Early-life exercise induces immunometabolic epigenetic modification enhancing anti-inflammatory immunity in middle-aged male mice.

Exercise is usually regarded to have short-term beneficial effects on immune health. Here we show that early-life regular exercise exerts long-term beneficial effects on inflammatory immunity. Swimming training for 3 months in male mice starting from 1-month-old curbs cytokine response and mitigates sepsis when exposed to lipopolysaccharide challenge, even after an 11-month interval of detraining. Metabolomics analysis of serum and liver identifies pipecolic acid, a non-encoded amino acid, as a pivotal metabolite responding to early-life regular exercise. Importantly, pipecolic acid reduces inflammatory cytokines in bone marrow-derived macrophages and alleviates sepsis via inhibiting mTOR complex 1 signaling. Moreover, early-life exercise increases histone 3 lysine 4 trimethylation at the promoter of Crym in the liver, an enzyme responsible for catalyzing pipecolic acid production. Liver-specific knockdown of Crym in adult mice abolishes this early exercise-induced protective effects. Our findings demonstrate that early-life regular exercise enhances anti-inflammatory immunity during middle-aged phase in male mice via epigenetic immunometabolic modulation, in which hepatic pipecolic acid production has a pivotal function.

Phenotypic Investigation and RNA-seq of KN1 Involved in Leaf Angle Formation in Maize (Zea mays L.).

Leaf angle (LA) is one of the core agronomic traits of maize, which controls maize yield by affecting planting density. Previous studies have shown that the KN1 gene is closely related to the formation of maize LA, but its specific mechanism has not been fully studied. In this study, phenotype investigation and transcriptomic sequencing were combined to explore the mechanism of LA changes in wild type maize B73 and mutant kn1 under exogenous auxin (IAA) and abscisic acid (ABA) treatment. The results showed that the effect of exogenous phytohormones had a greater impact on the LA of kn1 compared to B73. Transcriptome sequencing showed that genes involved in IAA, gibberellins (GAs) and brassinosteroids (BRs) showed different differential expression patterns in kn1 and B73. This study provides new insights into the mechanism of KN1 involved in the formation of maize LA, and provides a theoretical basis for breeding maize varieties with suitable LA.

FARS2 Deficiency Causes Cardiomyopathy by Disrupting Mitochondrial Homeostasis and the Mitochondrial Quality Control System.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a common heritable heart disease. Although HCM has been reported to be associated with many variants of genes involved in sarcomeric protein biomechanics, pathogenic genes have not been identified in patients with partial HCM. FARS2 (the mitochondrial phenylalanyl-tRNA synthetase), a type of mitochondrial aminoacyl-tRNA synthetase, plays a role in the mitochondrial translation machinery. Several variants of FARS2 have been suggested to cause neurological disorders; however, FARS2-associated diseases involving other organs have not been reported. We identified FARS2 as a potential novel pathogenic gene in cardiomyopathy and investigated its effects on mitochondrial homeostasis and the cardiomyopathy phenotype. METHODS: FARS2 variants in patients with HCM were identified using whole-exome sequencing, Sanger sequencing, molecular docking analyses, and cell model investigation. Fars2 conditional mutant (p.R415L) or knockout mice, fars2-knockdown zebrafish, and Fars2-knockdown neonatal rat ventricular myocytes were engineered to construct FARS2 deficiency models both in vivo and in vitro. The effects of FARS2 and its role in mitochondrial homeostasis were subsequently evaluated using RNA sequencing and mitochondrial functional analyses. Myocardial tissues from patients were used for further verification. RESULTS: We identified 7 unreported FARS2 variants in patients with HCM. Heart-specific Fars2-deficient mice presented cardiac hypertrophy, left ventricular dilation, progressive heart failure accompanied by myocardial and mitochondrial dysfunction, and a short life span. Heterozygous cardiac-specific Fars2R415L mice displayed a tendency to cardiac hypertrophy at age 4 weeks, accompanied by myocardial dysfunction. In addition, fars2-knockdown zebrafish presented pericardial edema and heart failure. FARS2 deficiency impaired mitochondrial homeostasis by directly blocking the aminoacylation of mt-tRNAPhe and inhibiting the synthesis of mitochondrial proteins, ultimately contributing to an imbalanced mitochondrial quality control system by accelerating mitochondrial hyperfragmentation and disrupting mitochondrion-related autophagy. Interfering with the mitochondrial quality control system using adeno-associated virus 9 or specific inhibitors mitigated the cardiac and mitochondrial dysfunction triggered by FARS2 deficiency by restoring mitochondrial homeostasis. CONCLUSIONS: Our findings unveil the previously unrecognized role of FARS2 in heart and mitochondrial homeostasis. This study may provide new insights into the molecular diagnosis and prevention of heritable cardiomyopathy as well as therapeutic options for FARS2-associated cardiomyopathy.

