RA-Net: reverse attention for generalizing residual learning.

Since residual learning was proposed, identity mapping has been widely utilized in various neural networks. The method enables information transfer without any attenuation, which plays a significant role in training deeper networks. However, interference with unhindered transmission also affects the network's performance. Accordingly, we propose a generalized residual learning architecture called reverse attention (RA), which applies high-level semantic features to supervise low-level information in the identity mapping branch. It means that higher semantic features selectively transmit low-level information to deeper layers. In addition, we propose a Modified Global Response Normalization(M-GRN) to implement reverse attention. RA-Net is derived by embedding M-GRN in the residual learning framework. The experiments show that the RA-Net brings significant improvements over residual networks on typical computer vision tasks. For classification on ImageNet-1K, compared with resnet101, RA-Net improves the Top-1 accuracy by 1.7% with comparable parameters and computational cost. For COCO detection, on Faster R-CNN, reverse attention improves box AP by 1.9%. Meanwhile, reverse attention improves UpperNet's mIoU by 0.7% on ADE20K segmentation.

Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Ultrasound-assisted polysaccharide extraction from Fritillaria ussuriensis Maxim. and its structural characterization, antioxidant and immunological activity.

Fritillaria ussuriensis Maxim. (F.M.) has been widely used in both food and medication for more than 2000 years. In order to achieve its comprehensive utilization and investigate the structural characterization and biology activity, response surface methodology (RSM) was used to optimize the ultrasound-assisted extraction conditions of F.M. polysaccharides. The optimal extraction conditions were ultrasonic power of 174.2 W, ratio of liquid to material of 40.7 mL/g and ultrasonic time of 82.0 min. In addition, a neutral polysaccharide F-1 was obtained, and its structure characterization, antioxidant and immunological activity were evaluated. The structural properties of the polysaccharide were characterized by UV, IR, GC-MS, NMR and AFM. Monosaccharide composition of F-1 (MW 18.11 kDa) was rhamnose, arabinose, glucosamine hydrochloride, galactose, and glucose which under the ratio of 0.9: 3.8: 0.2: 2.9: 92.2. The fractions of F-1 were mainly linked by â†’ 6)-α-D-Glcp-(1 â†’ with branch chain α-D-Glcp-(1 â†’ 4)-α-D-Glcp-(1 â†’ and 4,6)-α-D-Glcp-(1 â†’ residues. Moreover, F-1 has a significant scavenging activity, which can clear hydroxyl radicals, superoxide anion, DPPH and ABTS. In addition, the immunological activity showed that F-1 had an effect on macrophage phagocytic activity. And it can increase the release of inflammatory factors including TNF-α, IL-1ß and IL-6. F-1 is a novel polysaccharide with significant activity in antioxidant and immunological activity, which has great potential for antioxidant and immunizer in food, pharmaceutical and cosmetic industries. The study can provide a methodological basis for polysaccharide research and theoretical basis for the industrialized production and practical application.

Modified Buyang Huanwu Decoction alleviates diabetic liver injury via inhibiting oxidative stress in db/db mice.

OBJECTIVES: In diabetes, chronic hyperglycemia increases the overactivation of oxidative phosphorylation of mitochondria in the liver, resulting in oxidative stress (OS) damage. The Nrf2 signaling pathway plays a key role in preventing hepatic oxidative injury and inflammation. This study aims to investigate the therapeutic effect and mechanism of Modified Buyang Huanwu Decoction (mBYHWD) on diabetic liver injury (DLI) by regulating oxidative stress mediated by Nrf2 signaling pathway. METHODS: The experiment was divided into three groups: a control group (db/m mice, Con), a diabetes model group (db/db mice, Mod), and a traditional Chinese medicine group (db/m mice, mBYHWD). Post-treatment, serum from each group was analyzed to assess changes of blood glucose, blood lipid, and liver function. These results were combined with data mining to explore the possible pathogenesis of DLI. Liver tissues were collected to observe the pathological morphology and detect related proteins. RESULTS: The results demonstrated that mBYHWD significantly reduced blood lipids and improved liver function following diabetic liver injury. The histopathological results demonstrated that mBYHWD could significantly ameliorate damage of diabetic hepatocytes. Protein analysis revealed that mBYHWD treatment significantly increased the expression of antioxidant proteins in diabetic liver tissue and inhibited inflammation. CONCLUSIONS: The therapeutic mechanism of mBYHWD on DLI may involve activating the Nrf2 signaling pathway to improve oxidative stress, inhibit inflammation, and reduce liver tissue fibrosis.

