Effect of ST36 electroacupuncture on the switch of skeletal muscle fibres in mice with sciatic nerve dissociation.

Skeletal muscle is striated muscle that moves autonomously and is innervated by peripheral nerves. Peripheral nerve injury is very common in clinical treatment. However, the commonly used treatment methods often focus on the regeneration of the injured nerve but overlook the pathological changes in the injured skeletal muscle. Acupuncture, as the main treatment for denervated skeletal muscle atrophy, is used extensively in clinical practice. In the present study, a mouse model of lower limb sciatic nerve detachment was constructed and treated with electroacupuncture Stomach 36 to observe the atrophy of lower limb skeletal muscle and changes in skeletal muscle fibre types before and after electroacupuncture Stomach 36 treatment. Mice with skeletal muscle denervation showed a decrease in the proportion of IIa muscle fibres and an increase in the proportion of IIb muscle fibres, after electroacupuncture Stomach 36. The changes were reversed by specific activators of p38 MAPK, which increased IIa myofibre ratio. The results suggest that electroacupuncture Stomach 36 can reverse the change of muscle fibre type from IIb to IIa after denervation of skeletal muscle by inhibiting p38 MAPK. The results provide an important theoretical basis for the treatment of clinical peripheral nerve injury diseases with electroacupuncture, in addition to novel insights that could facilitate the study of pathological changes of denervated skeletal muscle.

The Impact of Dysregulated microRNA Biogenesis Machinery and microRNA Sorting on Neurodegenerative Diseases.

MicroRNAs (miRNAs) are 22-nucleotide noncoding RNAs involved in the differentiation, development, and function of cells in the body by targeting the 3'- untranslated regions (UTR) of mRNAs for degradation or translational inhibition. miRNAs not only affect gene expression inside the cells but also, when sorted into exosomes, systemically mediate the communication between different types of cells. Neurodegenerative diseases (NDs) are age-associated, chronic neurological diseases characterized by the aggregation of misfolded proteins, which results in the progressive degeneration of selected neuronal population(s). The dysregulation of biogenesis and/or sorting of miRNAs into exosomes was reported in several NDs, including Huntington's disease (HD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD). Many studies support the possible roles of dysregulated miRNAs in NDs as biomarkers and therapeutic treatments. Understanding the molecular mechanisms underlying the dysregulated miRNAs in NDs is therefore timely and important for the development of diagnostic and therapeutic interventions. In this review, we focus on the dysregulated miRNA machinery and the role of RNA-binding proteins (RBPs) in NDs. The tools that are available to identify the target miRNA-mRNA axes in NDs in an unbiased manner are also discussed.

Use of Artificial Intelligence to Improve the Calculation of Percent Adhesion for Transdermal and Topical Delivery Systems.

Adhesion is a critical quality attribute and performance characteristic for transdermal and topical delivery systems (TDS). Regulatory agencies recommend in vivo skin adhesion studies to support the approval of TDS in both new drug applications and abbreviated new drug applications. The current assessment approach in such studies is based on the visual observation of the percent adhesion, defined as the ratio of the area of TDS attached to the skin to the total area of the TDS. Visually estimated percent adhesion by trained clinicians or trial participants creates variability and bias. In addition, trial participants are typically confined to clinical centers during the entire product wear period, which may lead to challenges when translating adhesion performance to the real world setting. In this work we propose to use artificial intelligence and mobile technologies to aid and automate the collection of photographic evidence and estimation of percent adhesion. We trained state-of-art deep learning models with advanced techniques and in-house curated data. Results indicate good performance from the trained models and the potential use of such models in clinical practice is further explored.

A novel splice site variant in the POPDC3 causes autosomal recessive limb-girdle muscular dystrophy type 26.

