Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid.

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that eventually develops into cirrhosis and even liver cancer. In recent years, the incidence rate has been increasing, and the early diagnosis and treatment of PBC are crucial. In the early diagnosis method of PBC, anti-mitochondrial antibodies (AMAs) are an important diagnostic basis, especially the M2 subtype (AMA-M2) with almost 100% specificity. We selected the BCOADC-E2 protein, a mitochondrial autoantigen that reacts specifically with AMA-M2 antibodies, and carried out DNA recombination and protein mutation experiments by cloning in vitro the homologous target gene sequence BCKD that expresses the antigenic epitope of BCOADC-E2 protein, to provide experience for later exploring the effect of mutations of amino acids around the lysine in the active center of BCOADC-E2 protein on its specific binding to AMA-M2, and to lay the foundation for determining the key amino acids of BCOADC-E2 for the diagnosis and treatment of PBC. In addition, we apply this scientific research content to graduate course teaching. Experimental technology of microbial molecular ecology is a course with the cross-integration of multidisciplinary knowledge and experimental skills offered at our college since 2018. This article derives from the part of this course on the construction of recombinant plasmids. The students first constructed the recombinant plasmid pGEX-BCKD using the vector plasmid pGEX-4T1 and the target gene fragment BCKD provided by the laboratory and used this as a template to construct the pGEX-BCKD-E4A point mutation plasmid by the overlap extension PCR (SOE PCR) technique to achieve the effect of mutating the fifth amino acid glutamate in front of lysine, the active centre of the BCOADC-E2 lipid acyl binding domain, to alanine for subsequent studies. Through the research experiment, combining theoretical knowledge and experimental operation, we aim to deepen the student's understanding of DNA recombination technology, let them feel the practical application prospect of experimental technology, stimulate students' interest in professional knowledge learning, and cultivate students' scientific thinking and innovation consciousness. We examined the quality of the teaching through the process and summative evaluation of the students. In this study, the students successfully completed the construction of pGEX-BCKD-E4A point mutant plasmid, and the average test score increased from 40.4% before teaching to 91.1%. The teaching effect was remarkable. This kind of research experimental teaching mode has good application prospects, and other education and teachers can refer to and reference it.

FUCA2 and TSTA3 expression in gastric cancer: candidate biomarkers of malignant transformation.

INTRODUCTION: Aberrant fucosylation is closely related to malignant transformation, cancer detection, and evaluation of treatment efficacy. The fucosylation process requires GDP-L-fucose, fucosyltransferases, and fucosidases. In gastric cancer (GC), fucosylation alterations were associated with tumor formation, metastasis inhibition, and multi-drug resistance. It is not clear whether tissue-specific transplantation antigen P35B (TSTA3) and alpha-L-fucosidase 2 (FUCA2) have any effect on the development of GC. MATERIALS AND METHODS: We used immunohistochemistry to assess the expression of TSTA3 and FUCA2 in 71 gastric adenocarcinoma samples and their relationship with clinicopathological parameters. RESULTS: TSTA3 expression was associated with lower histological grade I and II (P = 0.0120) and intestinal type Lauren classification (P = 0.0120). TSTA3 immunopositivity could predict Lauren's classification. Analysis of mRNA expression in GC validation cohorts corroborates the significant TSTA3 association with histological grade observed in our study. However, no associations were found between TSTA3 staining and overall survival. FUCA2 expression was markedly increased in GC tissues compared with non-tumoral tissues (P < 0.0001) and was associated with surgical staging III and IV (P = 0.0417) and advanced histological grade tumor states (P = 0.0125). CONCLUSIONS: Alterations of FUCA2 and TSAT3 immunoexpression could lay the basis for future studies using cell glycosylation as a biomarker for the planning of therapeutic strategy in primary gastric cancer.

A novel PDK1 inhibitor, JX06, inhibits glycolysis and induces apoptosis in multiple myeloma cells.

