Comparative effectiveness of exercise on cardiorespiratory function or exercise efficiency after stroke: A network meta-analysis of randomized control trials.

OBJECTIVE: The network meta-analysis (NMA) was to compare and rank the effectiveness of different exercises on cardiorespiratory function or exercise efficiency in post-stroke patients. DESIGN: A NMA of randomized controlled trials (RCTs) was conducted. PubMed, Embase, Cochrane Library, and Web of Science were searched. The impact of exercises including individual and combination of aerobic exercise (AE), resistance exercise (RE), task-oriented training (TOT), gait training (GT), breathing exercise (BE), and regular rehabilitation training (RRT) on 6-min walk test (6MWT), peak oxygen consumption (VO2peak), maximum oxygen consumption (VO2max), resting heart rate (HRrest), resting systolic blood pressure (SBPrest), and resting diastolic blood pressure (DBPrest) were assessed. RESULTS: In total, 36 studies were included in the meta-analysis. AEGT (AE + GT) (63.06%) had the highest likelihood of improving 6MWT performance in post-stroke patients. RRTRE (RRT + RE) was the most favourable exercise in terms of 6MWT performance assessing by minimum clinically significant difference (MCID). AERE (AE + RE) had the highest likelihood of improving VO2peak and reducing HRrest in post-stroke patients. CONCLUSION: Different types of exercise demonstrated the benefits of improving cardiorespiratory function in stroke patients. Further research is needed to determine the best exercise regimen to maximize the benefits of rehabilitation interventions for post-stroke patients.

Electroacupuncture attenuates middle cerebral artery occlusion-induced learning and memory impairment by regulating microglial polarization in hippocampus.

BACKGROUND: As a traditional medical therapy, electroacupuncture (EA) has been demonstrated to have beneficial effects on ischemic stroke-induced cognitive impairment. However, the underlying mechanism is largely unclear. METHODS: Adult rats received occlusion of the middle cerebral artery and reperfusion (MCAO/R) to establish the ischemic stroke model. Morris water maze test was performed following EA stimulation at the GV20, PC6, and KI1 acupoints in rats to test the learning and memory ability. Western blot, immunofluorescent staining, and enzyme-linked immunosorbent assay were conducted to assess the cellular and molecular mechanisms. RESULTS: EA stimulation attenuated neurological deficits. In the Morris water maze test, EA treatment ameliorated the MCAO/R-induced learning and memory impairment. Moreover, we observed that MCAO/R induced microglial activation and polarization in the ischemic hippocampus, whereas, EA treatment dampened microglial activation and inhibited M1 microglial polarization but enhanced M2 microglial polarization. EA treatment inhibited the increased expression of proinflammatory cytokines and enhanced the increased expression of anti-inflammatory cytokines. Finally, we found that EA treatment dampened microglial p38 mitogen-activated protein kinase (MAPK) phosphorylation. CONCLUSION: Collectively, our data suggested that EA treatment ameliorated cognitive impairment induced by MCAO/R and the underlying mechanism may be p38-mediated microglia polarization and neuroinflammation.

In silico and in vitro analyses of a novel FoxO1 agonist reducing Aß levels via downregulation of BACE1.

AIMS: FoxO1 is an important target in the treatment of Alzheimer's disease (AD). However, FoxO1-specific agonists and their effects on AD have not yet been reported. This study aimed to identify small molecules that upregulate the activity of FoxO1 to attenuate the symptoms of AD. METHODS: FoxO1 agonists were identified by in silico screening and molecular dynamics simulation. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were used to assess protein and gene expression levels of P21, BIM, and PPARγ downstream of FoxO1 in SH-SY5Y cells, respectively. Western blotting and enzyme-linked immunoassays were performed to explore the effect of FoxO1 agonists on APP metabolism. RESULTS: N-(3-methylisothiazol-5-yl)-2-(2-oxobenzo[d]oxazol-3(2H)-yl) acetamide (compound D) had the highest affinity for FoxO1. Compound D activated FoxO1 and regulated the expression of its downstream target genes, P21, BIM, and PPARγ. In SH-SY5Y cells treated with compound D, BACE1 expression levels were downregulated, and the levels of Aß1-40 and Aß1-42 were also reduced. CONCLUSIONS: We present a novel small-molecule FoxO1 agonist with good anti-AD effects. This study highlights a promising strategy for new drug discovery for AD.

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells.