Limb-Girdle muscular dystrophy (LGMD) is a group of muscle disorders with highly heterogeneous genetic patterns and clinical phenotypes, and this group includes multiple subtypes. Different LGMD subtypes have similar phenotypes and clinical overlaps, these subtypes are difficult to distinguish by clinical symptoms alone and can only be accurately diagnosed by analysis in combination with definitive genetic test results. Here, we report a female presenting features of LGMD. After analysis of whole-exome sequencing data, a novel homozygous POPDC3 variant c.486-1G>A (rs113419658) located in the acceptor splice site of intron 2 was identified in the proband. The variant effect on splicing were analyzed by genetic analysis based on cDNA synthesized by the patient's RNA. cDNA analysis indicated that the novel homozygous POPDC3 splice variant disrupted original acceptor splice site, which can cause a frameshift in the mRNA of the POPDC3 gene, thereby producing a truncated POPDC3 protein and ultimately affecting its normal function. POPDC3 variant was recently associated with recessive limb-girdle muscular dystrophy type 26 (LGMDR26). Based on the above results, we hypothesize that this variant is probably a pathogenic variant, and expand the gene variant spectrum of POPDC3.

TRAX Provides Neuroprotection for Huntington's Disease Via Modulating a Novel Subset of MicroRNAs.

BACKGROUND: Huntington's disease (HD) is a neurodegenerative disease caused by CAG-repeat expansions (>36) in exon 1 of HTT, which dysregulates multiple cellular machineries. Translin-associated protein X (TRAX) is a scaffold protein with diverse functions, including suppressing the microRNA (miRNA)-mediated silencing by degrading pre-miRNA. To date, the role of TRAX in neurodegenerative diseases remains unknown. OBJECTIVES: We delineated the role of TRAX upregulation during HD progression. METHODS: Expression of TRAX in the brains of humans and three mouse models with HD were analyzed by immunohistochemistry staining, western blot, and quantitative reverse transcription-polymerase chain reaction. Adeno-associated viruses harboring TRAX short hairpin RNA were intrastriatally injected into HD mice to downregulate TRAX. HD-like symptoms were analyzed by behavioral and biochemical assessments. The miRNA-sequencing and RNA-sequencing analyses were used to identify the TRAX- regulated miRNA-messenger RNA (mRNA) axis during HD progression. The identified gene targets were validated biochemically in mouse and human striatal cells. RESULTS: We discovered that TRAX was upregulated in the brains of HD patients and three HD mouse models. Downregulation of TRAX enhanced 83 miRNAs (including miR-330-3p, miR-496a-3p) and subsequently changed the corresponding mRNA networks critical for HD pathogenesis (eg, DARPP-32 and brain-derived neurotrophic factor). Disruption of the TRAX-mediated miRNA-mRNA axis accelerated the progression of HD-like symptoms, including the degeneration of motor function, accumulation of mHTT aggregates, and shortened neurite outgrowth. CONCLUSIONS: We demonstrated that TRAX upregulation is authentic and protective in HD. Our study provides a novel layer of regulation for HD pathogenesis and may lead to the development of new therapeutic strategies for HD. © 2022 International Parkinson and Movement Disorder Society.

Multiscale Physics-Informed Neural Networks for Stiff Chemical Kinetics.

In this paper, a multiscale physics-informed neural network (MPINN) approach is proposed based on the regular physics-informed neural network (PINN) for solving stiff chemical kinetic problems with governing equations of stiff ordinary differential equations (ODEs). In MPINNs, chemical species with different time scales are grouped and trained by multiple corresponding neural networks with the same structure. The adaptive weight based on a key performance indicator is assigned to each loss term when calculating the summation of loss residues. With this structure, MPINNs provide a framework to solve challenging stiff chemical kinetic problems without any stiffness-removal artifacts before training. In addition, by introducing a small number of ground truth data (GTD) points (less than 10% of the number required for residual loss calculation) and adding data loss terms into loss functions, MPINNs show superior ability to represent stiff ODE solutions at any desired time. The accuracy of MPINNs is tested with classical chemical kinetic problems, by comparing with the regular PINN and other state-of-the-art methods with special consideration for solving stiff chemical kinetic problems with PINNs. The validation results show that MPINNs can effectively avoid the influence of stiffness on neural network optimization. Compared with the traditional deep neural network only trained by GTD, MPINNs can use no data or a relatively small amount of data to achieve high-precision prediction of stiff chemical ODEs. The proposed approach is very promising for solving stiff chemical kinetics, opening up possibilities of MPINN application in different fields involving stiff chemical dynamics.

microRNA-128 mediates CB1 expression and regulates NF-KB/p-JNK axis to influence the occurrence of diabetic bladder disease.

