Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

Both epilepsy and anti-seizure medications affect bone metabolism in children with self-limited epilepsy with centrotemporal spikes.

OBJECTIVE: Bone metabolism can be influenced by a range of factors. We selected children with self-limited epilepsy with centrotemporal spikes (SeLECTS) and lifestyles similar to those of healthy children to control for the confounding factors that may influence bone metabolism. We aimed to identify the specific effects of epilepsy and/or anti-seizure medications (ASMs) on bone metabolism. METHODS: Patients with SeLECTS were divided into an untreated group and a monotherapy group, and the third group was a healthy control group. We determined the levels of various biochemical markers of bone metabolism, including procollagen type I nitrogenous propeptide (PINP), alkaline phosphatase (ALP), osteocalcin (OC), collagen type I cross-linked C-telopeptide (CTX), calcium, magnesium, phosphorus, parathyroid hormone (PTH), and vitamin D3 (VD3 ). RESULTS: A total of 1487 patients (from 19 centers) were diagnosed with SeLECTS; 1032 were analyzed, including 117 patients who did not receive any ASMs (untreated group), 643 patients who received only one ASM (monotherapy group), and 272 children in the healthy control group. Except for VD3 , other bone metabolism of the three groups were different (p < .001). Bone metabolism was significantly lower in the untreated group than the healthy control group (p < .05). There were significant differences between the monotherapy and healthy control group in the level of many markers. However, when comparing the monotherapy and untreated groups, the results were different; oxcarbazepine, levetiracetam, and topiramate had no significant effect on bone metabolism. Phosphorus and magnesium were significantly lower in the valproic acid group than the untreated group (adjusted p < .05, Cliff's delta .282-.768). CTX was significantly higher in the lamotrigine group than in the untreated group (adjusted p = .012, Cliff's delta = .316). SIGNIFICANCE: Epilepsy can affect many aspects of bone metabolism. After controlling epilepsy and other confounders that affect bone metabolism, we found that the effects of ASMs on bone metabolism differed. Oxcarbazepine, levetiracetam, and topiramate did not affect bone metabolism, and lamotrigine corrected some of the abnormal markers of bone metabolism in patients with epilepsy.

Evolutionary and biochemical characterization of a Chromochloris zofingiensis MBOAT with wax synthase and diacylglycerol acyltransferase activity.

Wax synthase (WS) catalyzes the last step in wax ester biosynthesis in green plants. Two unrelated sub-families of WS, including the bifunctional acyltransferase and plant-like WS have been reported, but the latter is largely uncharacterized in microalgae. Here, we functionally characterized a putative plant-like WS (CzWS1) from the emerging model green microalga Chromochloris zofingiensis. Our results showed that plant-like WS evolved under different selection constraints in plants and microalgae, with positive selection likely contributing to functional divergence. Unlike jojoba with high amounts of wax ester in seeds and a highly active WS enzyme, C. zofingiensis has no detectable wax ester but a high abundance of WS transcripts. Co-expression analysis showed that C. zofingiensis WS has different expression correlation with lipid biosynthetic genes from jojoba, and may have a divergent function. In vitro characterization indicated that CzWS1 had diacylglycerol acyltransferase activity along with WS activity, and overexpression of CzWS1 in yeast and Chlamydomonas reinhardtii affected triacylglycerol accumulation. Moreover, biochemical and bioinformatic analyses revealed the relevance of the C-terminal region of CzWS1 in enzyme function. Taken together, our results indicated a functional divergence of plant-like WS in plants and microalgae, and the importance of its C-terminal region in specialization of enzyme function.

The influence of fatty acid supply and aldehyde reductase deletion on cyanobacteria alkane generating pathway in Escherichia coli.

