Cytochrome B Research Articles

Steroidogenic electron-transfer factors and their diseases.

Most steroidogenesis disorders are caused by mutations in genes encoding the steroidogenic enzymes, but work in the past 20 years has identified related disorders caused by mutations in the genes encoding the cofactors that transport electrons from NADPH to P450 enzymes. Most P450s are microsomal and require electron donation by P450 oxidoreductase (POR); by contrast, mitochondrial P450s require electron donation via ferredoxin reductase (FdxR) and ferredoxin (Fdx). POR deficiency is the most common and best-described of these new forms of congenital adrenal hyperplasia. Severe POR deficiency is characterized by the Antley-Bixler skeletal malformation syndrome and genital ambiguity in both sexes, and hence is easily recognized, but mild forms may present only with infertility and subtle disorders of steroidogenesis. The common POR polymorphism A503V reduces catalysis by P450c17 (17-hydroxylase/17,20-lyase) and the principal drugmetabolizing P450 enzymes. The 17,20-lyase activity of P450c17 requires the allosteric action of cytochrome b5, which promotes interaction of P450c17 with POR, with consequent electron transfer. Rare b5 mutations are one of several causes of 17,20-lyase deficiency. In addition to their roles with steroidogenic mitochondrial P450s, Fdx and FdxR participate in the synthesis of iron-sulfur clusters used by many enzymes. Disruptions in the assembly of Fe-S clusters is associated with Friedreich ataxia and Parkinson disease. Recent work has identified mutations in FdxR in patients with neuropathic hearing loss and visual impairment, somewhat resembling the global neurologic disorders seen with mitochondrial diseases. Impaired steroidogenesis is to be expected in such individuals, but this has not yet been studied.

Mitochondrial DNA indicate paraphyletic relationships and resolve disjunct distributions in the Neotoma mexicana species group

Woodrats (genus Neotoma) comprise 24 species found primarily in the US and Mexico. The Neotoma mexicana species group reaches its southernmost distribution in the highlands of southern Mexico and Central America. Previous research suggested that N. mexicana has a disjunct distribution, and it, N. ferruginea and N. picta have allopatric distributions and limit each other around the lowlands of the Isthmus of Tehuantepec. However, these hypotheses were suggested with incomplete subspecific sampling near the isthmus. We used new samples of N. m. parvidens from the Oaxacan Sierra Madre del Sur and N. m. tropicalis from the Sierra Norte de Oaxaca to assess their taxonomic affinity. With new sequences of the mitochondrial cytochrome-b gene, we found that both subspecies belong to N. ferruginea, not N. mexicana. We therefore suggest that N. mexicana is continuously distributed from the US to the Transmexican Volcanic Belt, N. picta inhabits the Guerreran Sierra Madre del Sur, and N. ferruginea ranges from the Oaxacan Sierra Madre del Sur to Central America. Our findings indicate that the Isthmus of Tehuantepec did not promote speciation in these woodrats, but we did detect intraspecific genetic differentiation between samples of N. ferruginea from east and west of the isthmus.

A genome-wide association study reveals that the cytochrome b5 involved in seed reserve mobilization during seed germination in rice.

A candidate gene cytochrome b5 for the major QTL qSRMP9 for rice seed reserve mobilization was validated during seed germination using a genome-wide association study approach. Seed reserve mobilization plays important roles in the early seedling growth in rice. However, the genetic basis underlying this process is poorly understood. In this study, the genetic architecture of variation in seed reserve mobilization during seed germination was studied using a genome-wide association study approach in rice. Three quantitative trait loci (QTL) including qSRMP6, qSRMP9, and qSRMP12 for seed reserve mobilization percentage were identified. In which, the candidate gene cytochrome b5 (OsCyb5) for the major QTL qSRMP9 was validated. Disruption of this gene in Oscyb5 mutants reduced the seed reserve mobilization and seedling growth compared with wild-type (WT) in rice. There were no significant differences of grain size, starch, protein and total soluble sugar content in the mature grains between Oscyb5 mutants and WT. However, the α-amylase activity in the germinating seeds of Oscyb5 mutants was significantly decreased compared to that of WT, and then, the starch and sugar mobilization and the glucose accumulation during seed germination were significantly decreased in Oscyb5 mutants. Two elite haplotypes of OsCyb5 associated with the higher seed reserve mobilization percentage and its elite single nucleotide polymorphism variations were mainly existed in the INDICA and AUS accessions. The natural variation of OsCyb5 contributing to seed reserve mobilization might be useful for the future rice breeding.

