The Effect of Fermentation on the Chemical Constituents of Gastrodia Tuber Hallimasch Powder (GTHP) Estimated by UHPLC-Q-Orbitrap HRMS and HPLC.

OBJECTIVE: To compare the effect of fermentation on the chemical constituents of Gastrodia Tuder Halimasch Powder (GTHP), to establish its fingerprinting and multicomponent content determination, and to provide a basis for the processing, handling, and clinical application of this herb. METHODS: Ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to conduct a preliminary analysis of the chemical constituents in GTHP before and after fermentation. High-performance liquid chromatography (HPLC) was used to determine some major differential components of GTHP and establish fingerprints. Cluster analysis (CA), and principal component analysis (PCA) were employed for comprehensive evaluation. RESULTS: Seventy-nine compounds were identified, including flavonoids, organic acids, nucleosides, terpenoids, and others. The CA and PCA results showed that ten samples were divided into three groups. Through standard control and HPLC analysis, 10 compounds were identified from 22 peaks, namely uracil, guanosine, adenosine, 5-hydroxymethylfurfural (5-HMF), daidzin, genistin, glycitein, daidzein, genistein, and ergosterol. After fermentation, GTHP exhibited significantly higher contents of uracil, guanosine, adenosine, 5-hydroxymethylfurfural, and ergosterol and significantly lower genistein and daidzein contents. CONCLUSIONS: The UHPLC-Q-Orbitrap HRMS and HPLC methods can effectively identify a variety of chemical components before and after the fermentation of GTHP. This study provides a valuable reference for further research on the rational clinical application and quality control improvement of GTHP.

A comparison of clinical features between neurobrucellosis and tuberculous meningitis.

BACKGROUD: This study aims to compare the clinical manifestations, imaging findings, routine tests, biochemistry indicators and cerebrospinal fluid cytology between neurobrucellosis and tuberculous meningitis. The objective is to evaluate the similarities and differences of these two diseases and improve early diagnosis. METHODS: A comprehensive evaluation was conducted by comparing clinical data, imaging results, routine tests findings, biochemistry indicators and cerebrospinal fluid cytology of patients admitted to the Department of Neurology, the Second Hospital of Hebei Medical University from 2019 to 2021. Statistical analysis was applied to identify significant differences and similarities between the two diseases. RESULTS: Preliminary analysis demonstrated both diseases commonly present with symptoms such as fever, headache. However, there were no statistical differences between neurobrucellosis and tuberculous meningitis in early clinical data, imaging results, routine tests findings, biochemistry indicators. Further analysis indicates there is a statistically significantly difference in the lymphocyte ratio and neutrophil ratio in the cerebrospinal fluid between the two groups. CONCLUSIONS: Neurobrucellosis and tuberculous meningitis share similarities in early clinical manifestations, imaging findings and initial cerebrospinal fluid parametes, making early-stage differentiation challenging. The ratio of lymphocytes and neutrophil in the cerebrospinal fluid and a detailed medical history investigation can provide clues for early clinical diagnosis. So the examination of CSF cytology might be a potential to distinguish these two diseases and become a powerful tool in the future.

[Comparison of chemical compositions of different parts of Xanthoceras sorbifolium seeds based on UHPLC-Q-Orbitrap HRMS].

