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Pichia pastoris, a methylotrophic yeast, can utilize methanol as a carbon source and energy source to synthesize high-value chemicals, and is an ideal host for biomanufacturing. Constructing the P. pastoris cell factory is somewhat impeded due to the absence of genetic tools for manipulating multi-gene biosynthetic pathways. To broaden its application in the field of metabolic engineering, this study identified and screened 15 novel integration sites in P. pastoris using CRISPR-Cpf1 genome editing technology, with EGFP serving the reporter protein. These integration sites have integration efficiencies of 10–100 % and varying expression strengths, which allow for selection based on the expression levels of genes as needed. Additionally, these integrated sites are applied in the heterologous biosynthesis of P. pastoris, such as the astaxanthin biosynthetic pathway and the carbon dioxide fixation pathway of the Calvin-Benson-Bassham (CBB) cycle. During the three-site integration process, the 8 genes of the CBB cycle were integrated into the genome of P. pastoris. This indicates the potential of these integration sites for integrating large fragments and suggests their successful application in metabolic engineering of P. pastoris. This may lead to improved efficiency of genetic engineering in P. pastoris.

Tourette Syndrome (TS) is a neurodevelopmental disorder characterized by multiple motor and vocal tics, often accompanied by comorbid disorders. Optional treatments for patients with TS include behavioral therapy, pharmacotherapy, and neurostimulation techniques. Deep brain stimulation (DBS) has been considered a therapeutic approach for refractory TS and its comorbid symptoms. However, systematic comparison is necessary to understand the therapeutic effect of DBS among patients with TS with various comorbid symptoms, demographic characteristics, or stimulation targets. Consequently, our research aimed to assess the clinical efficacy of DBS in alleviating the symptoms of TS and its comorbidities. A systematic literature search was conducted across five databases: PubMed, Web of Science, MEDLINE, Embase, and PsycINFO. The primary outcome was the mean change in the global score of the Yale Global Tic Severity Scale (YGTSS), which assesses the severity of tics. The secondary outcomes included mean improvement of comorbid symptoms, such as obsessive-compulsive behaviors (OCB), depression symptoms and anxiety symptoms. In total, 51 studies with 673 participants were included in this meta-analysis. Overall, the DBS led to a significant improvement in tic symptoms (p ​

ABSTRACT Introduction Hypersalivation has been associated with Rolandic epilepsy and other childhood epilepsy syndromes. However, pure salivatory seizures are a rare type of focal seizure in which ictal hypersalivation is the dominant feature throughout the seizures. Case presentation We present a case of pure salivatory seizures originating from the right post‐central operculum cortex, confirmed by the favorable surgical outcome. We attempt to analyze the symptom from behavioral and neural network perspectives and propose a possible mechanism to generate ictal hypersalivation and pure salivatory seizures. Conclusion Based on previous reports in the literature and our case, we emphasize the importance of the operculum in patients with ictal hypersalivation, particularly in patients with pure salivatory seizures.

Abstract Researchers have widely acknowledged the therapeutic value of epilepsy surgery for drug-resistant epilepsy. Nonetheless, there is a substantial gap in the surgical treatment for appropriate candidates owing to several factors, particularly in the population of young children. To standardize the protocols of preoperative evaluation and surgery of young children for epilepsy surgery, the China Association Against Epilepsy has appointed an expert task force to standardize the protocols of preoperative evaluation and surgery in pediatric epilepsy patients. It adopted the modified Delphi method and performed two rounds of surveys through an anonymous inquiry among 75 experts from four subgroups including pediatric neurologists, epileptologists, pediatric epilepsy surgeons, and functional neurosurgeons. The survey contents contained: (1) the participants, comprising children aged ≤ 6 years; (2) adopted DRE definition proposed by the International League Against Epilepsy in 2010; and (3) investigated epilepsy surgery, principally referring to curative epilepsy surgeries. The neuromodulation therapies were excluded because of the differences in treatment mechanisms from the above-mentioned surgeries. According to the Delphi process, a consensus was achieved for most aspects by incorporating two rounds of surveys including preoperative assessment, surgical strategies and techniques, and perioperative and long-term postoperative management, despite controversial opinions on certain items. We hope the results of this consensus will improve the level of surgical treatment and management of intractable epilepsy in young children.

