Quorum-quenching enzyme Est816 assisted antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in rats.

Introduction: Quorum-quenching enzyme Est816 hydrolyzes the lactone rings of N-acyl homoserine lactones, effectively blocking the biofilm formation and development of Gram-negative bacteria. However, its applications in the oral field is limited. This study aimed to evaluate the efficacy of enzyme Est816 in combination with antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in vitro and in vivo. Methods: The antimicrobial efficacy of enzyme Est816 in combination with minocycline, metronidazole, and amoxicillin was determined using the minimum inhibitory concentration test. The anti-biofilm effect of enzyme Est816 was assessed using scanning electron microscopy, live/dead bacterial staining, crystal violet staining, and real-time quantitative PCR. Biocompatibility of enzyme Est816 was assessed in human gingival fibroblasts (HGF) by staining. A rat model of periodontitis was established to evaluate the effect of enzyme Est816 combined with minocycline using micro-computed tomography and histological staining. Results: Compared to minocycline, metronidazole, and amoxicillin treatment alone, simultaneous treatment with enzyme Est816 increased the sensitivity of biofilm bacteria to antibiotics. Enzyme Est816 with minocycline exhibited the highest rate of biofilm clearance and high biocompatibility. Moreover, the combination of enzyme Est816 with antibiotics improved the antibiofilm effects of the antibiotics synergistically, reducing the expression of the virulence factor leukotoxin gene (ltxA) and fimbria-associated gene (rcpA). Likewise, the combination of enzyme Est816 with minocycline exhibited a remarkable inhibitory effect on bone resorption and inflammation damage in a rat model of periodontitis. Discussion: The combination of enzyme Est816 with antibiotics represents a prospective anti-biofilm strategy with the potential to treat periodontitis.

Current evidence of synaptic dysfunction after stroke: Cellular and molecular mechanisms.

BACKGROUND: Stroke is an acute cerebrovascular disease in which brain tissue is damaged due to sudden obstruction of blood flow to the brain or the rupture of blood vessels in the brain, which can prompt ischemic or hemorrhagic stroke. After stroke onset, ischemia, hypoxia, infiltration of blood components into the brain parenchyma, and lysed cell fragments, among other factors, invariably increase blood-brain barrier (BBB) permeability, the inflammatory response, and brain edema. These changes lead to neuronal cell death and synaptic dysfunction, the latter of which poses a significant challenge to stroke treatment. RESULTS: Synaptic dysfunction occurs in various ways after stroke and includes the following: damage to neuronal structures, accumulation of pathologic proteins in the cell body, decreased fluidity and release of synaptic vesicles, disruption of mitochondrial transport in synapses, activation of synaptic phagocytosis by microglia/macrophages and astrocytes, and a reduction in synapse formation. CONCLUSIONS: This review summarizes the cellular and molecular mechanisms related to synapses and the protective effects of drugs or compounds and rehabilitation therapy on synapses in stroke according to recent research. Such an exploration will help to elucidate the relationship between stroke and synaptic damage and provide new insights into protecting synapses and restoring neurologic function.

Predictive value of Cmmi-MHR combined with thromboelastography parameters in acute cerebral infarction.

Cerebral infarction is a common neurological disease with high rates of morbidity, mortality, and recurrence, posing a great threat to human life and health. Cerebral infarction is the second leading cause of death in the world and the leading cause of long-term disability in humans. The results of the third national retrospective sampling survey on causes of death in 2008 showed that cerebral infarction has become the leading cause of death in China and its mortality rate is 4-5 times that of European and American countries. Therefore, this article proposed a study on the predictive value of Cmmi-MHR combined with thromboelastography parameters that was performed for acute cerebral infarction. This paper mainly proposed a high frame rate imaging technology and analyzed its algorithm. In this article, in the experimental part, an in-depth analysis of the predictive value of the Monocyte-to-high-density lipoprotein cholesterol ratio (MHR) combined with thromboelastography parameters was performed for acute cerebral infarction. The final experimental results showed that HDL (OR = 1.695%, P-trend = 0.049) had a probability of death within 90 days of hospitalization (OR = 0.81, 95% CI = 1.06-3.11, P-trend = 0.523). There were no significant differences in mortality rate after 90 days. Regardless of adjusting for confounders such as age, gender, and NIHSS score, there was no significant difference in the risk of MHR or monocyte count within 90 days of hospitalization. The conclusion indicates that the combination of Cmmi-MHR and thromboelastography parameters provides a new perspective and method for the diagnosis and treatment of cerebral infarction, and provides important support for personalized treatment and management of cerebral infarction.