Xanthoceras sorbifolium seeds have a wide range of applications in the food and pharmaceutical industries. To compare and analyze the chemical compositions of different parts of X. sorbifolium seeds and explore the potential value and research prospects of non-medicinal parts, this study used ultra-high-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS) to detect the chemical composition of various parts of the seeds. A total of 82 components were preliminary identified from X. sorbifolium seeds, including 5 amino acids, 4 polyphenols, 3 phenylpropionic acids, 7 organic acids, 15 flavonoids, 6 glycosides, and 23 saponins. Mass spectrometry molecular networking(MN) analysis was conducted on the results from different parts of the seeds, revealing significant differences in the components of the seed kernel, seed coat, and seed shell. The saponins and flavonoids in the seed kernel were superior in terms of variety and content to those in the seed coat and shell. Based on the chromatographic peaks of different parts from multiple batches of samples, multivariate statistical analysis was carried out. Four differential components were determined using HPLC, and the average content of these components in the seed kernel, seed coat, and seed shell were as follows: 0.183 6, 0.887 4, and 1.440 1 mg·g~(-1) for fraxin; 0.035 8, 0.124 1, and 0.044 5 mg·g~(-1) for catechin; 0.032 9, 0.072 0, and 0.221 5 mg·g~(-1) for fraxetin; 0.435 9, 2.114 7, and 0.259 7 mg·g~(-1) for epicatechin. The results showed that catechin and fraxetin had relatively low content in all parts, while fraxin had higher content in the seed coat and seed shell, and epicatechin had higher content in the seed kernel and seed coat. Therefore, the seed coat and seed shell possess certain development value. This study provides rapid analysis and comparison of the chemical compositions of different parts of X. sorbifolium seeds, which offers an experimental basis for the research and clinical application of medicinal substances in X. sorbifolium seeds.

Paeoniflorin Ameliorates Colonic Fibrosis in Rats with Postinfectious Irritable Bowel Syndrome by Inhibiting the Leptin/LepRb Pathway.

Postinfectious irritable bowel syndrome (PI-IBS) is a highly prevalent gastrointestinal disorder associated with immune dysregulation and depression- and anxiety-like behaviors. Through traditional medicine, the active ingredient of Paeoniae Radix called paeoniflorin (PF) was previously found to prevent the symptoms of PI-IBS. However, there is limited information on the effects of PF on intestinal function and depression- and anxiety-like symptoms in PI-IBS animal models. Here, we aimed to determine the effects of PF treatment on the symptoms of PI-IBS in a rat model. The PI-IBS rat model was established via early postnatal sibling deprivation (EPSD), trinitrobenzenesulfonic acid (TNBS), and chronic unpredictable mild stress (CUMS) stimulation and then treated with different dosages of PF (10, 20, and 40 mg/kg) and leptin (1 and 10 mg/kg). The fecal water content and body weight were measured to evaluate the intestinal function, while the two-bottle test for sucrose intake, open field test (OFT), and elevated plus maze test (EMT) were performed to assess behavioral changes. The serum leptin levels were also measured using an enzyme-linked immunosorbent assay. Furthermore, the expressions of leptin and its receptor, LepRb, were detected in colonic mucosal tissues through an immunohistochemical assay. The activation of the PI3K/AKT signaling pathway and the expression of brain-derived neurotrophic factor (BDNF) were also detected via western blotting. After the experimental period, the PI-IBS rats presented decreased body weight and increased fecal water content, which coincided with elevated leptin levels and heightened depression- and anxiety-like behaviors (e.g., low sucrose intake, less frequency in the center areas during OFT, and fewer activities in the open arms during EMT). However, the PF treatment ameliorated these observed symptoms. Furthermore, PF not only inhibited leptin/LepRb expression but also reduced the PI3K/AKT phosphorylation and BDNF expression in PI-IBS rats. Notably, cotreatment with leptin (10 mg/kg) reduced the effects of PF (20 mg/kg) on colonic fibrosis, leptin/LepRb expression, and PI3K/AKT activation. Therefore, our findings suggest that leptin is targeted by PF via the leptin/LepRb pathway, consequently ameliorating the symptoms of PI-IBS. Our study also contributes novel insights for elucidating the pharmacological action of PF on gastrointestinal disorders and may be used for the clinical treatment of PI-IBS in the future.

Clinically available testing options resulting in diagnosis in post-exome clinic at one medical center.

Exome sequencing (ES) became clinically available in 2011 and promised an agnostic, unbiased next-generation sequencing (NGS) platform for patients with symptoms believed to have a genetic etiology. The diagnostic yield of ES has been estimated to be between 25-40% and may be higher in specific clinical scenarios. Those who remain undiagnosed may have no molecular findings of interest on ES, variants of uncertain significance in genes that are linked to human disease, or variants of uncertain significance in candidate genes that are not definitively tied to human disease. Recent evidence suggests that a post-exome evaluation consisting of clinical re-phenotyping, functional studies of candidate variants in known genes, and variant reevaluation can lead to a diagnosis in 5-15% of additional cases. In this brief research study, we present our experience on post-exome evaluations in a cohort of patients who are believed to have a genetic etiology for their symptoms. We have reached a full or partial diagnosis in approximately 18% (6/33) of cases that have completed evaluations to date. We accomplished this by utilizing NGS-based methods that are available on a clinical basis. A sample of these cases highlights the utility of ES reanalysis with updated phenotyping allowing for the discovery of new genes, re-adjudication of known variants, incorporating updated phenotypic information, utilizing functional testing such as targeted RNA sequencing, and deploying other NGS-based testing methods such as gene panels and genome sequencing to reach a diagnosis.