Recombinant antibodies are rapidly developing therapeutic agents; approximately 40 novel antibody molecules enter clinical trials each year, most of which are produced from Chinese hamster ovary (CHO) cells. However, one of the major bottlenecks restricting the development of antibody drugs is how to perform high-level expression and production of recombinant antibodies. The high-efficiency expression and quality of recombinant antibodies in CHO cells is determined by multiple factors. This review provides a comprehensive overview of several state-of-the-art approaches, such as optimization of gene sequence of antibody, construction and optimization of high-efficiency expression vector, using antibody expression system, transformation of host cell lines, and glycosylation modification. Finally, the authors discuss the potential of large-scale production of recombinant antibodies and development of culture processes for biopharmaceutical manufacturing in the future.

Notoginsenoside R1, An Active Compound from Panax notoginseng, Inhibits Hepatic Stellate Cell Activation and Liver Fibrosis via MAPK Signaling Pathway.

Activation of the hepatic stellate cell is implicated in pathological vascularization during development of liver fibrosis. MAPK signaling is involved in the activation of hepatic stellate cell. Oxidative stress and inflammation are also involved in the pathogenesis of liver fibrosis. Notoginsenoside R1 is an effective saponin isolated from the roots of Panax notoginseng (Burk) F. H. Chen and exerts anti-oxidant, anti-inflammatory and anti-fibrotic roles in various diseases. However, the role of Notoginsenoside R1 in liver fibrosis has not been investigated yet. First, a rat model with liver fibrosis was established through oral gavage administration with carbon tetrachloride. Data from hematoxylin and eosin (H&E) and Masson's trichrome stainings showed that carbon tetrachloride induced severe hepatic damages, including inflammatory cell infiltration, lipid droplets deposition in hepatocytes and liver centrilobular necrosis. Meanwhile, the rats were also intraperitoneal injected with different concentrations of Notoginsenoside R1. Results demonstrated that Notoginsenoside R1 treatment suppressed the pathological changes in the livers with enhanced levels of ALB and TP, and reduced levels of ALP, AST and ALT. Second, Notoginsenoside R1 also significantly attenuated carbon tetrachloride-induced decrease in PPAR-[Formula: see text] and increase in Coll-a1, [Formula: see text]-SMA and TIMP1 in liver tissues ([Formula: see text][Formula: see text] 0.001). Third, the decrease in GSH, SOD and GST and increase in MDA, IL-1[Formula: see text], IL-6 and TNF-[Formula: see text] induced by carbon tetrachloride were markedly restored by Notoginsenoside R1 ([Formula: see text][Formula: see text] 0.001). Lastly, Notoginsenoside R1 counteracted with the promotive effects of carbon tetrachloride on levels of proteins involved in MAPK signaling, including phosphorylated p65 (p-p65), p-ERK, p-JNK and p-p38. In conclusion, Notoginsenoside R1 suppressed the activation of hepatic stellate cells and exerted anti- oxidant and anti-inflammatory to attenuate carbon tetrachloride-induced liver fibrosis through inactivation of NF-[Formula: see text]B and MAPK signaling.

MiR-125b Inhibits Cell Proliferation and Induces Apoptosis in Human Colon Cancer SW480 Cells via Targeting STAT3.

BACKGROUND: Colon cancer is one of the most common types of cancer worldwide. Multiple studies have unveiled the key role of microRNAs (miRNAs) in the development of various types of cancer. However, the mechanism of action of miR-125b in the development and progression of colon cancer remains unknown. OBJECTIVES: In this study, we explored the association of miR-125b and signal transducer and activator of transcription 3 (STAT3) and its role in the proliferation and apoptosis of SW480 colon cancer cells. METHODS: The miR-125b expression in NCM460, SW480, HT29, and HCT8 cells was detected using quantitative real-time polymerase chain reaction (qRT-PCR). SW480 cells were transfected with lentiviruses of GFP-miR-125b and GFP-NC to establish a stable miR-125b overexpression colon cancer cell model and a control model. The targeting relationship between miR-125b and STAT3 was analyzed using bioinformatics and verified by the dual-luciferase reporter gene assay. Cell proliferation and apoptosis were assessed using the Cell Counting Kit-8 assay and TUNEL staining. The expression levels of STAT3, Bcl-2, and Bax were analyzed using Western blot analysis. RESULTS: It was found that the relative mRNA expression of miR-125b was decreased in SW480, HT29, and HCT8 cells compared with that in NCM460 cells (P<0.05). The luciferase reporter gene assay confirmed that miR-125b downregulated the STAT3 gene expression (P<0.05). Overexpression of miR-125b inhibited proliferation and promoted apoptosis in SW480 colon cancer cells and was accompanied by upregulated Bax expression and downregulated Bcl-2 expression (P<0.05). Re-expression of STAT3 promoted cell proliferation and inhibited cell apoptosis, whereas Bcl-2 expression increased, and Bax expression decreased (P<0.05). CONCLUSION: The miR-125b regulates the expression of Bax and Bcl-2 by downregulating the expression of STAT3, thereby inhibiting proliferation and inducing apoptosis of SW480 colon cancer cells.

