Investigation of monacolin K, yellow pigments, and citrinin production capabilities of Monascus purpureus and Monascus ruber (Monascus pilosus).

Red mold rice (RMR) is a traditional Chinese medicine prepared using Monascus fermentation. Monascus ruber ( pilosus) and Monascus purpureus have a long history of use as food and medicine. As an economically important starter culture, the relationship between the taxonomy of Monascus and production capabilities of secondary metabolites is crucial for the Monascus food industry. In this study, monacolin K, monascin, ankaflavin, and citrinin production by M. purpureus and M. ruber were genomically and chemically investigated. Our findings suggest that M. purpureus can produce monascin and ankaflavin in a correlated manner, whereas M. ruber produces monascin with minimum ankaflavin. M. purpureus is capable of producing citrinin; however, it is unlikely able to produce monacolin K. In contrast, M. ruber produces monacolin K, but not citrinin. We suggest that the current monacolin K content-related regulation of Monascus food should be revised, and labeling of Monascus species should be considered.

Microencapsulated rhEGF to facilitate epithelial healing and prevent scar formation of cesarean wound: A randomized controlled trial.

OBJECTIVE: Cesarean section (CS) is a major surgical intervention that affects women at childbearing age. Scarring from CS potentially causes discomfort and psychological distress. Emerging evidence indicates that epidermal growth factor (EGF) plays crucial roles in wound healing with the potential of minimizing scar formation. This study aims to investigate the effect of microencapsulated recombinant human EGF (Me-EGF) in scar prevention. Silicone gel was incorporated as part of the routine scar treatment. MATERIALS AND METHODS: Healthy women scheduled for cesarean delivery were enrolled and randomized to three groups: (1) no scar treatment, (2) silicone gel only, or (3) silicone gel plus Me-EGF. Vancouver Scar Scale (VSS: vascularity, pigmentation, elasticity, and height) was used for scar assessment at the 6th month and 9th month after CS. RESULTS: A total of 60 women were enrolled, but one patient withdrew due to noncompliance with the follow-up visit requirement. Me-EGF-containing treatment group consistently scored the lowest on every parameter in the VSS scale, followed by silicone gel group, and the group with no scar treatment. Kruskal-Wallis tests indicated significant differences (p < 0.05) between Me-EGF-containing treatment group and the other two groups in vascularity, pigmentation, elasticity, and the VSS total score, at either 6th month, 9th month, or both time points. The only parameter not showing any significant between-group difference was scar height, but the pattern still remained the same, in which Me-EGF group scored better in both month 6 and 9. CONCLUSION: Surgical incisions in lower abdomen posed challenge in scar management. Our findings suggest that Me-EGF is a potential therapeutic option for better wound healing and scar prevention.

Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects.

Development of specific antiviral agents is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infection. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validate cleavage at a putative furin substrate motif at SARS-CoV-2 spikes by expressing it in VeroE6 cells and find prominent syncytium formation. Cleavage and the syncytium are abolished by treatment with the furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein, but not by the transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein show antiviral effects on SARS-CoV-2-infected cells by decreasing virus production and cytopathic effects. Further analysis reveals that, similar to camostat, CMK blocks virus entry, but it further suppresses cleavage of spikes and the syncytium. Naphthofluorescein acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may be promising antiviral agents for prevention and treatment of SARS-CoV-2 infection.

Treatment with the herbal formulation Eefooton slows the progression of chronic kidney disease: A case report.

