A multi-cell model for the C4 photosynthetic pathway in developing wheat grains based upon tissue-specific transcriptome data.

A non-Kranz C4 photosynthesis of the NAD-ME subtype, specifically in developing wheat grains (14 dpa, days post-anthesis) was originally demonstrated using transcriptome-based RNA-seq. Here we present a re-examination of evidence for C4 photosynthesis in the developing grains of wheat and, more broadly, the Pooideae and an investigation of the evolutionary processes and implications. The expression profiles for the genes associated with C4 photosynthesis (C4- and C3-specific) were evaluated using published transcriptome data for the outer pericarp, inner pericarp, and endosperm tissues of the developing wheat grains. The expression of the C4-specific genes across these three tissues revealed the involvement of all three tissues in an orderly fashion to accomplish the non-Kranz NAD-ME-dependent C4 photosynthesis. Based on their expression levels in RPKM (reads per kilobase per million mapped reads) values, a model involving multiple cell- and tissue-types is proposed for C4 photosynthesis involved in the refixation of the respired CO2 from the endosperm tissues in the developing wheat grains. This multi-cell C4 model, proposed to involve more than two cell types, requires further biochemical validation.

Dysconnectivity of the brain functional network and abnormally expressed peripheral transcriptional profiles in patients with anxious depression.

BACKGROUND: Major depressive disorder (MDD) is a heterogeneous mental disorder, and accompanying anxiety symptoms, known as anxious depression (AD), are the most common subtype. However, the pathophysiology of AD may be distinct in depressed patients without anxiety (NAD) and remains unknown. This study aimed to investigate the relationship between functional connectivity and peripheral transcriptional profiles in patients with AD and NAD. METHODS: Functional imaging data were collected to identify differences in functional networks among patients with AD (n = 66), patients with NAD (n = 115), and healthy controls (HC, n = 200). The peripheral transcriptional data were clustered as co-expression modules, and their associations with AD, AND, and HC were analyzed. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses of the genes in the significant module were performed. Correlation analysis was performed to identify functional network-associated gene co-expression modules. RESULTS: A network was identified which consisted of 23 nodes and 28 edges that were significantly different among three sample groups. The regions of the network were located in temporal and occipital lobe. Two gene co-expression modules were shown to be associated with NAD, and one of which was correlated with the disrupted network in the AD group. The biological function of this module was enriched in immune regulation pathways. CONCLUSION: The results suggested that immune-related mechanisms were associated with functional networks in AD.

The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC derived Cortical Organoid of Alpers' Disease.

Alpers' syndrome is an early-onset neurodegenerative disorder usually caused by biallelic pathogenic variants in the gene encoding the catalytic subunit of polymerase-gamma (POLG), which is essential for mitochondrial DNA (mtDNA) replication. The disease is progressive, incurable, and inevitably it leads to death from drug-resistant status epilepticus. The neurological features of Alpers' syndrome are intractable epilepsy and developmental regression, with no effective treatment; the underlying mechanisms are still elusive, partially due to lack of good experimental models. Here, we generated the patient derived induced pluripotent stem cells (iPSCs) from one Alpers' patient carrying the compound heterozygous mutations of A467T (c.1399G>A) and P589L (c.1766C>T), and further differentiated them into cortical organoids and neural stem cells (NSCs) for mechanistic studies of neural dysfunction in Alpers' syndrome. Patient cortical organoids exhibited a phenotype that faithfully replicated the molecular changes found in patient postmortem brain tissue, as evidenced by cortical neuronal loss and depletion of mtDNA and complex I (CI). Patient NSCs showed mitochondrial dysfunction leading to ROS overproduction and downregulation of the NADH pathway. More importantly, the NAD+ precursor nicotinamide riboside (NR) significantly ameliorated mitochondrial defects in patient brain organoids. Our findings demonstrate that the iPSC model and brain organoids are good in vitro models of Alpers' disease; this first-in-its-kind stem cell platform for Alpers' syndrome enables therapeutic exploration and has identified NR as a viable drug candidate for Alpers' disease and, potentially, other mitochondrial diseases with similar causes.

