A method for the systematic selection of enzyme panel candidates by solving the maximum diversity problem.

Enzymes are being increasingly exploited for their potential as industrial biocatalysts. Establishing a portfolio of useful biocatalysts from large and diverse protein family is challenging and a systematic method for candidate selection promises to aid in this task. Moreover, accurate enzyme functional annotation can only be confidently guaranteed through experimental characterisation in the laboratory. The selection of catalytically diverse enzyme panels for experimental characterisation is also an important step for shedding light on the currently unannotated proteins in enzyme families. Current selection methods often lack efficiency and scalability, and are usually non-systematic. We present a novel algorithm for the automatic selection of subsets from enzyme families. A tabu search algorithm solving the maximum diversity problem for sequence identity was designed and implemented, and applied to three diverse enzyme families. We show that this approach automatically selects panels of enzymes that contain high richness and relative abundance of the known catalytic functions, and outperforms other methods such as k-medoids.

Esophageal stenosis after chemotherapy for breast cancer: A case report.

RATIONALE: Esophageal stenosis after chemotherapy in breast cancer patients is rare. Distinguishing esophageal stenosis from esophageal metastasis caused by breast cancer is important. PATIENT CONCERNS AND DIAGNOSIS: A 62-year-old woman diagnosed with advanced breast cancer and no distant metastases gradually developed skin changes, oral ulcers and mucosal injures after four cycles of chemotherapy. Dysphagia was the most severe symptom that greatly affected the patient's quality of life. Ultimately, esophageal stenosis and ulceration were confirmed by serial radiological examinations and endoscopic biopsy. INTERVENTIONS: Due to difficulties in eating orally, the patient was initially placed on a nasogastric tube in order to improve her nutritional status. Simultaneously, she was administered powerful proton pump inhibitors. She underwent modified radical mastectomy for breast cancer after her nutritional status improved. However, the patient was still suffering from severe dysphagia after more than 4 months of follow-up. Subsequently, she underwent removable esophageal stent implantation after after unsuccessful attempts to dilate her esophagus. OUTCOMES: The dysphagia symptoms were immediately alleviated to a certain degree, and the dilated cavity of the upper esophagus showed slight retraction. LESSONS: Esophageal stenosis is very infrequent in patients with breast cancer after chemotherapy. It needs to be. distinguished from esophageal metastasis caused by breast cancer. Esophageal stent implantation may provide benefits in terms of both symptom control and survival in patients with severe esophageal structures.

Environmental neurotoxicants and inflammasome activation in Parkinson's disease - A focus on the gut-brain axis.

Inflammasomes are multi-protein complexes expressed in immune cells that function as intracellular sensors of environmental, metabolic and cellular stress. Inflammasome activation in the brain, has been shown to drive neuropathology and disease progression by multiple mechanisms, making it one of the most attractive therapeutic targets for disease modification in Parkinson's Disease (PD). Extensive inflammasome activation is evident in the brains of people with PD at the sites of dopaminergic degeneration and synuclein aggregation. While substantial progress has been made on validating inflammasome activation as a therapeutic target for PD, the mechanisms by which inflammasome activation is triggered and sustained over the disease course remain poorly understood. A growing body of evidence point to environmental and occupational chemical exposures as possible triggers of inflammasome activation in PD. The involvement of the gastrointestinal system and gut microbiota in PD pathophysiology is beginning to be elucidated, especially the profound link between gut dysbiosis and immune activation. While large cohort studies confirmed specific changes in the gut microbiota in PD patients compared to age-matched healthy controls, recent research suggest that synuclein pathology could be initiated in the gastrointestinal tract. In this review, we present a summarized perspective on current understanding on inflammasome activation and the gut-brain-axis link during PD pathophysiology. We discuss multiple environmental toxicants that are implicated as the etiological agents in causing idiopathic PD and their mechanistic underpinnings during neuroinflammatory events. We additionally present future directions that needs to address the research questions related to the gut-microbiome-brain mechanisms in PD.

Exploring reward-related attention selectivity deficits in Parkinson's disease.

An important aspect of managing a limited cognitive resource like attention is to use the reward value of stimuli to prioritize the allocation of attention to higher-value over lower-value stimuli. Recent evidence suggests this depends on dopaminergic signaling of reward. In Parkinson's disease, both reward sensitivity and attention are impaired, but whether these deficits are directly related to one another is unknown. We tested whether Parkinson's patients use reward information when automatically allocating their attention and whether this is modulated by dopamine replacement. We compared patients, tested both ON and OFF dopamine replacement medication, to older controls using a standard attention capture task. First, participants learned the different reward values of stimuli. Then, these reward-associated stimuli were used as distractors in a visual search task. We found that patients were generally distracted by the presence of the distractors but that the degree of distraction caused by the high-value and low-value distractors was similar. Furthermore, we found no evidence to support the possibility that dopamine replacement modulates the effect of reward on automatic attention allocation. Our results suggest a possible inability in Parkinson's patients to use the reward value of stimuli when automatically allocating their attention, and raise the possibility that reward-driven allocation of resources may affect the adaptive modulation of other cognitive processes.

