Novel NARS2 variants in a patient with early-onset status epilepticus: case study and literature review.

BACKGROUND: NARS2 as a member of aminoacyl-tRNA synthetases was necessary to covalently join a specific tRNA to its cognate amino acid. Biallelic variants in NARS2 were reported with disorders such as Leigh syndrome, deafness, epilepsy, and severe myopathy. CASE PRESENTATION: Detailed clinical phenotypes were collected and the NARS2 variants were discovered by whole exome sequencing and verified by Sanger sequencing. Additionally, 3D protein structure visualization was performed by UCSF Chimera. The proband in our study had early-onset status epilepticus with abnormal EEG and MRI results. She also performed global developmental delay (GDD) and myocardial dysfunction. Next-generation sequencing (NGS) and Sanger sequencing revealed compound heterozygous missense variants [NM_024678.6:exon14: c.1352G > A(p.Arg451His); c.707T > C(p.Phe236Ser)] of the NARS2 gene. The proband develops refractory epilepsy with GDD and hyperlactatemia. Unfortunately, she finally died for status seizures two months later. CONCLUSION: We discovered two novel missense variants of NARS2 in a patient with early-onset status epilepticus and myocardial dysfunction. The NGS enables the patient to be clearly diagnosed as combined oxidative phosphorylation deficiency 24 (COXPD24, OMIM:616,239), and our findings expands the spectrum of gene variants in COXPD24.

Engineering Escherichia coli BL21 (DE3) for high-yield production of germacrene A, a precursor of ß-elemene via combinatorial metabolic engineering strategies.

ß-elemene is one of the most commonly used antineoplastic drugs in cancer treatment. As a plant-derived natural chemical, biologically engineering microorganisms to produce germacrene A to be converted to ß-elemene harbors great expectations since chemical synthesis and plant isolation methods come with their production deficiencies. In this study, we report the design of an Escherichia coli cell factory for the de novo production of germacrene A to be converted to ß-elemene from a simple carbon source. A series of systematic approaches of engineering the isoprenoid and central carbon pathways, translational and protein engineering of the sesquiterpene synthase, and exporter engineering yielded high-efficient ß-elemene production. Specifically, deleting competing pathways in the central carbon pathway ensured the availability of acetyl-coA, pyruvate, and glyceraldehyde-3-phosphate for the isoprenoid pathways. Adopting lycopene color as a high throughput screening method, an optimized NSY305N was obtained via error-prone polymerase chain reaction mutagenesis. Further overexpression of key pathway enzymes, exporter genes, and translational engineering produced 1161.09 mg/L of ß-elemene in a shake flask. Finally, we detected the highest reported titer of 3.52 g/L of ß-elemene and 2.13 g/L germacrene A produced by an E. coli cell factory in a 4-L fed-batch fermentation. The systematic engineering reported here generally applies to microbial production of a broader range of chemicals. This illustrates that rewiring E. coli central metabolism is viable for producing acetyl-coA-derived and pyruvate-derived molecules cost-effectively.

Application of two glycosylated Lactobacillus surface layer proteins in coating cationic liposomes.

The ability of isolated surface layer proteins (SLPs) to reassemble on suitable surfaces enables the application of SLPs in various fields of nanotechnology. In this work, SLPs from Lactobacillus buchneri BNCC 187,964 and L. kefir BNCC 190,565 were extracted and verified as glycosylated proteins. They were applied to coat on the surface of cationic liposomes. The absorption of the two SLPs on liposomes induced the zeta potential reduction and particle size increase. The two kinds of SLP-coated liposomes demonstrated better thermal, light and pH stability than the control liposomes. And the L. kefir SLP showed better protective effects than the L. buchneri SLP. Moreover, both of the SLPs could endow liposomes with the function of binding ferritin as observed by transmission electron microscope. Fourier transform infrared spectroscopy illustrated that the interaction between the two SLPs and liposomes was similar. The recrystallization of the two SLPs on the liposomes might drive the lipid into a higher order state and hydrogen bonds were formed between the two SLPs and the liposomes. All the findings demonstrated that L. kefir SLP and L. buchneri SLP had great potential to be explored as effective coating agents to improve the stability and function of cationic liposomes.Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.Yes, all have been checked.