Pyruvate dehydrogenase kinase 1 (PDK1) is a Ser/Thr kinase that inactivates mitochondrial pyruvate dehydrogenase (PDH), leading to switch of glucose metabolism from mitochondrial oxidation to aerobic glycolysis. We previously reported that PDK1 inhibition is a potent therapeutic strategy in multiple myeloma (MM). However, availability of PDK1 inhibitors, which are effective at low concentrations, are limited at present, making PDK1 inhibition difficult to apply in the clinic. In the present study, we examined the efficacy and mechanism of action of JX06, a novel PDK1 inhibitor, against MM cells. We confirmed that PDK1 is highly expressed in normal plasma cells and MM cells using publicly available gene expression datasets. JX06 suppressed cell growth and induced apoptosis against MM cells from approximately 0.5 µM JX06 treatment reduced PDH phosphorylation, suggesting that JX06 is indeed inhibiting PDK1. Intracellular metabolite analysis revealed that JX06 treatment reduced metabolites associated with glucose metabolism of MM cells. Additionally, JX06 in combination with a well-known proteasome inhibitor, bortezomib, significantly increased MM cell death, which raises the possibility of combination use of JX06 with proteasome inhibitors in the clinic. These findings demonstrate that PDK1 can be potentially targeted by JX06 in MM through glycolysis inhibition, leading to a novel therapeutic strategy in MM.

Atypical presentation of Charcot-Marie-Tooth disease type 2Q by mutations on DHTKD1 and NTRK2 genes.

Background: Charcot-Marie-Tooth disease type 2Q (CMT2Q) is a rare disorder (< 1/1,000,000 individuals worldwide) linked to chromosome 10p14 in the DHTKD1 gene. This phenotype is characterized by an adolescent or adulthood-onset, slowly progressive distal muscle weakness and symmetrical atrophy associated with reduced or absent deep tendon reflexes. Currently, only two familiar cases from China have been reported: one familiar case of eight individuals affected by isolated DHTKD1 gene mutation and one familiar case of two individuals affected by DHTKD1 gene mutation and GJB1 gene mutation. Case report: We present the case of a 10-year-old male patient with obesity, frequent falls, swollen legs and thighs, and pain in the lower and upper limbs. We performed the clinical evaluation and a clinical targeted exome test, which reported mutations on DHTKD1 y NTRK2 genes. Conclusions: Due to scientific and technological advances, genetic dysfunctions that can cause different diseases have been identified with greater sensitivity. Globally, this is the eleventh case reported of DHTKD1 gene mutation linked to CMT2Q. Moreover, this is the first case related to NTRK2 gene mutation (linked to obesity, hyperphagia, and delayed development). The patient showed an atypical CMT2Q phenotype additional to obesity. Therefore, we propose to study metabolic disorders linked to hereditary peripheral neuropathies.

Introducción: La enfermedad de Charcot-Marie-Tooth tipo 2Q (CMT2Q) es una alteración poco frecuente (< 1/1,000,000 habitantes en todo el mundo) condicionada por mutaciones en el gen DHTKD1, localizado en el cromosoma 10p14. El padecimiento inicia en la adolescencia o la edad adulta de manera lenta y progresiva, con debilidad muscular y atrofia distal simétrica, y afecta predominantemente las extremidades inferiores y los reflejos tendinosos profundos, que se encuentran reducidos o ausentes. Solo se ha reportado un caso familiar de ocho personas afectadas con la mutación aislada en el gen DHTKD1 y un caso familiar de dos personas afectadas con mutaciones en los genes DHTKD1 y GJB1, ambas familias de China. Caso clínico: Se presenta el caso de un paciente de sexo masculino de 10 años y 11 meses de edad con obesidad, caídas frecuentes, edema de miembros pélvicos y dolor en las extremidades inferiores y superiores. Se realizaron valoración clínica y estudio genético molecular de exoma dirigido, el cual reportó mutaciones en los genes DHTKD1 y NTRK2. Conclusiones: Gracias al avance científico y tecnológico se han podido identificar con mayor precisión las alteraciones genéticas causantes de diferentes enfermedades. Este es el undécimo caso reportado en el mundo de una mutación en el gen DHTKD1 asociada con la enfermedad de CMT2Q. También es el primer caso relacionado con una mutación del gen NTRK2 (asociada con obesidad, hiperfagia y retraso en el desarrollo). El paciente presentó un cuadro clínico atípico de enfermedad de CMT2Q agregado a obesidad. Por ello, se sugiere estudiar a fondo la conexión entre trastornos metabólicos y neuropatías periféricas hereditarias.