Peptide set test: a peptide-centric strategy to infer differentially expressed proteins.

MOTIVATION: The clinical translation of mass spectrometry-based proteomics has been challenging due to limited statistical power caused by large technical variability and inter-patient heterogeneity. Bottom-up proteomics provides an indirect measurement of proteins through digested peptides. This raises the question whether peptide measurements can be used directly to better distinguish differentially expressed proteins. RESULTS: We present a novel method called the peptide set test, which detects coordinated changes in the expression of peptides originating from the same protein and compares them to the rest of the peptidome. Applying our method to data from a published spike-in experiment and simulations demonstrates improved sensitivity without compromising precision, compared to aggregation-based approaches. Additionally, applying the peptide set test to compare the tumor proteomes of tamoxifen-sensitive and tamoxifen-resistant breast cancer patients reveals significant alterations in peptide levels of collagen XII, suggesting an association between collagen XII-mediated matrix reassembly and tamoxifen resistance. Our study establishes the peptide set test as a powerful peptide-centric strategy to infer differential expression in proteomics studies. AVAILABILITY AND IMPLEMENTATION: Peptide set test (PepSetTest) is publicly available at https://github.com/JmWangBio/PepSetTest.

A multiple linear regression analysis identifies factors associated with fear of cancer recurrence in postoperative patients with gastric cancer.

To investigate the risk factors of fear of cancer recurrence (FCR) in postoperative patients with gastric cancer (GC) and provide references for targeted nursing intervention development. A total of 84 patients who underwent GC surgery were included in this study. The fear of progression questionnaire-short form and social support rating scale were conducted, and multiple linear regression was performed to identify risk factors of FCR. The score of the fear of progression questionnaire-short form in patients with GC surgery was 39.1 ±â€…7.6. The results of multiple linear regression showed that age, education level, occupational status, course of the disease, Tumor node metastasis staging, and social support were the influencing factors of FCR in patients with GC (P < .05). The current situation of FCR in patients with GC surgery is not optimistic. The medical staff should pay more attention to patients with low age, low education level, unemployment, short course, high tumor node metastasis staging, low social support level, and other high-risk groups, and provide social support resources to reduce the level of FCR.

Role of dendritic cells in spinal cord injury.

BACKGROUND: Inflammation can worsen spinal cord injury (SCI), with dendritic cells (DCs) playing a crucial role in the inflammatory response. They mediate T lymphocyte differentiation, activate microglia, and release cytokines like NT-3. Moreover, DCs can promote neural stem cell survival and guide them toward neuron differentiation, positively impacting SCI outcomes. OBJECTIVE: This review aims to summarize the role of DCs in SCI-related inflammation and identify potential therapeutic targets for treating SCI. METHODS: Literature in PubMed and Web of Science was reviewed using critical terms related to DCs and SCI. RESULTS: The study indicates that DCs can activate microglia and astrocytes, promote T-cell differentiation, increase neurotrophin release at the injury site, and subsequently reduce secondary brain injury and enhance functional recovery in the spinal cord. CONCLUSIONS: This review highlights the repair mechanisms of DCs and their potential therapeutic potential for SCI.

