mortality among patients with TAFI. AIM: To study the spectrum of tropical acute febrile illness; the proportion, spectrum and staging of acute kidney injury; Renal Replacement Therapy (RRT) initiation and in-hospital mortality. MATERIALS AND METHODS: A total of 600 TAFI patients were prospectively studied at a tertiary care centre in coastal Karnataka between September 2012 and September 2014 for the aetiology of TAFI; the development and staging of AKI based on Kidney disease: Improving global outcomes (KDIGO) guidelines; the initiation of RRT and in-hospital mortality. STATISTICAL ANALYSIS: Data analysis was done using SPSS version 17.0 with statistical significance calculated using chi-square and Fisher's exact t-test for which p-value <0.05 was considered significant. RESULTS: The spectrum of TAFI, in decreasing order, was vivax malaria, leptospirosis, dengue fever, falciparum malaria, mixed malaria, enteric fever, scrub typhus and the most common aetiology was malaria. The proportion of AKI was 54%. The most common cause of AKI, its stages 2 and 3, RRT initiation and in-hospital mortality was leptospirosis; and AKI stage 1 was dengue fever. KDIGO AKI stage 1, 2 and 3 was seen in 46.9%, 31.2% and 21.9% of AKI patients, respectively. RRT initiation was required in 10.2% of AKI patients and in-hospital mortality was 3% among all patients. AKI, RRT initiationand in-hospital mortality were significantly associated with older age, fever duration and other presenting complaints, examination findings, renal function and other parameters, leptospirosis, dengue fever, falciparum malaria. CONCLUSION: The aetiology in about half of TAFI patients in coastal Karnataka was malaria. More than 50% develop AKI with greater than one-fifth of them progressing to AKI stage 3 and one-tenth requiring RRT. The most common cause of AKI, AKI stage 2, 3, RRT initiation and in-hospital mortality was leptospirosis. AKI was present in almost all patients with leptospirosis. Therefore leptospirosis was the most nephrotoxic acute febrile illness in the present study population. Dengue fever was the most common cause of AKI stage 1. Vivax malaria was the third most common cause of AKI. The factors like age, presenting complaints, examination findings, renal function and other parameters, aetiology and RRT initiation may be used to predict AKI and in-hospital mortality.

Oxidative Homeostasis Regulates the Response to Reductive Endoplasmic Reticulum Stress through Translation Control.

Reductive stress leads to the loss of disulfide bond formation and induces the unfolded protein response of the endoplasmic reticulum (UPR(ER)), necessary to regain proteostasis in the compartment. Here we show that peroxide accumulation during reductive stress attenuates UPR(ER) amplitude by altering translation without any discernible effect on transcription. Through a comprehensive genetic screen in Saccharomyces cerevisiae, we identify modulators of reductive stress-induced UPR(ER) and demonstrate that oxidative quality control (OQC) genes modulate this cellular response in the presence of chronic but not acute reductive stress. Using a combination of microarray and relative quantitative proteomics, we uncover a non-canonical translation attenuation mechanism that acts in a bipartite manner to selectively downregulate highly expressed proteins, decoupling the cell's transcriptional and translational response during reductive ER stress. Finally, we demonstrate that PERK, a canonical translation attenuator in higher eukaryotes, helps in bypassing a ROS-dependent, non-canonical mode of translation attenuation.

In-depth comparative proteomic analysis of yeast proteome using iTRAQ and SWATH based MS.

Quantitative proteomics using LC-MS has emerged as an essential tool for addressing different biological questions. Various labelling methods have been effectively employed for quantitative proteomics studies. However, these are fraught with several challenges, including reproducibility and the number of samples that can be analysed at a given time. To this end, unlabelled proteomics is a promising field, and the recently developed sequential window acquisition of all theoretical fragment ion spectra (SWATH-MS) method aims to address these limitations. In this study, we compared SWATH-MS to isobaric tag for relative and absolute quantitation (iTRAQ), a widely used labelled method for relative quantitation. For this, we used yeast, Saccharomyces cerevisiae, since almost all its proteins are identified. More importantly, the abundance of each protein is well documented. We found that although a similar number of proteins could be quantitated using the two techniques, SWATH had the advantage of quantifying a larger percentage of low abundance proteins (below 60 ppm). Thus, based on our analysis, we believe that these two techniques are complementary and can synergistically improve the number of quantifiable proteins. SWATH's ability to quantify low abundant proteins could be an asset in biomarker discovery studies.

Cross-compartment proteostasis regulation during redox imbalance induced ER stress.

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle-specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ-based LC-MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross-talk between ER- and mitochondrial proteostasis exemplified by an Ire1p-dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross-talk during stress.

Tunable superlattice in graphene to control the number of Dirac points.

Superlattice in graphene generates extra Dirac points in the band structure and their number depends on the superlattice potential strength. Here, we have created a lateral superlattice in a graphene device with a tunable barrier height using a combination of two gates. In this Letter, we demonstrate the use of lateral superlattice to modify the band structure of graphene leading to the emergence of new Dirac cones. This controlled modification of the band structure persists up to 100 K.

Regulation of homocysteine metabolism by Mycobacterium tuberculosis S-adenosylhomocysteine hydrolase.

Mycobacterium tuberculosis modulates expression of various metabolism-related genes to adapt in the adverse host environment. The gene coding for M. tuberculosis S-adenosylhomocysteine hydrolase (Mtb-SahH) is essential for optimal growth and the protein product is involved in intermediary metabolism. However, the relevance of SahH in mycobacterial physiology is unknown. In this study, we analyze the role of Mtb-SahH in regulating homocysteine concentration in surrogate host Mycobacterium smegmatis. Mtb-SahH catalyzes reversible hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine and we demonstrate that the conserved His363 residue is critical for bi-directional catalysis. Mtb-SahH is regulated by serine/threonine phosphorylation of multiple residues by M. tuberculosis PknB. Major phosphorylation events occur at contiguous residues Thr219, Thr220 and Thr221, which make pivotal contacts with cofactor NAD⁺. Consequently, phosphorylation negatively modulates affinity of enzyme towards NAD⁺ as well as SAH-synthesis. Thr219, Thr220 and Thr221 are essential for enzyme activity, and therefore, responsible for SahH-mediated regulation of homocysteine.

Peripheral insensate neuropathy-is height a risk factor?

INTRODUCTION: Peripheral insensate neuropathy is one of the most commonest and the earliest forms of peripheral neuropathy. It is one of the leading causes of the disability in working population who are at risk. METHODS: A study was conducted in Kasturba medical college (Manipal university) in the year 2009-12, which included examination of 818 people of more than 30yrs of age by random sampling method who were attending the outpatient clinic. A monofilament was used to determine the peripheral insensate neuropathy, which was defined by the presence of one or more insensate areas. RESULTS: In our study , the prevalence of peripheral insensate neuropathy was 16.2 % ( p-0.0001), among which 9.7% were males and 7.5% were females. The males were 1.27 times significantly at a higher risk than the females , even after a height adjustment to the gender difference in height. As the height increased, the prevalence of peripheral insensate neuropathy increased, irrespective of the diabetic and hypertensive statuses. The risk of the peripheral insensate neuropathy increases at a height of >167 cm in males and at a height of >159 cm in females. CONCLUSION: The authors conclude that body height is an important and an independent risk factor for peripheral insensate neuropathy, irrespective of co morbidities. Height as a marker, helps the health care professionals in identifying the people who are at risk for peripheral insensate neuropathy.