Two stripe rust effectors impair wheat resistance by suppressing import of host Fe-S protein into chloroplasts.

Several effectors from phytopathogens usually target various cell organelles to interfere with plant defenses, and they generally contain sequences that direct their translocation into organelles, such as chloroplasts. In this study, we characterized a different mechanism for effectors to attack chloroplasts in wheat (Triticum aestivum). Two effectors from Puccinia striiformis f. sp. tritici (Pst), Pst_4, and Pst_5, inhibit Bax-mediated cell death and plant immune responses, such as callose deposition and reactive oxygen species (ROS) accumulation. Gene silencing of the two effectors induced significant resistance to Pst, demonstrating that both effectors function as virulence factors of Pst. Although these two effectors have low sequence similarities and lack chloroplast transit peptides, they both interact with TaISP (wheat cytochrome b6-f complex iron-sulfur subunit, a chloroplast protein encoded by nuclear gene) in the cytoplasm. Silencing of TaISP impaired wheat resistance to avirulent Pst and resulted in less accumulation of ROS. Heterogeneous expression of TaISP enhanced chloroplast-derived ROS accumulation in Nicotiana benthamiana. Co-localization in N. benthamiana and western blot assay of TaISP content in wheat chloroplasts show that both effectors suppressed TaISP from entering chloroplasts. We conclude that these biotrophic fungal effectors suppress plant defenses by disrupting the sorting of chloroplast protein, thereby limiting host ROS accumulation and promoting fungal pathogenicity.

Smooth muscle cell CYB5R3 preserves cardiac and vascular function under chronic hypoxic stress

Chronic hypoxia is a major driver of cardiovascular complications, including heart failure. The nitric oxide (NO) – soluble guanylyl cyclase (sGC) – cyclic guanosine monophosphate (cGMP) pathway is integral to vascular tone maintenance. Specifically, NO binds its receptor sGC within vascular smooth muscle cells (SMC) in its reduced heme (Fe2+) form to increase intracellular cGMP production, activate protein kinase G (PKG) signaling, and induce vessel relaxation. Under chronic hypoxia, oxidative stress drives oxidation of sGC heme (Fe2+→Fe3+), rendering it NO-insensitive. We previously showed that cytochrome b5 reductase 3 (CYB5R3) in SMC is a sGC reductase important for maintaining NO-dependent vasodilation and conferring resilience to systemic hypertension and sickle cell disease-associated pulmonary hypertension. To test whether CYB5R3 may be protective in the context of chronic hypoxia, we subjected SMC-specific CYB5R3 knockout mice (SMC CYB5R3 KO) to 3 weeks hypoxia and assessed vascular and cardiac function using echocardiography, pressure volume loops and wire myography. Hypoxic stress caused 1) biventricular hypertrophy in both WT and SMC CYB5R3 KO, but to a larger degree in KO mice, 2) blunted vasodilation to NO-dependent activation of sGC in coronary and pulmonary arteries of KO mice, and 3) decreased, albeit still normal, cardiac function in KO mice. Overall, these data indicate that SMC CYB5R3 deficiency potentiates bilateral ventricular hypertrophy and blunts NO-dependent vasodilation under chronic hypoxia conditions. This implicates that SMC CYB5R3 KO mice post 3-week hypoxia have early stages of cardiac remodeling and functional changes that could foretell significantly impaired cardiac function with longer exposure to hypoxia.