Identification of Uncaria rhynchophylla in the Potential Treatment of Alzheimer's Disease by Integrating Virtual Screening and In Vitro Validation.

Uncaria rhynchophylla (Gouteng in Chinese, GT) is the main medicine in many traditional recipes in China. It is commonly used to alleviate central nervous system (CNS) disorders, although its mechanism in Alzheimer's disease is still unknown. This study was designed to predict and validate the underlying mechanism in AD treatment, thus illustrating the biological mechanisms of GT in treating AD. In this study, a PPI network was constructed, KEGG analysis and GO analysis were performed, and an "active ingredient-target-pathway" network for the treatment of Alzheimer's disease was constructed. The active ingredients of GT were screened out, and the key targets were performed by molecular docking. UHPLC-Q-Exactive Orbitrap MS was used to screen the main active ingredients and was compared with the network pharmacology results, which verified that GT did contain the above ingredients. A total of targets were found to be significantly bound up with tau, Aß, or Aß and tau through the network pharmacology study. Three SH-SY5Y cell models induced by okadaic acid (OA), Na2S2O4, and H2O2 were established for in vitro validation. We first found that GT can reverse the increase in the hyperphosphorylation of tau induced by OA to some extent, protecting against ROS damage. Moreover, the results also indicated that GT has significant neuroprotective effects. This study provides a basis for studying the potential mechanisms of GT in the treatment of AD.

Abnormal Spontaneous Discharges of Primary Sensory Neurons and Pain Behavior in a Rat Model of Vascular Dementia.

Patients with vascular dementia experience more pain than healthy elders, potentially due to the presence of central neuropathic pain. However, the mechanisms underlying neuropathic pain in vascular dementia remain poorly understood, and there is currently a lack of effective treatment available. In this study, a rat model of vascular dementia was induced by permanently occluding the common carotid arteries bilaterally (2-VO). The cognitive impairments in the 2-VO rats were evaluated using the Morris Water Maze test, while HE and LBF staining were employed to assess brain tissue lesions in the hippocampal, cerebral cortex, and white matter regions known to be associated with severe memory and learning deficits. Furthermore, pain-related behavioral tests, including mechanical and thermal stimuli assessments, were conducted, and in vivo electrophysiological recordings of primary sensory neurons were performed. Compared to sham-operated and pre-operative rats, rats with vascular dementia exhibited mechanical allodynia and thermal hyperalgesia 30 days after surgery. Furthermore, in vivo electrophysiology revealed a significant increase in the occurrence of spontaneous activity of Aß- and C-fiber sensory neurons in the rat model of vascular dementia. These results indicate that neuropathic pain behaviors developed in the rat model of vascular dementia, and abnormal spontaneous discharges of primary sensory neurons may play a crucial role in the development of pain after vascular dementia.

Propofol-Induced miR-493-3p Inhibits Growth and Invasion of Gastric Cancer through Suppression of DKK1-Mediated Wnt/ß-Catenin Signaling Activation.