D-histidine combated biofilm formation and enhanced the effect of amikacin against Pseudomonas aeruginosa in vitro.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogenic microorganism that poses a significant challenge in clinical treatment. Antibiotics exhibit limited efficacy against mature biofilm, culminating in an increase in the number of antibiotic-resistant strains. Therefore, novel strategies are essential to enhance the effectiveness of antibiotics against Pseudomonas aeruginosa biofilms. D-histidine has been previously identified as a prospective anti-biofilm agent. However, limited attention has been directed towards its impact on Pseudomonas aeruginosa. Therefore, this study was undertaken to explore the effect of D-histidine on Pseudomonas aeruginosa in vitro. Our results demonstrated that D-histidine downregulated the mRNA expression of virulence and quorum sensing (QS)-associated genes in Pseudomonas aeruginosa PAO1 without affecting bacterial growth. Swarming and swimming motility tests revealed that D-histidine significantly reduced the motility and pathogenicity of PAO1. Moreover, crystal violet staining and confocal laser scanning microscopy demonstrated that D-histidine inhibited biofilm formation and triggered the disassembly of mature biofilms. Notably, D-histidine increased the susceptibility of PAO1 to amikacin compared to that in the amikacin-alone group. These findings underscore the efficacy of D-histidine in combating Pseudomonas aeruginosa by reducing biofilm formation and increasing biofilm disassembly. Moreover, the combination of amikacin and D-histidine induced a synergistic effect against Pseudomonas aeruginosa biofilms, suggesting the potential utility of D-histidine as a preventive strategy against biofilm-associated infections caused by Pseudomonas aeruginosa.

Biofortified Rice Provides Rich Sakuranetin in Endosperm.

Sakuranetin plays a key role as a phytoalexin in plant resistance to biotic and abiotic stresses, and possesses diverse health-promoting benefits. However, mature rice seeds do not contain detectable levels of sakuranetin. In the present study, a transgenic rice plant was developed in which the promoter of an endosperm-specific glutelin gene OsGluD-1 drives the expression of a specific enzyme naringenin 7-O-methyltransferase (NOMT) for sakuranetin biosynthesis. The presence of naringenin, which serves as the biosynthetic precursor of sakuranetin made this modification feasible in theory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) validated that the seeds of transgenic rice accumulated remarkable sakuranetin at the mature stage, and higher at the filling stage. In addition, the panicle blast resistance of transgenic rice was significantly higher than that of the wild type. Specially, the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging was performed to detect the content and spatial distribution of sakuranetin and other nutritional metabolites in transgenic rice seeds. Notably, this genetic modification also did not change the nutritional and quality indicators such as soluble sugars, total amino acids, total flavonoids, amylose, total protein, and free amino acid content in rice. Meanwhile, the phenotypes of the transgenic plant during the whole growth and developmental periods and agricultural traits such as grain width, grain length, and 1000-grain weight exhibited no significant differences from the wild type. Collectively, the study provides a conceptual advance on cultivating sakuranetin-rich biofortified rice by metabolic engineering. This new breeding idea may not only enhance the disease resistance of cereal crop seeds but also improve the nutritional value of grains for human health benefits.

Research progress, challenges and perspectives of phospholipids metabolism in the LXR­LPCAT3 signaling pathway and its relation to NAFLD (Review).

Phospholipids (PLs) are principle constituents of biofilms, with their fatty acyl chain composition significantly impacting the biophysical properties of membranes, thereby influencing biological processes. Recent studies have elucidated that fatty acyl chains, under the enzymatic action of lyso­phosphatidyl­choline acyltransferases (LPCATs), expedite incorporation into the sn­2 site of phosphatidyl­choline (PC), profoundly affecting pathophysiology. Accumulating evidence suggests that alterations in LPCAT activity are implicated in various diseases, including non­alcoholic fatty liver disease (NAFLD), hepatitis C, atherosclerosis and cancer. Specifically, LPCAT3 is instrumental in maintaining systemic lipid homeostasis through its roles in hepatic lipogenesis, intestinal lipid absorption and lipoprotein secretion. The liver X receptor (LXR), pivotal in lipid homeostasis, modulates cholesterol, fatty acid (FA) and PL metabolism. LXR's capacity to modify PL composition in response to cellular sterol fluctuations is a vital mechanism for protecting biofilms against lipid stress. Concurrently, LXR activation enhances LPCAT3 expression on cell membranes and elevates polyunsaturated PL levels. This activation can ameliorate saturated free FA effects in vitro or endoplasmic reticulum stress in vivo due to lipid accumulation in hepatic cells. Pharmacological interventions targeting LXR, LPCAT and membrane PL components could offer novel therapeutic directions for NAFLD management. The present review primarily focused on recent advancements in understanding the LPCAT3 signaling pathway's role in lipid metabolism related to NAFLD, aiming to identify new treatment targets for the disease.