NAD+ precursors and bile acid sequestration treat preclinical refractory environmental enteric dysfunction.

Environmental enteric dysfunction (EED) is a diffuse small bowel disorder associated with poor growth, inadequate responses to oral vaccines, and nutrient malabsorption in millions of children worldwide. We identify loss of the small intestinal Paneth and goblet cells that are critical for innate immunity, reduced villous height, increased bile acids, and dysregulated nicotinamide adenine dinucleotide (NAD+) synthesis signaling as potential mechanisms underlying EED and which also correlated with diminished length-for-age z score. Isocaloric low-protein diet (LPD) consumption in mice recapitulated EED histopathology and transcriptomic changes in a microbiota-independent manner, as well as increases in serum and fecal bile acids. Children with refractory EED harbor single-nucleotide polymorphisms in key enzymes involved in NAD+ synthesis. In mice, deletion of Nampt, the gene encoding the rate-limiting enzyme in the NAD+ salvage pathway, from intestinal epithelium also reduced Paneth cell function, a deficiency that was further aggravated by LPD. Separate supplementation with NAD+ precursors or bile acid sequestrant partially restored LPD-associated Paneth cell defects and, when combined, fully restored all histopathology defects in LPD-fed mice. Therapeutic regimens that increase protein and NAD+ contents while reducing excessive bile acids may benefit children with refractory EED.

Tumor cell-induced macrophage senescence plays a pivotal role in tumor initiation followed by stable growth in immunocompetent condition.

BACKGROUND: The cancer stem cell theory proposes that tumor formation in vivo is driven only by specific tumor-initiating cells having stemness; however, clinical trials conducted to test drugs that target the tumor stemness provided unsatisfactory results thus far. Recent studies showed clear involvement of immunity in tumors; however, the requirements of tumor-initiation followed by stable growth in immunocompetent individuals remain largely unknown. METHODS: To clarify this, we used two similarly induced glioblastoma lines, 8B and 9G. They were both established by overexpression of an oncogenic H-RasL61 in p53-deficient neural stem cells. In immunocompromised animals in an orthotopic transplantation model using 1000 cells, both show tumor-forming potential. On the other hand, although in immunocompetent animals, 8B shows similar tumor-forming potential but that of 9G's are very poor. This suggests that 8B cells are tumor-initiating cells in immunocompetent animals. Therefore, we hypothesized that the differences in the interaction properties of 8B and 9G with immune cells could be used to identify the factors responsible for its tumor forming potential in immunocompetent animals and performed analysis. RESULTS: Different from 9G, 8B cells induced senescence-like state of macrophages around tumors. We investigated the senescence-inducing factor of macrophages by 8B cells and found that it was interleukin 6. Such senescence-like macrophages produced Arginase-1, an immunosuppressive molecule known to contribute to T-cell hyporesponsiveness. The senescence-like macrophages highly expressed CD38, a nicotinamide adenine dinucleotide (NAD) glycohydrolase associated with NAD shortage in senescent cells. The addition of nicotinamide mononucleotide (NMN), an NAD precursor, in vitro inhibited to the induction of macrophage senescence-like phenotype and inhibited Arginase-1 expression resulting in retaining T-cell function. Moreover, exogenous in vivo administration of NMN after tumor inoculation inhibited tumor-initiation followed by stable growth in the immunocompetent mouse tumor model. CONCLUSIONS: We identified one of the requirements for tumor-initiating cells in immunocompetent animals. In addition, we have shown that tumor growth can be inhibited by externally administered NMN against macrophage senescence-like state that occurs in the very early stages of tumor-initiating cell development. This therapy targeting the immunosuppressive environment formed by macrophage senescence-like state is expected to be a novel promising cancer therapeutic strategy.