Cyanobacteria alkane synthetic pathway has been heterologously constructed in many microbial hosts. It is by far the most studied and reliable alkane generating pathway. Aldehyde deformylating oxygenase (i.e., ADO, key enzyme in this pathway) obtained from different cyanobacteria species showed diverse catalytic abilities. This work indicated that single aldehyde reductase deletions were beneficial to Nostoc punctiforme ADO-depended alkane production in Escherichia coli even better than double deletions. Fatty acid metabolism regulator (FadR) overexpression and low temperature increased C18:1 fatty acid supply, and in turn stimulated C18:1-derived heptadecene production, suggesting that supplying ADO with preferred substrate was important to overall alkane yield improvement. Using combinational methods, 1 g/L alkane was obtained in fed-batch fermentation with heptadecene accounting for nearly 84% of total alkane.

Identification of novel ATP7A mutations and prenatal diagnosis in Chinese patients with Menkes disease.

Menkes disease (MD) is a fatal X-linked multisystem disease caused by mutations in ATP7A. In this study, clinical and genetic analysis was performed in 24 male MD patients. Development delay, seizures, kinky coarse hair, and dystonia were found in 24, 22, 24, and 24 patients, respectively. Serum ceruloplasmin/copper tested in 19 patients was low. Abnormal classic features of MD presented in the MRI/MRA of 19 patients. Seventeen mutations of ATP7A were identified in 22 patients. Twelve were novel mutations including three small deletion/insertion, one missense mutation, two nonsense mutations, three splicing-site mutations, and three gross deletions. Twenty-two patients were genetically diagnosed; neither point mutation nor deletion/duplication was found in two of them. c.2179G > A found in five patients might be a hot-spot mutation. Prenatal molecular diagnosis was performed for five unrelated fetuses (1 female and 4 male), which found four fetuses to be wild type and one male carried the same mutation as the proband. This study of the largest sample of Chinese MD patients examined to date discovered the unique phenotype and genotype spectrum in Chinese patients with 12 novel mutations of ATP7A, and that c.2179G > A might be a hot-spot mutation in MD patients. Five successful prenatal diagnosis contributed important information for MD families.

Alu-minating the Mechanisms Underlying Primate Cortex Evolution.

The higher-order cognitive functions observed in primates correlate with the evolutionary enhancement of cortical volume and folding, which in turn are driven by the primate-specific expansion of cellular diversity in the developing cortex. Underlying these changes is the diversification of molecular features including the creation of human and/or primate-specific genes, the activation of specific molecular pathways, and the interplay of diverse layers of gene regulation. We review and discuss evidence for connections between Alu elements and primate brain evolution, the evolutionary milestones of which are known to coincide along primate lineages. Alus are repetitive elements that contribute extensively to the acquisition of novel genes and the expansion of diverse gene regulatory layers, including enhancers, alternative splicing, RNA editing, and microRNA pathways. By reviewing the impact of Alus on molecular features linked to cortical expansions or gyrification or implications in cognitive deficits, we suggest that future research focusing on the role of Alu-derived molecular events in the context of brain development may greatly advance our understanding of higher-order cognitive functions and neurologic disorders.

Current progress in lipid-based biofuels: Feedstocks and production technologies.

The expanding use of fossil fuels has caused concern in terms of both energy security and environmental issues. Therefore, attempts have been made worldwide to promote the development of renewable energy sources, among which biofuel is especially attractive. Compared to other biofuels, lipid-derived biofuels have a higher energy density and better compatibility with existing infrastructure, and their performance can be readily improved by adjusting the chemical composition of lipid feedstocks. This review thus addresses the intrinsic interactions between lipid feedstocks and lipid-based biofuels, including biodiesel, and renewable equivalents to conventional gasoline, diesel, and jet fuel. Advancements in lipid-associated biofuel technology, as well as the properties and applicability of various lipid sources in terms of biofuel production, are also discussed. Furthermore, current progress in lipid production and profile optimization in the context of plant lipids, microbial lipids, and animal fats are presented to provide a wider context of lipid-based biofuel technology.

The Influence of Telemedicine and Compassionate Care on the Quality of Life and Mental Health of Patients with Epilepsy in Northeastern China During the COVID-19 Crisis.