BACKGROUND: Diabetic bladder disease is common complications of diabetes, its symptoms are diverse, can be due to different stages. In this study we investigate the mechanism of miR-128 targeting CB1 expression to mediate the occurrence of diabetic bladder disease. METHODS: Bioinformatics analysis predicts related regulatory factors of miR-128 in diabetic bladder disease. Models of diabetic bladder lesions were constructed in male SD rats by intraperitoneal injection of streptozotocin at 65 mg/kg body weight. The expression of miR-128 and CB1 mRNA in bladder tissues of each group was detected by RT-qPCR, and CB1, NF-KB, p-JNK and Bcl2 protein expression was detected by Western Blotting. We tested the function of the bladder by urodynamics, detected the pathological characteristics of the bladder tissue by HE staining, and verified the targeting relationship between miR-128 and CB1 through the prediction of the biological website, dual luciferase reporter gene assay and RIP. RESULTS: miR-128 was highly expressed in the bladder tissue of diabetic rats. Inhibition of miR-128 could improve the occurrence of diabetic bladder lesions in rats. miR-128 could target the inhibition of CB1 expression, and high expression of CB1 could antagonize miR-128 against diabetic bladder. In the diabetic bladder, miR-128 can regulate the expression of NF-KB and p-JNK through CB1 and affect the level of apoptosis. miR-128 regulates NF-KB/p-JNK through CB1, thus affecting the occurrence of diabetic bladder disease. CONCLUSION: The high expression of miR-128 can down-regulate the expression of CB1, promote the activation of NF-KB and p-JNK, increase the level of apoptosis and promote the occurrence of diabetic bladder disease.

Methods and applications of percentile estimation.

Percentile is ubiquitous in statistics and plays a significant role in the day-to-day statistical application. FDA Guidance for Industry: Assay Development for Immunogenicity Testing of Therapeutic Protein Products (2016) recommends the use of a lower confidence limit of the percentile of the negative subject population as the cut point to guarantee a pre-specified false-positive rate with high confidence. Shen proposed and compared an exact t approach with some approximated approaches. However, the exact t approach might be compromised by computational time and complexity. In this article, we proposed to use a UMOVER method as a potential alternative for percentile estimation for one application to screening and confirmatory cut point determination due to its easy implementation and similar performance to the exact t approach. The applications and performance comparison with different approaches are investigated and discussed. Furthermore, we extended the proposed method for the comparison of the percentile of the test product and percentile of the reference product followed by numerical studies.

Inactivation of APC Induces CD34 Upregulation to Promote Epithelial-Mesenchymal Transition and Cancer Stem Cell Traits in Pancreatic Cancer.

Pancreatic cancer (PC) is a highly lethal malignancy due to the cancer routinely being diagnosed late and having a limited response to chemotherapy. Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic malignant tumor, representing more than 85% of all pancreatic cancers. In the present study, we characterized the phenotypes of concomitant P53 and APC mutations in pancreatic neoplasms driven by the oncogene KRAS in genetically modified mice (GEMM). In this GEMM setting, APC haploinsufficiency coupled with P53 deletion and KRASG12D activation resulted in an earlier appearance of pancreatic intraepithelial neoplasia (PanIN) lesions and progressed rapidly to highly invasive and metastatic PDAC. Through a microarray analysis of murine PDAC cells derived from our APC-deficient PDAC model, we observed that APC loss leads to upregulated CD34 expression in PDAC. CD34 is a member of a family of single-pass transmembrane proteins and is selectively expressed in hematopoietic progenitor cells, vascular endothelial cells, interstitial precursor cells, and various interstitial tumor cells. However, the functional roles of CD34 in pancreatic cancer remain unclear. Thus, in this study, we explored the mechanisms regarding how CD34 promotes the deterioration of pancreatic malignancy. Our results demonstrated that the increased expression of CD34 induced by APC inactivation promotes the invasion and migration of PDAC cells, which may relate to PDAC metastasis in vivo. Collectively, our study provides first-line evidence to delineate the association between CD34 and the APC/Wnt pathway in PDAC, and reveals the potential roles of CD34 in PDAC progression.