RATIONALE: Patients with end-stage kidney disease (ESKD) receiving maintenance dialysis experience an overall burden of physical and emotional symptoms. However, there were limited alternative treatments to dialysis. PATIENT CONCERNS: A 79-year-old woman with chronic kidney disease stage 5 (CKD5) and gout had refused to be on dialysis. She also had hypoglycemia, hypertension, and heart disease. DIAGNOSES: The patient had received the ultrasonography, the renal biopsy and biochemical examinations, confirming the diagnosis of renal impairment, primary hypertension, and chronic nephritic syndrome with unspecified morphologic changes. INTERVENTIONS: She was administered with 20 mL Eefooton (a liquid formula of herbal extracts: Astragalus membranaceus 3 g, Codonopsis pilosula 3 g, Ligustrum lucidum 3 g, Panax quinquefolius 1.3 g, and Rhodiola sacra 1.3 g) orally twice a day for 6 months in addition to her regular medications. OUTCOMES: The patient was followed up for 3 months after the completion of the Eefooton adjuvant treatment. The patient's renal function was improved, and CKD progression was alleviated. After Eefooton treatment, the sizes of both kidneys in the patient increased by 8% while blood urea nitrogen (BUN) and serum creatinine concentrations were decreased. In addition, further reduction in BUN concentration was observed 2 months posttreatment. LESSONS: This case demonstrated that Eefooton has potential therapeutic significance in patients with CKD5 who chose conservative treatment over dialysis.

Novel Neovaginoplasty Using Rudimentary Uterine Horn Serosa and Pelvic Peritoneum as a Graft in Müllerian Anomalies with Vaginal Agenesis.

STUDY OBJECTIVE: To study the outcome of a novel method of laparoscopic neovaginal reconstruction using rudimentary uterine horn serosa and the pelvic peritoneum as a graft. DESIGN: Canadian Task Force classification II-1. SETTING: A university hospital. PATIENTS: A retrospective study of 14 patients from 2000 to 2014 of patients with vaginal agenesis who underwent laparoscopic neovagina reconstruction using rudimentary uterine horn serosa and the pelvic peritoneum as a graft. INTERVENTION: Patients with vaginal agenesis associated with müllerian agenesis who requested surgery. Tertiary referral center and laparoscopic unit. The creation of a neovagina using rudimentary uterine horn serosa and the pelvic peritoneum as a graft via a combined laparoscopic and vaginal route. MEASUREMENTS AND MAIN RESULTS: Data were collected retrospectively including postoperative vaginal length and width, complications, stenosis or reoperations, dyspareunia, and sexual satisfaction. There were no major complications from the surgery with no rectal perforation or bladder or ureteric injury. The postoperative mean (±SD) vaginal length was 6.0±0.7 cm and a width of 2 fingerbreadths. The mean operation time was 142.7±45.9 min. Median blood loss was 100 ml (range: 10 to 300 mL). The mean duration of the hospital stay was 6.6±1.6 days. The follow-up period ranged from 3 to 84 months with a median follow-up of 11 months. CONCLUSION: Lee's method of neovaginoplasty using rudimentary uterine horn serosa and the pelvic peritoneum as a graft is a good method for neovagina creation with minimal morbidity, fast recovery, and minimal complications. This method results in good anatomic and functional outcome and can be a method that is widely used.

The implication of probiotics in the prevention of dental caries.

The current oral health crisis, whose causes are varied and complex, necessitates timely oral evaluation and early detection and treatment of oral health problems. Dramatic changes in eating habits and lifestyles are associated with the recent decline in oral health. Probiotics are "good" bacteria that support digestion and a healthy immune system and offer various health benefits to the host. Traditionally, probiotics have been used to improve gut health; the most common uses have historically been as a treatment or prevention of gastrointestinal infections and disease. During the last decade, studies have additionally suggested the intake of probiotics for oral health purposes. Probiotic use provides an effective strategy to combat oral disease, including the development of dental caries and periodontal infection. The aim of this review is to describe the beneficial roles of probiotic bacteria in the oral cavity and the potential mechanisms by which these bacteria exert their effects on oral health.

A "Ct contrast"-based strain-specific real-time quantitative PCR system for Lactobacilllus paracasei subsp. paracasei NTU 101.