Background: Low-grade epilepsy-associated brain tumors (LEATs) are found to be the second most common lesion-related epilepsy. Malignant potential of LEATs is very low and the overall survival is good, so the focus of treatment is focused more on seizure outcome rather than oncological prognosis. Objectives: This study was conducted to evaluate the risk factors of seizure outcomes after resection in patients with LEATs. Design: A retrospective study. Methods: A retrospective analysis of patients with LEATs who underwent resective surgery in our three epilepsy centers between October 2010 and April 2023 with a minimum follow-up of 1 year. Demography, clinical characters, neurophysiology, and molecular neuropathology were assessed for association with postoperative seizure outcomes at 1-, 2-, and 5-year follow-up. Synthetic minority oversampling technique (SMOTE) algorithm model was performed to handle the imbalance of data distribution. Gaussian Naïve Bayes (GNB) algorithms were created as a basis for classifying outcomes according to observation indicators. Results: A total of 111 patients were enrolled in the cohort. The most common pathology was ganglioglioma ( n = 37, 33.3%). The percentage of patients with seizure freedom was 91.0% (101/111) at 1-year follow-up, 87.5% (77/88) at 2-year follow-up, and 79.1% (53/67) at 5-year follow-up. Partial resection had a significantly poor seizure outcome compared to total resection and supratotal resection ( p

BackgroundAutism Spectrum Disorders (ASD) are a collection of neurodevelopmental diseases characterized by poor social interaction and communication, a limited range of interests, and stereotyped behavior. High-functioning autism (HFA) indicates a subgroup of individuals with autism who possess cognitive and/or language skills that are within the average to above-normal range for their age. Transcutaneous auricular vagus nerve stimulation (taVNS) holds promise in children with HFA. However, few studies have used randomized controlled trials to validate the effectiveness of taVNS. Therefore, in this study, we intend to provide a study protocol to examine the therapeutic effects of taVNS in individuals diagnosed with HFA and to investigate the process of brain network remodeling in individuals with ASD using functional imaging techniques to observe alterations in large-scale neural networks.Methods and designWe planned to employ a randomized, double-blind experimental design, including 40 children receiving sham stimulation and 40 children receiving real stimulation. We will assess clinical scales and perform functional imaging examinations before and after the stimulation. Additionally, we will include age- and gender-matched healthy children as controls and conduct functional imaging examinations. We plan first to observe the therapeutic effects of taVNS. Furthermore, we will observe the impact of taVNS stimulation on the brain network.DiscussiontaVNS was a low-risk, easy-to-administer, low-cost, and portable option to modulate the vagus system. taVNS may improve the social performance of HFA. Changes in the network properties of the large-scale brain network may be related to the efficacy of taVNS.Clinical trial registrationhttp://www.chictr.org.cn, identifier ChiCTR2300074035.

Abstract Flexible wearable sweat sensors allow continuous, real-time, noninvasive detection of sweat analytes, provide insight into human physiology at the molecular level, and have received significant attention for their promising applications in personalized health monitoring. Electrochemical sensors are the best choice for wearable sweat sensors due to their high performance, low cost, miniaturization, and wide applicability. Recent developments in soft microfluidics, multiplexed biosensing, energy harvesting devices, and materials have advanced the compatibility of wearable electrochemical sweat-sensing platforms. In this review, we summarize the potential of sweat for medical detection and methods for sweat stimulation and collection. This paper provides an overview of the components of wearable sweat sensors and recent developments in materials and power supply technologies and highlights some typical sensing platforms for different types of analytes. Finally, the paper ends with a discussion of the challenges and a view of the prospective development of this exciting field.

Abstract Ficus hirta Vahl (FHV), a traditional herbal ingredient of the tonic diet, receives increasing popularity in southern China. However, it is largely unknown that how a FHV diet (FHVD) affects the human gut microbiome. In this exploratory study, a total of 43 healthy individuals were randomized into the FHVD (n = 25) and Control (n = 18) groups to receive diet intervention for 8 weeks. 16S rRNA gene sequencing, metagenomic sequencing and metabolic profile of participants were measured to assess the association between FHV diet and gut microbiome. A preservation effect of Faecalibacterium and enrichment of Dialister, Veillonella, Clostridium, and Lachnospiraceae were found during the FHVD. Accordingly, the pathway of amino acid synthesis, citrate cycle, coenzyme synthesis, and partial B vitamin synthesis were found to be more abundant in the FHVD. In addition, serine, glutamine, gamma-aminobutyric acid, tryptamine, and short-chain fatty acids (SCFAs) were higher after the FHVD. The conjoint analysis of FHV components and in-vitro fermentation confirmed that the improved SCFAs concentration was collectively contributed by the increasing abundance of key enzyme genes and available substrates. In conclusion, the muti-omics analysis showed that the FHVD optimized the structure of the gut microbial community and its metabolic profile, leading to a healthy tendency, with a small cluster of bacteria driving the variation rather than a single taxon.