TMT-Based proteomics analysis of LPS-induced acute lung injury

Purpose: The proteome during lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice is unclear. Materials and Methods: In this study, eight-week-old male C57BL/6 mice were intraperitoneally injected with LPS and sacrificed 18 hours after LPS administration to identify protein expression levels in lung tissue using tandem mass tag (TMT) analysis for relative quantification. Hematoxylin-eosin (HE) staining was used to evaluate lung injury in mice. Immunohistochemical staining was used to calculate the production of myeloperoxidase (MPO) and TUNEL staining was performed to detect apoptosis. GO functional clustering and KEGG pathway enrichment analyses were performed to determine functions of differentially expressed proteins (DEPs) and transduction pathways. Domain annotation and subcellular localization analysis of the DEPs were also performed. Furthermore, parallel reaction monitoring (PRM) analysis was used to verify the top 30 DEPs. Results: A total of 5188 proteins were found to be expressed in lung tissues from LPS- and saline-treated mice. Among these proteins, 293 were differentially expressed between the two groups; 255 proteins were upregulated in the LPS-treated ALI mice, while 38 were downregulated. GO analysis showed that the DEPs are mainly extracellular, and KEGG analysis suggested that the DEPs are mainly enriched in the NOD-like receptor signaling pathway, complement and coagulation cascades and natural killer cell-mediated cytotoxicity. Enrichment of the DEPs is mainly peptidase S1A, serine proteases, peptidase S1, and the serpin domain. 26.6% of the DEPs are in the nucleus, 24.6% are in the cytosol, 19.1% are in the extracellular space, and 18.8% are in the plasma membrane. PRM validation showed that the trend of 30 DEPs was same with TMT analysis. Among these, Cytochrome b-245 heavy chain (Cybb), Monocyte differentiation antigen CD14 (Cd14) and Neutrophil gelatinase-associated lipocalin (NGAL) were the most obvious change. Conclusions: Our results may help to identify markers and therapeutic targets for LPS-induced ALI.

Modification of sesame (Sesamum indicum L.) for Triacylglycerol accumulation in plant biomass for biofuel applications

Sesame is considered as the queen of oil seeds owing to its high oil content of about 56–62% and good quality oil. Sesame oil alone or in combination with other vegetable oils can yield good quality biodiesel. Sesame biodiesel blends up to 20% yields fuel efficiency and power output on par with mineral diesel but superior in environmental performance. Though biodiesel from edible oil is highly criticized, the demand for renewable energy compels the development of high-performance sesame plants. Triacylglycerol synthesis in flowering plants follows an acyl CoA-dependent and independent manner. This study envisages transgenic approaches to enhance oil production in sesame biomass. The genes of choice for oil enhancement includes DGAT1, PDAT1, FAD3 and cytochrome b5F. Diacylglycerol acyltransferase (DGAT) and phospholipid diacylglycerol acyltransferases (PDAT) are key enzymes in TAG synthesis. Fatty acid desaturases (FAD) has the ability to enhance specific fatty acids, whereas cytochrome b5 genes augment the process by donating electrons. A combination of the above categories of genes which performed well in terms of oil content in the yeast expression system from our earlier studies is used in Agrobacterium-mediated sesame transformation experiments to evaluate the biodiesel potential of transgenic sesame plants. The transgenic construct with PDAT1 and FAD3 combination yielded a 10% increase in TAG content. The possibility of transgenic sesame as a biodiesel plant is discussed.

Structural and functional effects of single nucleotide polymorphism on canine cytochrome b 5 reductase - in silico

The objectives of this study were to investigate the effects of single nucleotide polymorphism in Canine cytochrome b5 reductase using computational methods. Data was obtained from database of National Centre for Biotechnology Information (db SNP) and computational software was used for the analysis. The 3D protein structure was predicted using phyre 2 server. PANTHER analysis predicted the effect of single nucleotide polymorphism (substitution of Isoleucine for Leucine at position 194) as damaging. Analysis using the Mutpred 2 web application also indicated deleterious effects of the amino acid substitution. Molecular mechanisms of structural changes in the amino acid were determined using Mutpred 2 to be altered ordered interface, gain of allosteric sites and altered metal binding. The study indicated that the substitution of Isoleucine by Leucine at position 194 of the amino acid sequence (Ile 194 Leu) resulted in the destabilization of the amino acid structure leading to functional deviation in canine cytochrome b5 reductase.