BACKGROUND/PURPOSES: Routine cell number determination for specific Lactobacillus strain by cultivation requires at least 4-7 days. Thus rapid and specific cell number determine methods such as strain-specific quantitative PCR (qPCR) are valuable. However, qPCR method is vulnerable to difficult PCR target such as dimer/secondary structure forming sequence. METHODS: In this study, a two-component, "Ct contrast" approach was applied to strain-specific qPCR system following the development of Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) strain-specific PCR with random amplification of polymorphic DNA (RAPD)-derived strain-specific sequences. RESULTS: The quantitative range of the NTU 101 strain-specific qPCR system was 3.0 × 101 to 3.0 × 105 copies for pure cultures, and 3.0 × 102 to 3.0 × 105 copies for multi-strain or unknown food samples. The results of spike in test and real sample testing suggested that non-specific weak background signals did not compromise test specificity, and demonstrated the potential of the NTU 101 strain-specific qPCR system in food samples. CONCLUSION: The two-component, "Ct contrast" approach is useful for qPCR discrimination when no ideal PCR target is available or the variance of the target site is unpredictable. The Ct contrast approach might provide a simple and robust solution for other challenging qPCR targets.

Alleviation of metabolic syndrome by monascin and ankaflavin: the perspective of Monascus functional foods.

The metabolites of Monascus with multiple benefits are popular subjects for the development of functional foods. The yellow pigments, monascin and ankaflavin, which are the constituent metabolites of M. purpureus, M. pilosus and M. ruber, are becoming the focus of research on Monascus. Monascin and ankaflavin are azaphilone compounds with similar structures that exhibit multiple beneficial effects including anti-inflammation, anti-oxidation, anti-diabetes, immunomodulation, attenuation of Alzheimer's disease risk factor, and anti-tumorigenic effects. Monascin and ankaflavin not only possess pleiotropic bioactivities, but are also more potent than monacolin K in lowering lipid levels and have lower toxicity. Monascin and ankaflavin act as the activators of PPARγ agonist/Nrf-2 that subsequently ameliorate metabolic syndrome. Following the intensive exploration of Monascus bioactivities in recent years, the focus of research on Monascus-functional foods has shifted from whole fermented products/extracts to specific bioactive compounds. Therefore, the production of monascin and ankaflavin is an important topic with respect to Monascus-functional foods. Although several genomic studies have paved the way for understanding the production of secondary metabolites in Monascus, efforts are still required to effectively manipulate the biosynthesis of secondary metabolites with genetic engineering and/or culture techniques.

Effects of deep sea water and Lactobacillus paracasei subsp. paracasei NTU 101 on hypercholesterolemia hamsters gut microbiota.

Hypercholesterolemia is a common metabolic syndrome in modern human society. Despite that the alteration of host gut microbiota has been linked to hypercholesterolemia in previous studies, the key host-microbiota interaction of hypercholesterolemia remains elusive. Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) and deep sea water (DSW) were known for cholesterol-lowering potential. The impact of NTU 101 and DSW on hamster gut microbiota was investigated side-by-side using denaturing gradient gel electrophoresis (DGGE) and metagenomic analysis in this study. These two cholesterol-lowering substances altered hamster cecal microbiota in a very different way with similar cholesterol-lowering effects. Bacteroidetes was the only bacterial population that significantly correlated to host lipid profile (serum total cholesterol and serum low-density lipoprotein). Allobaculum and Clostridium XIVa were associated with beneficial effect of NTU 101. Parasutterella was only associated with consumption of DSW. The major bacterial taxa Akkermansia is associated with high-cholesterol diet but not host cholesterol level. This phenomenon suggested that cholesterol-lowering effect is not necessarily linked to specific bacteria-host interaction, and the conclusion of causal relationships among bacterial abundance, diet, and host physiology should be more rigorously investigated.

Clinical Relevance and Treatment Selection of Ureteral Injury After Cesarean Section.