Future Perspectives for the Identification and Sequencing of Nicotinamide Adenine Dinucleotide-Capped RNAs.

Ribonucleic acid (RNA) is composed primarily of four canonical building blocks. In addition, more than 170 modifications contribute to its stability and function. Metabolites like nicotinamide adenine dinucleotide (NAD) were found to function as 5'-cap structures of RNA, just like 7-methylguanosine (m7G). The identification of NAD-capped RNA sequences was first made possible by NAD captureSeq, a multistep protocol for the specific targeting, purification, and sequencing of NAD-capped RNAs, developed in the authors' laboratory in the year 2015. In recent years, a number of NAD-RNA identification protocols have been developed by researchers around the world. They have enabled the discovery and identification of NAD-RNAs in bacteria, archaea, yeast, plants, mice, and human cells, and they play a key role in studying the biological functions of NAD capping. We introduce the four parameters of yield, specificity, evaluability, and throughput and describe to the reader how an ideal NAD-RNA identification protocol would perform in each of these disciplines. These parameters are further used to describe and analyze existing protocols that follow two general methodologies: the capture approach and the decapping approach. Capture protocols introduce an exogenous moiety into the NAD-cap structure in order to either specifically purify or sequence NAD-capped RNAs. In decapping protocols, the NAD cap is digested to 5'-monophosphate RNA, which is then specifically targeted and sequenced. Both approaches, as well as the different protocols within them, have advantages and challenges that we evaluate based on the aforementioned parameters. In addition, we suggest improvements in order to meet the future needs of research on NAD-modified RNAs, which is beginning to emerge in the area of cell-type specific samples. A limiting factor of the capture approach is the need for large amounts of input RNA. Here we see a high potential for innovation within the key targeting step: The enzymatic modification reaction of the NAD-cap structure catalyzed by ADP-ribosyl cyclase (ADPRC) is a major contributor to the parameters of yield and specificity but has mostly seen minor changes since the pioneering protocol of NAD captureSeq and needs to be more stringently analyzed. The major challenge of the decapping approach remains the specificity of the decapping enzymes, many of which act on a variety of 5'-cap structures. Exploration of new decapping enzymes or engineering of already known enzymes could lead to improvements in NAD-specific protocols. The use of a curated set of decapping enzymes in a combinatorial approach could allow for the simultaneous detection of multiple 5'-caps. The throughput of both approaches could be greatly improved by early sample pooling. We propose that this could be achieved by introducing a barcode RNA sequence before or immediately after the NAD-RNA targeting steps. With increased processing capacity and a potential decrease in the cost per sample, protocols will gain the potential to analyze large numbers of samples from different growth conditions and treatments. This will support the search for biological roles of NAD-capped RNAs in all types of organisms.

Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera.

Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO4 treatments), seven different tissues of B. papyrifera, as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB, HSP, NADH, and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera. In addition, HSP, rRNA, NADH, and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera. The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera.

LncRNA like NMRK2 mRNA functions as a key molecular scaffold to enhance mitochondrial respiration of NONO-TFE3 rearranged renal cell carcinoma in an NAD+ kinase-independent manner.