GSK3ß negatively regulates TRAX, a scaffold protein implicated in mental disorders, for NHEJ-mediated DNA repair in neurons.

Translin-associated protein X (TRAX) is a scaffold protein with various functions and has been associated with mental illnesses, including schizophrenia. We have previously demonstrated that TRAX interacts with a Gsα protein-coupled receptor, the A2A adenosine receptor (A2AR), and mediates the function of this receptor in neuritogenesis. In addition, stimulation of the A2AR markedly ameliorates DNA damage evoked by elevated oxidative stress in neurons derived from induced pluripotent stem cells (iPSCs). Here, we report that glycogen synthase kinase 3 beta (GSK3ß) and disrupted-in-schizophrenia 1 (DISC1) are two novel interacting proteins of TRAX. We present evidence to suggest that the stimulation of A2AR markedly facilitated DNA repair through the TRAX/DISC1/GSK3ß complex in a rat neuronal cell line (PC12), primary mouse neurons, and human medium spiny neurons derived from iPSCs. A2AR stimulation led to the inhibition of GSK3ß, thus dissociating the TRAX/DISC1/GSK3ß complex and facilitating the non-homologous end-joining pathway (NHEJ) by enhancing the activation of a DNA-dependent protein kinase via phosphorylation at Thr2609. Similarly, pharmacological inhibition of GSK3ß by SB216763 also facilitated the TRAX-mediated repair of oxidative DNA damage. Collectively, GSK3ß binds with TRAX and negatively affects its ability to facilitate NHEJ repair. The suppression of GSK3ß by A2AR activation or a GSK3ß inhibitor releases TRAX for the repair of oxidative DNA damage. Our findings shed new light on the molecular mechanisms underlying diseases associated with DNA damage and provides a novel target (i.e., the TRAX/DISC1/GSK3ß complex) for future therapeutic development for mental disorders.

A modified Wald test for reference scaled assessment of analytical equivalence.

For the reference scaled equivalence hypothesis to reduce the deficiency of the current practice in analytical equivalence assessment, the Wald test with Constrained Maximum Likelihood Estimate (CMLE) of the standard error was proposed to improve the efficiency when the sample sizes of test and reference product lots are small, and variances are unequal. However, by using the Wald test with CMLE standard error, simulations show that the type I error rate is below the nominal significance level. We proposed the Modified Wald test with CMLE standard error by replacing the maximum likelihood estimate of reference standard deviation with the sample estimate (MWCMLE), resulting in further improvement of type I error rate and power over the Wald test with CMLE standard error. In this paper, we further compare the proposed MWCMLE method to the Exact-test-Based (EB) method and the Generalized Pivotal Quantity (GPQ) method with equal or unequal variances, or equal or unequal sample sizes of both product lots. The simulations show that the proposed MWCMLE method outperforms the other two methods in type I error rate control and power improvement.

The TRAX, DISC1, and GSK3 complex in mental disorders and therapeutic interventions.

Psychiatric disorders (such as bipolar disorder, depression, and schizophrenia) affect the lives of millions of individuals worldwide. Despite the tremendous efforts devoted to various types of psychiatric studies and rapidly accumulating genetic information, the molecular mechanisms underlying psychiatric disorder development remain elusive. Among the genes that have been implicated in schizophrenia and other mental disorders, disrupted in schizophrenia 1 (DISC1) and glycogen synthase kinase 3 (GSK3) have been intensively investigated. DISC1 binds directly to GSK3 and modulates many cellular functions by negatively inhibiting GSK3 activity. The human DISC1 gene is located on chromosome 1 and is highly associated with schizophrenia and other mental disorders. A recent study demonstrated that a neighboring gene of DISC1, translin-associated factor X (TRAX), binds to the DISC1/GSK3ß complex and at least partly mediates the actions of the DISC1/GSK3ß complex. Previous studies also demonstrate that TRAX and most of its interacting proteins that have been identified so far are risk genes and/or markers of mental disorders. In the present review, we will focus on the emerging roles of TRAX and its interacting proteins (including DISC1 and GSK3ß) in psychiatric disorders and the potential implications for developing therapeutic interventions.