N-acyl homoserine lactones lactonase est816 suppresses biofilm formation and periodontitis in rats mediated by Aggregatibacter actinomycetemcomitans.

Aims: The current study aimed to explore the adjuvant therapeutic effect of N-acyl homoserine lactones (AHLs)-lactonase est816 on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) biological behaviors and periodontitis progression. Methods: The inhibitory properties of est816 were detected by live/dead bacterial staining, scanning electron microscope (SEM), crystal-violet staining and reverse-transcription quantitative PCR (RT-qPCR). Biocompatibility of est816 on human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGEs) was evaluated by CCK8 and ELISA. The ligature-induced periodontitis model was established in rats. Micro computed tomography and immunohistochemical and histological staining served to evaluate the effect of est816 on the prevention of periodontitis in vivo. Results: est816 significantly attenuated biofilm formation, reduced the mRNA expression of cytolethal distending toxin, leukotoxin and poly-N-acetyl glucosamine (PNAG) and downregulated expressions of interleukin-6 and tumor necrosis factor-α with low cell toxicity. In vivo investigations revealed est816 decreased alveolar bone resorption, suppressed matrix metalloproteinase-9 expression and increased osteoprotegerin expression. Conclusion: est816 inhibited A. actinomycetemcomitans biofilm formation and virulence release, resulting in anti-inflammation and soothing of periodontitis in rats, indicating that est816 could be investigated in further research on periodontal diseases.

Labial inverse dilaceration of bilateral maxillary central incisors: A case report.

BACKGROUND: Dilaceration is a rare dental developmental anomaly characterized by an abrupt deviation along the longitudinal axis of the root in which an angulation forms between the root and the crown. Here, we report on dilacerated bilateral maxillary central incisors in mixed dentition. CASE SUMMARY: A 10-year-old girl presented with a chief complaint of unerupted central incisors. An oral examination and radiography provided the basis for a diagnosis of dilaceration of the maxillary central incisors. After surgical exposure of the impacted teeth, a button with an attached chain was applied to the palatal surface of teeth 11 and 21. After 8 mo, a button was bonded to the labial surface of the crown to fix an elastic chain and move the teeth toward the maxillary arch. Finally, a fixed appliance was applied to tooth alignment to Class 1 malocclusion using a 0.019 × 0.025-inch nickel-titanium wire. After 3 years of follow-up, the clinical findings and radiographic assessment showed that the roots had developed with vital dental pulp and healthy periodontium, were acceptable aesthetically, and showed no resorption. CONCLUSION: The rare occurrences of dilacerated bilateral maxillary central incisors can be successfully treated through surgical exposure and orthodontics.

Post-synthetic modification of covalent organic framework for efficient adsorption of organochlorine pesticides from cattle's milk.

Analysis of organochlorine pesticides (OCPs) residues in milk faces a significant challenge. Herein, a sea urchin structured covalent organic framework bearing boric acid groups named COF-B(OH)2 was synthesized and applied as a coating material for solid-phase microextraction (SPME) of the OCPs in cattle's milk. Its performance was superior to that of three commonly used commercial SPME fibers, which could be due to the coexistence of hydrogen bonding, halogen bonding, π-stacking and electrostatic interactions. Besides, the fiber coating displayed good stability and reusability. After optimization, a COF-B(OH)2 based SPME coupled with gas chromatography-electron capture detection was established for the sensitive detection of the OCPs from milk samples. The limits of detection (S/N = 3) were between 0.04 and 1.00 µg kg-1. Satisfactory accuracy was achieved with the method recoveries in the range of 87.5 % to 112.5 %. These results manifest the feasibility of the COF-B(OH)2 coated fiber for the enrichment of the trace OCPs from milk samples.