PURPOSE: To understand the influence of telemedicine and compassionate care on the quality of life and mental health of patients with epilepsy (PWE) in northeastern China during the COVID-19 crisis. PATIENTS AND METHODS: Physicians in the epilepsy department conducted a questionnaire survey on PWE on February 2020. The Quality Of Life In Epilepsy-31 (QOLIE-31), Generalized Anxiety Disorder 7-Item Scale (GAD-7) and Patient Health Questionnaire-9 (PHQ-9) were used. The intervention (IG) group received compassionate care and follow-up through telemedicine equipment every week, while the nonintervention (NIG) group did not receive. The questionnaire survey was conducted again three month later. RESULTS: Ninety patients were recruited: mean age 39.91±15.57 in the IG, 37.39±11.69 in the NIG, 46 (51.1%) were men. Twenty patients had difficulty in purchasing antiepileptic drugs (AEDs). Seven patients reported seizure in the last 1 month. Only 1 patient (2.2%) consulted the emergency department. Up to 84 patients' lives were affected. Fifteen (33.3%) of the IG and 20 (44.4%) of the NIG patients stated that their family income had decreased, and among them, 13 (28.9%) in the IG group and 10 (22.2%) in the NIG group stated that they were unemployed. 3 months later, the interaction between groups and time of QOLIE-31 was significant, F (1, 88) = 16.996, p<0.001; the interaction between group and time on the PHQ-9 was significant, F (1, 88)= 14.992, p<0.001; the interaction between group and time on the QAD-7 was significant, F (1, 88)= 6.026, p<0.001. CONCLUSION: Our study found that during the COVID-19 outbreak, when patients were in a lockdown, telemedicine and compassionate care were effective and successful in managing PWE in northeastern China. It is a valid method to decrease anxiety and depression and improve the patients' quality of life. Further research is necessary about compassionate care methods for PWE.

Improving the Production of Punicic Acid in Baker's Yeast by Engineering Genes in Acyl Channeling Processes and Adjusting Precursor Supply.

Punicic acid (PuA) is a high-value edible conjugated fatty acid with strong bioactivities and has important potential applications in nutraceutical, pharmaceutical, feeding, and oleochemical industries. Since the production of PuA is severely limited by the fact that its natural source (pomegranate seed oil) is not readily available on a large scale, there is considerable interest in understanding the biosynthesis and accumulation of this plant-based unusual fatty acid in transgenic microorganisms to support the rational design of biotechnological approaches for PuA production via fermentation. Here, we tested the effectiveness of genetic engineering and precursor supply in PuA production in the model yeast strain Saccharomyces cerevisiae. The results revealed that the combination of precursor feeding and co-expression of selected genes in acyl channeling processes created an effective "push-pull" approach to increase PuA content, which could prove valuable in future efforts to produce PuA in industrial yeast and other microorganisms via fermentation.

