Recent advances in LC-MS-based metabolomics for clinical biomarker discovery.

The employment of liquid chromatography-mass spectrometry (LC-MS) untargeted and targeted metabolomics has led to the discovery of novel biomarkers and improved the understanding of various disease mechanisms. Numerous strategies have been reported to expand the metabolite coverage in LC-MS-untargeted and targeted metabolomics. To improve the sensitivity of low-abundance or poor-ionized metabolites for reducing the amount of clinical sample, chemical derivatization methods are used to target different functional groups. Proper sample preparation is beneficial for reducing the matrix effect, maintaining the stability of the LC-MS system, and increasing the metabolite coverage. Machine learning has recently been integrated into the workflow of LC-MS metabolomics to accelerate metabolite identification and data-processing automation, and increase the accuracy of disease classification and clinical outcome prediction. Due to the rapidly growing utility of LC-MS metabolomics in discovering disease markers, this review will address the recent advances in the field and offer perspectives on various strategies for expanding metabolite coverage, chemical derivatization, sample preparation, clinical disease markers, and machining learning for disease modeling.

[Enhancing Understanding Among Intensive Care Unit Nurses of Lower Back Musculoskeletal Disorders and Associated Risks].

BACKGROUND & PROBLEMS: Work-related musculoskeletal disorders (WMSDs) have received the most attention worldwide of the various diseases addressed by the field of occupational medicine. In intensive care units (ICUs), patients with critical illness typically rely heavily on assistance provided by nurses to engage in daily life and rehabilitation activities. This dependence increases the risk of nurses experiencing WMSDs. An injury screening revealed that 56.4% of the nurses working in the ICU of the case hospital faced a mild risk of lower back musculoskeletal disorders and that the main contributor to this risk was lack of understanding among these nurses of lower-back-related WMSDs. PURPOSE: This project was designed to enhance understanding of lower back WMSDs among the ICU nurses and to reduce the percentage of nurses facing a mild risk of contracting WMSDs. RESOLUTIONS: 1. Organize integrated courses to introduce human-induced hazards and enhance nurses' understanding and prevention of WMSDs. 2. Design slogans, posters, and teaching videos to promote awareness of patient turning tips and procedures to prevent nurses from experiencing WMSDs due to incorrect force application. 3. Design illustrations highlighting risky postures commonly performed by nurses in ICUs that may cause lower back WMSDs to prevent the occurrence of human-induced injuries. RESULTS: The rate of correct understanding of lower back WMSDs in the target nurse population improved from 73.8% to 96.2%. In addition, the percentage of those assessed with a mild risk of contracting lower back musculoskeletal injuries decreased from 56.4% to 25.5%. CONCLUSIONS: This project promoted multifaceted improvement measures based on the WMSD screening and risk classification and management processes stipulated by Taiwan's Ministry of Labor to increase understanding of lower back WMSDs among ICU nurses and reduce the percentage of those facing a mild risk of contracting WMSDs.

Evaluating the advantages of treating acute cholecystitis by following the Tokyo Guidelines 2018 (TG18): a study emphasizing clinical outcomes and medical expenditures.