ObjectiveTo investigate the effect of current resistance on therapeutic outcomes, and the mechanism of current conduction treatment in a rat model of temporal lobe epilepsy (TLE).MethodsRats were randomly divided into four groups: normal control, epileptic group, low-resistance conduction (LRC) and high-resistance conduction (HRC) group. The content of glutamate (Glu) and gamma-amino butyric acid (GABA) in the hippocampus was determined using a neurotransmitter analyzer. mRNA and protein expression of interleukin 1β (IL-1β) /IL-1 receptor 1(IL-1R1) and high mobility group protein B1 (HMGB-1)/toll-like receptor-4 (TLR-4) in hippocampal neurons were tested. Video electroencephalogram monitoring was used to record seizures and EEG discharges. Cognitive function in the rats was tested using the Morris water maze.ResultsGlu/GABA ratio in the epileptic control and HRC groups was significant differences from LRC group. The levels of HMGB1/TLR4 and IL-1β/IL-1R1 in the LRC group and normal control group were significantly lower than those in epileptic control group (p

Acetaldehyde is an important carbonyl compound commonly detected in wines. A high concentration of acetaldehyde can affect the flavor of wines and result in adverse effects on human health. Alcohol dehydrogenase I (ADH1) in Saccharomyces cerevisiae catalyzes the reduction reaction of acetaldehyde into ethanol in the presence of cofactors, showing the potential to reduce the content of acetaldehyde in wines. In this study, ADH1 was successfully expressed in Pichia pastoris GS115 based on codon optimization. Then, the expression level of ADH1 was enhanced by replacing its promoter with optimized promoters and increasing the copy number of the expression cassette, with ADH1 being purified using nickel column affinity chromatography. The enzymatic activity of purified ADH1 reached 605.44 ± 44.30 U/mg. The results of the effect of ADH1 on the content of acetaldehyde in wine revealed that the acetaldehyde content of wine samples was reduced from 168.05 ± 0.55 to 113.17 ± 6.08 mg/L with the addition of 5 mM NADH and the catalysis of ADH1, and from 135.53 ± 4.08 to 52.89 ± 2.20 mg/L through cofactor regeneration. Our study provides a novel approach to reducing the content of acetaldehyde in wines through enzymatic catalysis.

ABSTRACT Tuberous sclerosis complex (TSC) is a rare disease that involves multiple organs, including the brain; approximately 80%–90% of TSC patients exhibit TSC‐associated epilepsy. Independent temporal lobe epilepsy (TLE), TSC‐unrelated epilepsy, is particularly rare in patients with TSC. Here, we describe three patients with TSC with independent TLEs that were confirmed by stereo‐electroencephalography (EEG), postoperative pathological findings, and seizure outcome at follow‐up. The patients were retrospectively enrolled at two centers; their ictal epileptiform discharge onsets were determined using electrode contacts in the hippocampus during stereo‐EEG. The three patients underwent anterior temporal lobectomies and remained seizure‐free at 1–5 years after surgery. Postoperative pathological examinations confirmed hippocampal sclerosis in all three patients. Furthermore, postoperative intelligence quotient improvement was evident in one patient, while the quality of life was improved in two patients at 12 months after surgery.

Seizures are a common symptom of craniocerebral diseases, and epilepsy is one of the comorbidities of craniocerebral diseases. However, how to rationally use anti-seizure medications (ASMs) in the perioperative period of craniocerebral surgery to control or avoid seizures and reduce their associated harm is a problem. The China Association Against Epilepsy (CAAE) united with the Trauma Group of the Chinese Neurosurgery Society, Glioma Professional Committee of the Chinese Anti-Cancer Association, Neuro-Oncology Branch of the Chinese Neuroscience Society, and Neurotraumatic Group of Chinese Trauma Society, and selected experts for consultancy regarding outcomes from evidence-based medicine in domestic and foreign literature. These experts referred to the existing research evidence, drug characteristics, Chinese FDA-approved indications, and expert experience, and finished the current guideline on the application of ASMs during the perioperative period of craniocerebral surgery, aiming to guide relevant clinical practice. This guideline consists of six sections: application scope of guideline, concepts of craniocerebral surgery-related seizures and epilepsy, postoperative application of ASMs in patients without seizures before surgery, application of ASMs in patients with seizures associated with lesions before surgery, emergency treatment of postoperative seizures, and 16 recommendations.

Epilepsy is a chronic disorder of the nervous system characterized by recurrent seizures. Inflammation is one of the six major causes of epilepsy, and its role in the pathogenesis of epilepsy is gaining increasing attention. Two signaling pathways, the high mobility group box-1 (HMGB1)/toll-like receptor 4 (TLR4) and interleukin-1β (IL-1β)/interleukin-1 receptor 1 (IL-1R1) pathways, have become the focus of research in recent years. These two signaling pathways have potential as biomarkers in the prediction, prognosis, and targeted therapy of epilepsy. This review focuses on the association between epilepsy and the neuroinflammatory responses mediated by these two signaling pathways. We hope to contribute further in-depth studies on the role of HMGB1/TLR4 and IL-1β/IL-1R1 signaling in epileptogenesis and provide insights into the development of specific agents targeting these two pathways.