INTRODUCTION: The aim of this case series was to report the clinical relevance and management outcomes of ureteral injuries acquired secondary to cesarean section. METHODS: This was a retrospective case series from January 2007 to September 2014. Description of the patients' characteristics, diagnostic tools for investigation, management, and postoperative follow-up was conducted on postcesarean section patients who developed symptoms of urine leakage after cesarean section and necessitated secondary surgery for ureteral injury. Descriptive statistics was used for demographics and operative data. RESULTS: A total of 5619 cases were managed by cesarean section during the study period. Six (0.107%; 95% confidence interval [CI], 0.1069%-0.1071%) patients had ureteral injury related to the cesarean section. Of 6 cases, 3 (0.053%; 95% CI, 0.0529%-0.0531%) had ureterouterine fistula. Three cases were managed by ureteroneocystostomy, 1 by ureteroneocystostomy with Boari flap, 1 by transureteroureterostomy, and the other one by ureteral stenting via ureterocystoscopy. Three patients had immediate operation because of an acute abdomen and 3 patients had delayed operation. The left ureter was the most common site of ureteral injury (5/6). The postoperative course was uneventful for all cases. CONCLUSIONS: Continuous urinary leakage and acute abdominal distention associated with fluid accumulation after emergency cesarean section should be considered as "red flag" symptoms of ureteral injury and ureterouterine fistulae complications. Delayed management for ureteral repair may not be associated with bad outcomes for management of ureterouterine fistula. Delayed management was associated with less blood loss, less operating time, and acceptable outcome among patients with ureterouterine fistulae when the renal function is not compromised.

Perspectives on genetically modified crops and food detection.

Genetically modified (GM) crops are a major product of the global food industry. From 1996 to 2014, 357 GM crops were approved and the global value of the GM crop market reached 35% of the global commercial seed market in 2014. However, the rapid growth of the GM crop-based industry has also created controversies in many regions, including the European Union, Egypt, and Taiwan. The effective detection and regulation of GM crops/foods are necessary to reduce the impact of these controversies. In this review, the status of GM crops and the technology for their detection are discussed. As the primary gap in GM crop regulation exists in the application of detection technology to field regulation, efforts should be made to develop an integrated, standardized, and high-throughput GM crop detection system. We propose the development of an integrated GM crop detection system, to be used in combination with a standardized international database, a decision support system, high-throughput DNA analysis, and automated sample processing. By integrating these technologies, we hope that the proposed GM crop detection system will provide a method to facilitate comprehensive GM crop regulation.

Proteomic insight into the effect of ethanol on citrinin biosynthesis pathway in Monascus purpureus NTU 568.

Monascus species produce several potent bioactive metabolites through polyketide secondary metabolic pathways; however, little is known of the regulation of metabolic processes in this organism. Therefore, we investigated the effect of extreme growth conditions on the production of secondary metabolites by Monascus purpureus strain NTU 568 and identified pathways that plausibly regulate the polyketide pathways by using proteomic analysis. Citrinin, a type of antibiotic, is synthesized through the polyketide pathway in M. purpureus NTU 568. We hypothesized that production of citrinin might be inhibited by ethanol. When M. purpureus NTU 568 was cultured in the medium containing 4% ethanol, the secretion of secondary metabolites was inhibited, but the dry cell weight was increased. We also found that branched chain amino acid degradation and the expression level of aldehyde dehydrogenase (ALDH) were downregulated, but proteins related to the heat shock response were induced. Furthermore, polyketide synthesis-related proteins, fatty acid synthase, epoxide hydrolase, and proteins involved in the shikimate secondary metabolic pathway were inhibited by ethanol treatment. These results suggested that reduced production of polyketide metabolites resulted not only from the expression levels of proteins in the polyketide synthesis pathway but also from reduction in the concentration of primary metabolism-generated molecules (e.g., acetyl-CoA, fatty acids) that are used as substrates for polyketide syntheses. This study provides insights into the polyketide secondary metabolism of Monascus as well as into improvement of the production of bioactive secondary metabolites in Monascus species.

Safety and Efficacy of Tien-Hsien Liquid Practical in Patients with Refractory Metastatic Breast Cancer: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group, Phase IIa Trial.