Purpose: Gastric cancer (GC) is one of the most common malignant tumors and also one of the most deadly tumors. In recent years, studies have shown that propofol can inhibit the proliferation and metastasis of many tumor cells. In the present study, we aimed to investigate the underlying mechanism of propofol inhibition of the growth and invasion of GC cells. Methods: Human gastric cancer cell line SGC-7901 and human normal gastric epithelial cell GES-1 were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Propofol of different concentrations (0, 2, 5, and 10 µg/mL) was used to treat SGC-7901, and miR-493-3p inhibitor was transfected into SGC-7901. The cell proliferation of SGC-7901 was analyzed by MTT as well as colony formation assay. The qRT-PCR was used to assess the expression of mRNA for key genes. We examined the protein expression of DKK1 and relative markers with western blot. Putative binding places of miR-493-3p on the 3'-untranslated area of DKK1 were predicted using bioinformatics and dual-luciferase method. Results: Propofol prohibited phenotypic features of GC, according to our findings. Furthermore, research into the underlying mechanisms of propofol's suppressive effects in GC cell proved that propofol therapy improved the degrees of expression of the potential tumor suppressor miR-493-3p. The inhibiting properties of propofol on GC cell development, migration, and invasion were abolished when propofol-induced miR493-3p was silenced with anti-miR-493-3p. We also found that this drug reversed epithelial-mesenchymal transformation in SGC-7901 cells via inducing miR-493-3p. Propofol-induced miR-493-3p decreases GC cell development via targeting DKK1 and hence inhibits Wnt/ß-catenin signaling, according to these findings. Conclusion: Propofol-induced miR-493-3p decreased GC cell development via targeting DKK1 and hence inhibited Wnt/ß-catenin signaling, according to these findings.

Network Pharmacology Approach to Investigate the Mechanism of Danggui-Shaoyao-San against Diabetic Kidney Disease.

Background: Danggui-Shaoyao-San (DSS) is a traditional Chinese medicine formula that has been widely used to treat a variety of disorders, including renal diseases. Despite being well-established in clinical practice, the mechanisms behind the therapeutic effects of DSS on diabetic nephropathy (DN) remain elusive. Methods: To explore the therapeutic mechanism, we explored the action mechanism of DSS on DN using network pharmacology strategies. All ingredients were selected from the relevant databases, and active ingredients were chosen on the basis of their oral bioavailability prediction and drug-likeness evaluation. The putative proteins of DSS were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, whereas the potential genes of DN were obtained from the GeneCards and OMIM databases. Enrichment analysis using gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) was performed to discover possible hub targets and gene-related pathways. Afterwards, the underlying molecular mechanisms of DSS against DN were validated experimentally in vivo against db/db mice. Results: We identified 91 phytochemicals using the comprehensive network pharmacology technique, 51 of which were chosen as bioactive components. There were 40 proteins and 20 pathways in the target-pathway network. The experimental validation results demonstrated that DSS may reduce the expression of TNF-α, IL-6, and ICAM-1, as well as extracellular matrix deposition, by blocking the JNK pathway activation, which protects against kidney injury. Conclusion: This study discovered the putative molecular mechanisms of action of DSS against diabetic kidney damage through a network pharmacology approach and experimental validation.

Hydrophosphorylation of electron-deficient alkenes and alkynes mediated by convergent paired electrolysis.

A straightforward and practical strategy for hydrophosphorylation of electron-deficient alkenes and alkynes to access γ-ketophosphine oxides, enabled by a convergent paired electrolysis (CPE) in the absence of a metal, base, and redox reagent, has been described. Mechanistic studies have revealed that the diarylphosphane oxides play the dual role of a phosphorus radical precursor and hydrogen donor in this transformation.

BzATP Activates Satellite Glial Cells and Increases the Excitability of Dorsal Root Ganglia Neurons In Vivo.

The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3'-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability.

[Clinical observation on different frequency of auricular bloodletting combined with auricular point sticking for acne vulgaris].