Identifying hypothermia death in a mouse model by ATR-FTIR.

The identification of hypothermia death (HD) is difficult for cadavers, especially the distinction from death due to alternative causes. A large number of studies have shown that brown adipose tissue (BAT) plays critical roles in thermoregulation of mammals. In this study, BAT of mice was used for the discrimination of HD using attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). A modified mouse HD model conducted by Feeney DM was used in this study to obtain infrared spectra of BAT. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to establish discrimination models. The PLS-DA and OPLS-DA models exhibit prominent discriminative efficiency, and the accuracy of HD identification using fingerprint regions and ratios of absorption intensity is near 100% in both the calibration and validation sets. Our preliminary study suggests that BAT may be an extremely effective target tissue for identification of cadavers of HD, and ATR-FTIR spectra combined with chemometrics have also shown potential for cadaver identification in forensic practice in a fast and accurate manner.

Serine protease mediates Ovomermis sinensis-inhibited host immune responses by inducing apoptosis: implications for successful parasitism and host mortality.

BACKGROUND: Mermithid nematodes are entomopathogens that parasitize and kill insect hosts and are used for biological control. It is widely believed that mermithid nematodes kill their host upon nematode emergence, unlike other parasites that depend on virulence factors. In this study, we disproved this theory by demonstrating that the mermithid nematode Ovomermis sinensis mediates host mortality by serine protease-induced apoptosis. RESULTS: Successful parasitism of O. sinensis increased with the infection rate, and the inhibition of host immunity by O. sinensis increased with the parasitic load. A serine protease was identified from the host hemolymph. This protease belongs to the trypsin-like serine protease family, which is an apoptosis-inducing serine protease. Specifically, Os-sp was highly expressed only during the parasitic stage and could be induced by host hemocytes and the fat body. Importantly, host immune effectors (melanization, phenoloxidase activity, and encapsulation) were suppressed by the recombinant protein rOs-sp that induced apoptosis of hemocytes and fat body in a dose-dependent manner, which contributes to host death. CONCLUSION: Serine protease mediates O. sinensis-inhibited host immune responses by inducing apoptosis that is lethal to the insect host. Our findings have broader implications for understanding the mechanism of successful parasitism and killing of host by nematodes. © 2023 Society of Chemical Industry.

Cholesterol homeostasis confers glioma malignancy triggered by hnRNPA2B1-dependent regulation of SREBP2 and LDLR.