An endophytic fungus interacts with the defensin-like protein OsCAL1 to regulate cadmium allocation in rice.

Defensin-like proteins are conserved in multicellular organisms and contribute to innate immune responses against fungal pathogens. In rice, defensins play a novel role in regulating cadmium (Cd) efflux from the cytosol. However, whether the antifungal activity of defensins correlates with Cd-efflux function remains unknown. In this study, we isolated an endophytic Fusarium, designed Fo10, by a comparative microbiome analysis of rice plants grown in a paddy contaminated with Cd. Fo10 is tolerant to high levels of Cd, but is sensitive to the defensin-like protein OsCAL1, which mediates Cd efflux to the apoplast. We found that Fo10 symbiosis in rice is regulated by OsCAL1 dynamics, and Fo10 coordinates multiple plant processes, including Cd uptake, vacuolar sequestration, efflux to the environment, and formation of Fe plaques in the rhizosphere. These processes are dependent on the salicylic acid signaling pathway to keep Cd levels low in the cytosol of rice cells and to decrease Cd levels in rice grains without any yield penalty. Fo10 also plays a role in Cd tolerance in the poaceous crop maize and wheat, but has no observed effects in the eudicot plants Arabidopsis and tomato. Taken together, these findings provide insights into the mechanistic basis underlying how a fungal endophyte and host plant interact to control Cd accumulation in host plants by adapting defense responses to promote the establishment of a symbiosis that permits adaptation to high-Cd environments.

Environmental Enrichment for Stroke and Traumatic Brain Injury: Mechanisms and Translational Implications.

Acquired brain injuries, such as ischemic stroke, intracerebral hemorrhage (ICH), and traumatic brain injury (TBI), can cause severe neurologic damage and even death. Unfortunately, currently, there are no effective and safe treatments to reduce the high disability and mortality rates associated with these brain injuries. However, environmental enrichment (EE) is an emerging approach to treating and rehabilitating acquired brain injuries by promoting motor, sensory, and social stimulation. Multiple preclinical studies have shown that EE benefits functional recovery, including improved motor and cognitive function and psychological benefits mediated by complex protective signaling pathways. This article provides an overview of the enriched environment protocols used in animal models of ischemic stroke, ICH, and TBI, as well as relevant clinical studies, with a particular focus on ischemic stroke. Additionally, we explored studies of animals with stroke and TBI exposed to EE alone or in combination with multiple drugs and other rehabilitation modalities. Finally, we discuss the potential clinical applications of EE in future brain rehabilitation therapy and the molecular and cellular changes caused by EE in rodents with stroke or TBI. This article aims to advance preclinical and clinical research on EE rehabilitation therapy for acquired brain injury. © 2024 American Physiological Society. Compr Physiol 14:5291-5323, 2024.

FRIZZLE PANICLE (FZP) regulates rice spikelets development through modulating cytokinin metabolism.

BACKGROUND: The number of grains per panicle is an important factor in determining rice yield. The DST-OsCKX2 module has been demonstrated to regulate panicle development in rice by controlling cytokinin content. However, to date, how the function of DST-OsCKX2 module is regulated during panicle development remains obscure. RESULT: In this study, the ABNORMAL PANICLE 1 (ABP1), a severely allele of FRIZZY PANICLE (FZP), exhibits abnormal spikelets morphology. We show that FZP can repress the expression of DST via directly binding to its promotor. Consistently, the expression level of OsCKX2 increased and the cytokinin content decreased in the fzp mutant, suggesting that the FZP acts upstream of the DST-OsCKX2 to maintain cytokinin homeostasis in the inflorescence meristem. CONCLUSIONS: Our results indicate that FZP plays an important role in regulating spikelet development and grain number through mediating cytokinin metabolism.

DOSE-L1000: unveiling the intricate landscape of compound-induced transcriptional changes.