BACKGROUND: NONO-TFE3 rearranged renal cell carcinoma (NONO-TFE3 rRCC) is one of a subtype of TFE3 rRCCs with high malignancy and poor prognosis. Compared with clear cell RCC, NONO-TFE3 rRCC shows a preference for mitochondrial respiration. We recently identified that the upregulation of nicotinamide ribokinase 2 (NMRK2) was associated with enhanced mitochondrial respiration and tumor progression in TFE3 rRCC. METHODS: A tumor-bearing mouse model was established to verify the pro-oncogenic effect of NMRK2 on NONO-TFE3 rRCC. Then the expression of NMRK2 RNA and protein was detected in cell lines and patient specimens. The NMRK2 transcripts were Sanger-sequenced and blasted at NCBI website. We constructed dCas13b-HA system to investigate the factors binding with NMRK2 RNA. We also used molecular experiments like RIP-seq, IP-MS, FISH and fluorescence techniques to explore the mechanisms that long non-coding RNA (lncRNA) like NMRK2 mRNA promoted the mitochondrial respiration of NONO-TFE3 rRCC. The efficacy of the combination of shRNA (NMRK2)-lentivirus and metformin on NONO-TFE3 rRCC was assessed by CCK-8 assay. RESULTS: In this study, we confirmed that NMRK2 showed transcriptional-translational conflict and functioned as lncRNA like mRNA in the NONO-TFE3 rRCC. Furthermore, we revealed the molecular mechanism that NONO-TFE3 fusion suppressed the translation of NMRK2 mRNA. Most importantly, three major pathways were shown to explain the facilitation effects of lncRNA like NMRK2 mRNA on the mitochondrial respiration of NONO-TFE3 rRCC in an NAD+ kinase-independent manner. Finally, the efficacy of combination of shRNA (NMRK2)-lentivirus and metformin on NONO-TFE3 rRCC was demonstrated to be superior than either agent alone. CONCLUSIONS: Overall, our data comprehensively demonstrated the mechanisms for the enhanced mitochondrial respiration in NONO-TFE3 rRCC and proposed lncRNA like NMRK2 mRNA as a therapy target for NONO-TFE3 rRCC.

DNMT3A mutation promotes leukemia development through NAM-NAD metabolic reprogramming.

BACKGROUND: DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia (AML) with Arg882His (R882H) as the hotspot mutation. It has been reported that DNMT3A mutation plays a key role in leukemogenesis through hypomethylation of some target genes associated with cell growth and differentiation. In this study, we investigated the function of DNMT3A R882H in the malignant progression of AML by regulating metabolic reprogramming. METHODS: Ultra-High Performance Liquid Chromatography-High Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS) was used to detect metabolites in the serum of mice harboring Dnmt3a R878H mutation and the wild-type Dnmt3a. Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) and RNA sequencing (RNA-seq) were used to analyze the levels of DNA methylation and mRNA expression of genes in mouse Gr1+ bone marrow cells respectively. The TCGA and GO databases were used to analyze the differential genes between human samples carrying the DNMT3A R882 mutation and the wild-type DNMT3A. Co-immunoprecipitation and immunoblotting were used to illustrate the binding levels of Cyclins-CDKs and CDK inhibitors including CDKN1A and CDKN1B. Flow cytometry was used to analyze the cell differentiation, division, apoptosis and cell cycle. The effect of NAMPT inhibition on leukemia was evaluated by using in vivo fluorescence imaging in NOG mouse model bearing OCI-AML3 cells. RESULTS: DNMT3A mutation caused high expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the nicotinamide adenine dinucleotide (NAD) salvage synthetic pathway, through DNA hypomethylation, and finally led to abnormal nicotinamide (NAM) metabolism and NAD synthesis. The NAM-NAD metabolic abnormalities caused accelerated cell cycle progression. Inhibition of NAMPT can reduce the binding degree between Cyclins-CDKs, and increase the binding interaction of the CDK inhibitors with Cyclins-CDKs complexes. Moreover, cells with high expression of NAMPT were more sensitive to the NAMPT inhibitor FK866 with a lower IC50. The inhibition of NAMPT can remarkably extend the survival time of tumor-bearing mice and reduce the infiltration of tumor cells. CONCLUSIONS: Taken together, our data showed that DNMT3A mutation caused NAMPT overexpression to induce the reprogramming of NAM-NAD metabolism and contribute to abnormal proliferation, which provided a potential direction for targeted therapy at the metabolic level in AML with DNMT3A mutation.

DMT1 differentially regulates mitochondrial complex activities to reduce glutathione loss and mitigate ferroptosis.