ObjectiveTo analyze the interictal discharge (IID) patterns on pre-operative scalp electroencephalogram (EEG) and compare the changes in IID patterns after removal of epileptogenic tubers in preschool children with tuberous sclerosis complex (TSC)-related epilepsy.MethodsThirty-five preschool children who underwent resective surgery for TSC-related epilepsy were enrolled retrospectively, and their EEG data collected before surgery to 3 years after surgery were analyzed.ResultsTwenty-three (65.7%) patients were seizure-free post-operatively at 1-year follow-up, and 37–40% of post-operative patients rendered non-IID on scalp EEGs, and patients with focal IIDs or generalized IID patterns on pre-operative EEG presented a high percentage of normal post-operative scalp EEGs. IID patterns on pre-operative scalp EEGs did not influence the outcomes of post-operative seizure controls, while patients with non-IID and focal IID on post-operative EEGs were likely to achieve post-operative seizure freedom. Patients with new focal IIDs presented a significantly lower percentage of seizure freedom than those without new focal IIDs on post-operative EEGs at 3-year follow-up.ConclusionOver 1/3 children with TSC presented normal scalp EEGs after resective epileptsy surgery. Patients with post-operative seizure freedom were more likely to have non-IIDs on post-operative EEGs. New focal IIDs were negative factors for seizure freedom at the 3-year follow-up.

Abstract Background Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. In this study, the functions of potential GPI proteins in Pichia pastoris were explored by gene knockout approaches. Results Through an extensive knockout of GPI proteins in P. pastoris, a single-gene deletion library was constructed for 45 predicted GPI proteins. The knockout of proteins may lead to the activation of a cellular response named the ‘compensatory mechanism’, which is characterized by changes in the content and relationship between cell wall polysaccharides and surface proteins. Among the 45 deletion strains, five showed obvious methanol tolerance, four owned high content of cell wall polysaccharides, and four had a high surface hydrophobicity. Some advantages of these strains as production hosts were revealed. Furthermore, the deletion strains with high surface hydrophobicity were used as hosts to display Candida antarctica lipase B (CALB). The strain gcw22Δ/CALB-GCW61 showed excellent fermentation characteristics, including a faster growth rate and higher hydrolytic activity. Conclusions This GPI deletion library has some potential applications for production strains and offers a valuable resource for studying the precise functions of GPI proteins, especially their putative functions.

As a natural sweetener and sucrose substitute, the biosynthesis and application of steviol glycosides containing the component rebaudioside D have attracted worldwide attention. Here, a glycosyltransferase PgUGT from Panax ginseng was first reported for the biosynthesis of rebaudioside D. With the three-dimensional structures built by homology modeling and deep-learning–based modeling, PgUGT was semi-rationally designed by FireProt. After detecting 16 site-directed variants, eight of them were combined in a mutant Mut8 with both improved enzyme activity and thermostability. The enzyme activity of Mut8 was 3.2-fold higher than that of the wild type, with an increased optimum reaction temperature from 35 to 40°C. The activity of this mutant remained over 93% when incubated at 35°C for 2 h, which was 2.42 times higher than that of the wild type. Meanwhile, when the enzymes were incubated at 40°C, where the wild type was completely inactivated after 1 h, the residual activity of Mut8 retained 59.0% after 2 h. This study would provide a novel glycosyltransferase with great potential for the industrial production of rebaudioside D and other steviol glycosides.

The large-scale fermentation of Pichia pastoris for recombinant protein production would be time consuming and produce a large amount of waste yeast. Here we introduce a novel semi-continuous fermentation process for P. pastoris GS115 that can separate vitality cells from broth and recycle the cells to produce high-secretory recombinant pectate lyase. It is based on differences in cell sedimentation coefficients with the formation of salt bridges between metal ions and various cell states. Compared to batch-fed cultivation and general semi-continuous culture, the novel process has significant advantages, such as consuming fewer resources, taking less time, and producing less waste yeast. Sedimentation with the addition of Fe3+ metal ions consumed 14.8 ± 0.0% glycerol, 97.8 ± 1.3% methanol, 55.0 ± 0.9 inorganic salts, 81.5 ± 0.0% time cost, and 77.0 ± 0.1% waste yeast versus batch-fed cultivation to produce an equal amount of protein; in addition, the cost of solid–liquid separation was lower for cells in the collected fermentation broth. The process is economically and environmentally efficient for producing recombinant proteins.

Abstract Background The methylotrophic yeast Pichia pastoris is well-known for the production of a broad spectrum of functional types of heterologous proteins including enzymes, antigens, engineered antibody fragments, and next gen protein scaffolds and many transcription factors are utilized to address the burden caused by the high expression of heterologous proteins. In this article, a novel P. pastoris transcription factor currently annotated as Fhl1p, an activator of ribosome biosynthesis processing, was investigated for promoting the expression of the recombinant proteins. Results The function of Fhl1p of P. pastoris for improving the expression of recombinant proteins was verified in strains expressing phytase, pectinase and mRFP, showing that the productivity was increased by 20–35%. RNA-Seq was used to study the Fhl1p regulation mechanism in detail, confirming Fhl1p involved in the regulation of rRNA processing genes, ribosomal small/large subunit biogenesis genes, Golgi vesicle transport genes, etc., which contributed to boosting the expression of foreign proteins. The overexpressed Fhl1p strain exhibited increases in the polysome and monosome levels, showing improved translation activities. Conclusion This study illustrated that the transcription factor Fhl1p could effectively enhance recombinant protein expression in P. pastoris. Furthermore, we provided the evidence that overexpressed Fhl1p was related to more active translation state.