Genetic Testing in Patients with Neurodevelopmental Disorders: Experience of 511 Patients at Cincinnati Children's Hospital Medical Center.

Our institution developed and continuously improved a Neurodevelopmental Reflex (NDR) algorithm to help physicians with genetic test ordering for neurodevelopmental disorders (NDDs). To assess its performance, we performed a retrospective study of 511 patients tested through NDR from 2018 to 2019. SNP Microarray identified pathogenic/likely pathogenic copy number variations in 27/511 cases (5.28%). Among the 484 patients tested for Fragile X FMR1 CGG repeats, a diagnosis (0.20%) was established for one male mosaic for a full mutation, a premutation, and a one-CGG allele. Within the 101 normocephalic female patients tested for MECP2, two patients were found to carry pathogenic variants (1.98%). This retrospective study suggested the NDR algorithm effectively established diagnoses for patients with NDDs with a yield of 5.87%.

Application of Empirical Mode Decomposition for Decoding Perception of Faces Using Magnetoencephalography.

Neural decoding is useful to explore the timing and source location in which the brain encodes information. Higher classification accuracy means that an analysis is more likely to succeed in extracting useful information from noises. In this paper, we present the application of a nonlinear, nonstationary signal decomposition technique-the empirical mode decomposition (EMD), on MEG data. We discuss the fundamental concepts and importance of nonlinear methods when it comes to analyzing brainwave signals and demonstrate the procedure on a set of open-source MEG facial recognition task dataset. The improved clarity of data allowed further decoding analysis to capture distinguishing features between conditions that were formerly over-looked in the existing literature, while raising interesting questions concerning hemispheric dominance to the encoding process of facial and identity information.

Effects of Phonological Consistency and Semantic Radical Combinability on N170 and P200 in the Reading of Chinese Phonograms.

Studies have suggested that visually presented words are obligatorily decomposed into constituents that could be mapped to language representations. The present study aims to elucidate how orthographic processing of one constituent affects the other and vice versa during a word recognition task. Chinese orthographic system has characters representing syllables and meanings instead of suffixation roles, and the majority of Chinese characters are phonograms that can be further decomposed into phonetic radical and semantic radical. We propose that semantic radical combinability indexed by semantic radicals and the effect of phonological consistency indexed by phonetic radicals would interact with each other during the reading of Chinese phonograms. Twenty-six right-handed native Chinese speakers were recruited to the study. Participants were presented with phonograms divided into four conditions following their semantic radical combinability (large vs. small) and phonological consistency (high vs. low). EEG signals were recorded throughout the covert naming task. Our results show that there is an interaction effect between phonological consistency and semantic radical combinability on the right hemisphere N170 activity while reading phonograms. Semantic radical combinability influenced the right hemisphere N170 during the process of low-consistency character reading but not high-consistency character reading. On the other hand, the left hemisphere N170 revealed a more significant activity during reading high-consistency characters and was not affected by radical combinability. In addition, while low-consistency characters revealed a larger P200 than high-consistency characters, the semantic radical combinability effect on P200 was only significant when participants were reading high-consistency characters but not low-consistency characters. These results provide new information about how ERPs are involved in word recognition within the context of interaction among orthographic and phonological dimensions.

Triptolide Attenuates Vascular Calcification by Upregulating Expression of miRNA-204.