MED28 Regulates Epithelial-Mesenchymal Transition Through NFκB in Human Breast Cancer Cells.

MED28, a mammalian Mediator subunit, was found highly expressed in several types of malignancy, including breast cancer. Recently, we have identified a role of MED28 in regulating both cell growth and migration in human breast cancer cells. In epithelium-derived solid tumor, migration and invasion are preceded by the progression of epithelial-mesenchymal transition (EMT) which calls for downregulation of epithelial markers as well as upregulation of mesenchymal markers, among other features. The objective of this study was to investigate a putative role of MED28 in the progression of EMT in human breast cancer cells. In fibroblast-like MDA-MB-231 cells, suppression of MED28 attenuated the mesenchymal morphology, concomitantly with a reduction of several mesenchymal biomarkers and Snail, a transcriptional repressor of E-cadherin. The suppression effect was also accompanied by downregulation of p-NFκB/p65. However, overexpression of MED28 exhibited in an opposite manner. In epithelial MCF7 cells, administration of Adriamycin®, an experimental EMT induction system, led to a mesenchyme-like appearance correlated with increased expression of MED28, p-p65, and Snail, and a reciprocal change of epithelial and mesenchymal markers. Furthermore, suppression of MED28 attenuated the experimental EMT effect and restored the original expression status of E-cadherin and MMP9 in MCF7 cells. Our data indicate that MED28 modulates the development of EMT through NFκB in human breast cancer cells, further reinforcing the significance of MED28 in the progression of breast cancer on top of its role in cell growth and migration. J. Cell. Physiol. 232: 1337-1345, 2017. © 2016 Wiley Periodicals, Inc.

Adjustment for unbalanced sample size for analytical biosimilar equivalence assessment.

Large sample size imbalance is not uncommon in the biosimilar development. At the beginning of a product development, sample sizes of a biosimilar and a reference product may be limited. Thus, a sample size calculation may not be feasible. During the development stage, more batches of reference products may be added at a later stage to have a more reliable estimate of the reference variability. On the other hand, we also need a sufficient number of biosimilar batches in order to have a better understanding of the product. Those challenges lead to a potential sample size imbalance. In this paper, we show that large sample size imbalance may increase the power of the equivalence test in an unfavorable way, giving higher power for less similar products when the sample size of biosimilar is much smaller than that of the reference product. Thus, it is necessary to make some sample size imbalance adjustments to motivate sufficient sample size for biosimilar as well. This paper discusses two adjustment methods for the equivalence test in analytical biosimilarity studies. Please keep in mind that sufficient sample sizes for both biosimilar and reference products (if feasible) are desired during the planning stage.

Wald tests for variance-adjusted equivalence assessment with normal endpoints.

Equivalence tests may be tested with mean difference against a margin adjusted for variance. The justification of using variance adjusted non-inferiority or equivalence margin is for the consideration that a larger margin should be used with large measurement variability. However, under the null hypothesis, the test statistic does not follow a t-distribution or any well-known distribution even when the measurement is normally distributed. In this study, we investigate asymptotic tests for testing the equivalence hypothesis. We apply the Wald test statistic and construct three Wald tests that differ in their estimates of variances. These estimates of variances include the maximum likelihood estimate (MLE), the uniformly minimum variance unbiased estimate (UMVUE), and the constrained maximum likelihood estimate (CMLE). We evaluate the performance of these three tests in terms of type I error rate control and power using simulations under a variety of settings. Our empirical results show that the asymptotic normalized tests are conservative in most settings, while the Wald tests based on ML- and UMVU-method could produce inflated significance levels when group sizes are unequal. However, the Wald test based on CML-method provides an improvement in power over the other two Wald tests for medium and small sample size studies.