Mitochondria are vital for energy production and redox homeostasis, yet knowledge of relevant mechanisms remains limited. Here, through a genome-wide CRISPR-Cas9 knockout screening, we have identified DMT1 as a major regulator of mitochondria membrane potential. Our findings demonstrate that DMT1 deficiency increases the activity of mitochondrial complex I and reduces that of complex III. Enhanced complex I activity leads to increased NAD+ production, which activates IDH2 by promoting its deacetylation via SIRT3. This results in higher levels of NADPH and GSH, which improve antioxidant capacity during Erastin-induced ferroptosis. Meanwhile, loss of complex III activity impairs mitochondrial biogenesis and promotes mitophagy, contributing to suppression of ferroptosis. Thus, DMT1 differentially regulates activities of mitochondrial complex I and III to cooperatly suppress Erastin-induced ferroptosis. Furthermore, NMN, an alternative method of increasing mitochondrial NAD+, exhibits similar protective effects against ferroptosis by boosting GSH in a manner similar to DMT1 deficiency, shedding a light on potential therapeutic strategy for ferroptosis-related pathologies.

Complete Mitochondrial Genome Sequence and Phylogenetic Analysis of Procambarus clarkii and Cambaroides dauricus from China.

To enhance the management and protection of crayfish genetic diversity and germplasm resources in Cambaroides dauricus (C. dauricus), a common species of Procambarus clarkii (P. clarkii) was used as a control group to compare the whole mitochondrial genome sequence using Illumina sequencing technology. This study found that the mitochondrial genome of C. dauricus is 15580 bp in length, with a base composition of A (31.84%), G (17.66%), C (9.42%), and T (41.08%) and a C + G content of 27.08%. The C + G in the D-loop is rich in 17.06%, indicating a significant preference. The mitochondrial genome of C. dauricus contains 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes, with most of the genes labeled in the negative direction, except for a few genes that are labeled in the positive direction. The start codons of the ten coding sequences are ATG, and the quintessential TAA and TAG are the stop codons. This study also found that the Ka/Ks ratios of most protein-coding genes in the mitochondria of both shrimps are lower than 1, indicating weak natural selection, except for nad 2, nad 5, and cox 1. The Ka/Ks ratio of cox 3 is the lowest (less than 0.1), indicating that this protein-coding gene bears strong natural selection pressure and functional constraint in the process of mitochondrial genetic evolution of both shrimps. Furthermore, we constructed phylogenetic analyses based on the entire sequence, which effectively distinguishes the high body from other shrimp species of the genus based on the mitochondrial genome. This study provides molecular genetic data for the diversity investigation and protection of fishery resources with Chinese characteristics and a scientific reference for the evolutionary study of Procambarus.

Symbiosis of Carpenter Bees with Uncharacterized Lactic Acid Bacteria Showing NAD Auxotrophy.

Eusocial bees (such as honey bees and bumble bees) harbor core gut microbiomes that are transmitted through social interaction between nestmates. Carpenter bees are not eusocial; however, recent microbiome analyses found that Xylocopa species harbor distinctive core gut microbiomes. In this study, we analyzed the gut microbiomes of three Xylocopa species in Japan between 2016 and 2021 by V1 to V2 region-based 16S rDNA amplicon sequencing, and 14 candidate novel species were detected based on the full-length 16S rRNA gene sequences. All Xylocopa species harbor core gut microbiomes consisting of primarily lactic acid bacteria (LAB) that were phylogenetically distant from known species. Although they were difficult to cultivate, two LAB species from two different Xylocopa species were isolated by supplementing bacterial culture supernatants. Both genomes exhibited an average LAB genome size with a large set of genes for carbohydrate utilization but lacked genes to synthesize an essential coenzyme NAD, which is unique among known insect symbionts. Our findings of phylogenetically distinct core LAB of NAD auxotrophy reflected the evolution of Xylocopa-restricted bacteria retention and maintenance through vertical transmission of microbes during solitary life. We propose five candidate novel species belonging to the families Lactobacillaceae and Bifidobacteriaceae, including a novel genus, and their potential functions in carbohydrate utilization. IMPORTANCE Recent investigations found unique microbiomes in carpenter bees, but the description of individual microbes, including isolation and genomics, remains largely unknown. Here, we found that the Japanese Xylocopa species also harbor core gut microbiomes. Although most of them were difficult to isolate a pure colony, we successfully isolated several strains. We performed whole-genome sequencing of the isolated candidate novel species and found that the two Lactobacillaceae strains belonging to the Xylocopa-specific novel LAB clade lack the genes for synthesizing NAD, a coenzyme central to metabolism in all living organisms. Here, we propose a novel genus for the two LAB species based on very low 16S rRNA gene sequence similarities and genotypic characters.