Identification of hub genes with prognostic values in multiple myeloma by bioinformatics analysis.

PURPOSE: Multiple myeloma (MM) is a malignant disease with abnormal proliferation of clonal plasma cells. Hypoxia is an important factor in the pathogenesis and development of MM. However, the underlying mechanisms are not fully understood. MATERIAL & METHODS: To determine hub genes related to hypoxia in MM, this study took integrated bioinformatics analysis with two expression datasets (GSE80140 and GSE80545) downloaded from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were filtrated under the condition of both p-value < 0.05 and [log2FoldChange (log2FC)] > 1. Then, gene ontology (GO) and Kyoto encyclopedia of genes and genomes enrichment (KEGG) analysis, and protein-protein interaction (PPI) network construction were utilized to further explore these DEGs. PrognoScan evaluated all the candidate hub genes for survival analysis. RESULTS: In total, three hub genes, including FH, TSTA3, and POLR3G, were screened out to be related to hypoxia in MM. Patients with the lower expression level of FH, TSTA3, and POLR3G have statistically significantly longer disease- specific survival (Cox p < 0.05). CONCLUSION: We identified FH, TSTA3, and POLR3G as hub genes which can affect MM patients'outcome and new biomarkers for diagnosis and prognosis of MM. Further functional and mechanistic studies are need to develop in order to make them as potential target for clinical treatment.

Tissue-specific transplantation antigen P35B functions as an oncogene and is regulated by microRNA-125a-5p in lung cancer.

Tissue­specific transplantation antigen P35B (TSTA3) expression is upregulated in esophageal squamous cell carcinoma and breast cancer, and functions as an oncogene in breast cancer. However, the roles and underlying mechanisms of TSTA3 in lung cancer have not been fully elucidated. The current study aimed to reveal the role of TSTA3 in lung cancer and explore whether TSTA3 may be modulated by microRNA (miR)­125a­5p to activate ß­catenin signaling. Immunohistochemical staining and western blotting were used to analyze TSTA3 expression in lung cancer tissues and cells. Cell functions were assessed via Cell Counting Kit­8, flow cytometry, wound­healing, Transwell and in vivo tumor formation assays. The effect of TSTA3 on the activation of ß­catenin signaling was determined using western blot and immunofluorescence analyses. The association between miR­125a­5p and TSTA3 was determined by western blotting and luciferase gene reporter assay. The present study revealed that, compared with normal tissues and cells, TSTA3 expression was significantly increased in lung cancer tissues and cell lines, and high TSTA3 expression predicted a poor prognosis and more malignant clinical features in patients with lung cancer. TSTA3 upregulation significantly enhanced ß­catenin expression and promoted its nuclear accumulation. In addition, TSTA3 expression was negatively regulated by miR­125a­5p, which was downregulated in lung cancer. Furthermore, TSTA3 overexpression markedly promoted cell proliferation, migration, invasion and tumorigenesis, and suppressed cell apoptosis. TSTA3 downregulation abolished the effects of miR­125a­5p downregulation on promoting lung cancer cell malignant transformation. Overall, the current study demonstrates that TSTA3 is regulated by miR­125a­5p and functions as an oncogene in lung cancer via promoting the activation of ß­catenin signaling.