BACKGROUND: Dysregulation of cholesterol metabolism is a significant characteristic of glioma, yet the underlying mechanisms are largely unknown. N6-methyladenosine (m6A) modification has been implicated in promoting tumor development and progression. The aim of this study was to determine the key m6A regulatory proteins involved in the progression of glioma, which is potentially associated with the reprogramming of cholesterol homeostasis. METHODS: Bioinformatics analysis was performed to determine the association of m6A modification with glioma malignancy from TCGA and GTEx datasets. GSC self-renewal was determined by tumor sphere formation and bioluminescence image assay. RNA sequencing and lipidomic analysis were performed for cholesterol homeostasis analysis. RNA immunoprecipitation and luciferase reporter assay were performed to determine hnRNPA2B1-dependent regulation of SREBP2 and LDLR mRNA. The methylation status of hnRNPA2B1 promoter was determined by bioinformatic analysis and methylation-specific PCR assay. RESULTS: Among the m6A-regulatory proteins, hnRNPA2B1 was demonstrated the most important independent prognostic risk factor for glioma. hnRNPA2B1 ablation exhibited a significant tumor-suppressive effect on glioma cell proliferation, GSC self-renewal and tumorigenesis. hnRNPA2B1 triggers de novo cholesterol synthesis by inducing HMGCR through the stabilization of SREBP2 mRNA. m6A modification of SREBP2 or LDLR mRNA is required for hnRNPA2B1-mediated mRNA stability. The hypomethylation of cg21815882 site on hnRNPA2B1 promoter confers elevated expression of hnRNPA2B1 in glioma tissues. The combination of targeting hnRNPA2B1 and cholesterol metabolism exhibited remarkable anti-tumor effects, suggesting valuable clinical implications for glioma treatment. CONCLUSIONS: hnRNPA2B1 facilitates cholesterol uptake and de novo synthesis, thereby contributing to glioma stemness and malignancy.

Frequency and polymorphism of acetylcholinesterase gene involved in the organophosphate resistance of Musca domestica in Guizhou Province, China.

Organophosphate (OP) resistance has been prevalent in Musca domestica populations worldwide since 1960s. Previous studies have demonstrated that point mutations of the acetylcholinesterase gene (Ace) are one of the important molecular mechanisms underlying OP resistance. However, few studies have investigated the molecular mechanisms of OP resistance in the past 10 years in China. In this study, we investigated the status of OP resistance and genetic diversity of Ace in the field populations of houseflies in Guizhou Province of China. The bioassays showed that the houseflies had 142-304-fold resistance to dichlorvos (DDVP) and 122-364-fold resistance to temephos, compared to the susceptible houseflies. Five nonsynonymous mutations (Y226F, V260L, G342A/V, F407Y) in Ace were detected among the 7 field populations, with an average frequency of 5.4%, 55%, 68%, 32%, and 94%, respectively, of which the Y226F mutation had not been reported previously. Eleven combinations of triple mutations (at positions 260, 342, and 407) were observed, of which the combination 260L/V+342A/V+407Y was predominant. The ZY and AS populations showed greatest diversity of allelic combination and the other five populations showed different distributions among different regions. These results indicate that the resistance to OPs is prevalent among the housefly populations and target-site insensitivity is the main cause of resistance in Guizhou Province. The difference in distribution and the allelic diversity of Ace in field populations may be due to the complexity and variability of insecticide application. It is necessary to monitor resistance to insecticides and conduct management of houseflies in Guizhou Province.

Non-invasive prenatal testing for fetal Ss, Kidd, and CTL2 blood group prediction by multiplex digital droplet PCR.

Background: Some blood groups, such as S and s blood groups in the MNS blood group system, and Kidd and CTL2 blood group systems, can cause severe fetal and newborn alloimmune disorders. Non-invasive prenatal testing (NIPT) to predict fetal blood groups and knowledge of local blood group gene frequency are both important for pregnancy management decisions. Droplet digital PCR (ddPCR) has high specificity and sensitivity in detecting fetal single nucleotide variation. Objectives: The objective is to predict fetal Ss, Kidd, and CTL2 blood groups using multiplex ddPCR. The gene frequencies of three blood groups were detected by ddPCR in northwest China. Design: This is a prospective study. Methods: Cell-free fetal DNA isolated from 26 healthy single pregnant women at different gestational stages was tested with QX200 Droplet Digital PCR. Results were compared with fetal genotypes. DNA samples purified from 20 blood pools containing a total of 1000 donors in northwest China were subjected to ddPCR to detect the gene frequency of three blood groups. Results: Ss, Kidd, and CTL2 blood groups of 26 pregnant fetuses were accurately detected by multiplex ddPCR. The multiplex ddPCR results were consistent with the Sanger sequencing results of 26 fetal blood samples after birth. The gene frequencies of the three blood groups detected by ddPCR were 9.30% for S, 90.70% for s, 48.43% for Jka, 51.57% for Jkb, 66.57% for HNA-3A, and 33.43% for HNA-3B. Conclusions: It is reliable to predict fetal Ss, Kidd, and CTL2 blood groups by multiplex ddPCR. Meanwhile, we designed a simple and efficient method for inferring the gene frequency of three blood groups based on ddPCR.

