Germline status and micronutrient availability regulate a somatic mitochondrial quality control pathway via short-chain fatty acid metabolism.

Reproductive status, such as pregnancy and menopause in women, profoundly influences metabolism of the body. Mitochondria likely orchestrate many of these metabolic changes. However, the influence of reproductive status on somatic mitochondria and the underlying mechanisms remain largely unexplored. We demonstrate that reproductive signals modulate mitochondria in the Caenorhabditis elegans soma. We show that the germline acts via an RNA endonuclease, HOE-1, which despite its housekeeping role in tRNA maturation, selectively regulates the mitochondrial unfolded protein response (UPRmt). Mechanistically, we uncover a fatty acid metabolism pathway acting upstream of HOE-1 to convey germline status. Furthermore, we link vitamin B12's dietary intake to the germline's regulatory impact on HOE-1-driven UPRmt. Combined, our study uncovers a germline-somatic mitochondrial connection, reveals the underlying mechanism, and highlights the importance of micronutrients in modulating this connection. Our findings provide insights into the interplay between reproductive biology and metabolic regulation.

RNA: De-silencing to the rescue.

The fate of transcribed RNA dictates cellular function. A new study finds that mutations in specific RNA processing machinery genes result in de-silencing of a transcript encoding a subunit of the mitochondrial electron transport chain and rescue of a mitochondrial respiratory complex I defect.

Production of a Bacteriocin Like Protein PEG 446 from Clostridium tyrobutyricum NRRL B-67062.

Clostridium tyrobutyricum strain NRRL B-67062 was previously isolated from an ethanol production facility and shown to produce high yields of butyric acid. In addition, the cell-free supernatant of the fermentation broth from NRRL B-67062 contained antibacterial activity against certain Gram-positive bacteria. To determine the source of this antibacterial activity, we report the genome and genome mining of this strain. The complete genome of NRRL B-67062 showed one circular chromosome of 3,242,608 nucleotides, 3114 predicted coding sequences, 79 RNA genes, and a G+C content of 31.0%. Analyses of the genome data for genes potentially associated with antimicrobial features were sought after by using BAGEL-4 and anti-SMASH databases. Among the leads, a polypeptide of 66 amino acids (PEG 446) contains the DUF4177 domain, which is an uncharacterized highly conserved domain (pfam13783). The cloning and expression of the peg446 gene in Escherichia coli and Bacillus subtilis confirmed the antibacterial property against Lactococcus lactis LM 0230, Limosilactobacillus fermentum 0315-25, and Listeria innocua NRRL B-33088 by gel overlay and well diffusion assays. Molecular modeling suggested that PEG 446 contains one alpha-helix and three anti-parallel short beta-sheets. These results will aid further functional studies and facilitate simultaneously fermentative production of both butyric acid and a putative bacteriocin from agricultural waste and lignocellulosic biomass materials.

HIF-PHD inhibitor desidustat ameliorates iron deficiency anemia.

Iron deficiency anemia is caused by many pathological conditions like chronic kidney disease (CKD), inflammation, malnutrition and gastrointestinal abnormality. Current treatments that are erythropoiesis stimulating agents (ESAs) and iron supplementation are inadequate and often lead to tolerance and/or toxicity. Desidustat, a prolyl hydroxylase (PHD) inhibitor, is clinically used for the treatment of anemia with CKD. In this study, we investigated the effect of desidustat on iron deficiency anemia (IDA). IDA was induced in C57BL6/J mice by iron deficient diet feeding. These mice were then treated with desidustat (15 mg/kg, PO) and FeSO4 (20 mg/kg) for five weeks and effect of the treatment on hematology, iron homeostasis, and bone marrow histology was observed. Effect of desidustat on iron metabolism in inflammation (LPS)-induced iron deficiency was also assessed. Both, Desidustat and FeSO4, increased MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), hemoglobin, and HCT (hematocrit) in blood and increased iron in serum, liver, and spleen. Desidustat increased MCHC (mean corpuscular hemoglobin concentration) while FeSO4 treatment did not alter it. FeSO4 treatment significantly increased iron deposition in liver, and spleen, while desidustat increased iron in circulation and demonstrated efficient iron utilization. Desidustat increased iron absorption, serum iron and decreased hepcidin without altering tissue iron, while FeSO4 increased serum and tissue iron by increasing hepcidin in LPS-induced iron deficiency. Desidustat increased erythroid population, especially iron-dependent polychromatic normoblasts and orthochromatic normoblasts, while FeSO4 did not improve cell architecture. PHD inhibition by desidustat improved iron utilization in iron deficiency anemia, by efficient erythropoiesis.