Change in the active component of processed Tetradium ruticarpum extracts leads to improvement in efficacy and toxicity attenuation.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried and nearly ripe fruits of Tetradium ruticarpum (A. Juss.) T.G. Hartley (TR) have long been used in treating headache and gastrointestinal disorders in oriental medicine. TR is usually processed by stir-frying with licorice extract before use. Although processing procedure is considered as the way to relieve pungent smell, reduce toxicity, and improve efficacy, its effects on TR's toxicity and efficacy and bioactive compound profiles are largely unknown. AIM OF THE STUDY: The purposes of the study are to evaluate the acute toxicity, efficacy and variation of toxic and effective components of TR before and after processing, and to explore the possible mechanism of how the processing procedure affect the quality of TR as a herbal medicine. MATERIALS AND METHODS: Volatile oil, aqueous extract and ethanol extract of raw and processed TR were tested for their acute toxicity, analgesic, and anti-inflammatory effects in mouse models, respectively. To identify potential toxic and effective components, the extracts were analyzed with gas chromatography-mass spectrometry and ultra-performance liquid chromatography - quadrupole time-of-flight mass spectrometry, followed by fold-change-filtering analysis. RESULTS: LD50 and LD5 tests indicated that although the aqueous extract has higher toxicity than volatile oil and ethanol extract, the use of TR is safe under the recommended does. The processing procedure could effectively decrease the toxicity of all three extracts with the largest decrease in volatile oil, which is likely due to the loss of volatile compounds during processing. Analgesic and anti-inflammatory studies suggested that volatile oil and ethanol extract of TR have better efficacy than the aqueous extract and the processing procedure significantly enhanced the efficacy of these two former extracts, whereas processing showed no substantially effects on the bioactivities of aqueous extract. Integrated analysis of animal trial and chromatographic analyses indicated that indole and quinolone type alkaloids, limonoids, amides and 18ß-glycyrrhetinic acid were identified as the potential main contributors of TR's efficacy, whereas hydroxy or acetoxy limonoid derivates and coumarins could be the major causes of toxicity. Moreover, the reduced toxicity and improved efficacy of the processed TR are liked due to the licorice ingredients and altered alkaloids with better solubility. CONCLUSIONS: In summary, the integrated toxicity and efficacy analyses of volatile, aqueous and ethanol extracts of TR indicated that the processing procedure could effectively reduce its acute toxicity in all three extracts and enhance its analgesic and anti-inflammatory effects in volatile and ethanol extracts. The promising candidate compounds related to the toxicity and efficacy of TR were also identified. The results could expand our understanding of the value of the standard processing procedure of TR, be valuable to the quality control of TR manufacturing and administration, as well as support clinical rational and safety applications of this medicinal plant.

A randomized trial in the investigation of anxiety and depression in patients with coronavirus disease 2019 (COVID-19).

BACKGROUND: In March 2020, the World Health Organization (WHO) declared COVID-19 a public health emergency of international concern. A small proportion of patients infected with COVID-19 go on to develop pneumonia. We speculated that COVID-19 may be likely to result in psychological disorders such as anxiety and depression. In this study, we conducted an investigation of anxiety and depression in patients with COVID-19. METHODS: Sixty-five COVID-19 patients were randomly enrolled into this study. Anxiety and depression among participants were measured through the completion of anonymous Chinese-language Zung self-rating anxiety scale and self-rating depression scale questionnaires. Data were analyzed using independent samples t-tests, Mann-Whitney U-tests, and χ2 tests. RESULTS: The questionnaire results showed that 26.15% and 41.54% of participants suffered from anxiety and depression, respectively, although there was no significantly statistical difference between the proportions of COVID-19 patients with anxiety and depression. Statistically significant differences in employment status, partial pressure of oxygen, and corticosteroid application existed between moderate- and severe COVID-19 patients (P<0.05). In particular, the partial pressure of oxygen was significantly lower in severe COVID-19 patients than in their moderate counter parts (71.31±23.54 vs. 101.06±34.43, U=156, P=0.006). Total lymphocytes was lower in severe group than in moderate group [1.659±0.643 vs. 0.745 (0.645, 0.928), U=109, P=0.000]. Also, a higher proportion of female than male patients had anxiety (χ2=5.388, P=0.02). COVID-19 patients who received antiviral medications also displayed a higher rate of anxiety (χ2=4.481, P=0.034). Total lymphocytes between the non-anxiety and anxiety had statistical difference (U=321, P=0.019). Meanwhile, total lymphocytes between the non-depression and depression also had statistical difference (U=389.5, P=0.01). CONCLUSIONS: Among patients with COVID-19, females and those treated with antiviral medications were more likely to experience anxiety. In addition, our findings reflected the effect of anxiety and depression on immune system.

The Role of Triacylglycerol in Plant Stress Response.