Investigation of sex expression profiles and the cantharidin biosynthesis genes in two blister beetles.

Cantharidin (CTD) is a well-established defensive toxin synthesized by blister beetles, displaying both therapeutic potential and toxicity. Among these beetles, Hycleus cichorii and Hycleus phaleratus are the two most commercially significant species due to their capacity to produce CTD in males. In this investigation, we conducted a gene expression profiling analysis of male and female individuals of these two species, utilizing the Illumina Hiseq4000 platform. We identified 7,983 expressed genes, including 2,823 differentially expressed genes (DEGs) shared by both male and female blister beetles. Nineteen genes related to CTD biosynthesis in the terpenoid backbone biosynthesis pathway were identified, including hydroxymethylglutaryl-CoA reductase (HMGR; EC:1.1.1.34), which demonstrated a significant correlation with CTD content. Furthermore, hydroxymethylglutaryl-CoA synthase (HMGS; EC:2.3.3.10) and isopentenyl-diphosphate Delta-isomerase (IDI; EC:5.3.3.2) were also found to be significantly up-regulated in males. Comparative analysis revealed that NADP+-dependent farnesol dehydrogenase (FOHSDR; EC:1.1.1.216) and farnesyl diphosphate synthase (FDPS; EC:2.5.1.1) had the highest copy number in these beetles, significantly higher than the copy number of the other four non-Meloidae insects. The analysis of the protein-protein interaction network of genes related to CTD biosynthesis revealed that the acetyl-CoA C-acetyltransferase (ACAT; EC:2.3.1.9) gene was the central gene, exhibiting greater expression in male blister beetles than in females. This study offers novel insights into the mechanisms of CTD biosynthesis in blister beetles and enhances our comprehensions of the association between particular genes and CTD content.

Regulating Cardiolipin Biosynthesis for Efficient Production of Colanic Acid in Escherichia coli.

Colanic acid has broad application prospects in the food and healthcare market due to its excellent physical properties and biological activities. In this study, we discovered that colonic acid production in Escherichia coli could be enhanced by regulating cardiolipin biosynthesis. Single deletion of clsA, clsB, or clsC related to cardiolipin biosynthesis in E. coli MG1655 only slightly increased colonic acid production, but double or triple deletion of these three genes in E. coli MG1655 increased colonic acid production up to 2.48-fold. Previously, we have discovered that truncating lipopolysaccharide by deletion of the waaLUZYROBSPGQ gene cluster and enhancing RcsA by deletion of genes lon and hns can increase colonic acid production in E. coli. Therefore, these genes together with clsA, clsB, or/and clsC were deleted in E. coli, and all the resulting mutants showed increased colonic acid production. The best colonic acid production was observed in the mutant WWM16, which is 126-fold higher than in the control MG1655. To further improve colonic acid production, the genes rcsA and rcsD1-466 were overexpressed in WWM16, and the resulting recombinant E. coli WWM16/pWADT could produce 44.9 g/L colonic acid, which is the highest titer reported to date.

Application of droplet digital PCR combined with TaqMan real-time PCR in Dombrock blood group genotyping in Northwest China.