Ante-mortem and Post-mortem Diagnosis Modalities and Phylogenetic Analysis of Rabies Virus in Domestic and Wild Animals of Gujarat, India.

In the present study, total of 32 ante-mortem (AM) samples (saliva = 18 and corneal smears = 14) from six animal species (cattle = 5; camel = 1; goat = 1; horse = 1; buffalo = 4; dog = 6) and 28 post-mortem (PM) samples of domestic (cattle = 6; camel = 1; goat = 1; buffalo = 5; dog = 7) and wild animals (lion = 4, mongoose = 2; bear = 1; leopard = 1) were examined for rabies diagnosis in Gujarat, India. Direct fluorescent antibody test (dFAT) and reverse transcriptase polymerase chain reaction (RT-PCR) were applied on AM samples, whereas along with dFAT and RT-PCR, histopathological examination, immunohistochemistry (IHC) and real time PCR (qPCR) were used for PM diagnosis. Nucleotide sequencing of full nucleoprotein (N) and glycoprotein (G) genes were carried out upon representative amplicons. In AM examination, 7/18 saliva and 5/14 corneal impressions samples were found positive in dFAT and 8/18 saliva samples were found positive in RT-PCR. In PM examination, 14/28 samples showed positive results in dFAT and IHC with unusual large fluorescent foci in two samples. In histopathology, 11/28 samples showed appreciable lesion and Negri bodies were visible in 6 samples, only. Out of 23 brain samples examined. 12 samples were found positive in N gene RT-PCR and qPCR, and 10 samples in G gene RT-PCR. Phylogenetic analysis of N gene revealed that test isolates (except sample ID: lion-1; lion, Gir) form a close group with sequence ID, KM099393.1 (Mongoose, Hyderabad) and KF660246.1 (Water Buffalo, Hyderabad) which was far from some south Indian and Sri Lankan isolates but similar to Indian isolates from rest of India and neighboring countries. In G gene analysis, the test isolates form a close group with sequence ID, KP019943.1. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01126-0.

Novel endolysin LysMP for control of Limosilactobacillus fermentum contamination in small-scale corn mash fermentation.

BACKGROUND: Traditional bioethanol fermentation industries are not operated under strict sterile conditions and are prone to microbial contamination. Lactic acid bacteria (LAB) are often pervasive in fermentation tanks, competing for nutrients and producing inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. Antibiotics are frequently used to combat contamination, but antibiotic stewardship has resulted in a shift to alternative antimicrobials. RESULTS: We demonstrate that endolysin LysMP, a bacteriophage-encoded peptidoglycan hydrolase, is an effective method for controlling growth of LAB. The LysMP gene was synthesized based on the prophage sequence in the genome of Limosilactobacillus fermentum KGL7. Analysis of the recombinant enzyme expressed in E. coli and purified by immobilized metal chelate affinity chromatography (IMAC) showed an optimal lysis activity against various LAB species at pH 6, with stability from pH 4 to 8 and from 20 to 40 °C up to 48 h. Moreover, it retains more than 80% of its activity at 10% ethanol (v/v) for up to 48 h. When LysMP was added at 250 µg/mL to yeast corn mash fermentations containing L. fermentum, it reduced bacterial load by at least 4-log fold compared to the untreated controls and prevented stuck fermentation. In comparison, untreated controls with contamination increased from an initial bacterial load of 1.50 × 107 CFU/mL to 2.25 × 109 CFU/mL and 1.89 × 109 CFU/mL after 24 h and 48 h, respectively. Glucose in the treated samples was fully utilized, while untreated controls with contamination had more than 4% (w/v) remaining at 48 h. Furthermore, there was at least a fivefold reduction in lactic acid (0.085 M untreated contamination controls compared to 0.016 M treated), and a fourfold reduction in acetic acid (0.027 M untreated contamination controls vs. 0.007 M treated), when LysMP was used to treat contaminated corn mash fermentations. Most importantly, final ethanol yields increased from 6.3% (w/v) in untreated contamination samples to 9.3% (w/v) in treated contamination samples, an approximate 50% increase to levels comparable to uncontaminated controls 9.3% (w/v). CONCLUSION: LysMP could be a good alternative to replace antibiotics for mitigation of LAB contamination in biofuel refineries.