A novel 7-base pair deletion at a splice site in MS-2 impairs male fertility via premature tapetum degradation in common bean (Phaseolis vulgaris L.).

KEY MESSAGE: A novel splice-site mutation in the P. vulgarisgene for TETRAKETIDE α-PYRONE REDUCTASE 2 impairs male fertility, and parthenocarpic pod development can be improved by external application of IAA. Snap bean (Phaseolus vulgaris L.) is an important vegetable crop in many parts of the world, and the main edible part is the fresh pod. Here, we report the characterization of the genic male sterility (ms-2) mutant in common bean. Loss of function of MS-2 accelerates degradation of the tapetum, resulting in a complete male sterility. Through fine-mapping, co-segregation, and re-sequencing analysis, we identified Phvul.003G032100, which encodes the TETRAKETIDE α-PYRONE REDUCTASE 2 (PvTKPR2) protein in common bean, as the causal gene for MS-2. PvTKPR2 is predominantly expressed at the early stages of flower development. A novel 7-bp (+ 6028 bp to + 6034 bp) deletion mutation spans the splice site between the fourth intron and fifth exon, leading to a 9-bp deletion in transcribed mRNA and a 3-amino acid (G210M211V212) deletion in the protein coding sequence of the PvTKPR2ms-2 gene. The 3-D structural changes in the protein due to the mutation may impair the activities of NAD-dependent epimerase/dehydratase and the NAD(P)-binding domains of PvTKPR2ms-2 protein. The ms-2 mutant plants produce many small parthenocarpic pods, and the size of the pods can be doubled by external application of 2 mM indole-3-acetic acid (IAA). Our results demonstrate that a novel mutation in PvTKPR2 impairs male fertility through premature degradation of the tapetum.

Haplotypes of Dirofilaria repens from Poland and selected countries of Central, North-Eastern Europe and the Middle East: An evaluation on the relation between the genetic diversity and the geographic distribution of the fast-spreading parasite.

Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1-5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1-4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1-5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes.

Mitochondrial dysfunction in human hypertrophic cardiomyopathy is linked to cardiomyocyte architecture disruption and corrected by improving NADH-driven mitochondrial respiration.

AIMS: Genetic hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein-encoding genes (i.e. genotype-positive HCM). In an increasing number of patients, HCM occurs in the absence of a mutation (i.e. genotype-negative HCM). Mitochondrial dysfunction is thought to be a key driver of pathological remodelling in HCM. Reports of mitochondrial respiratory function and specific disease-modifying treatment options in patients with HCM are scarce. METHODS AND RESULTS: Respirometry was performed on septal myectomy tissue from patients with HCM (n = 59) to evaluate oxidative phosphorylation and fatty acid oxidation. Mitochondrial dysfunction was most notably reflected by impaired NADH-linked respiration. In genotype-negative patients, but not genotype-positive patients, NADH-linked respiration was markedly depressed in patients with an indexed septal thickness ≥10 compared with <10. Mitochondrial dysfunction was not explained by reduced abundance or fragmentation of mitochondria, as evaluated by transmission electron microscopy. Rather, improper organization of mitochondria relative to myofibrils (expressed as a percentage of disorganized mitochondria) was strongly associated with mitochondrial dysfunction. Pre-incubation with the cardiolipin-stabilizing drug elamipretide and raising mitochondrial NAD+ levels both boosted NADH-linked respiration. CONCLUSION: Mitochondrial dysfunction is explained by cardiomyocyte architecture disruption and is linked to septal hypertrophy in genotype-negative HCM. Despite severe myocardial remodelling mitochondria were responsive to treatments aimed at restoring respiratory function, eliciting the mitochondria as a drug target to prevent and ameliorate cardiac disease in HCM. Mitochondria-targeting therapy may particularly benefit genotype-negative patients with HCM, given the tight link between mitochondrial impairment and septal thickening in this subpopulation.