Protective function of surface layer protein from Lactobacillus casei fb05 against intestinal pathogens in vitro.

Escherichia coli and Salmonella are common pathogenic bacteria in human intestine, which can infect epithelial cells and cause diseases. Adhesion to intestinal tissue is the first step of pathogen infection. This work was to investigate the protective function of surface layer protein (SLP) from Lactobacillus casei fb05 against the harmful effects of E. coli and Salmonella on intestinal tissue (collagen and HT-29 cells). The SLP of L. casei fb05 was identified by transmission electron microscopy and SDS-PAGE. The purified SLP could reduce the adhesion of E. coli and Salmonella to collagen and HT-29 cells as observed by light microscope. The flow cytometry results showed that the L. casei fb05 SLP decreased the two pathogens-induced apoptosis of HT-29 cells by about 45%-49%. In addition, the activation of caspase-9 and caspase-3 caused by the two pathogens was significantly declined by the interference of the L. casei fb05 SLP. All the findings demonstrated that the L. casei fb05 SLP could decrease the deleterious effects of E. coli and Salmonella on intestinal tract in two ways: reducing pathogen adhesion and inhibiting pathogen-induced apoptosis. The potential of L. casei fb05 SLP in the treatment of intestinal diseases might be explored in this work.

Human Papillomavirus E6/E7 and Long Noncoding RNA TMPOP2 Mutually Upregulated Gene Expression in Cervical Cancer Cells.

TMPOP2 was previously suggested to be an oncogenic long noncoding RNA which is excessively expressed in cervical cancer cells and inhibits E-cadherin gene expression by recruiting transcription repressor EZH2 to the gene promoter. So far, the function and regulation of TMPOP2 in cervical cancer remain largely unknown. Herein, we found that TMPOP2 expression was correlated with human papillomavirus 16/18 (HPV16/18) E6 and E7 in cervical cancer cell lines CaSki and HeLa. Tumor suppressor p53, which is targeted for degradation by HPV16/18, was demonstrated to associate with two p53 response elements in the TMPOP2 promoter to repress the transcription of the TMPOP2 gene. Reciprocally, ectopic expression of TMPOP2 was demonstrated to sequester tumor repressor microRNAs (miRNAs) miR-375 and miR-139 which target HPV16/18 E6/E7 mRNA and resulted in an upregulation of HPV16/18 E6/E7 genes. Thereby, HPV16/18 E6/E7 and the long noncoding RNA (lncRNA) TMPOP2 form a positive feedback loop to mutually derepress gene expression in cervical cancer cells. Moreover, results of RNA sequencing and cell cycle analysis showed that knockdown of TMPOP2 impaired the expression of cell cycle genes, induced cell cycle arrest, and inhibited HeLa cell proliferation. Together, our results indicate that TMPOP2 and HPV16/18 E6/E7 mutually strengthen their expression in cervical cancer cells to enhance tumorigenic activities.IMPORTANCE Human papillomaviruses 16 and 18 (HPV16/18) are the main causative agents of cervical cancer. Viral proteins HPV16/18 E6 and E7 are constitutively expressed in cancer cells to maintain oncogenic phenotypes. Accumulating evidences suggest that HPVs are correlated with the deregulation of long noncoding RNAs (lncRNAs) in cervical cancer, although the mechanism was unexplored in most cases. TMPOP2 is a newly identified lncRNA excessively expressed in cervical cancer. However, the mechanism for the upregulation of TMPOP2 in cervical cancer cells remains largely unknown and its relationship with HPVs is still elusive. The significance of our research is in revealing the mutual upregulation of HPV16/18 E6/E7 and TMPOP2 with the molecular mechanisms explored. This study will expand our understandings of the oncogenic activities of human papillomaviruses and lncRNAs.

Synergistic effects of Rho kinase inhibitor Y-27632 and Noggin overexpression on the proliferation and neuron-like cell differentiation of stem cells derived from human exfoliated deciduous teeth.