BACKGROUND AND OBJECTIVES: The Dombrock blood group system is based on the DO gene. DO*A and DO*B antigens are the result of a single-nucleotide polymorphism (SNP) on this gene. The introduction of Do antigens through blood transfusion or other invasive factors like infection may result in the production of Do antibodies, which may cause serious haemolytic transfusion reactions. In this study, TaqMan real-time PCR and droplet digital PCR were used to detect rare DO*A allele, guide the search for rare DO*A allele donors, and calculate DO alleles frequencies in mixed populations in Northwest China. MATERIALS AND METHODS: In this study, the highly sensitive and accurate TaqMan real-time polymerase chain reaction (PCR) method was used to detect and screen DO genotype SNPs in combination with droplet digital PCR. We also searched for rare DO*A allele donors and calculated the frequencies of DO alleles in mixed populations. RESULTS: A total of 1202 donor DNA samples were collected from Northwest China, of which 202 were used to detect DO allele SNPs using TaqMan real-time PCR. The rare DO*A allele was detected in the other 1000 blood donors by droplet digital PCR, and gene frequencies were inferred from dual channel droplet digital PCR data. Among 1202 donors from Northwest China, the allele frequencies of DO*A and DO*B were 0.1128 and 0.8872, respectively. CONCLUSION: The sequencing results confirmed that this new way of detecting DO alleles by droplet digital PCR with specific probes can detect rare DO*A allele to predict the presence of the rare antigen Doa and infer DO allele frequencies. This method is highly sensitive and specific.

Metabolic engineering of Escherichia coli to efficiently produce monophosphoryl lipid A.

Monophosphoryl lipid A (MPL), mainly isolated from Salmonella minnesota R595, has been used as adjuvant in several vaccines. In this study, an Escherichia coli strain that can efficiently produce the MPL has been constructed. The gene clusters related to the biosynthesis of O-antigen, core oligosaccharide, enterobacterial common antigen, and colanic acid were sequentially removed to save the carbon source and to increase the activity of PagP in E. coli MG1655. Then, the genes pldA, mlaA, and mlaC related to the phospholipid transport system were further deleted, resulting in the strain MW012. Finally, the genes lpxE from Francisella novicida and pagP and pagL from Salmonella were overexpressed in MW012 to modify the structure of lipid A, resulting in the strain MW012/pWEPL. Lipid A species were isolated from MW012/pWEPL and analyzed by thin-layer chromatography and liquid chromatography-mass spectrometry. The results showed that mainly two MPL species were produced in E. coli MW012/pWEPL, one is hexa-acylated, and the other is penta-acylated. More importantly, the proportion of the hexa-acylated MPL, which is the most effective component of lipid A vaccine adjuvant, reached 75%. E. coli MW012/pWEPL constructed in this study provided a good alternative for the production of lipid A vaccine adjuvant MPL.

Overexpression of the key genes in the biosynthetic pathways of lipid A and peptidoglycan in Escherichia coli.

Gram-negative bacterium Escherichia coli has a tripartite cell envelope with a cytoplasmic membrane, a peptidoglycan layer, and an asymmetric outer membrane containing lipopolysaccharide in its outer leaflet. The biogenesis of peptidoglycan and lipopolysaccharide shares the same substrate UDP-GlcNAc. From UDP-GlcNAc, MurA catalyzes the first reaction for peptidoglycan biosynthesis, while LpxA catalyzes the first reaction for lipopolysaccharide biosynthesis. This study demonstrates that murA overexpression in E. coli MG1655 inhibited the cell growth and increased the cell length, whereas lpxA overexpression in MG1655 neither inhibited the cell growth nor increased the cell length. Further study showed that individual overexpression of the other eight genes encoding the enzymes to catalyze the initial reactions in the biosynthetic pathway of lipopolysaccharide did not inhibit the cell growth. When MG1655/pBad-lpxA, MG1655/pBad-lpxD, and MG1655/pBad-lpxH were transformed with pFW01-thrA*BC-rhtC that contains the key genes for L-threonine biosynthesis and transport, the L-threonine production was increased. The L-threonine production in MG1655/pFW01-thrA*BC-rhtC/pBad-lpxH increased 46.1% as compared to the control MG1655/pFW01-thrA*BC-rhtC/pBad.

Hedgehog pathway is negatively regulated during the development of Drosophila melanogaster PheRS-m (Drosophila homologs gene of human FARS2) mutants.