OBJECTIVE: To observe the clinical efficacy of different treatment frequency of auricular bloodletting combined with auricular point sticking for acne vulgaris. METHODS: A total of 90 patients with acne vulgaris were randomized into a treatment group 1 (30 cases, 2 cases dropped off), a treatment group 2 (30 cases, 4 cases dropped off) and a treatment group 3 (30 cases, 5 cases dropped off). Combination therapy of auricular bloodletting and auricular point sticking at Fei (CO14), Shenmen (TF4), Neifenmi (CO18) and Shenshangxian (TG2p) were given once a week, twice a week and 3 times a week in the treatment group 1, the treatment group 2 and the treatment group 3 respectively, 4 weeks were as one course and totally 3 courses were required in the 3 groups. Before treatment and after 1, 2, 3 courses of treatment, the scores of global acne grading system (GAGS), skin lesion and quality of life-acne (QoL-Acne) were observed, the clinical efficacy was evaluated after 3 courses of treatment and the recurrence rate was evaluated in follow-up of 1 month after treatment in the 3 groups. RESULTS: Compared before treatment, the scores of GAGS and skin lesion were decreased at each time point in the 3 groups (P<0.05), the QoL-Acne scores were increased after 1 course of treatment in the treatment group 3 and after 2, 3 courses of treatment in the 3 groups (P<0.05). There were no statistical differences in scores of GAGS, skin lesion and QoL-Acne among the 3 groups (P>0.05). There were no statistical differences in effective rate and recurrence rate among the 3 groups (P>0.05). CONCLUSION: Different frequency of auricular bloodletting combined with auricular point sticking have similar efficacy in treating acne vulgaris, all can improve the skin lesion and quality of life in patients with acne vulgaris, and have a cumulative effect and good long-term curative effect.

Purinergic signaling between neurons and satellite glial cells of mouse dorsal root ganglia modulates neuronal excitability in vivo.

ABSTRACT: Primary sensory neurons in dorsal root ganglia (DRG) are wrapped by satellite glial cells (SGCs), and neuron-SGC interaction may affect somatosensation, especially nociceptive transmission. P2-purinergic receptors (P2Rs) are key elements in the two-way interactions between DRG neurons and SGCs. However, because the cell types are in such close proximity, conventional approaches such as in vitro culture and electrophysiologic recordings are not adequate to investigate the physiologically relevant responses of these cells at a population level. Here, we performed in vivo calcium imaging to survey the activation of hundreds of DRG neurons in Pirt-GCaMP6s mice and to assess SGC activation in GFAP-GCaMP6s mice in situ. By combining pharmacologic and electrophysiologic techniques, we investigated how ganglionic purinergic signaling initiated by α,ß-methyleneadenosine 5'-triphosphate (α,ß-MeATP) modulates neuronal activity and excitability at a population level. We found that α,ß-MeATP induced robust activation of small neurons-likely nociceptors-through activation of P2X3R. Large neurons, which are likely non-nociceptive, were also activated by α,ß-MeATP, but with a delay. Blocking pannexin 1 channels attenuated the late phase response of DRG neurons, indicating that P2R stimulation may subsequently induce paracrine ATP release, which could further activate cells in the ganglion. Moreover, ganglionic α,ß-MeATP treatment in vivo sensitized small neurons and enhanced responses of spinal wide-dynamic-range neurons to subsequent C-fiber inputs, suggesting that modulation via ganglionic P2R signaling could significantly affect nociceptive neuron excitability and pain transmission. Therefore, targeting functional P2Rs within ganglia may represent an important new strategy for pain modulation.

[Comparative study on the academic thought of bloodletting for expelling pathogens between Jin-Yuan Dynasties and Ming-Qing Dynasties].