Abstract Background Glioma‐related epilepsy (GRE) is defined as symptomatic epileptic seizures secondary to gliomas, it brings both heavy financial and psychosocial burdens to patients with diffuse glioma and significantly decreases their quality of life. To date, there have been no clinical guidelines that provide recommendations for the optimal diagnostic and therapeutic procedures for GRE patients. Methods In March 2017, the Joint Task Force for GRE of China Association Against Epilepsy and Society for Neuro‐Oncology of China launched the guideline committee for the diagnosis and treatment of GRE. The guideline committee conducted a comprehensive review of relevant domestic and international literatures that were evaluated and graded based on the Oxford Centre for Evidence‐Based Medicine Levels of Evidence, and then held three consensus meetings to discuss relevant recommendations. The recommendations were eventually given according to those relevant literatures, together with the experiences in the diagnosis and treatment of over 3000 GRE cases from 24 tertiary level hospitals that specialize in clinical research of epilepsy, glioma, and GRE in China. Results The manuscript presented the current standard recommendations for the diagnostic and therapeutic procedures of GRE. Conclusions The current work will provide a framework and assurance for the diagnosis and treatment strategy of GRE to reduce complications and costs caused by unnecessary treatment. Additionally, it can serve as a reference for all professionals involved in the management of patients with GRE.

Abstract Background Translational regulation played an important role in the correct folding of heterologous proteins to form bioactive conformations during biogenesis. Translational pausing coordinates protein translation and co-translational folding. Decelerating translation elongation speed has been shown to improve the soluble protein yield when expressing heterologous proteins in industrial expression hosts. However, rational redesign of translational pausing via synonymous mutations may not be feasible in many cases. Our goal was to develop a general and convenient strategy to improve heterologous protein synthesis in Pichia pastoris without mutating the expressed genes. Results Here, a large-scale deletion library of ribosomal protein (RP) genes was constructed for heterologous protein expression in Pichia pastoris, and 59% (16/27) RP deletants have significantly increased heterologous protein yield. This is due to the delay of 60S subunit assembly by deleting non-essential ribosomal protein genes or 60S subunit processing factors, thus globally decreased the translation elongation speed and improved the co-translational folding, without perturbing the relative transcription level and translation initiation. Conclusion Global decrease in the translation elongation speed by RP deletion enhanced co-translational folding efficiency of nascent chains and decreased protein aggregates to improve heterologous protein yield. A potential expression platform for efficient pharmaceutical proteins and industrial enzymes production was provided without synonymous mutation.

Objectives: To evaluate the value of fast ripples (FRs) (200–500 Hz) recorded with stereo-electroencephalograph (SEEG) in the localization of epileptogenic tubers in patients with tuberous sclerosis complex (TSC).Methods: Seventeen TSC patients who underwent preoperative SEEG examination and resective epilepsy surgery were retrospectively enrolled. They were divided into two groups according to the seizure control at 1-year postoperative follow-up. The occurrence frequencies of FRs were automatically counted, and the FR rate was calculated. The high FR rate was defined as FR rate ≧0.5. According to different positions, the contacts’ locations were divided into three groups: inner of the tubers, the junction region of the tubers, and out of the tubers. The influence factors of postoperative seizure freedom were also analyzed.Results: Twelve patients reached postoperative seizure freedom at 1-year follow-up. In total, FRs were found in 24.2% of the contacts and 67.1% of the tubers in all assessed patients. There were 47 high FR rate contacts localized in the junction region of the tubers, which was 62.7% of the 75 high FR rate contacts in total and was 8.4% of the total 561 contacts localized in the junction region of the tubers. Total removal of epileptogenic tubers and total resection of the high FR rate tubers/contacts were associated with postoperative seizure freedom (P < 0.05).Conclusion: FRs could be extensively detected in TSC patients using SEEG, and high FR rate contacts were mostly localized in the junction region of the epileptogenic tuber, which could aid in the localization of epileptogenic tubers.