BACKGROUND: Acute cholecystitis (AC) is a common surgical emergency. The Tokyo Guidelines 2018 (TG18) provides a reliable algorithm for the treatment of AC patients to achieve optimal outcomes. However, the economic benefits have not been validated. We hypothesize that good outcomes and cost savings can both be achieved if patients are treated according to the TG18. METHOD: This retrospective study included 275 patients who underwent cholecystectomy in a 15-month span. Patients were divided into three groups (group 1: mild AC; group 2: moderate AC with American Society of Anesthesiologists (ASA) physical status class ≤ 2 and Charlson Comorbidity Index (CCI) score ≤ 5; and group 3: moderate AC with ASA class ≥ 3, CCI score ≥ 6, or severe AC). Each group was further divided into two subgroups according to management (followed or deviated from the TG18). Patient demographics, clinical outcomes, and hospital costs were compared. RESULTS: For group 1 patients, 77 (81%) were treated according to the TG18 and had a significantly higher successful laparoscopic cholecystectomy (LC) rate (100%), lower hospital cost ($1896 vs $2388), and shorter hospital stay (2.9 vs 8 days) than those whose treatment deviated from the TG18. For group 2 patients, 50 (67%) were treated according to the TG18 and had a significantly lower hospital cost ($1926 vs $2856), shorter hospital stay (3.9 vs 9.9 days), and lower complication rate (0% vs 12.5%). For group 3 patients, 62 (58%) were treated according to the TG18 and had a significantly lower intensive care unit (ICU) admission rate (9.7% vs 25%), but a longer hospital stay (12.6 vs 7.8 days). However, their hospital costs were similar. Early LC in group 3 patients did not have economic benefits over gallbladder drainage and delayed LC. CONCLUSION: The TG18 are the state-of-the-art guidelines for the treatment of AC, achieving both satisfactory outcomes and cost-effectiveness.

Diagnosis-Related Group (DRG)-Based Prospective Hospital Payment System can be well adopted for Acute Care Surgery: Taiwanese Experience with Acute Cholecystitis.

BACKGROUND: Laparoscopic cholecystectomy (LC) is a common procedure for cholelithiasis paid by diagnostic-related groups (DRGs) systems. However, acute cholecystitis (AC) patients usually have heterogeneous conditions that compromise the successful implementation of DRGs. We evaluated the quality/efficiency of treating AC patients under the DRG system in Taiwan. METHODS: All AC patients who underwent LC between October 2015 and December 2016 were included. Patient demographics, treatment outcomes, and financial results were analyzed. Patients were reimbursed by one of the two DRG schemes based on their comorbidities/complications (CC): DRG-1, LC without CC; and DRG-2, LC with CC. Hospitals were reimbursed the costs incurred if they were below the lower threshold (balanced sector); with the outlier threshold if costs were between the lower and outlier thresholds (profitable sector); and with the outlier threshold plus 80% of the exceeding cost if costs were higher than the outlier threshold (profit-losing sector). RESULTS: Among 246 patients, 114 were paid by DRG-1, and 132 were by DRG-2. In total, 195 of 246 patients underwent LC within 1 day after admission, and patients with mild AC had shorter hospital stays than those with moderate or severe AC. The complication rate was 7.3% with only one mortality. In total, 92.1% of patients in DRG-1 and 90.9% of patients in DRG-2 were profitable. The average margin per patient was 11,032 TWD for DRG-1 and 24,993 TWD for DRG-2. CONCLUSIONS: DRGs can be well adopted for acute care surgery, and hospitals can still provide satisfactory services without losing profit.

Rethinking potentially inappropriate medication use in nursing homes within the Chinese population.

This study aimed to understand the prevalence of Chinese medicine and other potentially inappropriate medications and to examine if there are relationships with emergency room visits, hospital admissions, and falls in a Chinese nursing home population. This cross-sectional descriptive study was a secondary analysis of data from 531 nursing home residents in Taiwan. Cox proportional hazard regression models were used in the analysis. Use of Chinese medicine in combination with Western medicine was observed in approximately 1% of residents. For every additional Chinese medicine used, the hazard ratio was 3.09 (p=.26) for emergency room visits and 3.22 (p=.21) for hospital admissions. For every additional nonsteroidal antiinflammatory agent used, the hazard ratio for falls was 5.42 (p=.006). Further studies with larger sample sizes are required to understand the appropriate time intervals required between administration of Chinese and Western medicine as well as to understand the drug-drug interactions.

Identification of Urinary Metabolite Biomarkers of Type 2 Diabetes Nephropathy Using an Untargeted Metabolomic Approach.

Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus (DM). To discover early stage biomarkers of DN, untargeted liquid chromatography-mass spectrometry-based metabolomic analysis was performed in urine samples from healthy subjects and patients with micro- or macroalbuminuria due to nondiabetic disease (macro), type 2 DM without microalbuminuria (T2DM), and type 2 DM with microalbuminuria (T2DM+micro). Levels of four metabolites were significantly different among groups, and they were quantified in a larger group of 267 urine samples. Two metabolites were also discovered and validated in targeted metabolic study of amino acids. For diagnosis of nephropathy, N1-methylguanosine had the highest area-under-the-curve (AUC) value of 0.75 when compared to those of valine (0.68), xanthosine (0.67), and 7-methyluric acid (0.69). After combining fasting blood glucose and diastolic blood pressure (DBP) with N1-methylguanosine, the AUC increased to 0.987. To distinguish between T2DM and T2DM+micro conditions, xanthosine and N1-methylguanosine have AUC value of 0.612 and 0.624, respectively. After adjustment of HbA1c and DBP, AUC values of xanthosine and N1-methylguanosine increased to 0.716 and 0.723, respectively, and could be used to predict the development of nephropathy in T2DM patients.

Urethral proteomic analysis in ovariectomized mice administered 17ß-oestradiol replacement therapy.

The molecular mechanism by which 17ß-oestradiol (E2) increases urethral tone is unclear. As human tissue is limited in availability, we explored changes in the urethras of ovariectomized (OVX) mice. Twenty-four virgin female mice were randomised into three groups: mice with a sham operation only (control), OVX mice without E2 replacement (OVX), and OVX mice with E2 replacement (OVX + E2). Two weeks after the ovariectomy, mice received either E2 or placebo for 4 weeks. Leak point pressure (LPP) and maximum urethral closure pressure (MUCP) were assessed in these mice at 6 weeks after OVX, under anaesthesia. After measurements were recorded, the animals were sacrificed and the urethras were removed for proteomic and further analyses. LPP and MUCP values were significantly higher in OVX + E2 group than in OVX group. Fourteen differentially expressed proteins within the urethras of mice from OVX and OVX + E2 groups were identified; six proteins were upregulated and eight proteins were down-regulated. Most E2-induced proteins are involved in proteolysis, development, neurophysiological processes, transcription, and the cell cycle. Expression of survival motor neuron (SMN) protein in the urethra was significantly increased in OVX + E2 group compared to OVX group. OVX can impair urethral tone in female mice. E2 supplementation in OVX mice rescued urethral tone. E2-mediated increase in urethral tone in OVX mice involves overexpression of SMN, decreased proteolysis and promotion of development, neurophysiological processes, and transcription in the urethra. The urethra actively undergoes multiple biological processes in response to OVX and OVX with E2 stimuli. Impact statement Estrogens are known to modulate lower urinary tract trophicity. Although treatments with 17ß-oestradiol (E2) result in an increase in urethral tone, the mechanism by which E2 increases urethral tone is still not completely understood. Ovariectomy can impair urethral tone in female mice. E2 supplementation in ovariectomized mice rescued urethral tone. E2-mediated increase in urethral tone in ovariectomized mice involves overexpression of survival motor neuron, decreased proteolysis and promotion of development, neurophysiological processes, and transcription in the urethra. This information will offer clues about pathogenesis of stress urinary incontinence after menopause and will open additional avenues for novel research and potential therapies.

Proteomic analysis of urethral protein expression in an estrogen receptor α-deficient murine model of stress urinary incontinence.