Population connectivity of the euphausiid, Stylocheiron elongatum, in the Gulf Stream (NW Atlantic Ocean) in relation to COI barcode diversity of Stylocheiron species

Abstract The euphausiid genus Stylocheiron includes species with biogeographical distributions spanning multiple ocean basins. Despite their circumglobal distributions, the species show low levels of genetic diversity and little or no evidence of population structure based on the mitochondrial cytochrome oxidase I (COI) barcode region, with the exception of a possible cryptic species within Stylocheiron affine. Stylocheiron elongatum showed < 1% variation of the COI barcode region among populations in different ocean basins, but analysis of samples collected from the Florida Current (February, 1993) and Gulf Stream Meander Region (April, 1993) in the Northwest Atlantic Ocean revealed small-but-significant genetic differentiation between samples based on a different section of COI and mitochondrial cytochrome b (CYB). Both COI and CYB showed large haplotype and small nucleotide diversities, departures from neutral expectations, and haplotype networks consistent with persistent genetic structuring of the species population. These patterns of diversity indicate the presence of selection driving population divergence. We hypothesize that position-keeping by this deep-living, non-migrating euphausiid species may prevent genetic homogenization (panmixia) in the dynamic Gulf Stream System. This study demonstrates the importance of analyzing patterns of genetic diversity and structure at regional and global scales to understand the ecological and evolutionary processes impacting marine zooplankton.

Immune-Related Genes of Megalurothrips usitatus (Bagrall) Against Beauveria brongniartii and Akanthomyces attenuatus Identified Using RNA Sequencing.

Megalurothrips usitatus (Bagrall) is an important pest of legumes worldwide, causing great economic loss every year. Beauveria brongniartii and Akanthomyces attenuatus have shown considerable pathogenicity against M. usitatus in our previous studies. The medial lethal concentration (LC50) and the sublethal lethal concentration (LC25) of B. brongniartii isolate SB010 against M. usitatus were 8.38 × 105 and 1.73 × 105 conidia mL−1, respectively, whereas those of A. attenuatus isolate SCAUDCL-53 against M. usitatus were 4.37 × 105 and 2.97 × 104 conidia mL−1, respectively. This study reports the transcriptome-based explanation of the stress responses of M. usitatus following the application of B. brongniartii and A. attenuatus. The analysis of the transcriptomic data revealed the expression of 254, 207, 195, and 234 immunity-related unigenes by M. usitatus in response to B. brongniartii LC50 (SB1), B. brongniartii LC25 (SB2), A. attenuatus LC50 (V1), and A. attenuatus LC25 (V2), respectively. The biological function and metabolic pathway analyses showed that these unigenes were mainly related to pattern recognition receptors, information transduction factors, and reaction factors, such as scavenger receptor, cytochrome b5, cuticle protein, lysozyme, and serine protease.

Interaction of Smoking and Lead Exposure among Carriers of Genetic Variants Associated with a Higher Level of Oxidative Stress Indicators.

Smoking and lead (Pb) exposure increased oxidative stress in human body, and people with some gene variants may be susceptible to Pb and smoking via oxidative stress. The aim of this study is to evaluate oxidative stress by measuring thiobarbituric acid reactive substances (TBARS) and the relationship of lipid peroxidation markers in Pb workers with different gene polymorphisms (rs4673 and rs1050450) in both smokers and nonsmokers. Blood samples were collected from 267 Pb workers who received their annual health examination in the Kaohsiung Medical University Hospital. Glutathione peroxidase 1 (GPx-1) rs1050450 and cytochrome B-245 Alpha Chain (CYBA) rs4673 single-nucleotide polymorphisms (SNP) were analyzed by specific primer-probes using Real-Time PCR methods. The interaction between blood Pb and smoking increased serum levels of TBARS and the ratio of oxidative low-density lipoprotein and low-density lipoprotein (oxLDL/LDL). Analysis of workers with rs1050450 SNPs showed higher blood Pb levels in the workers with CC genotype than those with CT genotype. Smokers had significantly higher blood Pb, alanine transaminase (ALT), TBARS, and OxLDL levels than nonsmokers. TBARS increased 0.009 nmol/mL when blood Pb increased one µg/dL in smokers compared to nonsmokers. The ratio of OxLDL/LDL increased 0.223 when blood Pb increased one µg/dL in smokers compared to nonsmokers. TBARS levels and the ratio of OxLDL/LDL were positively correlated and interacted between blood Pb and smoking after the adjustment of confounders, suggesting that smoking cessation is an important issue in the Pb-exposed working environment.