Notch-Regulated Dendritic Cells Restrain Inflammation-Associated Colorectal Carcinogenesis.

Conventional dendritic cells (cDC) play a central role in T-cell antitumor responses. We studied the significance of Notch-regulated DC immune responses in a mouse model of colitis-associated colorectal cancer in which there is epithelial downregulation of Notch/Hes1 signaling. This defect phenocopies that caused by GMDS (GDP-mannose 4,6-dehydratase) mutation in human colorectal cancers. We found that, although wild-type immune cells restrained dysplasia progression and decreased the incidence of adenocarcinoma in chimeric mice, the immune system with Notch2 deleted in all blood lineages or in only DCs promoted inflammation-associated transformation. Notch2 signaling deficiency not only impaired cDC terminal differentiation, but also downregulated CCR7 expression, reduced DC migration, and suppressed antigen cross-presentation to CD8+ T cells. Transfer of Notch-primed DCs restrained inflammation-associated dysplasia progression. Consistent with the mouse data, we observed a correlation between infiltrating cDC1 and Notch2 signaling in human colorectal cancers and found that GMDS-mutant colorectal cancers showed decreased CCR7 expression and suppressed cDC1 signature gene expression. Suppressed cDC1 gene signature expression in human colorectal cancer was associated with a poor prognosis. In summary, our study supports an important role for Notch2 signaling in cDC1-mediated antitumor immunity and indicates that Notch2-controlled DCs restrain inflammation-associated colon cancer development in mice.

Generation of FX-/- and Gmds-/- CHOZN host cell lines for the production of afucosylated therapeutic antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8-/- Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)-fucose synthesis, GDP mannose-4,6-dehydratase (Gmds) and GDP-L-fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX-/- and Gmds-/-, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of two proprietary IgG1 mAbs in the engineered host cells, and found that the titers were comparable to CHOZN® cells. The mAbs generated from either KO cell line exhibited loss of fucose modification, leading to significantly boosted FcγRIIIa binding and ADCC effects. Our data demonstrated that both FX-/- and Gmds-/- host cells could replace Fut8-/- CHO cells for clinical manufacturing of antibody therapeutics.

TSTA3 facilitates esophageal squamous cell carcinoma progression through regulating fucosylation of LAMP2 and ERBB2.

Background: TSTA3 gene encodes an enzyme responsible for synthesis of GDP-L-fucose as the only donor in fucosylation. This study was designed to explore clinical value, function and underlying mechanism of TSTA3 in the development of esophageal squamous cell carcinoma (ESCC). Methods: Whole genomic sequencing data from 663 ESCC patients and RNA sequencing data from 155 ESCC patients were used to analyze the copy number variation and mRNA expression of TSTA3 respectively. Immunohistochemistry based or not based on the tissue microarrays was used to detect its protein expression. Transwell assay and in vivo metastasis assay were used to study the effect of TSTA3 on invasion and metastasis of ESCC. Immunofluorescence was used to analyze fucosylation level. N-glycoproteomics and proteomics analysis, Lens Culinaris Agglutinin (LCA) and Ulex Europaeus Agglutinin I (UEA-I) affinity chromatography, immunoprecipitation, glycosyltransferase activity kit and rescue assay were used to explore the mechanism of TSTA3. Results: TSTA3 was frequently amplified and overexpressed in ESCC. TSTA3 amplification and protein overexpression were significantly associated with malignant progression and poor prognosis of ESCC patients. TSTA3 knockdown significantly suppressed ESCC cells invasion and tumor dissemination by decreasing fucosylation level. Conversely, exogenous overexpression of TSTA3 led to increased invasion and tumor metastasis in vitro and in vivo by increasing fucosylation level. Moreover, core fucosylated LAMP2 and terminal fucosylated ERBB2 might be mediators of TSTA3-induced pro-invasion in ESCC and had a synergistic effect on the process. Peracetylated 2-F-Fuc, a fucosyltransferase activity inhibitor, reduced TSTA3 expression and fucosylation modification of LAMP2 and ERBB2, thereby inhibiting ESCC cell invasion. Conclusion: Our results indicate that TSTA3 may be a driver of ESCC metastasis through regulating fucosylation of LAMP2 and ERBB2. Fucosylation inhibitor may have prospect to suppress ESCC metastasis by blocking aberrant fucosylation.