PURPOSE: The roles of estrogen receptor α (ERα) in stress urinary incontinence (SUI) remain elusive. This study was conducted to understand the molecular mechanism of ERα against SUI. METHODS: Wild-type (ERα(+/+)) and ACTB-cre ERα knockout (ERα(-/-)) female mice were generated. Urethral function and protein expression were measured. Leak point pressures (LPP) and maximum urethral closure pressure (MUCP) were assessed in mice under urethane anesthesia. After the measurements, the urethras were removed for proteomic analysis using the two-dimensional differential gel electrophoresis and liquid chromatography-mass spectrometry technology. Interaction between these ERα pathway proteins was further analyzed by using MetaCore. Lastly, Western blot and immunochemistry (IHC) were used to confirm the candidate protein expression levels and locations, respectively. RESULTS: Compared with the ERα(+/+) group, the LPP and MUCP values of the ERα(-/-) group were significantly decreased. Additionally, we identified 11 differentially expressed proteins in the urethra of ERα(-/-) female mice; five proteins were down-regulated and six were up-regulated. The majority of the ERα knockout-modified proteins were involved in muscle development, contraction, and regulation, as well as immune response (amphoterin signaling and phagocytosis), proteolysis, and cell adhesion (platelet aggregation and integrin-mediated cell-matrix adhesion). IHC and Western blot confirmed the down-regulation of tropomyosin and up-regulation of myosin in urethra. CONCLUSIONS: This is the first study to estimate protein expression changes in urethras from ERα(-/-) female mice. These changes could be related to the molecular mechanism of ERα in SUI.

Synergistic effect of vaginal trauma and ovariectomy in a murine model of stress urinary incontinence: upregulation of urethral nitric oxide synthases and estrogen receptors.

The molecular mechanisms underlying stress urinary incontinence (SUI) are unclear. We aimed to evaluate the molecular alterations in mice urethras following vaginal trauma and ovariectomy (OVX). Twenty-four virgin female mice were equally distributed into four groups: noninstrumented control; vaginal distension (VD) group; OVX group; and VD + OVX group. Changes in leak point pressures (LPPs), genital tract morphology, body weight gain, plasma 17ß-estradiol level and expressions of neuronal nitric oxide synthase (nNOS), induced nitric oxide synthase (iNOS), and estrogen receptors (ERs-ERα and ERß) were analyzed. Three weeks after VD, the four groups differed significantly in genital size and body weight gain. Compared with the control group, the plasma estradiol levels were significantly decreased in the OVX and VD + OVX groups, and LPPs were significantly decreased in all three groups. nNOS, iNOS, and ERα expressions in the urethra were significantly increased in the VD and VD + OVX groups, whereas ERß expression was significantly increased only in the VD + OVX group. These results show that SUI following vaginal trauma and OVX involves urethral upregulations of nNOS, iNOS, and ERs, suggesting that NO- and ER-mediated signaling might play a role in the synergistic effect of birth trauma and OVX-related SUI pathogenesis.

Identification of a potential prognostic plasma biomarker of acute ischemic stroke via untargeted LC-MS metabolomics.

PURPOSE: Stroke is the sudden death of brain cells in a localized area due to an inadequate blood flow or blood vessel rupture, and it seriously affects the quality of life. The metabolite biomarkers are needed for predicting the functional outcome of acute ischemic stroke (AIS). EXPERIMENTAL DESIGN: To identify biomarkers for AIS, untargeted LC/MS metabolomics was performed on plasma samples from subjects with favorable prognosis (mRS ≤ 2) and unfavorable prognosis (mRS > 2). The identified markers were further absolutely quantified by a targeted MRM approach. RESULTS: There were 10 upregulated and 26 downregulated markers. Among these candidates, one was successfully identified as glycocholic acid and then absolutely quantified in plasma samples. Glycocholic acid could discriminate between subjects with favorable and unfavorable prognosis with an area under the curve (AUC) of 0.68 and odds ratio of 5.88. CONCLUSIONS AND CLINICAL RELEVANCE: Glycocholic acid was identified as a potential plasma metabolite marker of non-progressive outcomes after ischemic stroke and could serve as predictive prognostic markers for clinical acute stroke outcomes.

The identification of a potential plasma metabolite marker for Alzheimer's disease by LC-MS untargeted metabolomics.