Background: Triptolide (TP), a naturally derived compound from Tripterygium wilfordii, has been proven effective in protecting against cardiovascular system, but the molecular mechanisms underlying its protective effects are poorly understood. In the current study, we sought to test the potential protective role of TP in the regulation of vascular calcification in a rat model and explore whether TP attenuates medial vascular calcification by upregulating miRNA-204. Methods: Vitamin D3 plus nicotine (VDN) was used to induce a vascular calcification (VC) model of rat aorta. Von Kossa and Hematoxylin-Eosin staining were applied to assess the degree of calcification of rat aortas. Calcium content and alkaline phosphatase activity were measured. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was applied to quantify miRNA-204 expression. The localization of runt-related transcription factor-2 (RUNX2) and bone morphogenetic protein-2 (BMP2) expressions were detected by immunohistochemistry and western blotting. Results: Administration of TP greatly reduced vascular calcification in a dose-dependent manner compared with VC controls. The increase in ALP activity and calcium content was ameliorated by TP. Moreover, protein expression levels of BMP2 and RUNX2 were significantly reduced in calcified aortas. MiRNA-204 expression was increased in the TP-treated groups compared with VC controls and the effects of TP were reversed by the intravenous injection of miRNA-204-interfering lentivirus. However, the miRNA-204-overexpressing lentivirus had no additional effects on ALP activity, calcium content, BMP2 and RUNX2 expressions compared with those from TP group. Conclusion: TP inhibited BMP2 and RUNX2 expression and attenuated vascular calcification via upregulating the level of miRNA-204. TP appears to be a potential new therapeutic option for treating vascular calcification.

p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic cell reprogramming.

The generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming holds great potential for modeling human diseases. However, the reprogramming process remains very inefficient and a better understanding of its basic biology is required. The mesenchymal-to-epithelial transition (MET) has been recognized as a crucial step for the successful reprogramming of fibroblasts into iPSCs. It has been reported that the p53 tumor suppressor gene acts as a barrier of this process, while its homolog p63 acts as an enabling factor. In this regard, the information concerning the role of the third homolog, p73, during cell reprogramming is limited. Here, we derive total Trp73 knockout mouse embryonic fibroblasts, with or without Trp53, and examine their reprogramming capacity. We show that p73 is required for effective reprogramming by the Yamanaka factors, even in the absence of p53. Lack of p73 affects the early stages of reprogramming, impairing the MET and resulting in altered maturation and stabilization phases. Accordingly, the obtained p73-deficient iPSCs have a defective epithelial phenotype and alterations in the expression of pluripotency markers. We demonstrate that p73 deficiency impairs the MET, at least in part, by hindering BMP pathway activation. We report that p73 is a positive modulator of the BMP circuit, enhancing its activation by DNp73 repression of the Smad6 promoter. Collectively, these findings provide mechanistic insight into the MET process, proposing p73 as an enhancer of MET during cellular reprogramming.

A de novo interstitial deletion of 7q31.2q31.31 identified in a girl with developmental delay and hearing loss.

We report on a 4-year-old female who presented with unilateral sensorineural hearing loss and a concern for developmental delay. A genome-wide SNP array analysis was performed and revealed a de novo 3.2 Mb interstitial deletion of chromosome 7q31.2q31.31. This region contains thirteen protein-encoding genes. It is unknown whether haploinsufficiency of any of these genes is responsible for the clinical features of our patient. We reviewed, the clinical phenotype of a previously published 7q31.3 deletion patient and 18 additional patients with overlapping 7q31 deletions listed in the DECIPHER database. The most consistent feature in these patients and our proband is delayed speech and language development. Hearing loss is presented both in our proband and the published 7q31.3 patient. Our study suggests that a small region on chromosome 7q31.3 encompassing four genes, CFTR, CTTNBP2, NAA38, and ANKRD7, may represent a new locus for congenital hearing loss and/or speech development. © 2016 Wiley Periodicals, Inc.

Hearing loss, coloboma and left ventricular enlargement in a boy with an interstitial 10q26 deletion.