The relevant provisions of bloodletting for expelling pathogens are collected from the works of the medical representative scholars in Jin-Yuan Dynasties and Ming-Qing Dynasties respectively to construct the databases of bloodletting for expelling pathogens of Jin-Yuan Dynasties and Ming-Qing Dynasties. Using frequency analysis, the bloodletting device, bloodletting location, bloodletting volume, the related pathogens and indications are compared between these two times so that the evidences could be provided for the inheritance and development of the academic thought of bloodletting for expelling pathogens. It is found that the three-edge needle is the most commonly used device for bloodletting in Jin-Yuan Dynasties and Ming-Qing Dynasties and yang meridians and local affected area are generally selected for bloodletting. The range of meridian and acupoint selection in Ming-Qing Dynasties are more extensive than those in Jin-Yuan Dynasties, while bloodletting volume is less than that in Jin-Yuan Dynasties. In Jin-Yuan Dynasties, bloodletting therapy is mainly for expelling exogenous pathogens i.e. heat, fire and wind, while, in Ming-Qing Dynasties, this therapy is specially for clearing heat and removing stasis. The disorders of internal medicine are often treated with such therapy in these two dynasties. But, compared with Jin-Yuan Dynasties, the indication of bloodletting therapy is expanded gradually in Ming-Qing Dynasties. It is shown that the academic thought of bloodletting for expelling pathogens in Jin-Yuan Dynasties is inherited and developed from Ming-Qing Dynasties.

A colorimetric sensor for acid phosphatase activity detection based on acridone derivative as visible-light-stimulated oxidase mimic.

Currently, organic artificial enzymes as biocatalysts have been extensively used to construct various colorimetric sensors. However, exploiting a potential organic artificial enzyme with high catalytic efficiency still remains a challenge. To address this issue, herein, we synthesize an acridone derivative 10-benzyl-2-amino-acridone (BAA). The synthesized BAA exhibits an intrinsic visible-light-stimulated oxidase-like activity, which is capable of oxidizing various chromogenic substrates without destructive hydrogen peroxide (H2O2) under visible light stimulation, resulting in colored products. The reaction system can be regulated by switching light on and off, which is milder and more reliable means than others H2O2-dependent. The photocatalytic mechanism of BAA is investigated in detail. However, l-ascorbic acid (AA), an antioxidant generating from the acid phosphatase (ACP)-mediated hydrolysis of 2-phospho-l-ascorbic acid (AAP), is able to inhibit the catalytic activity of BAA. Based on the above properties, a facile, photo-switchable and low-cost colorimetric sensing strategy is developed for ACP detection. The linear range is 0.05-2.5 U/L (r = 0.9994), and the limit of detection (LOD) is 0.0415 U/L. Moreover, the proposed sensing system can be applied for monitoring ACP activity in practical samples, demonstrating promising applications in clinical analysis and biosensor platform.

[Changes of microcirculation in superficial tissues of acupoint regions of lower leg during menstrual cycle in healthy young girls].

OBJECTIVE: To observe changes of microcirculation in the superficial regions of acupoints of the three Yin meridians of foot during the menstrual cycle in young college students, so as to provide experimental evidence for explaining the saying of traditional Chinese medicine that acupoints reflect the state of physiological and pathological activities of the internal organs. METHODS: Ninety healthy female volunteer college students were recruited in the present study. The subjects were asked to take a supine position on an examination couch to expose the Yuan-primary acupoints Taixi (KI3), Taibai (SP3) and Taichong (LR3), and Xi-cleft acupoints Shuiquan (KI5), Diji (SP8) and Zhongdu (LR6) which are related to the uterus of the three Yin meridians of foot， the crossing acupoints of the three Yin meridians of foot Sanyinjiao (SP6), non-specific acupoint of the Spleen meridian Xuehai (SP10), non-related meridian acupoint Xuanzhong (GB39) and non-meridian-non-acupoint (being at the same level of GB39, between the Stomach and Gallbladder meridians on the lateral aspect of the lower leg). The laser speckle blood flow imaging technique was used to detect the state of microcirculation (average blood perfusion volume) during menstrual, follicular, ovulatory and luteal phases. RESULTS: The average blood perfusion volume (ABPV) of the right SP8 region was significantly lower in the menstrual phase than in the ovulation and luteal phases (P<0.05), and in the follicular phase than in the ovulation phase (P<0.05). In the left LR6 region, the ABPV was obviously lower in the menstrual period than in the follicular, ovulation and luteal phases (P<0.05). In the SP8, SP6, KI3 and KI5 regions, the ABPV was significantly higher in each of the 4 phases on the left side than on the right side (P<0.05). In the right GB39, the ABPV in each of the 4 phases was apparently higher on the right side than on the left side (P<0.05). In the SP10 region, the ABPV was considerably higher on the left side than on the right side in the ovulation period (P<0.05). The ABPV of SP3 in the menstrual phase and that of the non-acupoint in the luteal phase were significantly higher on the right side than on the left side (P<0.05). CONCLUSION: The blood perfusion of microcirculation in the superficial tissues of Xi-cleft acupoints SP8 and LR6 is obviously lower in the menstrual phase than in both of the ovulation and luteal phases in healthy young college students, which may specifically reflect the periodical activities of the uterus in the physiological state, and provide a basis of acupoint selection for menstrual healthy care.