Impacts of Endocrine Disruptor di-n-Butyl Phthalate Ester on Microalga Chlorella vulgaris Verified by Approaches of Proteomics and Gene Ontology.

Di-n-butyl phthalate (DBP) is an extensively used plasticizer. Most investigations on DBP have been concentrated on its environmental distribution and toxicity to humans. However, information on the effects of plasticizers on algal species is scarce. This study verified the impacts of endocrine disruptor di-n-butyl phthalate ester on microalga Chlorella vulgaris by approaches of proteomics and gene ontology. The algal acute biotoxicity results showed that the 24h-EC50 of DBP for C. vulgaris was 4.95 mg L-1, which caused a decrease in the chlorophyll a content and an increase in the DBP concentration of C. vulgaris. Proteomic analysis led to the identification of 1257 C. vulgaris proteins. Sixty-one more proteins showed increased expression, compared to proteins with decreased expression. This result illustrates that exposure to DBP generally enhances protein expression in C. vulgaris. GO annotation showed that both acetolactate synthase (ALS) and GDP-L-fucose synthase 2 (GER2) decreased more than 1.5-fold after exposure to DBP. These effects could inhibit both the valine biosynthetic process and the nucleotide-sugar metabolic process in C. vulgaris. The results of this study demonstrate that DBP could inhibit growth and cause significant changes to the biosynthesis-relevant proteins in C. vulgaris.

Structure-function analyses of the G729R 2-oxoadipate dehydrogenase genetic variant associated with a disorder of l-lysine metabolism.

2-Oxoadipate dehydrogenase (E1a, also known as DHTKD1, dehydrogenase E1, and transketolase domain-containing protein 1) is a thiamin diphosphate-dependent enzyme and part of the 2-oxoadipate dehydrogenase complex (OADHc) in l-lysine catabolism. Genetic findings have linked mutations in the DHTKD1 gene to several metabolic disorders. These include α-aminoadipic and α-ketoadipic aciduria (AMOXAD), a rare disorder of l-lysine, l-hydroxylysine, and l-tryptophan catabolism, associated with clinical presentations such as developmental delay, mild-to-severe intellectual disability, ataxia, epilepsy, and behavioral disorders that cannot currently be managed by available treatments. A heterozygous missense mutation, c.2185G→A (p.G729R), in DHTKD1 has been identified in most AMOXAD cases. Here, we report that the G729R E1a variant when assembled into OADHc in vitro displays a 50-fold decrease in catalytic efficiency for NADH production and a significantly reduced rate of glutaryl-CoA production by dihydrolipoamide succinyl-transferase (E2o). However, the G729R E1a substitution did not affect any of the three side-reactions associated solely with G729R E1a, prompting us to determine the structure-function effects of this mutation. A multipronged systematic analysis of the reaction rates in the OADHc pathway, supplemented with results from chemical cross-linking and hydrogen-deuterium exchange MS, revealed that the c.2185G→A DHTKD1 mutation affects E1a-E2o assembly, leading to impaired channeling of OADHc intermediates. Cross-linking between the C-terminal region of both E1a and G729R E1a with the E2o lipoyl and core domains suggested that correct positioning of the C-terminal E1a region is essential for the intermediate channeling. These findings may inform the development of interventions to counter the effects of pathogenic DHTKD1 mutations.

DHTKD1 and OGDH display substrate overlap in cultured cells and form a hybrid 2-oxo acid dehydrogenase complex in vivo.