Measurements and predictions of diffusible hydrogen escape and absorption in catholically charged 316LN austenitic stainless steel.

Hydrogen can have an impact on the service life of safety critical components, such as coolant pipes in nuclear reactors, where it may interact with other factors including irradiation. Hence, it is important to characterise such behaviour which in turn requires the capability to charge representative material specimens with hydrogen and to quantity the levels of hydrogen present. Hydrogen concentrations resulting from cathodic charging of 316LN stainless steel over short time periods (< 2 h) were estimated from hydrogen release rates obtained from potentiostatic discharge measurements and used to calibrate simulations based on Fick's second law of diffusion in order to predict the hydrogen concentration after 24 h of charging. Leave-one-out cross-validation was used to establish confidence in results which were also validated using measurements from the melt extraction technique. The success of Fick's second law for estimating escape rates showed that a majority of the absorbed hydrogen was diffusible rather than trapped. These results confirmed that the potentiostatic discharge technique can be used on materials with low diffusivity, and provide a new method through which hydrogen concentrations within a sample can be estimated after cathodic charging non-destructively without the need to remove samples from solution.

Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations.

Control of bacterial contamination in bioethanol fermentation facilities has traditionally relied on chemical-based products such as hop acids and use of antibiotics. Recent emphasis on antibiotic stewardship has prompted new research into the development of alternative approaches to microbial remediation strategies. We recently described a recombinant peptidoglycan hydrolase, endolysin LysKB317, which inhibited Limosilactobacillus fermentum strains in corn mash fermentation. Here, Saccharomyces cerevisiae EBY100 was used to anchor recombinant LysKB317 using cell surface display with the a-agglutinin proteins Aga1p-Aga2p. Immunostaining and confocal fluorescence were used for localization of the extracellular interface of the cells. Yeast surface-expressed endolysin demonstrated an 83.8% decrease in bacterial cell counts compared to a 9.5% decrease in control yeast. Recombinant S. cerevisiae expressing LysKB317 used for small-scale corn mash fermentation, when infected with L. fermentum, could proactively control bacterial infection for 72 h with at least 1-log fold reduction. Analysis of fermentation products showed improved ethanol concentrations from 3.4% to at least 5.9% compared to the infection-only control and reduced levels of lactic and acetic acid from 34.7 mM to 13.8 mM and 25.5 mM to 18.1 mM, respectively. In an optimized yeast surface display system, proactive treatment of bacterial contaminants by endolysin LysKB317 can improve fermentation efficiency in the presence of L. fermentum contamination.

A role for N6-methyldeoxyadenosine in C. elegans mitochondrial genome regulation.

Epigenetic modifications provide powerful means for transmitting information from parent to progeny. As a maternally inherited genome that encodes essential components of the electron transport chain, the mitochondrial genome (mtDNA) is ideally positioned to serve as a conduit for the transgenerational transmission of metabolic information. Here, we provide evidence that mtDNA of C. elegans contains the epigenetic mark N6-methyldeoxyadenosine (6mA). Bioinformatic analysis of SMRT sequencing data and methylated DNA IP sequencing data reveal that C. elegans mtDNA is methylated at high levels in a site-specific manner. We further confirmed that mtDNA contains 6mA by leveraging highly specific anti-6mA antibodies. Additionally, we find that mtDNA methylation is dynamically regulated in response to antimycin, a mitochondrial stressor. Further, 6mA is increased in nmad-1 mutants and is accompanied by a significant decrease in mtDNA copy number. Our discovery paves the way for future studies to investigate the regulation and inheritance of mitochondrial epigenetics.