Diastereoselective Synthesis of Fluorine-Containing Pyrrolizidines via Triphenylcarbenium Tetrafluoroborate-Promoted Carbofluorination of N-3-Arylpropargylpyrrolidine-Tethered Tertiary Allylic Alcohols.

Inexpensive and air stable triphenylcarbenium tetrafluoroborate efficiently promoted the carbofluorination of N-arylpropargylpyrrolidines bearing a tertiary allylic alcohol tether at the 2-position of the pyrrolidine ring to provide 1-isobutenyl-2-(fluoro(phenyl)methylenylhexahydro-1H-pyrrolizidines in a stereoselective fashion. When subjected to bis(trifluoromethane)sulfonamide, the same substrates underwent cycloisomerization reaction within minutes to generate 1-isobutenyl-2-benzoylhexahydro-1H-pyrrolizidines with excellent stereoselectivity.

Downregulation of proteins involved in the endoplasmic reticulum stress response and Nrf2-ARE signaling in lymphoblastoid cells of spinocerebellar ataxia type 17.

Spinocerebellar ataxia type 17 (SCA17) is caused by CAG repeat expansion in the TATA-box binding protein gene. Studies of several polyglutamine (polyQ) expansion diseases have suggested that the expanded polyQ proteins misfold and induce oxidative stress to contribute to cell death. Substantial deficits in peripheral tissues including lymphocytes have been shown and these peripheral abnormalities could also be found in neurons possessing polyQ disease proteins. In this study, we used a lymphoblastoid cell model to investigate the functional implication of SCA17 expanded alleles and assess the potential therapeutic strategies that may ameliorate the effects of expanded polyQ. Proteomics studies of patient/control pairs including two-dimensional (2-D) gel electrophoresis, mass spectrometry and immunoblotting were conducted. A total of 8 proteins with reduced expression changes greater than 1.3-fold were identified, including previously reported HSPA5 and HSPA8. Among 6 proteins further semi-quantified by immunoblotting and real-time PCR, the reduced expression of HYOU1, PDIA3, P4HB, NQO1 and HMOX1 was confirmed. Treatment with resveratrol and genipin up-regulated NQO1 and HMOX1 expression and reduced oxidative stress in patients' lymphoblastoid cells. The results illustrate downregulation of proteins involved in the endoplasmic reticulum stress response (HYOU1, HSPA5, PDIA3, and P4HB) and Nrf2-ARE signaling (NQO1 and HMOX1) in SCA17 lymphoblastoid cells. Compounds increasing anti-oxidative activity such as resveratrol and genipin may serve as a potential therapeutic strategy for SCA17.

Two genetic variants in the HIBCH and FTCDNL1 genes are associated with susceptibility to developmental dysplasia of the hips among the Han Chinese population of Southwest China.

BACKGROUND: Developmental dysplasia of the hip (DDH) is a common cause of childhood disability, and the incidence of DDH shows significant familial aggregation. As the genetic factors of DDH remain unknown, the correlation between five candidate single nucleotide polymorphisms (SNPs) and DDH was evaluated in the Han Chinese population of Southwest China. METHODS: A case‒control association study was conducted in 276 patients with DDH and 318 healthy controls. SNP genotyping in the case and control groups was performed by SNPshot and multiple PCR. SNPs were genotyped in the case and control groups by multiplex PCR. The relationship between DDH and candidate SNPs was evaluated using the χ2 test. RESULTS: The genotype distributions of rs291412 in HIBCH and rs769956 in FTCDNL1 were different between the case and control groups (P < 0.05). After genetic model analysis, logistic regression analysis revealed that the C allele of rs291412 had a protective effect on DDH (OR = 0.605, P = 0.010) and that the G allele of rs769956 was a risk factor (OR = 2.939, P = 0.010).s. CONCLUSION: These SNPs could be associated with susceptibility to DDH but larger population-based studies should confirm the current results.