MOTIVATION: The LINCS L1000 project has collected gene expression profiles for thousands of compounds across a wide array of concentrations, cell lines, and time points. However, conventional analysis methods often fall short in capturing the rich information encapsulated within the L1000 transcriptional dose-response data. RESULTS: We present DOSE-L1000, a database that unravels the potency and efficacy of compound-gene pairs and the intricate landscape of compound-induced transcriptional changes. Our study uses the fitting of over 140 million generalized additive models and robust linear models, spanning the complete spectrum of compounds and landmark genes within the LINCS L1000 database. This systematic approach provides quantitative insights into differential gene expression and the potency and efficacy of compound-gene pairs across diverse cellular contexts. Through examples, we showcase the application of DOSE-L1000 in tasks such as cell line and compound comparisons, along with clustering analyses and predictions of drug-target interactions. DOSE-L1000 fosters applications in drug discovery, accelerating the transition to omics-driven drug development. AVAILABILITY AND IMPLEMENTATION: DOSE-L1000 is publicly available at https://doi.org/10.5281/zenodo.8286375.

A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation.

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

Characterizing current noise of commercial constant-current sources by using an optically pumped rubidium atomic magnetometer.

This paper introduces a method for characterizing the current noise of commercial constant-current sources (CCSs) using a free-induction-decay (FID) type optically pumped rubidium atomic magnetometer driven by a radio frequency magnetic field. We convert the sensitivity of the atomic magnetometer into the current noise of CCS by calibrating the coil constant. At the same time, the current noise characteristics of six typical commercial low-noise CCSs are compared. The current noise level of the Keysight model B2961A is the lowest among the six tested CCSs, which is 36.233 ± 0.022 nA/Hz1/2 at 1-25 Hz and 133.905 ± 0.080 nA/Hz1/2 at 1-100 Hz. The sensitivity of the atomic magnetometer is dependent on the current noise level of the CCS. The CCS with low noise is of great significance for high-sensitivity atomic magnetometers. This research provides an important reference for promoting the development of high precision CCS, metrology, and basic physics research.

Whole-cage randomization for animal studies with unequal cage or group sizes.

Following good statistical practice, in vivo study investigators allocate animals into two or more treatment groups using a randomization routine to eliminate selection bias and balance known and unknown confounding factors. For some studies, however, randomization at the individual animal level cannot be implemented. For example, for studies that involve co-housed male mice, an animal-level randomization can place unfamiliar mice together in the same cage, which can trigger fighting. To meet the ethical obligations to enhance the welfare of an animal used in science, the experimental procedures are, therefore, often modified, and male mice, possibly from the same brood, may be housed together. It follows that animal allocation into groups must proceed at the whole-cage level. Given the small sample sizes in animal studies, controlling baseline variables can be quite challenging. The difficulty greatly increases with a whole-cage randomization restriction. When the number of animals per cage or the treatment group sizes are unequal, there is no algorithm in the literature to perform the task. We propose a novel, fast, and reliable algorithm to provide a whole-cage randomization that balances one or more baseline variables across groups. The algorithm was applied to a realistic example dataset.

Application of metabolomics in irritable bowel syndrome in recent 5 years.

Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders worldwide, characterized by chronic abdominal pain or discomfort and altered bowel habits. To date, the exact pathogenesis of IBS remains elusive, but is clearly multifactorial, including environmental and host factors. However, the management of patients with IBS is challenging and the current diagnostic and therapeutic modalities have unsatisfactory outcomes. Therefore, it is important to develop more effective methods to diagnose IBS early. Metabolomics studies the metabolites most closely related to patient characteristics, which can provide useful clinical biomarkers that can be applied to IBS and may open up new diagnostic approaches. Traditional Chinese medicine (TCM) can play a role in improving symptoms and protecting target organs, but its mechanism needs to be studied in depth. In this review, based on PubMed/MEDLINE and other databases, we searched metabolomics studies related to IBS in the past 5 years, including those related to clinical studies and animal studies, as well as literatures on TCM interventions in IBS, to provide an updated overview of the application of metabolomics to the diagnosis and treatment of IBS and the improvement of IBS by TCM.