Tissue-specific heteroplasmy segregation is accompanied by a sharp mtDNA decline in Caenorhabditis elegans soma.

Mutations in the mitochondrial genome (mtDNA) can be pathogenic. Owing to the multi-copy nature of mtDNA, wild-type copies can compensate for the effects of mutant mtDNA. Wild-type copies available for compensation vary depending on the mutant load and the total copy number. Here, we examine both mutant load and copy number in the tissues of Caenorhabditis elegans. We found that neurons, but not muscles, have modestly higher mutant load than rest of the soma. We also uncovered different effect of aak-2 knockout on the mutant load in the two tissues. The most surprising result was a sharp decline in somatic mtDNA content over time. The scale of the copy number decline surpasses the modest shifts in mutant load, suggesting that it may exert a substantial effect on mitochondrial function. In summary, measuring both the copy number and the mutant load provides a more comprehensive view of the mutant mtDNA dynamics.

Genomic and probiotic attributes of Lactobacillus strains from rice-based fermented foods of North Eastern India.

The probiotic attributes and genomic profiles of amylase-producing Lactobacillus strains from rice-based fermented foods of Meghalaya in the North-Eastern India were evaluated in the study. A preliminary screening of 17 lactic acid bacteria strains was performed based on their starch hydrolysis and glucoamylase activities. Out of 17 strains, 5 strains (L. fermentum KGL4, L. rhamnosus RNS4, L. fermentum WTS4, L. fermentum KGL2, and L. rhamnosus KGL3A) were selected for further characterization of different probiotic attributes. Whole-genome sequencing of two of the best strains was carried out using a shotgun sequencing platform based on their rich probiotic attributes. The EPS production was in the range of 2.89-3.92 mg/mL. KGL2 (41.5%) and KGL3A (41%) showed the highest antioxidant activity. The highest antibiotic susceptibility was exhibited by all the five Lactobacillus strains against ampicillin, ranging from 24.66 to 27.33 mm. The lactobacilli isolates used in the study could survive the simulated gastric/intestinal juices. Genomic characterization of KGL4 and KGL3A illustrated their possible adherence to the intestinal wall, specialized metabolic patterns, and possible role in boosting host immunity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05633-8.

Should I stay, or should I go? Gene retention in organellar genomes.

Mitochondria and plastids retain their own small but essential genomes. However, the evolutionary pressures that determine whether a gene is retained in organellar DNA or exported to the "host" nuclear genome remain unclear. A new study in Cell Systems addresses this knowledge gap using bioinformatic data and modeling to identify universal "rules" that determine organellar gene retention.

Independent regulation of mitochondrial DNA quantity and quality in Caenorhabditis elegans primordial germ cells.

Mitochondria harbor an independent genome, called mitochondrial DNA (mtDNA), which contains essential metabolic genes. Although mtDNA mutations occur at high frequency, they are inherited infrequently, indicating that germline mechanisms limit their accumulation. To determine how germline mtDNA is regulated, we examined the control of mtDNA quantity and quality in C. elegans primordial germ cells (PGCs). We show that PGCs combine strategies to generate a low point in mtDNA number by segregating mitochondria into lobe-like protrusions that are cannibalized by adjacent cells, and by concurrently eliminating mitochondria through autophagy, reducing overall mtDNA content twofold. As PGCs exit quiescence and divide, mtDNAs replicate to maintain a set point of ~200 mtDNAs per germline stem cell. Whereas cannibalism and autophagy eliminate mtDNAs stochastically, we show that the kinase PTEN-induced kinase 1 (PINK1), operating independently of Parkin and autophagy, preferentially reduces the fraction of mutant mtDNAs. Thus, PGCs employ parallel mechanisms to control both the quantity and quality of the founding population of germline mtDNAs.