Vegetable oil is mainly composed of triacylglycerol (TAG), a storage lipid that serves as a major commodity for food and industrial purposes, as well as an alternative biofuel source. While TAG is typically not produced at significant levels in vegetative tissues, emerging evidence suggests that its accumulation in such tissues may provide one mechanism by which plants cope with abiotic stress. Different types of abiotic stress induce lipid remodeling through the action of specific lipases, which results in various alterations in membrane lipid composition. This response induces the formation of toxic lipid intermediates that cause membrane damage or cell death. However, increased levels of TAG under stress conditions are believed to function, at least in part, as a means of sequestering these toxic lipid intermediates. Moreover, the lipid droplets (LDs) in which TAG is enclosed also function as a subcellular factory to provide binding sites and substrates for the biosynthesis of bioactive compounds that protect against insects and fungi. Though our knowledge concerning the role of TAG in stress tolerance is expanding, many gaps in our understanding of the mechanisms driving these processes are still evident. In this review, we highlight progress that has been made to decipher the role of TAG in plant stress response, and we discuss possible ways in which this information could be utilized to improve crops in the future.

A Novel PRRT2 Variant in Chinese Patients Suffering from Paroxysmal Kinesigenic Dyskinesia with Infantile Convulsion.

PRRT2 mutations are the major causative agent of paroxysmal kinesigenic dyskinesia with infantile convulsion (PKD/IC). The study is aimed at screening PRRT2 gene mutations in patients who suffered from PKD/IC in Chinese population. Thirteen Chinese patients with PKD/IC were screened randomly for coding exons of the PRRT2 gene mutation along with 50 ethnically coordinated control people. Nine (2 unaffected) and 4 of the patients showed familial PKD/IC and apparently sporadic cases, respectively. We identified 5 different PRRT2 mutations in 10 individuals, including 8 familial and 2 apparently sporadic cases. However, no mutations were found in the 50 ethnically matched controls. Unknown (novel) NM_145239.2:c.686G>A and previously reported NM_145239.2:c.743G>C variants were identified in two familial and sporadic patients. All affected members of family A showed mutation NM_145239.2:c.650_670delinsCAATGGTGCCACCACTGGGTTA. The previously identified NM_145239.2:c.412 C>G and NM_145239.2:c.709G>A variants are seen in two individuals assessed in family B. Other than the previously identified variants, some of the patients with PRRT2-PKD/IC showed a new PRRT2 substitution variant. Thus, the spectrum of PRRT2 variants is expanded. The possible role and probability of PRRT2 variants involved in PKD/IC are highlighted.

Multilayered control of exon acquisition permits the emergence of novel forms of regulatory control.

BACKGROUND: The long introns of mammals are pools of evolutionary potential due to the multiplicity of sequences that permit the acquisition of novel exons. However, the permissibility of genes to this type of acquisition and its influence on the evolution of cell regulation is poorly understood. RESULTS: Here, we observe that human genes are highly permissive to the inclusion of novel exonic regions permitting the emergence of novel regulatory features. Our analysis reveals the potential for novel exon acquisition to occur in over 30% of evaluated human genes. Regulatory processes including the rate of splicing efficiency and RNA polymerase II (RNAPII) elongation control this process by modulating the "window of opportunity" for spliceosomal recognition. DNA damage alters this window promoting the inclusion of repeat-derived novel exons that reduce the ribosomal engagement of cell cycle genes. Finally, we demonstrate that the inclusion of novel exons is suppressed in hematological cancer samples and can be reversed by drugs modulating the rate of RNAPII elongation. CONCLUSION: Our work demonstrates that the inclusion of repeat-associated novel intronic regions is a tightly controlled process capable of expanding the regulatory capacity of cells.

Employing metabolic engineered lipolytic microbial platform for 1-alkene one-step conversion.