BACKGROUND AND AIMS: Alzheimer's disease (AD), the most common type of dementia, is hard to recognize early, resulting in delayed treatment and poor outcome. At present, there is neither reliable, non-invasive methods to diagnose it accurately and nor effective drugs to recover it. Discovery and quantification of novel metabolite markers in plasma of AD patients and investigation of the correlation between the markers and AD assessment scores. MATERIALS AND METHODS: Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics with LC-quadrupole- time-of-flight (Q-TOF) was performed in plasma samples of age-matched AD patients and healthy controls. The potential markers were further quantified with targeted multiple reaction monitoring (MRM) approach. RESULTS: Among the candidates, progesterone, and 3-indoleacetic acid (3-IAA) were successfully identified and then validated in 50 plasma samples from 25 AD patients and 25 matched normal controls with MRM approach. As a result, 3-IAA was significantly altered in AD patients and correlated with some AD assessment scores. CONCLUSION: By using untargeted LC-MS metabolomic and LC-MRM approaches to analyze plasma metabolites of AD patients and normal subjects, 3-IAA was discovered and quantified to be significantly altered in AD patients and correlated with several AD assessment scores.

HSP90-regulated CHIP/TRIM21/p21 Axis Involves in the Senescence of Osteosarcoma Cells.

BACKGROUND: OS is the most frequent malignant bone tumor with a poor prognosis. TRIM21 has been reported to play a critical role in OS by regulating the expression of the TXNIP/p21 axis and inhibiting the senescence of OS cells. AIM: Investigation of the molecular mechanism of tripartite motif 21 (TRIM21) in osteosarcoma (OS) would shed light on the understanding of the pathogenesis of OS. OBJECTIVE: This study aimed to explore the mechanism regulating the protein stability of TRIM21 in the process of OS senescence. METHODS: Human U2 OS cells were used to establish stable cells overexpressing TRIM21 (induced by Dox) or knocking down TRIM21. The co-immunoprecipitation (co-IP) assay was used to examine the interaction between TRIM21 and HSP90. Immunofluorescence (IF) assay was used to observe colocalization in OS cells. Western blot analysis was applied to detect the protein expression, and quantitative real-time PCR (qRT-PCR) assay was used to test the mRNA expression of corresponding genes. SA-ß-gal staining was used to evaluate OS senescence. RESULTS: In this study, we verified the interaction between HSP90 and TRIM21 using a co-IP assay. Knockdown or inhibition of HSP90 with its inhibitor 17-AAG accelerated the degradation of TRIM21 by the proteasome in OS cells. CHIP E3 ligase mediated this degradation of TRIM21, with the knockdown of CHIP rescuing the downregulation of TRIM21 induced by 17-AAG. TRIM21 inhibited OS senescence and downregulated the expression of senescence marker p21, while CHIP exhibited an opposite regulatory role on p21 expression. CONCLUSION: Taken together, our results demonstrated that HSP90 is responsible for the stabilization of TRIM21 in OS and that the CHIP/TRIM21/p21 axis controlled by HSP90 affects the senescence of OS cells.

Baicalein Exerts Therapeutic Effects against Endotoxin-Induced Depression-like Behavior in Mice by Decreasing Inflammatory Cytokines and Increasing Brain-Derived Neurotrophic Factor Levels.