CircACAP2 promotes breast cancer proliferation and metastasis by targeting miR-29a/b-3p-COL5A1 axis.

BACKGROUND: Breast cancer is the most common cancer occurred in female around the world. The occurrence percentage of breast cancer among female who was over 50 years old is >80%. These factors may induce the chance of morbidity, including heavy drinking for a long term, smoking and postmenopausal obesity. Treatment of adjuvant chemotherapeutic and hormonal agents is mostly applied nowadays, which does contribute to the improvement of breast cancer, yet it is still an intractable disease for the side effects it brings. Emerging evidence has proved circular RNAs are involved in gene expression and modulation of biological behaviors. However, the mechanism and functions of circRNA ACAP2 (circACAP2) with respect to breast cancer remain unexplored. METHODS: qRT-PCR analysis was used to detect relevant expression. Bioinformatics analysis was applied to seek miRNAs which might have binding sites with circACAP2 and miR-29a/b-3p. Functional experiments including CCK-8, colony formation assay and flow cytometry assay detected gene function role in breast cancer cells. RIP, RNA pull down and Luciferase reporter assays were carried out to verify binding sites among circACAP2, miR-29a/b-3p and COL5A1. RESULTS: CircACAP2 expression was prominently elevated in tumorous tissues. Functionally and mechanistically, circACAP2 promoted breast cancer proliferation and motility by sponging miR-29a/b-3p and modulating COL5A1. CONCLUSION: Elevated expression of circACAP2 in breast cancer tissues leads to malignant phenotype upon cancerous cells. CircACAP2-miR-29a/b-3p-COL5A1 axis leads to breast cancer tumorigenesis and could hopefully be a novel method for diagnosis and treatment for breast cancer.

GATA-type transcriptional factor Gat1 regulates nitrogen uptake and polymalic acid biosynthesis in polyextremotolerant fungus Aureobasidium pullulans.

Polymalic acid (PMA) is a novel biopolymer produced by the polyextremotolerant fungus Aureobasidium pullulans. In this study, a GATA-family transcriptional factor, Gat1, which regulates nitrogen uptake and PMA biosynthesis, was investigated. PMA production increased to 11.2% in the mutant overexpressing gat1 but decreased to 49.1% of the PMA titre when gat1 was knocked out from the genome of A. pullulans. Comparative transcriptome analysis of wild-type and mutant strains (∆gat1 and OE::gat1) revealed that 23 common differentially expressed genes were related to oxidative phosphorylation, ribosome biogenesis, and nitrogen metabolism. Under nitrogen-limited stress, regardless of the preferred nitrogen (glutamine, Gln) or non-preferred nitrogen (proline, Pro), 70% of Gat1 in the cells was located in the nucleus-cytoplasm, which resulted in an increase in nitrogen uptake and PMA biosynthesis regulation. Quantitative RT-PCR revealed that glucosekinase (GLK) in the glycolytic pathway and malate synthase (MLS) in the glyoxylate shunt pathway may be cross-regulated by Gat1 and nitrogen concentration (Gln or Pro), Therefore, glk was overexpressed in mutant strain (OE::gat1), which resulted in an increased PMA titre and yield of 12.6% and 13.0% respectively. These findings indicate that Gat1 may play an important role in the dual regulation of the nitrogen and carbon metabolisms in PMA biosynthesis.