Designed Artificial Protein Heterodimers With Coupled Functions Constructed Using Bio-Orthogonal Chemistry.

The formation of protein complexes is central to biology, with oligomeric proteins more prevalent than monomers. The coupling of functionally and even structurally distinct protein units can lead to new functional properties not accessible by monomeric proteins alone. While such complexes are driven by evolutionally needs in biology, the ability to link normally functionally and structurally disparate proteins can lead to new emergent properties for use in synthetic biology and the nanosciences. Here we demonstrate how two disparate proteins, the haem binding helical bundle protein cytochrome b 562 and the β-barrel green fluorescent protein can be combined to form a heterodimer linked together by an unnatural triazole linkage. The complex was designed using computational docking approaches to predict compatible interfaces between the two proteins. Models of the complexes where then used to engineer residue coupling sites in each protein to link them together. Genetic code expansion was used to incorporate azide chemistry in cytochrome b 562 and alkyne chemistry in GFP so that a permanent triazole covalent linkage can be made between the two proteins. Two linkage sites with respect to GFP were sampled. Spectral analysis of the new heterodimer revealed that haem binding and fluorescent protein chromophore properties were retained. Functional coupling was confirmed through changes in GFP absorbance and fluorescence, with linkage site determining the extent of communication between the two proteins. We have thus shown here that is possible to design and build heterodimeric proteins that couple structurally and functionally disparate proteins to form a new complex with new functional properties.

Genome-Wide Association Study Using Whole-Genome Sequencing Identifies a Genomic Region on Chromosome 6 Associated With Comb Traits in Nandan-Yao Chicken.

Comb traits have potential economic value in the breeding of indigenous chickens in China. Identifying and understanding relevant molecular markers for comb traits can be beneficial for genetic improvement. The purpose of this study was to utilize genome-wide association studies (GWAS) to detect promising loci and candidate genes related to comb traits, namely, comb thickness (CT), comb weight (CW), comb height, comb length (CL), and comb area. Genome-wide single-nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) in 300 Nandan-Yao chickens were detected using whole-genome sequencing. In total, we identified 134 SNPs and 25 INDELs that were strongly associated with the five comb traits. A remarkable region spanning from 29.6 to 31.4 Mb on chromosome 6 was found to be significantly associated with comb traits in both SNP- and INDEL-based GWAS. In this region, two lead SNPs (6:30,354,876 for CW and CT and 6:30,264,318 for CL) and one lead INDEL (a deletion from 30,376,404 to 30,376,405 bp for CL and CT) were identified. Additionally, two genes were identified as potential candidates for comb development. The nearby gene fibroblast growth factor receptor 2 (FGFR2)—associated with epithelial cell migration and proliferation—and the gene cytochrome b5 reductase 2 (CYB5R2)—identified on chromosome 5 from INDEL-based GWAS—are significantly correlated with collagen maturation. The findings of this study could provide promising genes and biomarkers to accelerate genetic improvement of comb development based on molecular marker-assisted breeding in Nandan-Yao chickens.

Hematologically important mutations: The autosomal forms of chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. CGD patients suffer from severe, recurrent bacterial and fungal infections. The disease is caused by mutations in the genes encoding the components of the leukocyte NADPH oxidase. This enzyme produces superoxide, which is subsequently metabolized to hydrogen peroxide and other reactive oxygen species (ROS). These products are essential for intracellular killing of pathogens by phagocytic leukocytes (neutrophils, eosinophils, monocytes and macrophages). The leukocyte NADPH oxidase is composed of five subunits, four of which are encoded by autosomal genes. These are CYBA, encoding p22phox, NCF1, encoding p47phox, NCF2, encoding p67phox and NCF4, encoding p40phox. This article lists all mutations identified in these genes in CGD patients. In addition, cytochrome b558 chaperone-1 (CYBC1), recently recognized as an essential chaperone protein for the expression of the X-linked NADPH oxidase component gp91phox (also called Nox2), is encoded by the autosomal gene CYBC1. Mutations in this gene also lead to CGD. Finally, RAC2, a small GTPase of the Rho family, is needed for activation of the NADPH oxidase, and mutations in the RAC2 gene therefore also induce CGD-like symptoms. Mutations in these last two genes are also listed in this article.