To evaluate the safety and efficacy of Tien-Hsien Liquid Practical (THL-P), a Chinese herbal mixture, in patients with refractory metastatic breast cancer, we performed a randomized, double-blind, placebo-controlled, parallel-group, phase IIa pilot trial. Patients were randomly assigned to either receive THL-P or matching placebo and followed up every 4 weeks for 24 weeks. The primary endpoint was changes in the global health status/quality of life (GHS/QOL) scale. The secondary endpoints were changes in functional and symptom scales, immunomodulating effects, and adverse events. Sixty-three patients were enrolled between June 2009 and June 2011. The intent-to-treat population included 28 patients in the THL-P group and 11 patients in the placebo group. Compared to the placebo group, the THL-P group had significant improvement from baseline to last visit in GHS/QOL (41.7 versus -33.3; P < 0.05), CD3, CD4/CD8, CD19, CD16+56 positive cells (P < 0.05), and higher levels of physical, role, emotional, and cognitive functioning, as well as decreased fatigue and systemic side effects. Treatment-related adverse events were mild constipation and localized itching, and no serious adverse events were reported. THL-P appears to be a safe alternative adjuvant treatment for patients with refractory metastatic breast cancer, as it effectively improves QOL and palliates cancer-related symptoms.

Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

PCR-denaturing gradient gel electrophoresis analysis to assess the effects of a genetically modified cucumber mosaic virus-resistant tomato plant on soil microbial communities.

The effects of a genetically modified cucumber mosaic virus (CMV)-resistant tomato on soil microbial communities were evaluated in this study. Soil position and environmental factors played more dominant roles than the tomato genotype in the variation of soil microbial communities.

Allergenicity assessment of genetically modified cucumber mosaic virus (CMV) resistant tomato (Solanum lycopersicon).

Cucumber mosaic virus (CMV) has been identified as the causal agent of several disease epidemics in most countries of the world. Insect-mediated virus diseases, such as those caused by CMV, caused remarkable loss of tomato (Solanum lycopersicon) production in Taiwan. With expression of the CMV coat protein gene (Cmvcp) in a local popular tomato cultivar L4783, transgenic tomato line R8 has showed consistent CMV resistance through T(0) to T(8). In this report, the allergenicity of the CMV coat protein (CMV cp) expressed in transgenic tomato R8 was assessed by investigation of the expression of the transgene source of protein, sequence similarity with known allergens, and resistance to pepsin hydrolysis. There is no known account for either the CMV or its coat protein being an allergen. The result of a bioinformatic search also showed no significant homology between CMV cp and any known allergen. The pepsin-susceptible property of recombinant CMV cp was revealed by a simulated gastric fluid (SGF) assay. Following the most recent FAO/WHO decision tree, all results have indicated that CMV cp was a protein with low possibility to be an allergen and the transgenic tomato R8 should be considered as safe as its host.

Establishment of a system based on universal multiplex-PCR for screening genetically modified crops.

The rapid development of many genetically modified (GM) crops in the past two decades makes it necessary to introduce an alternative strategy for routine screening and identification. In this study, we established a universal multiplex PCR detection system which will effectively reduce the number of reactions needed for sample identification. The PCR targets of this system include the six most frequently used transgenic elements: cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptII) gene, the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 epsps) gene of Agrobacterium tumefaciens strain CP4, and the phosphinothricin N-acetyltransferase (pat) gene. According to the AGBIOS database, the coverage of this detection system is 93% of commercial GM crops. This detection system could detect all certified reference materials (CRMs) at the 1.0% level. The correct combination of all the CRM amplicon patterns proved the specificity of this multiplex PCR system. Furthermore, the amplicon patterns of this multiplex PCR detection system could be used as an index of classification which will narrow the range of possible GM products. The simulation result of this multiplex PCR detection system on all commercialized 139 GM products in the AGBIOS database showed that the maximum number of PCR reactions needed to identify an unknown sample can be reduced to 13. In this study, we established a high-throughput multiplex PCR detection system with feasible sensitivity, specificity, and cost. By incorporating this detection system, the routine GM crop-detection process will meet the challenges resulting from a rapid increase in the number of GM crops in the future.