Inflammation plays an important role in the pathophysiology of depression. This study aims to elucidate the antidepressant effect of baicalein, an anti-inflammatory component of a traditional Chinese herbal medicine (Scutellaria baicalensis), on lipopolysaccharide (LPS)-induced depression-like behavior in mice, and to investigate the underlying mechanisms. In vitro, baicalein exhibited antioxidant activity and protected macrophages from LPS-induced damage. The results of the tail suspension test and forced swimming test (tests for despair potential in mice) showed the antidepressant effect of baicalein on LPS-treated mice. It also substantially decreased the production of pro-inflammatory cytokines, including IL-6, TNF-α, MCP-1, and eotaxin, elicited by LPS in the plasma. Baicalein downregulated NF-κB-p65 and iNOS protein levels in the hippocampus, demonstrated its ability to mitigate neuroinflammation. Additionally, baicalein increased the levels of the mature brain-derived neurotrophic factor (mBDNF) in the hippocampus of LPS-treated mice, and elevated the ratio of mBDNF/proBDNF, which regulates neuronal survival and synaptic plasticity. Baicalein also promoted the expression of CREB, which plays a role in a variety of signaling pathways. In summary, the findings of this study demonstrate that the administration of baicalein can attenuate LPS-induced depression-like behavior by suppressing neuroinflammation and inflammation induced by the peripheral immune response.

Prediction of Gastric Gastrointestinal Stromal Tumors before Operation: A Retrospective Analysis of Gastric Subepithelial Tumors.

Gastrointestinal stromal tumors (GISTs), leiomyomas, and schwannomas are the most common gastric subepithelial tumors (GSETs) with similar endoscopic findings. Preoperative prediction of GSETs is difficult. This study analyzed and predicted GSET diagnosis through a retrospective review of 395 patients who underwent surgical resection of GISTs, leiomyomas, and schwannomas measuring 2-10 cm. GSETs were divided by size (group 2-5, >2 and ≤5 cm; group 5-10, >5 and ≤10 cm) for analysis. Demographics, clinical symptoms, and images were analyzed. A recursive partitioning analysis (RPA) was used to identify optimal classifications for specific GSET diagnoses. GIST patients were relatively older than other patients. Both groups had higher proportions of UGI bleeding, lower hemoglobin (Hb) levels, and a higher ratio of necrosis on their computed tomography (CT) scans. The RPA tree showed that (a) age ≤ 55, Hb ≥ 10.7, and CT necrosis; (b) age ≤ 55 and Hb < 10.7; (c) age >55 and Hb < 12.9; and (d) age >55 and CT hetero-/homogeneity can predict high GIST risk in group 2-5. Positive or negative CT necrosis, with age >55, can predict high GIST risk in group 5-10. GIST patients were older and presented with low Hb levels and tumor necrosis. In RPA, the accuracy reached 85% and 89% in groups 2-5 and 5-10, respectively.

Chinese Herbal Medicine Usage Reduces Overall Mortality in HIV-Infected Patients With Osteoporosis or Fractures.

The survival of patients with HIV has greatly improved, due to Anti-Retroviral Therapy (ART). However, long-term HIV survivors often develop serious bone abnormalities, possibly due to the interplay of osteoblasts, osteoclasts, HIV ad ART. We evaluated in a nation-wide study in Taiwan the effect of Chinese herbal medicine (CHM) on overall mortality in HIV patients with osteoporosis or fractures. Enrollment period was between 1998 and 2011. Patients with osteoporosis or fractures before the HIV infection, and those with less than 14 days CHM use, were excluded. This left 498 patients, 160 CHM users, 338 without CHM. Univariate Kaplan-Meier and multivariate Cox regression analysis were used to compare the overall mortality in these 2 groups. Due to the nature of Chinese medicine, CHMs inevitably varied. We therefore also used rule mining and network analysis to determine which major CHM clusters were prescribed to the patients. CHM users had a much Lower mortality (hazard ratio (HR) = 0.43, 95% confidence interval (CI): 0.24-0.77, p < 0.005) and higher survival (p = 0.004, log-rank test). Although the CHMs greatly varied, network analysis identified one main cluster of strongly related CHM combinations (Chuan-Xiong-Cha-Tiao-San (CXCTS), Gan-Cao (GC; Glycyrrhiza uralensis Fisch.), Liu-He-Tang (LHT), Huang-Qin-Tang (HQT), Jia-Wei-Ping-Wei-San (JWPWS), and Dang-Gui-Long-Hui-Wan (DGLHuiW)). CHM as an additional treatment strongly improves overall survival in HIV-infected patients with osteoporosis and fractures.