Investigation of phytochemical profile and in vivo anti-proliferative effect of Laetiporus versisporus (Lloyd) Imazeki mushroom against diethylnitrosamine-induced hepatocellular carcinoma

The purpose of the study is to explore the bioactive compounds present in Laetiporus versisporus (LVEE) Lloyd Imazeki ethanolic extract and its anticancer activity against hepatocellular carcinoma (HCC) induced by DEN in rats. The bioactive compounds present in the LVEE was analyzed by GC–MS. The anticancer activity of LVEE was analyzed with the DEN induced rats were orally treated with LVEE (250 mg/kg), cyclophosphamide (50 mg/kg) to the respective treatment groups for 45 days. The levels of tissue markers, phase-I metabolizing enzymes was measured calorimetrically. ELISA was used to determine the level of AFP and CEA. The expression of apoptotic genes Bcl-2, p53, caspase 3 and caspase 9 was assessed by real time RT-PCR. Results of the phytochemical screening analysis showed that 12 bioactive molecules were existing in LVEE. Results of pharmacodynamics analysis showed that the 45 days of treatment with LVEE and cyclophosphamide (CPA) therapy significantly (p < 0.05) decreased the levels of MDA, AFP, CEA, cytochrome P450 and cytochrome b5, and increased levels of tissue markers and bilirubin in serum. The findings of RT-PCR analysis showed that the LVEE and CPA were significantly (p < 0.05) downregulated the pro-apoptotic genes Bcl-2 and upregulated the p53, caspase 3 and caspase 9 mRNA expression through mechanisms involved in promoting the cell apoptosis, and preserving the functional integrity of the liver membrane. L. versisporus (LV) can be considered as new anticancer agent that offers new opportunities for anticancer therapy.

HSCT in two brothers with CGD arising from mutations in CYBC1 corrects the defect in neutrophil function

Homozygous mutations in cytochrome b-245 chaperone 1 (CYBC1) have been recently described as causing recurrent infections and inflammatory disease in an Icelandic cohort and a patient from Saudi Arabia, by destabilising the dimerisation of gp91phox with p22phox, manifesting as phenotypic chronic granulomatous disease (CGD). Haematopoietic stem cell transplantation is the treatment of choice in CGD, though experience of transplantation in this subtype of CGD is limited to a brief description in one patient.We provide clinical and transplant data for two Icelandic brothers with CGD due to homozygous p.Tyr2Ter mutations in CYBC1, demonstrating maintained cure of the immune defect 11 years post-transplant in one brother, and death in the peri-transplant period for the other.

Optimization of Multiparameters for Increased Yields of Cytochrome B5 in Bioreactors.

The production of recombinant proteins is gaining increasing importance as the market requests high quality proteins for several applications. However, several process parameters affect both the growth of cells and product yields. This study uses high throughput systems and statistical methods to assess the influence of fermentation conditions in lab-scale bioreactors. Using this methodology, it was possible to find the best conditions to produce cytochrome b5 with recombinant cells of Escherichia coli. Using partial least squares, the height-to-diameter ratio of the bioreactor, aeration rate, and PID controller parameters were found to contribute significantly to the final biomass and cytochrome concentrations. Hence, we could use this information to fine-tune the process parameters, which increased cytochrome production and yield several-fold. Using aeration of 1 vvm, a bioreactor with a height-to-ratio of 2.4 and tuned PID parameters, a production of 72.72 mg/L of cytochrome b5 in the culture media, and a maximum of product to biomass yield of 24.97 mg/g could be achieved.