1-Alkenes are traditionally used as basic chemicals with great importance. Biosynthetic 1-alkenes also have the potential to serve as biofuels. In this study, we engineered a Pseudomonas lipolytic microbial platform for 1-alkene production using hydrophobic substrate as sole carbon source. Fatty acid decarboxylase UndA and UndB were cloned and expressed, which successfully produced 1-alkenes. Optimal culturing temperature and the interruption of competitive pathway were proven to be beneficial to 1-alkene synthesis. Chromosomal integration of UndB conferred 177.8 mg/L 1-alkenes (mainly 1-undecene) in lauric acid medium and 128.9 mg/L 1-alkenes (mainly 1-pentadecene) in palm oil medium. Thioesterase expression, adjustments of fatty acid degradation pathway and a second copy of UndB improved 1-alkene titer to 1102.6 mg/L using lauric acid and 778.4 mg/L using palm oil. All in all, this study offers the first demonstration of lipolytic microbial 1-alkene producing platform with highest reported 1-alkene product titer up to date.

New variants in the CACNA1H gene identified in childhood absence epilepsy.

Childhood absence epilepsy (CAE) is a common form of idiopathic generalized epilepsy with polygenic inheritance. In our previous studies, relatively high frequent variants in the T-type calcium channel gene, CACNA1H, were identified in the Chinese Han population, most of which are located in exons 6-12. The goal of this study was to identify additional variants in this region of the CACNA1H gene. To this end, exons 6-12 were sequenced in 100 newly recruited CAE trios and 191 normal controls. Thirty-nine variants were identified in CAE trios or controls, 14 of which were found only in CAE patients, including two nonsynonymous variants that were newly found. Thirteen of the 39 variants were found in both CAE patients and controls, 11 were found only in parents of CAE trios, and one was found only in controls. Twenty-eight of these variants had not been previously reported. Both permutation test and transmission/disequilibrium test (TDT) indicated that a SNP-52037C>T in intron11 was significant in association with CAE. In conclusion, these data further support the hypothesis that CACNA1H is an important susceptibility gene for CAE in the Chinese Han population.

CACNA1I is not associated with childhood absence epilepsy in the Chinese Han population.

This study investigated whether the T-type calcium channel gene CACNA1I causes susceptibility in the Chinese Han population to childhood absence epilepsy, a form of idiopathic generalized seizure disorder. For this investigation, we searched for mutations in the 35 exons and exon-intron boundaries of the CACNA1I gene in 50 Han Chinese patients with childhood absence epilepsy. Seventeen single nucleotide polymorphisms were identified in the 35 exons. Using six single nucleotide polymorphisms as markers, the allele and genotype distributions of all of the identified single nucleotide polymorphisms were examined; there was no significant difference between the childhood absence epilepsy cases and the control groups. Thus, we do not consider the CACNA1I gene to be an important susceptibility gene for childhood absence epilepsy in the Chinese Han population.

[The effect of CACNA1H gene G773D mutation on calcium channel function].

OBJECTIVE: To study the effect of CACNA1H gene mutation G773D on calcium channel function. METHODS: By the overlap extension PCR we introduced G773D mutation into a human Cav3.2acDNA for constructing the mutant. And then using whole cell clamp technique, we studied the alterations of channel behavior in transfected HEK-293 cells. RESULTS: There were no difference in kinetics of activation and inactivation of calcium channel between wild type and mutant. However comparing with the wild-type Cav3.2 channel, G773D mutant could increase the calcium current density significantly. CONCLUSION: CACNA1H gene G773D mutation is able to increase calcium current and neuronal excitability.

[Advances of research in ion channels in idiopathic epilepsy].

Epilepsy is a kind of brain dysfunction syndrome caused by so many reasons instead of a certain disease. Abnormal neuron discharge can cause epilepsy. Idiopathic epilepsy refers to epilepsy or epilepsy syndrome without any latent reasons but inherited trait. Idiopathic epilepsy is confirmed as an ion channelopathy at present. The first genemutation was found in idiopathic epilepsy in 1995, and a lot of genes coding either voltage-gated or ligand-gated ion channels have been found since then. In the present review, some new advances in research on ion channels dysfunction in idiopathic epilepsy are summarized.