Mitochondria are the powerhouses of every cell in our bodies. These tiny structures convert energy from the food we eat into a form that cells are able to use. As well as being a separate organ-like structure within our cells, mitochondria even have their own DNA. Mitochondrial DNA contains genes for a small number of special enzymes that allow it to extract energy from food. In contrast, the rest of our cells' DNA is stored in another structure called the nucleus. Mitochondrial and nuclear DNA are also inherited differently. We inherit nuclear DNA from both our mother and father, but mitochondrial DNA is only passed down from our mothers. During reproduction, maternal DNA (including mitochondrial DNA) comes from the egg cell, which combines with sperm to produce offspring. Defects, or mutations, in mitochondrial genes often lead to mitochondrial diseases. These have a severe impact on health, especially during the very first stages of life. The lineage of precursor cells that gives rise to egg cells is thought to protect itself from mitochondrial mutations, but how it does this is still unclear. Schwartz et al. therefore set out to determine what molecular mechanisms preserve the integrity of mitochondrial DNA from one generation to the next. To address this question, C. elegans roundworms were used, as they are easy to manipulate genetically, and since they are small and transparent, their cells ­ as well as their mitochondria ­ are also easily viewed under a microscope. Tracking mitochondria in the worms' egg precursor cells (also called primordial germ cells, or PGCs) revealed that PGCs actively removed excess mitochondria. The PGCs did this either by internally breaking down mitochondria themselves, or by moving them into protruding lobe-like structures which surrounding cells then engulfed and 'digested'. Further genetic studies revealed that the PGCs also directly regulated the quality of mitochondrial DNA via a mechanism dependent on the protein PINK1. In worms lacking PINK1, mutant mitochondrial DNA remained in the PGCs at high levels, whereas normal worms successfully reduced the mutant DNA. Thus, the PGCs used parallel mechanisms to control both the quantity and quality of mitochondria passed to the next generation. These results contribute to our understanding of how organisms safeguard their offspring from inheriting mutant mitochondrial DNA. In the future, Schwartz et al. hope that this knowledge will help us treat inherited mitochondrial diseases in humans.

Small RNA sequencing and identification of papaya (Carica papaya L.) miRNAs with potential cross-kingdom human gene targets.

Several studies have demonstrated potential role of plant-derived miRNAs in cross-kingdom species relationships by transferring into non-plant host cells to regulate certain host cellular functions. How nutrient-rich plants regulate host cellular functions, which in turn alleviate physiological and disease conditions in the host remains to be explored in detail. This computational study explores the potential targets, putative role, and functional implications of miRNAs derived from Carica papaya L., one of the most cultivated tropical crops in the world and a rich source of phytochemicals and enzymes, in human diet. Using the next-generation sequencing, -Illumina HiSeq2500, ~ 30 million small RNA sequence reads were generated from C. papaya young leaves, resulting in the identification of a total of 1798 known and 49 novel miRNAs. Selected novel C. papaya miRNAs were predicted to regulate certain human targets, and subsequent annotation of gene functions indicated a probable role in various biological processes and pathways, such as MAPK, WNT, and GPCR signaling pathways, and platelet activation. These presumptive target gene in humans were predominantly linked to various diseases, including cancer, diabetes, mental illness, and platelet disorder. The computational finding of this study provides insights into how C. papaya-derived miRNAs may regulate certain conditions of human disease and provide a new perspective on human health. However, the therapeutic potential of C. papaya miRNA can be further explored through experimental studies.

Prolyl hydroxylase inhibitor desidustat improves anemia in erythropoietin hyporesponsive state.