Mechanisms underlying genetic susceptibility to multisystem inflammatory syndrome in children (MIS-C)

BackgroundMultisystem inflammatory syndrome in children (MIS-C) is a pediatric complication of severe acute respiratory syndrome coronavirus 2 infection that is characterized by multiorgan inflammation and frequently by cardiovascular dysfunction. It occurs predominantly in otherwise healthy children. We previously reported haploinsufficiency of suppressor of cytokine signaling 1 (SOCS1), a negative regulator of type I and II interferons, as a genetic risk factor for MIS-C.ObjectivesWe aimed to identify additional genetic mechanisms underlying susceptibility to severe acute respiratory syndrome coronavirus 2–associated MIS-C.MethodsIn a single-center, prospective cohort study, whole exome sequencing was performed on patients with MIS-C. The impact of candidate variants was tested by using patients’ PBMCs obtained at least 7 months after recovery.ResultsWe enrolled 18 patients with MIS-C (median age = 8 years; interquartile range = 5-12.25 years), of whom 89% had no conditions other than obesity. In 2 boys with no significant infection history, we identified and validated hemizygous deleterious defects in XIAP, encoding X-linked inhibitor of apoptosis, and CYBB, encoding cytochrome b-245, beta subunit. Including the previously reported SOCS1 haploinsufficiency, a genetic diagnosis was identified in 3 of 18 patients (17%). In contrast to patients with mild COVID-19, patients with defects in SOCS1, XIAP, or CYBB exhibit an inflammatory immune cell transcriptome with enrichment of differentially expressed genes in pathways downstream of IL-18, oncostatin M, and nuclear factor κB, even after recovery.ConclusionsAlthough inflammatory disorders are rare in the general population, our cohort of patients with MIS-C was enriched for monogenic susceptibility to inflammation. Our results support the use of next-generation sequencing in previously healthy children who develop MIS-C.

Identification of stable pollen development related reference genes for accurate qRT-PCR analysis and morphological variations in autotetraploid and diploid rice.

Autotetraploid rice exhibited hybrid vigor and greater genetic variation compared to diploid rice, but low pollen fertility is a major hindrance for its utilization. Our previous analysis revealed that large number of pollen fertility genes were exhibited down-regulation in autotetraploid rice. Hence, it is of utmost importance to reveal the expression patterns of pollen fertility genes with high accuracy. To find stable reference genes for autotetraploid rice, we compared the pollen development stages between diploid and autotetraploid rice, and 14 candidate genes were selected based on transcriptome analysis to evaluate their expression levels. Autotetraploid rice (i.e. Taichung65-4x) displayed lower seed set (40.40%) and higher percentage of abnormalities during the pollen development process than its diploid counterpart. To detect the candidate reference genes for pollen development of autotetraploid and diploid rice, we used five different algorithms, including NormFinder, BestKeeper, ΔCt method, geNorm and Re-Finder to evaluate their expression patterns stability. Consequently, we identified two genes, Cytochrome b5 and CPI, as the best candidate reference genes for qRT-PCR normalization in autotetraploid and diploid rice during pre-meiosis, meiosis, single microspore and bicellular pollen development stages. However, Cytochrome b5 was found to be the most stably expressed gene during different pollen development stages in autotetraploid rice. The results of our study provide a platform for subsequent gene expression analyses in autotetraploid rice, which could also be used in other polyploid plants.

Leaf transcriptome analysis of Lancaster versus other heterotic groups’ maize inbred lines revealed different regulation of cold‐responsive genes

AbstractOne of the strategies for overcoming global climate change threatening to decrease maize yield is early sowing. To contribute to the development of cold‐tolerant hybrids this research focused on the genetic background's comparative analysis in maize inbreds with good combining ability. Leaf whole‐transcriptome sequencing of 46 maize genotypes revealed 77 differentially expressed genes (DEGs) between Lancaster and other heterotic groups (i.e. BSSS, Iowa dent, Ohio), referred to as non‐Lancaster group, under optimal growing conditions. Cold test of the subset of four Lancaster and four non‐Lancaster lines showed that the former were cold sensitive and the latter cold tolerant. Cold‐induced expression analysis of seven DEGs in eight lines revealed different expression regulation dependent on the duration of cold exposure and genetic background for six out of seven analysed genes—chloroplast ATP‐sulphurylase, photosystem II cytochrome b559 alpha subunit, CIPK serine‐threonine protein kinase 15, glutamyl‐tRNA reductase, photosystem II reaction centre protein I and Calvin cycle CP12‐chloroplastic‐like encoding genes. The results imply that differently regulated basic processes between Lancaster and non‐Lancaster maize group involve, at least, photosynthesis and sulphate assimilation, contributing to their different cold response and different adaptation to low temperatures.