Stem cells from human exfoliated deciduous teeth (SHEDs) are highly proliferative, clonogenic, and multipotent stem cells with a neural crest cell origin. This property could be a desirable option for potential therapeutic applications. In this study, we focus on the effects of Rho kinase inhibitors Y-27632 and Noggin on the proliferation of SHEDs and their differentiation into neuron-like cells. SHEDs were extracted from 10 samples of deciduous teeth obtained from healthy children aged from 5 to 10. The passaged SHEDs were transfected with Noggin, Y-27632, or their combination. By means of MTT and colony formation assays, the effects of Y-27632 and Noggin on cell viability and colony formation were detected. Cellular morphology and neurosphere formation were observed under a microscope. Y-27632 transfection in SHEDs showed enhanced cell viability, colony formation, and neurosphere formation indicating that Y-27632 could promote cell proliferation of SHEDs. Furthermore, we observed that the SHEDs treated with Noggin in combination with Y-27632 displayed typical neuron-like cell morphology and reticular processes. Noggin or Y-27632 alone or in combination induced obviously increased NSE, Nestin, and GFAP levels, which were highest in SHEDs treated with the combination of Noggin and Y-27632. These findings suggest that Y-27632 promotes the proliferation of SHEDs, and Y-27632 and Noggin in combination have a synergistic effect on promoting differentiation of SHEDs into neuron-like cells.

Clinical characteristics, treatment, and long-term outcomes in children suffering from benign convulsions with mild gastroenteritis: a retrospective study.

BACKGROUND: Benign convulsions with mild gastroenteritis (BCWG) is a common condition in children in Asia and is generally not associated with pH or electrolyte imbalances. When BCWG is diagnosed, a lumbar puncture is usually recommended to rule out potential intracranial infections. This study examined the clinical characteristics of BCWG and evaluated the necessity of lumbar puncture. METHODS: Medical records of children admitted to the First Hospital of Jilin University with BCWG between January 2018 and May 2019 were reviewed and analyzed. Children were stratified by rotavirus positivity or lumbar puncture status. Clinical characteristics and long-term outcomes were compared between groups. RESULTS: A total of 51 children were included in the analyses (55.1% rotavirus [HRV] positive). The average age of convulsion onset was 21.12 ± 7.44 months, the male-to-female ratio was 1.8:1, and convulsions occurred primarily between October 2018 and April 2019. The main clinical presentations of BCWG were convulsions, vomiting, diarrhea, and fever. Convulsions occurred predominantly two days after diagnosis of gastroenteritis, were mainly generalized tonic-clonic with 88.2% of children having ≤ 3 convulsions per episode, and had a mean duration of 2.0 minutes (interquartile range [IQR]: 1.0, 3.0). Children with BCWG had mild metabolic acidosis (HCO3- 17.82 ± 3.63 mmol/L) with an elevated anion gap (AG; 20.98 ± 3.00 mmol/L), mild hyponatremia (134.56 ± 2.85 mmol/L), and slightly increased levels of creatine kinase myocardial band (CKMB). HRV + children had more severe acidosis and higher CKMB levels. Cerebrospinal fluid (CSF) samples collected via lumbar puncture were normal. No developmental abnormalities were noted as assessed by the Social Life Ability Scale. CONCLUSIONS: BCWG is a situation-related seizure, with clinical presentations of tonic-clonic or focal convulsions and mild gastroenteritis (vomiting, diarrhea). Mild metabolic acidosis and hyponatremia may exist. The prognosis of the disease is favorable; lumbar puncture and long-term antiepileptics are unnecessary and should not be recommended.

Randomized, double-blind, placebo-controlled trial of aripiprazole oral solution in children and adolescents with Tourette's disorder.