Complementary Chinese Herbal Medicine Therapy Improves Survival in Patients With Pemphigus: A Retrospective Study From a Taiwan-Based Registry.

Pemphigus is a life-threatening and skin-specific inflammatory autoimmune disease, characterized by intraepidermal blistering between the mucous membranes and skin. Chinese herbal medicine (CHM) has been used as an adjunct therapy for treating many diseases, including pemphigus. However, there are still limited studies in effects of CHM treatment in pemphigus, especially in Taiwan. To more comprehensively explore the effect of long-term CHM treatment on the overall mortality of pemphigus patients, we performed a retrospective analysis of 1,037 pemphigus patients identified from the Registry for Catastrophic Illness Patients database in Taiwan. Among them, 229 and 177 patients were defined as CHM users and non-users, respectively. CHM users were young, predominantly female, and had a lesser Charlson comorbidity index (CCI) than non-CHM users. After adjusting for age, sex, prednisolone use, and CCI, CHM users had a lower overall mortality risk than non-CHM users (multivariate model: hazard ratio (HR): 0.422, 95% confidence interval (CI): 0.242-0.735, p = 0.0023). The cumulative incidence of overall survival was significantly higher in CHM users than in non-users (p = 0.0025, log rank test). Association rule mining and network analysis showed that there was one main CHM cluster with Qi-Ju-Di-Huang-Wan (QJDHW), Dan-Shen (DanS; Radix Salviae miltiorrhizae; Salvia miltiorrhiza Bunge), Jia-Wei-Xiao-Yao--San (JWXYS), Huang-Lian (HL; Rhizoma coptidis; Coptis chinensis Franch.), and Di-Gu-Pi (DGP; Cortex lycii; Lycium barbarum L.), while the second CHM cluster included Jin-Yin-Hua (JYH; Flos lonicerae; Lonicera hypoglauca Miq.) and Lian-Qiao (LQ; Fructus forsythiae; Forsythia suspensa (Thunb.) Vahl). In Taiwan, CHMs used as an adjunctive therapy reduced the overall mortality to approximately 20% among pemphigus patients after a follow-up of more than 6 years. A comprehensive CHM list may be useful in future clinical trials and further scientific investigations to improve the overall survival in these patients.

Downregulation of tight junction protein zonula occludens-2 and urothelium damage in a cyclophosphamide-induced mouse model of cystitis.

OBJECTIVES: The cyclophosphamide (CYP)-induced model of cystitis in mice closely fits the symptoms of chronic bladder inflammation. Cystitis was recently found to be due to an altered gap junction protein in a rat model. Thus, this study was conducted to evaluate changes in protein expression and composition in the bladder of CYP-treated mice. MATERIALS AND METHODS: Administration of CYP induced cystitis-related symptoms in mice. Cystometry was assessed and cell junction-associated protein zonula occludens-2 (ZO-2) expression was measured. Voiding interval values (time between voids) were assessed in mice under anesthesia. The bladders were removed for proteomic analysis using label-free quantitative proteomics and liquid chromatography-mass spectrometry. Additionally, immunochemistry (IHC) and Western blot were used to confirm the location and level, respectively, of ZO-2 expression. RESULTS: Compared to the control group, the voiding interval values and urothelial thickness in the bladder in the CYP-treated group were significantly decreased. Additionally, we identified 105 differentially expressed proteins in the bladder of CYP-treated mice with proteomic analysis. These proteins were involved in cell-cell tight junctions, exocytosis, muscle development, contraction, and regulation, immune responses, proteolysis, and cell adhesion. IHC and Western blot confirmed the downregulation of the tight junction protein ZO-2 in the urothelium of bladder. CONCLUSIONS: Our results suggest that downregulation in tight junction protein ZO-2 and urothelium damage may have a role in cystitis-related OAB. These changes could be related to the molecular mechanism of cystitis-related OAB.