Hereditary spastic paraplegia (HSP) is a neurodegeneration disease, one of the reasons is caused by autosomal recessive missense mutation of the karyogene that encodes phenylalanyl-tRNA synthetase 2, mitochondrial (FARS2). However, the molecular mechanism underlying FARS2-mediated HSP progression is unknown. Mitochondrial phenylalanyl-tRNA synthetase gene (PheRS-m) is the Drosophila melanogaster homolog gene of human FARS2. This study constructed a Drosophila HSP missense mutation model and a PheRS-m knockout model. Some of the mutant fly phenotypes included developmental delay, shortened lifespan, wing-structure abnormalities and decreased mobility. RNA-sequencing results revealed a relationship between abnormal phenotypes and the hedgehog (Hh) pathway. A qRT-PCR assay was used to determine the key genes (ptc, hib, and slmb) of the Hh pathway that exhibited increased expression during different developmental stages. We demonstrated that Hh signaling transduction is negatively regulated during the developmental stages of PheRS-m mutants but positively regulated during adulthood. By inducing the agonist and inhibitor of Hh pathway in PheRS-m larvae, the developmental delay in mutants can be partly salvaged or postponed. Collectively, our findings indicate that Hh signaling negatively regulates the development of PheRS-m mutants, subsequently leading to developmental delay.

Aberrant activation of TGF-ß1 induces high bone turnover via Rho GTPases-mediated cytoskeletal remodeling in Camurati-Engelmann disease.

In the adult skeleton, the bone remodeling process involves a dynamic coordination between osteoblasts and osteoclasts, which is disrupted in diseases with high bone turnover rates and dysregulated transforming growth factor beta 1 (TGF-ß1). However, little is known about how TGF-ß1 signaling mediates bone resorption. Here, we described a pedigree with a heterozygous variant in TGF-ß1 (R218C) that resulted in aberrant activation of TGF-ß1 through an activating mechanism that caused Camurati-Engelmann disease (CED). We showed that CED patients have high levels of active Rho GTPases and the migration-related proteins Integrin ß1 and Integrin ß3 in their peripheral blood. HEK293T cells transfected with a plasmid encoding this mutant expressed high levels of TGF-ß1 and active Rho GTPases. Furthermore, activation of Rho by TGF-ß1 increased osteoclast formation and bone resorption, with increased migration of pre-osteoclasts, as well as cytoskeletal remodeling of pre-osteoclasts and mature osteoclasts. Importantly, pharmacological inhibition of Rho GTPases effectively rescued hyperactive TGF-ß1-induced osteoclastogenesis in vitro. Overall, we propose that Rho GTPases mediate TGF-ß1-induced osteoclastogenesis and suggest that Rho-TGF-ß1 crosstalk is associated with high bone turnover in CED.

TFG mutation induces haploinsufficiency and drives axonal Charcot-Marie-Tooth disease by causing neurite degeneration.

AIMS: TFG-related axonal Charcot-Marie-Tooth (CMT) disease is a late-onset, autosomal dominant, hereditary motor, and sensory neuropathy characterized by slowly progressive weakness and atrophy of the distal muscles. The objective of this study was to determine the common pathogenic mechanism of TFG-related CMT type 2 (CMT2) caused by different mutations and establish a direct association between TFG haploinsufficiency and neurodegeneration. METHODS: Three individuals carrying the TFG p.G269V mutation but with varying disease durations were studied. The effect of the p.G269V mutation was confirmed by analyzing protein samples extracted from the blood of two individuals. The functional consequences of both CMT2 mutant gene products were evaluated in vitro. The effect of TFG deficiency in the nervous system was examined using zebrafish models and cultured mouse neurons. RESULTS: Overexpression of p.G269V TFG failed to enhance soluble TFG levels by generating insoluble TFG aggregates. TFG deficiency disrupted neurite outgrowth and induced neuronal apoptosis both in vivo and in vitro and further impaired locomotor capacity in zebrafish, which was consistent with the phenotype in patients. Wnt signaling was activated as a protective factor in response to TFG deficiency. CONCLUSION: CMT2-related TFG mutation induces TFG haploinsufficiency within cells and drives disease by causing progressive neurite degeneration.