BACKGROUND: Aripiprazole is the most frequently recommended antipsychotic for the treatment of tics in children and adolescents with Tourette's disorder (TD). However, to date, a randomized controlled trial for aripiprazole oral solution has not been conducted despite being widely preferred by children. Therefore, we examined whether aripiprazole oral solution is effective for treating tics. METHODS: All patients received a flexible dose of aripiprazole oral solution (1 mg/mL, range: 2-20 mg) with a starting dose of 2 mg. The target dose for patients weighing < 50 kg was 2, 5, and 10 mg/day, and that for patients weighing ≥ 50 kg was 5, 10, 15, and 20 mg/day. The primary efficacy endpoint was the mean change in the Yale Global Tic Severity Scale-total tic score (YGTSS-TTS) from baseline to week 8. RESULTS: Of the 121 patients enrolled, 59 patients (96.7%) in the aripiprazole group and 53 patients (88.3%) in the placebo group completed the study. The aripiprazole group showed significantly greater improvement in the YGTSS-TTS from baseline to week 8 than the placebo group (least squares mean difference [95% confidence interval (CI)] -5.5 [95% CI - 8.4 to - 2.6]). At week 8, the response rate (i.e., percentage of patients with a Tourette's Syndrome Clinical Global Impression-Improvement score of 1 or 2) of the aripiprazole group (86.4%) was significantly higher than that of the placebo group (56.6%; odds ratio: 3.6, p < 0.001). The incidence of treatment-emergent adverse events (TEAEs) reported in at least one patient was 86.9% in the aripiprazole group and 65.5% in the placebo group. All TEAEs were mild or moderate in severity. No serious adverse events or deaths occurred during the study. CONCLUSIONS: Our findings suggest that aripiprazole oral solution is an effective, well-tolerated, and safe treatment for children and adolescents with TD. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03487783. Registered 4 April 2018.

Correlation analysis between gene expression profile of high-fat emulsion-induced non-alcoholic fatty liver and liver regeneration in rat.

To explore the relevance of non-alcoholic fatty liver disease (NAFLD) to liver regeneration (LR), rat models of non-alcoholic steatohepatitis (NASH) and LR were established, respectively, then Rat Genome 230 2.0 Array was used to detect the gene expression abundance of them, and the reliabilities of the array data were confirmed by real-time RT-PCR. As a result, the expression of 93 genes was significantly changed during NAFLD occurrence and 948 genes in LR. Hierarchical clustering indicated that the expression profiles of the above two events were quite different. K-means cluster classified their expression patterns into four clusters, and gene expression trends of clusters 1, 2 were similar in NAFLD and LR, while clusters 3, 4 were contrary with the gene expression changes of LR more abundant. DAVID classifications and functional enrichment analysis found that lipid metabolism and carbohydrate metabolism were stronger in NAFLD than in LR, but some other physiological activities including inflammation/immune response, cell adhesion, and migration, cell proliferation and differentiation in NAFLD were weaker than in LR. IPA further indicated that lipid metabolism, inflammation response, and cellular development were highly associated with NAFLD, and thus identified some potential biomarkers for NAFLD.

Design, synthesis and cytotoxicity of novel 3'-N-alkoxycarbonyl docetaxel analogs.

By-product 9a exhibited potent cytotoxicity against both SK-OV-3 and A549 cell lines. The structure of 9a was characterized using 1D and 2D NMR experiments and confirmed by synthesis to afford a diastereomeric mixture (16a) that was identical to 9a, as well as a pair of diastereomers (R)-16b and (S)-16c. The preliminary SAR study demonstrated that analogs with an (R)-configuration were slightly more potent than analogs with an (S)-configuration. In addition, α,α-gem-dimethyl analogs 16 g-i were the most potent analogs in this series, exhibiting similar potency to docetaxel and greater potency than Taxol against the SK-OV-3 cell line. For the A549 cell line, analogs 16 g-i were more potent (>65-fold) than both docetaxel and Taxol.

Synthesis and anti-HBV activity of novel 3'-N-phenylsulfonyl docetaxel analogs.

Nine new 3'-N-phenylsulfonyl docetaxel analogs were synthesized in good yields from the key intermediate N-phenylsulfonyl oxazolidine via a six-step route. These analogs were tested for anti-hepatitis B virus (HBV) activity in vitro. Compounds 3e, 3g and 3j showed more potent inhibitory activity against HBeAg secretion than the positive control lamivudine. Further extensive SAR and mechanistic studies will be reported in due course.

(3R,4S)-1-(4-Meth-oxy-phen-yl)-2-oxo-4-(3-vinyl-phen-yl)azetidin-3-yl acetate.

In the title compound, C20H19NO4, the absolute configuration (3R,4S) for the two chiral centres of the mol-ecule has been determined.

[Clinical efficacy of oxcarbazepine suspension in children with focal epilepsy].