Distal deletion of the long arm of chromosome 10 with breakpoints mapped at 10q26 is a well-recognized contiguous genomic disorder. A wide spectrum of clinical findings is seen in affected individuals and the common clinical features include craniofacial dysmorphia, developmental delay, intellectual disability, hypotonia, cardiovascular defects, and urogenital malformations. We report herein on a male patient with a 5.5 Mb interstitial deletion of 10q26.11q2613 and compare his clinical presentation to previously reported cases. Apart from characteristic phenotypes seen in 10q26 deletion syndrome, he presents with colobomas and left ventricle enlargement. These are cardiovascular and ophthalmological findings that have not been described in prior cases. © 2016 Wiley Periodicals, Inc.

Drosophila Nipped-B Mutants Model Cornelia de Lange Syndrome in Growth and Behavior.

Individuals with Cornelia de Lange Syndrome (CdLS) display diverse developmental deficits, including slow growth, multiple limb and organ abnormalities, and intellectual disabilities. Severely-affected individuals most often have dominant loss-of-function mutations in the Nipped-B-Like (NIPBL) gene, and milder cases often have missense or in-frame deletion mutations in genes encoding subunits of the cohesin complex. Cohesin mediates sister chromatid cohesion to facilitate accurate chromosome segregation, and NIPBL is required for cohesin to bind to chromosomes. Individuals with CdLS, however, do not display overt cohesion or segregation defects. Rather, studies in human cells and model organisms indicate that modest decreases in NIPBL and cohesin activity alter the transcription of many genes that regulate growth and development. Sister chromatid cohesion factors, including the Nipped-B ortholog of NIPBL, are also critical for gene expression and development in Drosophila melanogaster. Here we describe how a modest reduction in Nipped-B activity alters growth and neurological function in Drosophila. These studies reveal that Nipped-B heterozygous mutant Drosophila show reduced growth, learning, and memory, and altered circadian rhythms. Importantly, the growth deficits are not caused by changes in systemic growth controls, but reductions in cell number and size attributable in part to reduced expression of myc (diminutive) and other growth control genes. The learning, memory and circadian deficits are accompanied by morphological abnormalities in brain structure. These studies confirm that Drosophila Nipped-B mutants provide a useful model for understanding CdLS, and provide new insights into the origins of birth defects.

Enhancing xylanase production in the thermophilic fungus Myceliophthora thermophila by homologous overexpression of Mtxyr1.

The xylanase regulator 1 protein in Myceliophthora thermophila ATCC42464 (MtXyr1) is 60 % homologous with that of Trichoderma reesei. However, MtXyr1's regulatory role on cellulolytic and xylanolytic genes in M. thermophila is unknown. Herein, MtXyr1 was overexpressed under the control of the MtPpdc (pyruvate decarboxylase) promoter. Compared with the wild type, the extracellular xylanase activities of the transformant cultured in non-inducing and inducing media for 120 h were 25.19- and 9.04-fold higher, respectively. The Mtxyr1 mRNA level was 300-fold higher than in the wild type in corncob-containing medium. However, the filter paper activity and endoglucanase activities were unchanged in corncob-containing medium and glucose-containing medium. The different zymograms between the transformant and the wild type were analyzed and identified by mass spectrometry as three xylanases of the glycoside hydrolase (GH) family 11. Thus, overexpression of xyr1 resulted in enhanced xylanase activity in M. thermophila. Xylanase production could be improved by overexpressing Mtxyr1 in M. thermophila.

Clinical, developmental and molecular update on Cornelia de Lange syndrome and the cohesin complex: abstracts from the 2014 Scientific and Educational Symposium.

Cornelia de Lange Syndrome (CdLS) is the most common example of disorders of the cohesin complex, or cohesinopathies. There are a myriad of clinical issues facing individuals with CdLS, particularly in the neurodevelopmental system, which also have implications for the parents and caretakers, involved professionals, therapists, and schools. Basic research in developmental and cell biology on cohesin is showing significant progress, with improved understanding of the mechanisms and the possibility of potential therapeutics. The following abstracts are presentations from the 6th Cornelia de Lange Syndrome Scientific and Educational Symposium, which took place on June 25-26, 2014, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting in Costa Mesa, CA. The Research Committee of the CdLS Foundation organizes the meeting, reviews and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board. In addition to the scientific and clinical discussions, there were educationally focused talks related to practical aspects of behavior and development. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD.

Evaluation of salmon calcitonin (sCT) enteric-coated capsule for enhanced absorption and GI tolerability in rats.