Bisphenol A (BPA) and heavy metals are widespread contaminants in the environment. However, the combined toxicities of these contaminants are still unknown. In this study, the bioluminescent bacteria Vibrio qinghaiensis Q67 was used to detect the single and combined toxicities of BPA and heavy metals, then the joint effects of these contaminants were evaluated. The results show that chronic toxicities of chromium (Cr), cadmium (Cd), lead (Pb), arsenic (As), mercury (Hg), nickel (Ni), and BPA were time–dependent; in fact, the acute toxicities of these contaminants were stronger than the chronic toxicities. Furthermore, the combined toxicities of BPA and heavy metals displayed BPA + Hg > BPA + Cr > BPA + As > BPA + Ni > BPA + Pb > BPA + Cd in the acute test and BPA + Hg > BPA + Cd > BPA + As > BPA + Cd in the chronic test, which suggested that the combined toxicity of BPA and Hg was stronger than that of other mixtures in acute as well as chronic tests. Additionally, both CA and IA models underestimated the toxicities of mixtures at low concentrations but overestimated them at high concentrations, which indicates that CA and IA models were not suitable to predict the toxicities of mixtures of BPA and heavy metals. Moreover, the joint effects of BPA and heavy metals mainly showed antagonism and additive in the context of acute exposure but synergism and additive in the context of chronic exposure. Indeed, the difference in the joint effects on acute and chronic exposure can be explained by the possibility that mixtures inhibited cell growth and luminescence in chronic cultivation. The chronic toxicity of the mixture should be considered if the mixture results in the inhibition of the growth of cells.

Pectate lyases play an essential role in textiles, animal feed, and oil extraction industries. Pichia pastoris can be an ideal platform for pectate lyases production, and BspPel (a thermo-alkaline pectate lyase from Bacillus sp. RN1) was overexpressed by combined strategies, reaching 1859 U/mL in a 50 L fermentator. It displayed the highest activity at 80°C, and maintained more than 60% of the activity between 30 and 70°C for 1 h. It showed an optimal pH of 10.0, and exhibited remarkable stability over a wider pH range (3.0-11.0), retaining more than 80.0% of enzyme activity for 4 h. The Km and kcat of BspPel on PGA (polygalacturonic acid) was 2.19 g L–1 and 116.1 s–1, respectively. The activity was significantly enhanced by Ca2+, Mn2+, and Cu2+, and a slight increase was observed with the addition of Ba2+ and Mg2+. Scanning electron microscope was used to show the degumming efficiency of BspPel on ramie fibers. The loss weight was 9.2% when treated with crude enzyme supernatant and 20.8% when treated with the enzyme-chemical method, which was higher than the 14.2% weight loss in the positive control treated with 0.5% (w/v) NaOH alone. In conclusion, BspPel could be a good candidate for the ramie degumming industry.

This paper proposes a compact bioelectronics sensing platform, including a multi-channel electrode, intracranial electroencephalogram (iEEG) recorder, adjustable galvanometer, and shunt-current conduction circuit pathway. The developed implantable electrode made of polyurethane-insulated stainless-steel materials is capable of recording iEEG signals and shunt-current conduction. The electrochemical impedance of the conduction, ground/reference, and working electrode were characterized in phosphate buffer saline solution, revealing in vitro results of 517.2 Ω@1 kHz (length of 0.1 mm, diameter of 0.8 mm), 1.374 kΩ@1 kHz (length of 0.3 mm, diameter of 0.1 mm), and 3.188 kΩ@1 kHz (length of 0.1 mm, diameter of 0.1 mm), respectively. On-bench measurement of the system revealed that the input noise of the system is less than 2 μVrms, the signal frequency bandwidth range is 1 Hz~10 kHz, and the shunt-current detection range is 0.1~3000 μA with an accuracy of above 99.985%. The electrode was implanted in the CA1 region of the right hippocampus of rats for the in vivo experiments. Kainic acid (KA)-induced seizures were detected through iEEG monitoring, and the induced shunt-current was successfully measured and conducted out of the brain through the designed circuit-body path, which verifies the potential of current conduction for the treatment of epilepsy.

As a main inhibitory neurotransmitter in the central nervous system, γ-aminobutyric acid (GABA) activates chloride-permeable GABAa receptors (GABAa Rs) and induces chloride ion (Cl−) flow, which relies on the intracellular chloride concentration ([Cl−]i) of the postsynaptic neuron. The Na-K-2Cl cotransporter isoform 1 (NKCC1) and the K-Cl cotransporter isoform 2 (KCC2) are two main cation-chloride cotransporters (CCCs) that have been implicated in human epilepsy. NKCC1 and KCC2 reset [Cl−]i by accumulating and extruding Cl−, respectively. Previous studies have shown that the profile of NKCC1 and KCC2 in neonatal neurons may reappear in mature neurons under some pathophysiological conditions, such as epilepsy. Although increasing studies focusing on the expression of NKCC1 and KCC2 have suggested that impaired chloride plasticity may be closely related to epilepsy, additional neuroelectrophysiological research aimed at studying the functions of NKCC1 and KCC2 are needed to understand the exact mechanism by which they induce epileptogenesis. In this review, we aim to briefly summarize the current researches surrounding the expression and function of NKCC1 and KCC2 in epileptogenesis and its implications on the treatment of epilepsy. We will also explore the potential for NKCC1 and KCC2 to be therapeutic targets for the development of novel antiepileptic drugs.