Many anemic chronic kidney disease (CKD) patients are refractory to erythropoietin (EPO) effects due to inflammation, deranged iron utilization, and generation of EPO antibodies. This work assessed the effect of desidustat, an inhibitor of hypoxia inducible factor (HIF) prolyl hydroxylase (PHD), on EPO-refractory renal anemia. Sprague Dawley rats were made anemic by cisplatin (5 â€‹mg/kg, IP, single dose) and turpentine oil (5 â€‹mL/kg, SC, once a week). These rats were given recombinant human EPO (rhEPO, 1 â€‹µg/kg) and desidustat (15 or 30 â€‹mg/kg) for eight weeks. Separately, rhEPO (1-5 â€‹µg/kg) was given to anemic rats to sustain the normal hemoglobin levels and desidustat (15 â€‹mg/kg) for eight weeks. In another experiment, the anemic rats were treated rhEPO (5 â€‹µg/kg) for two weeks and then desidustat (15 â€‹mg/kg) for the next two weeks. Dosing of rhEPO was thrice a week, and for desidustat, it was on alternate days. Desidustat inhibited EPO-resistance caused by rhEPO treatment, decreased hepcidin, IL-6, IL-1ß, and increased iron and liver ferroportin. Desidustat reduced EPO requirement and anti-EPO antibodies. Desidustat also maintained normal hemoglobin levels after cessation of rhEPO treatment. Thus, novel prolyl hydroxylase inhibitor desidustat can treat EPO resistance via improved iron utilization and decreased inflammation.

Evolution of preclinical characterization and insights into clinical pharmacology of checkpoint inhibitors approved for cancer immunotherapy.

Cancer immunotherapy has significantly advanced the treatment paradigm in oncology, with approvals of immuno-oncology agents for over 16 indications, many of them first line. Checkpoint inhibitors (CPIs) are recognized as an essential backbone for a successful anticancer therapy regimen. This review focuses on the US Food and Drug Administration (FDA) regulatory approvals of major CPIs and the evolution of translational advances since their first approval close to a decade ago. In addition, critical preclinical and clinical pharmacology considerations, an overview of the pharmacokinetic and dose/regimen aspects, and a discussion of the future of CPI translational and clinical pharmacology as combination therapy becomes a mainstay of industrial immunotherapy development and in clinical practice are also discussed.

A tRNA processing enzyme is a key regulator of the mitochondrial unfolded protein response.

The mitochondrial unfolded protein response (UPRmt) has emerged as a predominant mechanism that preserves mitochondrial function. Consequently, multiple pathways likely exist to modulate UPRmt. We discovered that the tRNA processing enzyme, homolog of ELAC2 (HOE-1), is key to UPRmt regulation in Caenorhabditis elegans. We find that nuclear HOE-1 is necessary and sufficient to robustly activate UPRmt. We show that HOE-1 acts via transcription factors ATFS-1 and DVE-1 that are crucial for UPRmt. Mechanistically, we show that HOE-1 likely mediates its effects via tRNAs, as blocking tRNA export prevents HOE-1-induced UPRmt. Interestingly, we find that HOE-1 does not act via the integrated stress response, which can be activated by uncharged tRNAs, pointing toward its reliance on a new mechanism. Finally, we show that the subcellular localization of HOE-1 is responsive to mitochondrial stress and is subject to negative regulation via ATFS-1. Together, we have discovered a novel RNA-based cellular pathway that modulates UPRmt.

In vivo antidiabetic activity of nimesulide due to inhibition of amino acid transport.

Inhibiting the intestinal and renal neutral amino acid transporter B0AT1 by genetic means has improved insulin sensitivity in mice, but there are no antagonists available for preclinical or clinical use. Since the anti-inflammatory agent nimesulide selectively inhibited B0AT1 in vitro, we hypothesized that nimesulide exhibits in vivo potential to restrict neutral amino acid absorption and, therefore, may improve insulin sensitivity. The dose-related effect of nimesulide (10 to 100 mg/kg, PO) on intestinal absorption of neutral amino acids was estimated in C57 mice. The effect of nimesulide (50 mg/kg, PO) on renal resorption of amino acids was also assessed. The effect of chronic nimesulide (50 mg/kg, PO, BID for 14 days) was assessed in high protein diet-fed C57 mice, diet-induced obese mice and obese and diabetic db/db mice. Acute and chronic nimesulide treatment decreased absorption of neutral amino acids and increased their urinary excretion. Nimesulide treatment improved insulin sensitivity and glycemic control, increased GLP-1, decreased liver lipids and improved FGF-21 in serum. Nimesulide improved insulin sensitivity and glucose tolerance by inhibiting neutral amino acid transport in the intestine and kidneys. Thus, it can serve as a tool compound for in vivo B0AT1 inhibition.