OBJECTIVE: To investigate the clinical efficacy and safety of oxcarbazepine (OXC) suspension in children with focal epilepsy. METHODS: A total of 118 children aged 2-14 years, who were newly diagnosed with focal epilepsy between October 2009 and December 2011, were randomly divided into experimental group (n=60) and control group (n=58). The experimental group was treated with an orally suspension of OXC and the control group was orally administered with carbamazepine (CBZ) tablets. The two treatment regimens were compared in terms of clinical efficacy and safety. RESULTS: After 13 and 26 weeks of treatment, the experimental group had response rates of 75% and 72% respectively and seizure-free rates of 53% and 50%, and the control group had response rates of 71% and 66% and seizure-free rates of 50% and 43% respectively. There were no significant differences in the clinical efficacy between the two groups (P>0.05). After 26 weeks of treatment, the adverse event rates of the experimental and control groups were 18% and 40% respectively, with a significant difference between the two groups (P<0.05). CONCLUSIONS: OXC suspension has a comparable clinical efficacy to that of CBZ tablets in children aged 2-14 years who are newly diagnosed with focal epilepsy, but OXC suspension causes fewer adverse events and has higher safety.

Corrigendum: Effectiveness of mycophenolate mofetil in the treatment of pediatric anti-NMDAR encephalitis: A retrospective analysis of 6 cases.

[This corrects the article DOI: 10.3389/fneur.2020.584446.].

Lacticaseibacillus casei LH23 Suppressed HPV Gene Expression and Inhibited Cervical Cancer Cells.

Microbiota of lower female reproductive tract is special in its microorganism composition with Lactobacillus as the predominant bacteria. A few of Lactobacillus species have been identified to benefit the inhibition of inflammatory and malignant diseases. Lacticaseibacillus casei LH23 is a strain isolated from traditional fermented food and had been demonstrate to ameliorate DSS-induced colitis in mice. In the present study, effects of Lacticaseibacillus casei LH23 on cervical cancer cells were investigated. Supernatants of lysates and heat-inactivated Lacticaseibacillus casei LH23 were found to inhibit the expression of human papillomavirus genes E6/E7 which is the main causative factor of cervical cancer. With MTT, EdU staining, and TUNEL staining assays, Lacticaseibacillus casei LH23 was shown to suppress the proliferation and induced the apoptosis of cervical cancer cells. Additionally, with wound-healing and Western-blot assays, Lacticaseibacillus casei LH23 was shown to slowdown the migration of cervical cancer cells and altered the expression of metastasis-related genes. These results demonstrated the anti-cervical cancer potential of Lacticaseibacillus casei LH23.

Toward improved terpenoids biosynthesis: strategies to enhance the capabilities of cell factories.

Terpenoids form the most diversified class of natural products, which have gained application in the pharmaceutical, food, transportation, and fine and bulk chemical industries. Extraction from naturally occurring sources does not meet industrial demands, whereas chemical synthesis is often associated with poor enantio-selectivity, harsh working conditions, and environmental pollutions. Microbial cell factories come as a suitable replacement. However, designing efficient microbial platforms for isoprenoid synthesis is often a challenging task. This has to do with the cytotoxic effects of pathway intermediates and some end products, instability of expressed pathways, as well as high enzyme promiscuity. Also, the low enzymatic activity of some terpene synthases and prenyltransferases, and the lack of an efficient throughput system to screen improved high-performing strains are bottlenecks in strain development. Metabolic engineering and synthetic biology seek to overcome these issues through the provision of effective synthetic tools. This review sought to provide an in-depth description of novel strategies for improving cell factory performance. We focused on improving transcriptional and translational efficiencies through static and dynamic regulatory elements, enzyme engineering and high-throughput screening strategies, cellular function enhancement through chromosomal integration, metabolite tolerance, and modularization of pathways.