Glutaric aciduria type 1 (GA1) is an inborn error of lysine degradation characterized by a specific encephalopathy that is caused by toxic accumulation of lysine degradation intermediates. Substrate reduction through inhibition of DHTKD1, an enzyme upstream of the defective glutaryl-CoA dehydrogenase, has been investigated as a potential therapy, but revealed the existence of an alternative enzymatic source of glutaryl-CoA. Here, we show that loss of DHTKD1 in glutaryl-CoA dehydrogenase-deficient HEK-293 cells leads to a 2-fold decrease in the established GA1 clinical biomarker glutarylcarnitine and demonstrate that oxoglutarate dehydrogenase (OGDH) is responsible for this remaining glutarylcarnitine production. We furthermore show that DHTKD1 interacts with OGDH, dihydrolipoyl succinyltransferase and dihydrolipoamide dehydrogenase to form a hybrid 2-oxoglutaric and 2-oxoadipic acid dehydrogenase complex. In summary, 2-oxoadipic acid is a substrate for DHTKD1, but also for OGDH in a cell model system. The classical 2-oxoglutaric dehydrogenase complex can exist as a previously undiscovered hybrid containing DHTKD1 displaying improved kinetics towards 2-oxoadipic acid.

2-Aminoadipic acid protects against obesity and diabetes.

Obesity and type 2 diabetes (T2D) are both complicated endocrine disorders resulting from an interaction between multiple predisposing genes and environmental triggers, while diet and exercise have key influence on metabolic disorders. Previous reports demonstrated that 2-aminoadipic acid (2-AAA), an intermediate metabolite of lysine metabolism, could modulate insulin secretion and predict T2D, suggesting the role of 2-AAA in glycolipid metabolism. Here, we showed that treatment of diet-induced obesity (DIO) mice with 2-AAA significantly reduced body weight, decreased fat accumulation and lowered fasting glucose. Furthermore, Dhtkd1-/- mice, in which the substrate of DHTKD1 2-AAA increased to a significant high level, were resistant to DIO and obesity-related insulin resistance. Further study showed that 2-AAA induced higher energy expenditure due to increased adipocyte thermogenesis via upregulating PGC1α and UCP1 mediated by ß3AR activation, and stimulated lipolysis depending on enhanced expression of hormone-sensitive lipase (HSL) through activating ß3AR signaling. Moreover, 2-AAA could alleviate the diabetic symptoms of db/db mice. Our data showed that 2-AAA played an important role in regulating glycolipid metabolism independent of diet and exercise, implying that improving the level of 2-AAA in vivo could be developed as a strategy in the treatment of obesity or diabetes.

Obesity-associated, but not obesity-independent, tumors respond to insulin by increasing mitochondrial glucose oxidation.

Obesity is associated with increased incidence and worse prognosis of more than one dozen tumor types; however, the molecular mechanisms for this association remain under debate. We hypothesized that insulin, which is elevated in obesity-driven insulin resistance, would increase tumor glucose oxidation in obesity-associated tumors. To test this hypothesis, we applied and validated a stable isotope method to measure the ratio of pyruvate dehydrogenase flux to citrate synthase flux (VPDH/VCS, i.e. the percent of total mitochondrial oxidation fueled by glucose) in tumor cells. Using this method, we found that three tumor cell lines associated with obesity (colon cancer [MC38], breast cancer [4T1], and prostate cancer [TRAMP-C3] cells) increase VPDH/VCS in response to physiologic concentrations of insulin. In contrast, three tumor cell lines that are not associated with obesity (melanoma [YUMM1.7], B cell lymphoma [BCL1 clone 5B1b], and small cell lung cancer [NCI-H69] cells) exhibited no oxidative response to insulin. The observed increase in glucose oxidation in response to insulin correlated with a dose-dependent increase in cell division in obesity-associated tumor cell lines when grown in insulin, whereas no alteration in cell division was seen in tumor types not associated with obesity. These data reveal that a shift in substrate preference in the setting of physiologic insulin may comprise a metabolic signature of obesity-associated tumors that differs from that of those not associated with obesity.

PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice.

Skeletal muscle satellite cells (SMSCs), the major stem cells responsible for the regeneration of skeletal muscle, are normally cell cycle arrested but differentiate to generate myocytes upon muscle damage, forming new myofibers along with self-renewing stem cells in preparation for subsequent injury. In this study, we investigated which factors stimulate the proliferation and differentiation of SMSCs and found that pyruvate, the end product of glycolysis, stimulates their differentiation. Pyruvate antagonizes the effects of hypoxia on preferential self-renewal of SMSCs through dephosphorylation or activation of pyruvate dehydrogenase (PDH), which mediates opening of the gateway from glycolysis to the tricarboxylic acid (TCA) cycle by producing acetyl coenzyme A from pyruvate. PDH kinase 1, highly expressed under hypoxia, is down-regulated under normoxic conditions, leading to an increase in dephosphorylated PDH. Conditional deletion of PDH in SMSCs affects cell divisions generating myocytes and subsequent myotube formation, inefficient skeletal muscle regeneration upon injury, and aggravated pathogenesis of a dystrophin-deficient mouse model of Duchenne muscular dystrophy. Thus, the flow from glycolysis to the TCA cycle mediated by PDH plays a pivotal role in the differentiation of SMSCs, which is critical for the progression of skeletal muscle regeneration.-Hori, S., Hiramuki, Y., Nishimura, D., Sato, F., Sehara-Fujisawa, A. PDH-mediated metabolic flow is critical for skeletal muscle stem cell differentiation and myotube formation during regeneration in mice.

Combining Protein and Metabolic Engineering Strategies for High-Level Production of O-Acetylhomoserine in Escherichia coli.

O-acetylhomoserine (OAH) is a promising platform chemical for the production of l-methionine and other valuable compounds. However, the relative low titer and yield of OAH greatly limit its industrial production and cost-effective application. In this study, we successfully constructed an efficient OAH-producing strain with high titer and yield by combining protein and metabolic engineering strategies in E. coli. Initially, an OAH-producing strain was created by reconstruction of biosynthetic pathway and deletion of degradation and competitive pathways, which accumulated 1.68 g/L of OAH. Subsequently, several metabolic engineering strategies were implemented to improve the production of OAH. The pathway flux of OAH was enhanced by eliminating byproduct accumulation, increasing oxaloacetate supply and promoting the biosynthesis of precursor homoserine, resulting in a 1.79-fold increase in OAH production. Moreover, protein engineering was applied to improve the properties of the rate-limiting enzyme homoserine acetyltransferase (MetXlm) based on evolutionary conservation analysis and structure-guided engineering. The resulting triple F147L-M182I-M240A mutant of MetXlm exhibited a 12.15-fold increase in specific activity, and the optimized expression of the MetXlm mutant led to a 57.14% improvement in OAH production. Furthermore, the precursor acetyl-CoA supply and NADPH generation were also enhanced to facilitate the biosynthesis of OAH by promoting CoA biosynthesis, overexpressing heterogeneous acetyl-CoA synthetase (ACS), and introducing NADP-dependent pyruvate dehydrogenase (PDH). Finally, the engineered strain OAH-7 produced 62.7 g/L of OAH with yield and productivity values of 0.45 g/g glucose and 1.08 g/L/h, respectively, in a 7.5 L fed-batch fermenter, which was the highest OAH production ever reported.

A Chinese pedigree with a novel mutation in GJB1 gene and a rare variation in DHTKD1 gene for diverse Charcot­Marie­Tooth diseases.