Adjacent intact nociceptive neurons drive the acute outburst of pain following peripheral axotomy.

Injury of peripheral nerves may quickly induce severe pain, but the mechanism remains obscure. We observed a rapid onset of spontaneous pain and evoked pain hypersensitivity after acute transection of the L5 spinal nerve (SNT) in awake rats. The outburst of pain was associated with a rapid development of spontaneous activities and hyperexcitability of nociceptive neurons in the adjacent uninjured L4 dorsal root ganglion (DRG), as revealed by both in vivo electrophysiological recording and high-throughput calcium imaging in vivo. Transection of the L4 dorsal root or intrathecal infusion of aminobutyrate aminotransferase inhibitor attenuated the spontaneous activity, suggesting that retrograde signals from the spinal cord may contribute to the sensitization of L4 DRG neurons after L5 SNT. Electrical stimulation of low-threshold afferents proximal to the axotomized L5 spinal nerve attenuated the spontaneous activities in L4 DRG and pain behavior. These findings suggest that peripheral axotomy may quickly induce hyperexcitability of uninjured nociceptors in the adjacent DRG that drives an outburst of pain.

Biosynthesis of polymalic acid in fermentation: advances and prospects for industrial application.

Some microorganisms naturally produce ß-poly(l-malic acid) (PMA), which has excellent water solubility, biodegradability, and biocompatibility properties. PMA has broad prospective applications as novel biopolymeric materials and carriers in the drug, food, and biomedical fields. Malic acid, a four-carbon dicarboxylic acid, is widely used in foods and pharmaceuticals, as a platform chemical. Currently, malic acid produced through chemical synthesis and is available as a racemic mixture of l- and d-forms. The d-form malic acid exhibits safety concerns for human consumption. There is extensive interest to develop economical bioprocesses for l-malic acid and PMA production from renewable biomass feedstocks. In this review, we focus on PMA biosynthesis by Aureobasidium pullulans, a black yeast with a large genome containing genes encoding many hydrolases capable of degrading various plant materials. The metabolic and regulatory pathways for PMA biosynthesis, metabolic engineering strategies for strain development, process factors affecting fermentation kinetics and PMA production, and downstream processing for PMA recovery and purification are discussed. Prospects of microbial PMA and malic acid production are also considered.

[Complete genome sequencing of polymalic acid-producing strain Aureobasidium pullulans CCTCC M2012223].

Objective: To explore the genome sequence of Aureobasidium pullulans CCTCC M2012223, analyze the key genes related to the biosynthesis of important metabolites, and provide genetic background for metabolic engineering. Methods: Complete genome of A. pullulans CCTCC M2012223 was sequenced by Illumina HiSeq high throughput sequencing platform. Then, fragment assembly, gene prediction, functional annotation, and GO/COG cluster were analyzed in comparison with those of other five A. pullulans varieties. Results: The complete genome sequence of A. pullulans CCTCC M2012223 was 30756831 bp with an average GC content of 47.49%, and 9452 genes were successfully predicted. Genome-wide analysis showed that A. pullulans CCTCC M2012223 had the biggest genome assembly size. Protein sequences involved in the pullulan and polymalic acid pathway were highly conservative in all of six A. pullulans varieties. Although both A. pullulans CCTCC M2012223 and A. pullulans var. melanogenum have a close affinity, some point mutation and inserts were occurred in protein sequences involved in melanin biosynthesis. Conclusion: Genome information of A. pullulans CCTCC M2012223 was annotated and genes involved in melanin, pullulan and polymalic acid pathway were compared, which would provide a theoretical basis for genetic modification of metabolic pathway in A. pullulans.