Lysophosphatidylcholines and phosphatidylcholines as biomarkers for stroke recovery.

Stroke is a serious global public health issue, associated with severe disability and high mortality rates. Its early detection is challenging, and no effective biomarkers are available. To obtain a better understanding of stroke prevention, management, and recovery, we conducted lipidomic analyses to characterize plasma metabolic features. Lipid species were measured using an untargeted lipidomic analysis with liquid chromatography-tandem mass spectrometry. Sixty participants were recruited in this cohort study, including 20 healthy individuals and 40 patients with stroke. To investigate the association between lipids related to long-term functional recovery in stroke patients. The primary independent variable was activities of daily living (ADL) dependency upon admission to the stroke unit and at the 3-month follow-up appointment. ADL dependency was assessed using the Barthel Index. Eleven significantly altered lipid species between the stroke and healthy groups were detected and displayed in a hierarchically clustered heatmap. Acyl carnitine, triacylglycerol, and ceramides were detected as potential lipid markers. Regarding the association between lipid profiles and functional status of patients with stroke the results indicated, lysophosphatidylcholines (LPC) and phosphatidylcholines were closely associated with stroke recovery. LPC may contribute positively role in patient's rehabilitation process via an anti-inflammatory mechanism. Appropriate management or intervention for lipid levels is expected to lead to better clinical outcomes.

End-to-End Automatic Pronunciation Error Detection Based on Improved Hybrid CTC/Attention Architecture.

Advanced automatic pronunciation error detection (APED) algorithms are usually based on state-of-the-art automatic speech recognition (ASR) techniques. With the development of deep learning technology, end-to-end ASR technology has gradually matured and achieved positive practical results, which provides us with a new opportunity to update the APED algorithm. We first constructed an end-to-end ASR system based on the hybrid connectionist temporal classification and attention (CTC/attention) architecture. An adaptive parameter was used to enhance the complementarity of the connectionist temporal classification (CTC) model and the attention-based seq2seq model, further improving the performance of the ASR system. After this, the improved ASR system was used in the APED task of Mandarin, and good results were obtained. This new APED method makes force alignment and segmentation unnecessary, and it does not require multiple complex models, such as an acoustic model or a language model. It is convenient and straightforward, and will be a suitable general solution for L1-independent computer-assisted pronunciation training (CAPT). Furthermore, we find that find that in regards to accuracy metrics, our proposed system based on the improved hybrid CTC/attention architecture is close to the state-of-the-art ASR system based on the deep neural network-deep neural network (DNN-DNN) architecture, and has a stronger effect on the F-measure metrics, which are especially suitable for the requirements of the APED task.

Establishment of a DGKθ Endogenous Promoter Luciferase Reporter HepG2 Cell Line for Studying the Transcriptional Regulation of DGKθ Gene.

DGKθ protein expression levels are closely related to the development of diseases including diabetes, cancer, and neuronal disease. To investigate the transcriptional regulation of the DGKθ gene, we used CRISPR/Cas9 to generate a DGKθ endogenous promoter luciferase reporter HepG2 cell line, in which the endogenous DGKθ promoter controls the expression of the luciferase reporter gene. To test the cell line, FXR, the transcription factor for upregulating the expression of DGKθ gene, was used to validate the cell line. Furthermore, the reported agonists for the expression of DGKθ, cAMP and GW4064, the known inhibitor for DGKθ enzyme activity, R59949, and a potential regulator for DGKθ enzyme expression, EGCG (the major catechin in green tea), were applied to the reporter cell line. The results indicated that these reagents could significantly regulate the expression of reporter luciferase. Finally, four transcription factors (E2F1, c-Myc, USF1, and Bmal1) potentially binding to the DGKθ gene's upstream promoter region were tested. DGKθ expression was upregulated by c-Myc and downregulated by E2F1, which was also confirmed in wild-type HepG2 cells. We found that the cell line's luciferase activity was directly correlated with DGKθ endogenous promoter activity, suggesting that it is liable and sensitive for studying DGKθ transcriptional regulation. The study provides a useful tool for high-throughput drug screening for the treatment of DGKθ-involved diseases.

Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system.

The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.

Generation of apoptosis-resistant HEK293 cells with CRISPR/Cas mediated quadruple gene knockout for improved protein and virus production.

Apoptosis has important functions during pathophysiologic processes. However, from a biopharmaceutical point of view, active apoptosis of host cells is undesirable during viral packaging or protein expression, because it decreases the efficiency of viral or protein production. Here we used the CRISPR/Cas technique to knock out four pro-apoptotic genes, Caspase3, Caspase6, Caspase7 and AIF1, in HEK293 cells, and successfully produced an apoptosis-resistant cell line. Furthermore, this cell line showed higher expression levels of pro-apoptotic proteins and higher packaging efficiency for the virus carrying these proteins than control HEK293 cells. This study not only produced an apoptosis-resistant cell line that is useful in producing apoptosis-inducing proteins or viruses expressing these proteins, but also provides a methodology to build other apoptosis-resistant cell lines.

Combined venomics, antivenomics and venom gland transcriptome analysis of the monocoled cobra (Naja kaouthia) from China.

We conducted an omics-analysis of the venom of Naja kaouthia from China. Proteomics analysis revealed six protein families [three-finger toxins (3-FTx), phospholipase A2 (PLA2), nerve growth factor, snake venom metalloproteinase (SVMP), cysteine-rich secretory protein and ohanin], and venom-gland transcriptomics analysis revealed 28 protein families from 79 unigenes. 3-FTx (56.5% in proteome/82.0% in transcriptome) and PLA2 (26.9%/13.6%) were identified as the most abundant families in venom proteome and venom-gland transcriptome. Furthermore, N. kaouthia venom expressed strong lethality (i.p. LD50: 0.79µg/g) and myotoxicity (CK: 5939U/l) in mice, and showed notable activity in PLA2 but weak activity in SVMP, l-amino acid oxidase or 5' nucleotidase. Antivenomic assessment revealed that several venom components (nearly 17.5% of total venom) from N. kaouthia could not be thoroughly immunocaptured by commercial Naja atra antivenom. ELISA analysis revealed that there was no difference in the cross-reaction between N. kaouthia and N. atra venoms against the N. atra antivenom. The use of commercial N. atra antivenom in treatment of snakebites caused by N. kaouthia is reasonable, but design of novel antivenom with the attention on enhancing the immune response of non-immunocaptured components should be encouraged. BIOLOGICAL SIGNIFICANCE: The venomics, antivenomics and venom-gland transcriptome of the monocoled cobra (Naja kaouthia) from China have been elucidated. Quantitative and qualitative differences are evident when venom proteomic and venom-gland transcriptomic profiles are compared. Two protein families (3-FTx and PLA2) are found to be the predominated components in N. kaouthia venom, and considered as the major players in functional role of venom. Other protein families with relatively low abundance appear to be minor in the functional significance. Antivenomics and ELISA evaluation reveal that the N. kaouthia venom can be effectively immunorecognized by commercial N. atra antivenom, but still a small number of venom components could not be thoroughly immunocaptured. The findings indicate that exploring the precise composition of snake venom should be executed by an integrated omics-approach, and elucidating the venom composition is helpful in understanding composition-function relationships and will facilitate the clinical application of antivenoms.

Proteomic characterization and comparison of venoms from two elapid snakes (Bungarus multicinctus and Naja atra) from China.

Bungarus multicinctus (many-banded krait) and Naja atra (Chinese cobra) are widely distributed and medically important venomous snakes in China; however, their venom proteomic profiles have not been fully compared. Here, we fractionated crude venoms and analyzed them using a combination of proteomic techniques. Three-finger toxins (3-FTx) and phospholipase A2 (PLA2) were most abundant in both species, respectively accounting for 32.6% and 66.4% of total B. multicinctus venom, and 84.3% and 12.2% of total N. atra venom. Venoms from these two species contained one common protein family and six less abundant species-specific protein families. The proteomic profiles of B. multicinctus and N. atra venoms and analysis of toxicological activity in mice suggested that 3-FTx and PLA2 are the major contributors to clinical symptoms caused by envenomation. The venoms differed in enzymatic activity, likely the result of inter-specific variation in the amount of related venom components. Antivenomics assessment revealed that a small number of venom components (3-FTxs and PLA2s in B. multicinctus, and 3-FTxs in N. atra) could not be immunocaptured completely, suggesting that we should pay attention to enhancing the immune response of these components in designing commercial antivenoms for B. multicinctus and N. atra. BIOLOGICAL SIGNIFICANCE: The proteomic profiles of venoms from two medically important snake species - B. multicinctus and N. atra - have been explored. Quantitative and qualitative differences are evident in both venoms when proteomic profiles and transcriptomic results are compared; this is a reminder that combined approaches are needed to explore the precise composition of snake venom. Two protein families (3-FTx and PLA2) of high abundance in these snake venoms are major players in the biochemical and pharmacological effects of envenomation. Elucidation of the proteomic profiles of these snake venoms is helpful in understanding composition-function relationships and will facilitate the clinical application of antivenoms.