Neuropathy-associated Fars2 deficiency affects neuronal development and potentiates neuronal apoptosis by impairing mitochondrial function.

BACKGROUND: Neurodegenerative diseases encompass an extensive and heterogeneous group of nervous system disorders which are characterized by progressive degeneration and death of neurons. Many lines of evidence suggest the participation of mitochondria dysfunction in these diseases. Mitochondrial phenylalanyl-tRNA synthetase, encoded by FARS2, catalyzes the transfer of phenylalanine to its cognate tRNA for protein synthesis. As a member of mt-aaRSs genes, FARS2 missense homozygous mutation c.424G > T (p.D142Y) found in a Chinese consanguineous family first built the relationship between pure hereditary spastic paraplegia (HSP) and FARS2 gene. More FARS2 variations were subsequently found to cause heterogeneous group of neurologic disorders presenting three main phenotypic manifestations: infantile-onset epileptic mitochondrial encephalopathy, later-onset spastic paraplegia and juvenile onset refractory epilepsy. Studies showed that aminoacylation activity is frequently disrupt in cases with FARS2 mutations, indicating a loss-of-function mechanism. However, the underlying pathogenesis of neuropathy-associated Fars2 deficiency is still largely unknown. RESULTS: Early gestation lethality of global Fars2 knockout mice was observed prior to neurogenesis. The conditional Fars2 knockout-mouse model delayed lethality to late-gestation, resulting in a thinner cortex and an enlarged ventricle which is consist with the MRI results revealing cortical atrophy and reduced cerebral white matter volume in FARS2-deficient patients. Delayed development of neurite outgrowth followed by neuronal apoptosis was confirmed in Fars2-knockdown mouse primary cultured neurons. Zebrafish, in which fars2 was knocked down, exhibited aberrant motor neuron function including reduced locomotor capacity which well restored the spastic paraplegia phenotype of FARS2-deficient patients. Altered mitochondrial protein synthesis and reduced levels of oxidative phosphorylation complexes were detected in Fars2-deficient samples. And thus, reduced ATP, total NAD levels and mitochondrial membrane potential, together with increased ROS production, revealed mitochondrial dysfunction both in vitro and in vivo. Dctn3 is a potential downstream molecule in responds to Fars2 deficient in neurons, which may provide some evidence for the development of pathogenesis study and therapeutic schedule. CONCLUSIONS: The Fars2 deficiency genetic models developed in this study cover the typical clinical manifestations in FARS2 patients, and help clarify how neuropathy-associated Fars2 deficiency, by damaging the mitochondrial respiratory chain and impairing mitochondrial function, affects neuronal development and potentiates neuronal cell apoptosis.

Metabolic Engineering for Overproduction of Colanic Acid in Escherichia coli Mutant with Short Lipopolysaccharide.

Colanic acid is a major exopolysaccharide existing in most Enterobacteriaceae when exposed to an extreme environment. Colanic acid possesses excellent physical properties and biological activities, which makes it a candidate in the food and healthcare market. Previous strategies for colanic acid overproduction in E. coli mainly focus on removing the negative regulator on colanic acid biosynthesis or overexpressing the rcsA gene to up-regulate the cps operon. In this study, modifications in metabolic pathways were implemented in E. coli mutant strains with shortened lipopolysaccharides to improve colanic acid production. First, ackA was deleted to remove the byproduct acetate and the effect of accumulated acetyl-phosphate on colanic acid production was investigated. Second, 11 genes responsible for O-antigen synthesis were deleted to reduce its competition for glucose-1-phosphate and UDP-galactose with colanic acid production. Third, uppS was overexpressed to supply lipid carriers for synthesizing a colanic acid repeat unit. Colanic acid production in the final engineered strain WZM008/pTrcS reached 11.68 g/L in a 2.0 L bioreactor, 3.54 times the colanic acid production by the WQM001 strain. The results provide insights for further engineering E. coli to maximize CA production.