BACKGROUND: Considering the chronic and repeated nature of salmon calcitonin (sCT) therapy, the oral route is a preferred route of administration. But, the oral bioavailability of sCT is very low due to enzymatic degradation and poor permeation across intestinal epithelial cells. It was the aim of this study to investigate the pharmacodynamic (PD), pharmacokinetic (PK), and mucosal injury characteristic of sCT oral delivery system. METHOD: In this study, PD experiments were performed to find a suitable releasing region of sCT, an effect absorption enhancer, and an optimal mass ratio of sCT/enhancer. In addition, the PK experiments were designed to validate the absorption enhancement of this oral delivery system. Histopathological evaluations on the intestinal mucosa were carried out to assess any potential toxicity of the absorption enhancer. RESULTS: Through the PD research, we determined that oral sCT enteric-coated capsules containing sCT and citric acid (CA) with a ratio of 1:20 may be an adaptable delivery. PK study further proved that the oral absorption of sCT was enhanced from this delivery system. Finally, no damage on intestinal mucosa was observed when rats received the delivery system containing CA for up to 7 days. CONCLUSION: These results suggested that enteric-coated capsules with a certain amount of CA might give enhanced oral delivery of peptide drugs like sCT.

A Drosophila model for Angelman syndrome.

Angelman syndrome is a neurological disorder whose symptoms include severe mental retardation, loss of motor coordination, and sleep disturbances. The disease is caused by a loss of function of UBE3A, which encodes a HECT-domain ubiquitin ligase. Here, we generate a Drosophila model for the disease. The results of several experiments show that the functions of human UBE3A and its fly counterpart, dube3a, are similar. First, expression of Dube3a is enriched in the Drosophila nervous system, including mushroom bodies, the seat of learning and memory. Second, we have generated dube3a null mutants, and they appear normal externally, but display abnormal locomotive behavior and circadian rhythms, and defective long-term memory. Third, flies that overexpress Dube3a in the nervous system also display locomotion defects, dependent on the ubiquitin ligase activity. Finally, missense mutations in UBE3A alleles of Angelman syndrome patients alter amino acid residues conserved in the fly protein, and when introduced into dube3a, behave as loss-of-function mutations. The simplest model for Angelman syndrome is that in the absence of UBE3A, particular substrates fail to be ubiquitinated and proteasomally degraded, accumulate in the brain, and interfere with brain function. We have generated flies useful for genetic screens to identify Dube3a substrates. These flies overexpress Dube3a in the eye or wing and display morphological abnormalities, dependent on the critical catalytic cysteine. We conclude that dube3a mutants are a valid model for Angelman syndrome, with great potential for identifying the elusive UBE3A substrates relevant to the disease.

Drosophila klaroid encodes a SUN domain protein required for Klarsicht localization to the nuclear envelope and nuclear migration in the eye.

KASH (Klarsicht/Anc-1/Syne homology) domain proteins are cytoskeleton-associated proteins localized uniquely to the outer nuclear membrane. Klarsicht is a KASH protein required for nuclear migration in differentiating cells of the Drosophila eye. The C-terminal KASH domain of Klarsicht resides in the perinuclear space, and the cytoplasmic moiety connects to the microtubule organizing center. In C. elegans and vertebrate cells, SUN (Sad1/UNC-84) domain proteins reside in the inner nuclear membrane and tether KASH proteins to the outer nuclear membrane. Is there a Drosophila SUN protein that performs a similar function, and if so, is it like Klarsicht, obviously essential for nuclear positioning only in the eye? Here, we identify Drosophila Klaroid, a SUN protein that tethers Klarsicht. klaroid loss-of-function mutants are indistinguishable phenotypically from klarsicht mutants. Remarkably, neither gene is essential for Drosophila viability or fertility, and even in klaroid klorsicht double mutants, the only obvious external morphological defect is rough eyes. In addition, we find that klaroid and klarsicht are required for nuclear migration in differentiating neurons and in non-neural cells. Finally, while perinuclear Klaroid is ubiquitous in the eye, Klarsicht expression is limited to differentiating cells and may be part of the trigger for apical nuclear migration.