A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene.

Sox2 is an important transcriptional factor that has multiple functions in stem cell maintenance and tumorigenesis. To investigate the transcriptional regulation of the Sox2 gene, a luciferase knock-in reporter system was established in HEK293 cells by placing the luciferase gene in the genome under the control of the Sox2 gene promoter using a transcription activator-like effector nuclease (TALEN)-mediated genome editing technique. PCR and Southern blot results confirmed the site-specific integration of a single copy of the exogenous luciferase gene into the genome. To prove the reliability and sensitivity of this novel luciferase knock-in system, a CRISPR/Cas transcription activation system for the Sox2 gene was constructed and applied to the knock-in system. The results indicated that luciferase activity was directly correlated with the activity of the Sox2 endogenous promoter. This novel system will be a useful tool to study the transcriptional regulation of Sox2, and has great potential in medical and industrial applications.

Mifepristone modulates serotonin transporter function.

Regulating serotonin expression can be used to treat psychotic depression. Mifepristone, a glucocorticoid receptor antagonist, is an effective candidate for psychotic depression treatment. However, the underlying mechanism related to serotonin transporter expression is poorly understood. In this study, we cloned the human brain serotonin transporter into Xenopus oocytes, to establish an in vitro expression system. Two-electrode voltage clamp recordings were used to detect serotonin transporter activity. Our results show that mifepristone attenuates serotonin transporter activity by directly inhibiting the serotonin transporter, and suggests that the serotonin transporter is a pharmacological target of mifepristone for the treatment of psychotic depression.

Stable cell line of human SH-SY5Y uniformly expressing TWIK-related acid-sensitive potassium channel and eGFP fusion.

TWIK-related acid-sensitive potassium channels (TASK3) are pharmacological targets of CNS inflammation induced by acidification. They function as molecular switches between survival and death of neurons. In this report, TASK3 cloned from human brain cDNA was tagged with enhanced green fluorescent protein (eGFP), and the fusion gene was transiently expressed in human neuroblastoma SH-SY5Y cells. A cell line stably expressing TASK-eGFP fusion proteins was generated from transient expression cells by using fluorescence-activated cell sorting followed by antibiotic selection. The uniform expression of TASK3 fusion proteins was further confirmed by flow cytometry. Moreover, the localization of TASK3 tagged with eGFP was checked by confocal microcopy. TASK3-eGFP fusion proteins are observed on the SH-SY5Y cell membrane. The strategies using eGFP as a fusion tag facilitate the monitoring of the TASK3 expression and enable the successful employment of FACS for screening and construction of cell lines stably expressing TASK3. The TASK3 overexpression cell line will lay a fundamental for the in vitro evaluation of TASK3 function during hypoxic/ischemic injury.

Estradiol inhibits the activity of proton-coupled amino acid transporter PAT1 expressed in Xenopus oocytes.

Estrogen has great potential as a therapeutic agent in focal ischemic brain injury. Amino acids as energy resources and neurotransmitters in the central nervous system are crucial for proper neuronal function and excitability. The proton-coupled amino acid transporter PAT1 has clear potential in drug absorption. In this paper, human brain PAT1 was cloned and expressed in Xenopus oocytes. The effects of estradiol on the activity of PAT1 were investigated. Glycine-induced membrane currents mediated by PAT1 were measured using the two-electrode voltage clamp technique. The amplitude of the glycine-elicited current was decreased progressively with increasing concentrations of ß-estradiol. A concentration-dependent outwards current of PAT1 was also detected by the presence of ß-estradiol. We conclude that estrogen attenuates the activity of PAT1 by directly closing PAT1 channel. Our results may provide an additional mechanism for estrogen on neurotransmission and neuronal metabolism during ischemic injury.