Charcot­Marie­Tooth (CMT) disease is a group of motor and sensory neuropathies with a high degree of pathological and genetic heterogenicity. The present study described 2 patients with CMT in a Chinese Han pedigree. The proband exhibited the classic manifestation of CMT with slowly progressing muscular atrophy and weakness. Electrophysiological examination highlighted axonal and demyelinating features. His mother did not have any symptoms, but did exhibit abnormal electrophysiological results. Next­generation sequencing technology was employed to screen mutations in the genes associated with inherited motor never diseases. A novel mutation, c.528_530delAGT, in the gap junction protein beta 1 (GJB1) gene for CMTX, and a rare variation, c.2369C>T, in the dehydrogenase E1 and transketolase domain containing 1 (DHTKD1) gene for CMT disease type 2Q (CMT2Q), were identified in the proband and his mother. The results were verified by Sanger sequencing. Although the in silico analysis predicted no change in the 3­dimensional structure, the clinical and electrophysiological presentation in the pedigree and the high evolutionary conservation of the affected amino acid supported the hypothesis that the c.528_530delAGT mutation in the GJB1 gene may be pathogenic in this pedigree. In silico analysis and high evolutionary conservation suggested the pathogenicity of the c.2369C>T mutation in the DHTKD1 gene; however, the clinical and electrophysiological performances of the proband and his mother did not conform to those of CMT2Q caused by the DHTKD1 gene. The present study provided additional information concerning the range of mutations of the GJB1 gene, which facilitated the understanding of the genotype­phenotype association of CMT.

Mild inborn errors of metabolism in commonly used inbred mouse strains.

Inbred mouse strains are a cornerstone of translational research but paradoxically many strains carry mild inborn errors of metabolism. For example, α-aminoadipic acidemia and branched-chain ketoacid dehydrogenase deficiency are known in C57BL/6J mice. Using RNA sequencing, we now reveal the causal variants in Dhtkd1 and Bckdhb, and the molecular mechanism underlying these metabolic defects. C57BL/6J mice have decreased Dhtkd1 mRNA expression due to a solitary long terminal repeat (LTR) in intron 4 of Dhtkd1. This LTR harbors an alternate splice donor site leading to a partial splicing defect and as a consequence decreased total and functional Dhtkd1 mRNA, decreased DHTKD1 protein and α-aminoadipic acidemia. Similarly, C57BL/6J mice have decreased Bckdhb mRNA expression due to an LTR retrotransposon in intron 1 of Bckdhb. This transposable element encodes an alternative exon 1 causing aberrant splicing, decreased total and functional Bckdhb mRNA and decreased BCKDHB protein. Using a targeted metabolomics screen, we also reveal elevated plasma C5-carnitine in 129 substrains. This biochemical phenotype resembles isovaleric acidemia and is caused by an exonic splice mutation in Ivd leading to partial skipping of exon 10 and IVD protein deficiency. In summary, this study identifies three causal variants underlying mild inborn errors of metabolism in commonly used inbred mouse strains.

Engineering Corynebacterium glutamicum Mutants for 3-Methyl-1-butanol Production.

3-Methyl-1-butanol (3MB) is a promising biofuel that can be produced from 2-ketoisocaproate via the common L-leucine biosynthesis pathway. Corynebacterium glutamicum was chosen as a host bacterium because of its strong resistance to isobutanol. In the current study, several strategies were designed to overproduce 3MB in C. glutamicum through a non-fermentation pathway. The engineered C. glutamicum mutant was obtained by silencing the pyruvate dehydrogenase gene complex (aceE) and deleting the lactic dehydrogenase gene (ldh), followed by mutagenesis with diethyl sulfate (DES) and selection with Fmoc-3-4-thiazolyl-L-alanine (FTA). The mutant could produce 659 mg/L of 3MB after 12 h of incubation. To facilitate carbon flux to 3MB biosynthesis, the engineered recombinant was also constructed without branched-chain acid aminotransferase (ilvE) activity by deleting the ilvE gene. This recombinant could produce 697 mg/L of 3MB after 12 h of incubation.