Correlation analysis between gene expression profile of rat liver tissues and high-fat emulsion-induced nonalcoholic fatty liver.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is caused by fat metabolism disorders and thereby abnormal or excessive accumulation of fat in hepatocytes, and characterized by steatosis, inflammation, fibrosis, apoptosis or necrosis. AIM: This study was carried out to explore the correlation between gene expression profiles of rat livers and the occurrence and progression of NAFLD at the transcriptional level. METHODS: A rat model of nonalcoholic steatohepatitis (NASH) was established by feeding male rats with high-fat emulsion via gavage, and Rat Genome 230 2.0 Array was used to detect gene expression profiles of liver tissues obtained from male rats following 0, 2, 4, and 6 weeks of high-fat emulsion feeding. Methods of bioinformatics and systems biology were applied to analyze the correlation between gene expression changes and physiological activities involved in NAFLD. RESULTS: In total, 93 function-known genes, including 36 up-regulated and 57 down-regulated, differed significantly in expression compared to those of control rats, and 18 physiological activities were closely related to NAFLD. Especially, the activity of cell differentiation was decreased during the whole process of NAFLD, and the activities of inflammation response, stimulus response, cell migration and adhesion were attenuated in the second, fourth and sixth week, respectively. In the fourth and sixth weeks, lipid metabolism and cell apoptosis were augmented, and the former might be associated with the enhanced expression of plin, acsl6, scd2, elovl3, etc. CONCLUSION: These data provide useful information on the global gene expression changes due to high-fat emulsion feeding and bring important insights into the mechanisms of NAFLD.

Coating function and stabilizing effects of surface layer protein from Lactobacillus acidophilus ATCC 4356 on liposomes.

Surface layer proteins (SLPs) are crystalline arrays in the outermost layer of cell envelope in many archaea and bacteria. SLPs subunits have the ability to reassemble on the surface of lipid layers. In this work, the SLP from Lactobacillus acidophilus ATCC 4356 was extracted and reassembled on the surface of positively charged liposomes composed of dipalmitoyl phosphatidylcholine, cholesterol and octadecylamine. Zeta potentials and particle size were determined to describe the adsorption process of SLP on liposomes. The liposomes completely coated with SLP were observed by transmission electron microscope. To investigate the stabilizing effects of SLP on liposomes, carboxyfluorescein (CF) was encapsulated and its leakage was determined as an evaluation index. The results showed that the L. acidophilus ATCC 4356 SLP significantly (P < 0.05) increased the stability of the liposomes in the course of thermal challenge. Furthermore, SLP was able to reduce the aggregation of liposomes in serum. Storage stability of liposomes was performed at 25 °C, 4 °C and -20 °C for 90 days. And the SLP-coated liposomes released less CF than the control liposomes during storage at the three evaluated temperatures. Our findings extended the application field of Lactobacillus SLPs and introduced a novel nanocarrier system with good chemical stability.

Transfection of STAT3 overexpression plasmid mediated through recombinant lentivirus promotes differentiation of bone marrow mesenchymal stem cells into neural cells in fetal rats with spina bifida aperta.

We investigated the influence of signal transducer and activator of transcription-3 (STAT3) on the spinal cord tissue grafts of rat fetuses with spina bifida aperta. In particular, we hoped to identify whether transfection of the STAT3 overexpression plasmid increases the survival of spinal cord transplantation in order to improve therapeutic efficacy. The fetal rat model of spina bifida aperta was established using retinoic acid and treated with a microsurgical injection of bone marrow mesenchymal stem cells (BMSCs). The animals were divided into either the blank control group, negative control group or the experimental group. The optical density (OD) value of BMSCs viability was determined using the Cell Counting Kit-8 (CCK-8). The expression of STAT3, phosphorylated STAT3 (pSTAT3), neural markers and apoptosis-related factors were evaluated using real-time PCR and Western blot. The OD value in the experimental group was highest at eight hours after transplantation using CCK-8. The expression of pSTAT3, glial fibrillary acidic protein, neuron-specific enolase, neurofilament and nestin in the experimental group was significantly higher compared to the blank control group and negative control group (P<0.05). However, STAT3 expression in the experimental group was statistically significantly decreased (P<0.05). The relative expression of caspase-8 and bcl-2 in the experimental group were significantly lower compared to the blank control group and negative control group (P<0.05). Transfection of the recombinant lentivirus-mediated STAT3 overexpression plasmid with BMSCs can help improve the efficiency of transforming into neural cells and provide new seed cells for the treatment of congenital spina bifida aperta.