Molecular Dynamic Simulation Analysis of a Novel Missense Variant in CYB5R3 Gene in Patients with Methemoglobinemia.

Background and Objective: Mutations in the CYB5R3 gene cause reduced NADH-dependent cytochrome b5 reductase enzyme function and consequently lead to recessive congenital methemoglobinemia (RCM). RCM exists as RCM type I (RCM1) and RCM type II (RCM2). RCM1 leads to higher methemoglobin levels causing only cyanosis, while in RCM2, neurological complications are also present along with cyanosis. Materials and Methods: In the current study, a consanguineous Pakistani family with three individuals showing clinical manifestations of cyanosis, chest pain radiating to the left arm, dyspnea, orthopnea, and hemoptysis was studied. Following clinical assessment, a search for the causative gene was performed using whole exome sequencing (WES) and Sanger sequencing. Various variant effect prediction tools and ACMG criteria were applied to interpret the pathogenicity of the prioritized variants. Molecular dynamic simulation studies of wild and mutant systems were performed to determine the stability of the mutant CYB5R3 protein. Results: Data analysis of WES revealed a novel homozygous missense variant NM_001171660.2: c.670A > T: NP_001165131.1: p.(Ile224Phe) in exon 8 of the CYB5R3 gene located on chromosome 22q13.2. Sanger sequencing validated the segregation of the identified variant with the disease phenotype within the family. Bioinformatics prediction tools and ACMG guidelines predicted the identified variant p.(Ile224Phe) as disease-causing and likely pathogenic, respectively. Molecular dynamics study revealed that the variant p.(Ile224Phe) in the CYB5R3 resides in the NADH domain of the protein, the aberrant function of which is detrimental. Conclusions: The present study expanded the variant spectrum of the CYB5R3 gene. This will facilitate genetic counselling of the same and other similar families carrying mutations in the CYB5R3 gene.

Paving the way for synthetic C1 - Metabolism in Pseudomonas putida through the reductive glycine pathway.

One-carbon (C1) compounds such as methanol, formate, and CO2 are alternative, sustainable microbial feedstocks for the biobased production of chemicals and fuels. In this study, we engineered the carbon metabolism of the industrially important bacterium Pseudomonas putida to modularly assimilate these three substrates through the reductive glycine pathway. First, we demonstrated the functionality of the C1-assimilation module by coupling the growth of auxotrophic strains to formate assimilation. Next, we extended the module in the auxotrophic strains from formate to methanol-dependent growth using both NAD and PQQ-dependent methanol dehydrogenases. Finally, we demonstrated, for the first time, engineered CO2-dependent formation of part of the biomass through CO2 reduction to formate by the native formate dehydrogenase, which required short-term evolution to rebalance the cellular NADH/NAD + ratio. This research paves the way to further engineer P. putida towards full growth on formate, methanol, and CO2 as sole feedstocks, thereby substantially expanding its potential as a sustainable and versatile cell factory.

ONE-seq: epitranscriptome and gene-specific profiling of NAD-capped RNA.

The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Using ONE-seq, we discover more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveal epitranscriptome-wide dynamics of NAD-RNAs with age. ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification.

Genome-wide identification of glutamate synthase gene family and expression patterns analysis in response to carbon and nitrogen treatment in Populus.