Objectives: Patients with tuberous sclerosis complex (TSC) present multiple cortical tubers in the brain, which are responsible for epilepsy. It is still difficult to localize the epileptogenic tuber. The value of cortico-cortical evoked potentials (CCEPs) was assessed in epileptogenic tuber localization in patients with TSC using stereo-electroencephalography (SEEG).Methods: Patients with TSC who underwent SEEG and CCEP examination in preoperative evaluation during 2014–2017 and reached postoperative seizure freedom at 1-year follow-up were enrolled in this study (n = 11). CCEPs were conducted by stimulating every two adjacent contacts of SEEG electrodes and recording on other contacts of SEEG electrodes in one epileptogenic tuber and its early-stage propagating tuber, and their perituberal cortexes in each patient. The CCEP was defined as positive when N1 and/or N2 wave presented, and then the occurrence rates of positive CCEPs were then compared among different tubers and perituberal regions.Results: Occurrence rates of positive CCEP from epileptogenic tubers to early propagating tubers and epileptogenic tubers to perituberal cortexes were 100%, which were significantly higher than the occurrence rates of CCEP between other locations. The occurrence rates of CCEP from peripheral portions of epileptogenic tubers to peripheral portions of early propagating tubers or perituberal cortexes were 100%, which were significant higher than the occurrence rates of CCEP from peripheral regions of early propagating tubers to peripheral portions of epileptogenic tubers, from the central part of early propagating tuber to central portions of epileptogenic tubers, or from perituberal cortexes to the center part of epileptogenic tubers.Conclusion: Epileptogenic tubers presented much more diffusive connectivity with other tubers and perituberal cortexes than any other connectivity relationships across propagating tubers, and the peripheral region of epileptogenic tubers presented the greatest connectivity with propagating tubers and perituberal cortexes. CCEP can be an effective tool in epileptogenic tuber localization in patients with TSC.

Background: Deep brain stimulation (DBS) is considered an effective treatment option for Parkinson's disease (PD). Several studies have demonstrated the efficacy of neurostimulation in patients with advanced PD. The subthalamic nucleus (STN), the internal globus pallidus (GPi), ventral intermediate nucleus (Vim), and pedunculopontine nucleus (PPN) are reportedly effective DBS targets for control of Parkinsonian tremors. However, there is no consensus on the ideal target for DBS in patients with Parkinson's disease. Only a few studies have directly compared the efficacy of DBS of the Vim, STN, and GPi. Therefore, we searched PubMed, Embase, Cochrane Library, and other databases for observational studies, extracted data on unified Parkinson's disease rating scale (UPDRS) scores and performed a comprehensive network meta-analysis of different strategies of DBS and compared the efficiency of DBS at different targets.Methods: Forest plot was used to examine the overall efficiency of DBS; cumulative probability value was used to rank the strategies under examination. A node-splitting model was employed to assess consistency of reported outcomes inconsistency. A total of 16 studies which focused on UPDRS improvement were included in the network meta-analysis.Results: By comparing the overall efficiency associated with each target, we confirmed the efficacy of DBS therapy in PD. Our findings revealed similar efficacy of DBS targeted at GPi and STN in the on-medication phase [GPi-3.9 (95% CI −7.0 to −0.96); STN-3.1 (−5.9 to −0.38)]; however, in the off-medication phase, Vim-targeted DBS was associated with better improvement in UPDRS scores and could be a choice as a DBS target for tremor-dominant Parkinsonism.Conclusions: Our findings will help improve clinical application of DBS.

To analyze the incidence of epilepsy in adult patients with supratentorial glioblastoma, assess the factors influencing the development of epilepsy in these cases, and evaluate patients' response to antiepileptic drugs (AEDs) in a series of 184 patients.We retrospectively analyzed the 184 adult patients diagnosed with supratentorial glioblastoma. All subjects were treated within our hospital and subsequently died between 2003 and 2013. The incidence of epilepsy was assessed before and after initial resection and reexamined every 2 months thereafter. We evaluated the efficacy of prophylactic AEDs in this patient population based on the gathered incidence data.Of 184 patients, 43 (23.37%) were diagnosed with epilepsy before their initial resection. The total incidence of epilepsy (both pre- and postoperative) was 68.48%. The prevalence of active epilepsy reached over 80% in patients with epilepsy and survival of greater than 13 months postoperatively. Patients with glioblastoma in the frontal and/or temporal lobes had a higher prevalence of epilepsy. In the 43 patients with preoperative epilepsy, total resection of glioblastoma resulted in significantly lower seizure frequency. Patients who received epilepsy prophylaxis with AEDs for at least 6 months had significantly fewer seizures and higher Karnofsky scores than those receiving AEDs for less than one month or not at all.The incidence of epilepsy in adult patients with glioblastoma was high and responded poorly to AEDs in the short term. However, when taken for longer periods, AEDs can reduce the frequency of seizures in patients with glioblastoma.

Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT) from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), guaiacol, and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0-11.0 and thermostable at 40°C-90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.

Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.

PURPOSE: To examine the effects of current shunt on rats with temporal lobe epilepsy and neocortex epilepsy. EXPERIMENTAL DESIGN: A kainic acid (KA)-induced model of temporal lobe seizure and a penicillin-induced model of neocortical partial seizure were used in this study. Rats of each model were randomly allocated into two groups: control and model groups. The model group was further divided into the KA or penicillin group, sham conduction group and conduction group. The current shunt was realized through the implantation of a customized conduction electrode. After surgery, electroencephalogram (EEG) was recorded for two hours for each rat under anesthesia. Subsequently, the rats were video monitored for 72 h to detect the occurrence of behavioral seizures upon awakening. The average number and duration of seizures on EEG and the number of behavioral seizures were measured. RESULTS: In KA model, the number of total EEG seizures in conduction group (9.57±2.46) was significantly less than that in sham conduction group (15.13±3.45) (p

OBJECTIVE: To perform a systematic review and individual participant data meta-analysis to identify preoperative factors associated with a good seizure outcome in children with Tuberous Sclerosis Complex undergoing resective epilepsy surgery. DATA SOURCES: Electronic databases (MEDLINE, EMBASE, CINAHL and Web of Science), archives of major epilepsy and neurosurgery meetings, and bibliographies of relevant articles, with no language or date restrictions. STUDY SELECTION: We included case-control or cohort studies of consecutive participants undergoing resective epilepsy surgery that reported seizure outcomes. We performed title and abstract and full text screening independently and in duplicate. We resolved disagreements through discussion. DATA EXTRACTION: One author performed data extraction which was verified by a second author using predefined data fields including study quality assessment using a risk of bias instrument we developed. We recorded all preoperative factors that may plausibly predict seizure outcomes. DATA SYNTHESIS: To identify predictors of a good seizure outcome (i.e. Engel Class I or II) we used logistic regression adjusting for length of follow-up for each preoperative variable. RESULTS: Of 9863 citations, 20 articles reporting on 181 participants were eligible. Good seizure outcomes were observed in 126 (69%) participants (Engel Class I: 102(56%); Engel class II: 24(13%)). In univariable analyses, absence of generalized seizure semiology (OR = 3.1, 95%CI = 1.2-8.2, p = 0.022), no or mild developmental delay (OR = 7.3, 95%CI = 2.1-24.7, p = 0.001), unifocal ictal scalp electroencephalographic (EEG) abnormality (OR = 3.2, 95%CI = 1.4-7.6, p = 0.008) and EEG/Magnetic resonance imaging concordance (OR = 4.9, 95%CI = 1.8-13.5, p = 0.002) were associated with a good postoperative seizure outcome. CONCLUSIONS: Small retrospective cohort studies are inherently prone to bias, some of which are overcome using individual participant data. The best available evidence suggests four preoperative factors predictive of good seizure outcomes following resective epilepsy surgery. Large long-term prospective multicenter observational studies are required to further evaluate the risk factors identified in this review.

BACKGROUND: MicroRNAs (miRNAs) are important regulators that play key roles in tumorigenesis and tumor progression. A previous report has shown that let-7 family members can act as tumor suppressors in many cancers. Through miRNA array, we found that let-7f was downregulated in the highly metastatic potential gastric cancer cell lines GC9811-P and SGC7901-M, when compared with their parental cell lines, GC9811 and SGC7901-NM; however, the mechanism was not clear. In this study, we investigate whether let-7f acts as a tumor suppressor to inhibit invasion and metastasis in gastric cancers. METHODOLOGY/PRINCIPAL: Real-time PCR showed decreased levels of let-7f expression in metastatic gastric cancer tissues and cell lines that are potentially highly metastatic. Cell invasion and migration were significantly impaired in GC9811-P and SGC7901-M cell lines after transfection with let-7f-mimics. Nude mice with xenograft models of gastric cancer confirmed that let-7f could inhibit gastric cancer metastasis in vivo after transfection by the lentivirus pGCsil-GFP- let-7f. Luciferase reporter assays demonstrated that let-7f directly binds to the 3'UTR of MYH9, which codes for myosin IIA, and real-time PCR and Western blotting further indicated that let-7f downregulated the expression of myosin IIA at the mRNA and protein levels. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated that overexpression of let-7f in gastric cancer could inhibit invasion and migration of gastric cancer cells through directly targeting the tumor metastasis-associated gene MYH9. These data suggest that let-7f may be a novel therapeutic candidate for gastric cancer, given its ability to reduce cell invasion and metastasis.