High levels of circulating endothelial progenitor cells in patients with diabetic retinopathy are positively associated with ARHGAP22 expression.

Diabetic retinopathy (DR) is a common microvascular complication of diabetes. Circulating endothelial progenitor cells (EPCs) are derived from bone marrow and are characterized by pathological retinal neovascularization. Rho GTPase Activating Protein 22 (ARHGAP22) is a DR susceptibility gene that interacts with its downstream regulatory protein ras-related C3 botulinum toxin substrate 1 (Rac1), to assist in endothelial cell angiogenesis and increasing capillary permeability. The aim of this study was to elucidate the relationship between ARHGAP22 expression and EPC levels in type 2 diabetes (T2D) patients with DR. Fifty T2D patients with DR were recruited. Circulating EPCs were characterized as CD31+/vascular endothelial growth factor-2+/CD45dim/CD133+ and were quantified using triple staining flow cytometry. Real-time polymerase chain reaction tests were used to quantify ARHGAP22 expression. We found that T2D patients with proliferative DR had significantly lower EPC levels than those with non-proliferative DR (P = 0.028). T2D patients with EPC levels above the median value (> 4 cells/105 events) had higher levels of ARHGAP22 expression (P = 0.002). EPC levels were positively correlated with ARHGAP22 expression (r = 0.364, P = 0.009). Among T2D patients with DR, a higher expression of ARHGAP22 was associated with higher levels of EPCs. ARHGAP22 may be involved in the mobilization or active circulation of EPCs, thus contributing to neovascularization during DR development.

Treatment of stress urinary incontinence by ginsenoside Rh2.

Stress urinary incontinence (SUI) is a common disorder in middle-aged women and the elderly. Although surgical treatment of SUI has progressed, there are no effective pharmacological therapies without a side effect. We studied the effect of ginsenoside Rh2 against SUI. Here, we studied the effect of ginsenoside Rh2 on the contractile force of the urethra and blood vessels in an ex vivo organ bath assay. We further investigated the mechanisms and effects of Rh2 in cell culture and animal models. Ginsenoside Rh2 dose-dependently reduced lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells. In the vaginal distension (VD)-induced SUI mouse model, ginsenoside Rh2 significantly reversed the VD-induced SUI physical signs and reduced blood pressure. The modulation of several SUI-related proteins, including myosin, survival motor neuron (SMN) protein, α-adrenergic receptor 1a (AdR1a), and superoxide dismutase 3 (SOD3), may play some crucial roles in the therapeutic approaches against SUI. In conclusion, the ginsenoside Rh2 may offer therapeutic potential against SUI.

Treatment of stress urinary incontinence by cinnamaldehyde, the major constituent of the chinese medicinal herb ramulus cinnamomi.

Stress urinary incontinence (SUI) is a common disorder in middle-aged women and the elderly population. Although surgical treatment of SUI has progressed, pharmacological therapies remain unelucidated. We screened potential herbal medicines against SUI with an ex vivo organ bath assay. Ramulus Cinnamomi and its major constituent cinnamaldehyde cause a high contractile force of the urethra and a low contractile force of blood vessels. Cinnamaldehyde dose-dependently reduced lipopolysaccharide-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells. In the vaginal distension- (VD-) induced SUI model in mice, cinnamaldehyde significantly reversed the VD-induced SUI physical signs and reduced blood pressure. Cinnamaldehyde may offer therapeutic potential against SUI without the possible side effect of hypertension. The modulation of several SUI-related proteins including myosin, iNOS, survival motor neuron (SMN) protein, and superoxide dismutase 3 (SOD3) may play some crucial roles in the therapeutic approach against SUI. This information may offer clues to the pathogenesis of SUI and open additional avenues for potential therapy strategies.