SLC44A2 Frequency, a New TaqMan Real-Time Polymerase Chain Reaction Method for HNA-3A/3B Genotyping, and a New Application of Droplet Digital PCR.

Background: Human neutrophil antigen-3A (HNA-3A) and human neutrophil antigen-3B (HNA-3B) are generated by a single-nucleotide polymorphism (rs2288904, c.461G > A) in exon 7 of the choline transporter-like protein-2 gene (CTL2, also known as SLC44A2). Antibodies to HNA-3 can be generated following blood transfusion or other factors resulting in exposure to HNA-3 antigens. These antibodies can cause transfusion-related acute lung injury (TRALI) or neonatal alloimmune neutropenia (NAIN). This study describes a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) method to screen for the HNA-3 genotype using specific primers and probes designed to detect allelic polymorphisms. Considering the high sensitivity and accuracy of droplet digital PCR (ddPCR) in the identification of the rare SLC44A2*2 allele, we used this technique to identify blood donors with the rare HNA-3B antigen and calculate the allele frequency of SLC44A2 in mixed populations with different proportions. Methods: DNA samples purified from 208 donors in northwest China were subjected to TaqMan real-time PCR to detect allelic polymorphisms in SLC44A2. The results were confirmed by Sanger sequencing. The rare HNA-3B antigen was detected by ddPCR. SLC44A2 frequency was determined by two-channel ddPCR. Results: The genotypes of all DNA samples were detected by the TaqMan real-time PCR using specific probes for HNA-3, and the results were consistent with the Sanger sequencing results in respect to the HNA-3A and HNA-3B polymorphisms. The allele frequencies of SLC44A2*1 and SLC44A2*2 in the 208 donors in northwest China were 64.9% (95% confidence interval [CI], 59%-70.8%) and 35.1% (95% CI, 29.2%-41%), respectively. The ratio of SLC44A2*2 alleles was accurately detected in all blood pools by ddPCR but not by TaqMan real-time PCR. This allowed for the SLC44A2 frequency in the population to be accurately inferred. Conclusion: This new method of detecting SLC44A2 alleles was highly sensitive and specific, as confirmed by Sanger sequencing. ddPCR using the designed probes resulted in successful detection of the rare HNA-3B antigen. Furthermore, we successfully detected the rare HNA-3B antigen and inferred the SLC44A2 frequency by ddPCR using the probes that we designed.

A Novel Pseudogene Methylation Signature to Predict Temozolomide Outcome in Non-G-CIMP Glioblastomas.

Objective: Alterations in the methylation state of pseudogenes may serve as clinically useful biomarkers of glioblastomas (GBMs) that do not have glioma-CpG island methylator phenotype (G-CIMP). Methods: Non-G-CIMP GBM datasets were included for evaluation, and a RISK-score signature was determined from the methylation state of pseudogene loci. Both bioinformatic and experimental analyses were performed for biological validation. Results: By integrating clinical information with DNA methylation microarray data, we screened a panel of eight CpGs from discovery cohorts of non-G-CIMP GBMs. Each CpG could accurately and independently predict the prognosis of patients under a treatment regime that combined radiotherapy (RT) and temozolomide (TMZ). The 8-CpG signature appeared to show opposite prognostic correlations between patients treated with RT/TMZ and those treated with RT monotherapy. The analyses further indicated that this signature had predictive value for TMZ efficacy because different survival benefits between RT/TMZ and RT therapies were observed in each risk subgroup. The incorporation of other risk factors, such as age and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, with our pseudogene methylation signature could provide precise risk classification. In vitro experimental data revealed that two locus-specific pseudogenes (ZNF767P and CLEC4GP1) may modulate TMZ resistance via distinct mechanisms in GBM cells. Conclusion: The biologically and clinically relevant RISK-score signature, based on pseudogene methylation loci, may offer information for predicting TMZ responses of non-G-CIMP GBMs, that is independent from, but complementary to, MGMT-based approaches.