Glutamate synthase (GOGAT) is a key enzyme in glutamine synthetase (GS)/GOGAT cycle and at the hub of carbon and nitrogen metabolism, catalyzing the formation of glutamate from α-oxoglutarate and glutamine. In this study, members of GOGAT family in Populus trichocarpa were identified and analyzed by bioinformatics. The four PtGOGATs were divided into two subgroups: subgroup A (Fd-GOGAT1 and Fd-GOGAT2) and subgroup B (NADH-GOGAT1 and NADH-GOGAT2). Many important elements have been identified in the promoters of different PtGOGATs, including hormone- and light-responsive elements. Meanwhile, the transcript levels of PxGOGATs were affected by light and diurnal cycle. Quantitative real-time PCR showed PxFd-GOGATs and PxNADH-GOGATs were mainly expressed in leaves and roots in Populus × xiaohei T. S. Hwang et Liang, respectively. Under elevated CO2, PxGOGATs were suppressed in all tissues except the stem. And PxFd-GOGATs and PxNADH-GOGATs were strongly induced by nitrogen in leaves and roots, respectively. In addition, PxGOGATs were stimulated significantly in roots in response to NH4+and glutamine directly. Our results provide new insights about GOGATs in poplar and their expression patterns under exogenous substances, to lay molecular basis for studying gene function and provide a reference for exploring putative roles of GOGATs in carbon-nitrogen balance.

A novel lncRNA MDHDH suppresses glioblastoma multiforme by acting as a scaffold for MDH2 and PSMA1 to regulate NAD+ metabolism and autophagy.

BACKGROUND: To identify potential targets related to nicotinamide adenine dinucleotide (NAD+) metabolism in gliomas, we used RNA immunoprecipitation to identify a novel long noncoding RNA renamed malate dehydrogenase degradation helper (MDHDH) (NONCODE annotation ID: NONHSAT138800.2, NCBI Reference Sequence: NR_028345), which bound to MDH2 (malate dehydrogenase 2), that is downregulated in glioblastoma multiforme (GBM) and associated with metabolic regulation. However, its underlying mechanisms in the progression of GBM have not been well studied. METHODS: To investigate the clinical significance of MDHDH, we analyzed its expression levels in publicly available datasets and collected clinical samples from Shandong Provincial Hospital, affiliated with Shandong University. Functional assays, including FISH/CISH, CCK8, EdU, wound healing, and transwell assays, were used to determine the cellular/subcellular localization, tissue expression profile and anti-oncogenic role of MDHDH. Furthermore, RNA pulldown, mass spectrometry RNA immunoprecipitation, coimmunoprecipitation, JC-1 probe, and cell energy-production assays were used to determine the mechanisms of MDHDH in the development of GBM. Animal experiments were conducted to determine the antitumorigenic role of MDHDH in GBM in vivo. RESULTS: In public datasets, MDHDH expression was significantly downregulated in GBM and LGG compared with GTEx normal brain tissues. The results of the tissue microarray showed that the MDHDH expression level negatively correlated with the tumor grade. Altered MDHDH expression led to significant changes in the proliferation, migration and invasion of GBM cells both in vitro and in vivo. Mechanistically, we found that MDHDH directly bound to MDH2 and PSMA1 (20S proteasomal core subunit alpha-type 1) as a molecular scaffold and accelerated the degradation of MDH2 by promoting the binding of ubiquitinated MDH2 to the proteasome. The degradation of MDH2 subsequently led to changes in the mitochondrial membrane potential and NAD+/NADH ratio, which impeded glycolysis in glioma cells. CONCLUSIONS: In conclusion, this study broadened our understanding of the functions of lncRNAs in GBM. We demonstrated that the tumor suppressor MDHDH might act as a clinical biomarker and that the overexpression of MDHDH might be a novel synergistic strategy for enhancing metabolism-based, epigenetic-based, and autophagy regulation-based therapies with clinical benefits for glioblastoma multiforme patients.