Brain-derived neurotrophic factor in cerebrospinal fluid and plasma is not a biomarker for Huntington's disease.

Brain-derived neurotrophic factor (BDNF) is implicated in the survival of striatal neurons. BDNF function is reduced in Huntington's disease (HD), possibly because mutant huntingtin impairs its cortico-striatal transport, contributing to striatal neurodegeneration. The BDNF trophic pathway is a therapeutic target, and blood BDNF has been suggested as a potential biomarker for HD, but BDNF has not been quantified in cerebrospinal fluid (CSF) in HD. We quantified BDNF in CSF and plasma in the HD-CSF cohort (20 pre-manifest and 40 manifest HD mutation carriers and 20 age and gender-matched controls) using conventional ELISAs and an ultra-sensitive immunoassay. BDNF concentration was below the limit of detection of the conventional ELISAs, raising doubt about previous CSF reports in neurodegeneration. Using the ultra-sensitive method, BDNF concentration was quantifiable in all samples but did not differ between controls and HD mutation carriers in CSF or plasma, was not associated with clinical scores or MRI brain volumetric measures, and had poor ability to discriminate controls from HD mutation carriers, and premanifest from manifest HD. We conclude that BDNF in CSF and plasma is unlikely to be a biomarker of HD progression and urge caution in interpreting studies where conventional ELISA was used to quantify CSF BDNF.

Growth Media Affect Assessment of Antimicrobial Activity of Plant-Derived Polyphenols.

This study aimed to investigate the effects of different microbial growth media on the laboratory assessment of antimicrobial activity of natural polyphenolic compounds. The inhibition of the tea polyphenol EGCG on growth of selected oral microorganisms was evaluated in complex media and a protein-free chemically defined medium (CDM). Other antimicrobial agents (polyphenolic grape seed extract, plant alkaloid berberine, methyl salicylate, and chlorhexidine gluconate) were also tested in the study. The presence of proteins and their effects on the antimicrobial activity of EGCG were investigated by the addition of BSA to the CDM. The MICs of EGCG against test oral microorganisms were 4 to 64 times higher in complex media than in CDM. The polyphenolic grape seed extract exhibited similar discrepancies. However, the MICs of the nonpolyphenolic compounds (berberine, methyl salicylate, and chlorhexidine) were not significantly different between the two growth media. The MIC of EGCG against S. mutans UA159 in CDM with added BSA was 16 times higher than that in CDM alone. Therefore, nonproteinaceous CDM should be used to avoid interference of proteins with the active ingredients when testing the antimicrobial activity of plant-derived polyphenolic compounds against microorganisms. This will also minimize the discrepancies noted in results obtained by different investigators.

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.

Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects, including embryonic lethality, neurodegeneration and overt inflammation. The reasons for such drastic lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF, enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition, and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.

Use of the UPOINT phenotype system in treating Chinese patients with chronic prostatitis/chronic pelvic pain syndrome: a prospective study.

The urinary, psychosocial, organ-specific, infection, neurological/systemic and tenderness (UPOINT) phenotype system has been validated to be an effective phenotype system in classifying patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) in western populations. To validate the utility of the UPOINT system and evaluate the effect of multimodal therapy based on the UPOINT system in Chinese patients with CP/CPPS, we performed this study. Chinese patients with CP/CPPS were prospectively offered multimodal therapy using the UPOINT system and re-examined after 6 months. A minimum 6-point drop in National Institutes of Health-Chronic Prostatitis Symptoms Index (NIH-CPSI) was set to be the primary endpoint. Finally, 140 patients were enrolled in the study. The percentage of patients with each domain was 59.3%, 45.0%, 49.3%, 22.1%, 37.9%, and 56.4% for the UPOINT, respectively. The number of positive domains significantly correlated with symptom severity, which is measured by total NIH-CPSI scores (r = 0.796, P< 0.001). Symptom duration was associated with a greater number of positive domains (r = 0.589, P< 0.001). With 6 months follow-up at least, 75.0% (105/140) had at least a 6-point improvement in NIH-CPSI after taking the therapy. All NIH-CPSI scores were significantly improved from original ones: pain 10.14 ± 4.26 to 6.60 ± 3.39, urinary 6.29 ± 2.42 to 3.63 ± 1.52, quality of life 6.56 ± 2.44 to 4.06 ± 1.98, and total 22.99 ± 7.28 to 14.29 ± 5.70 (all P< 0.0001). Our study indicates that the UPOINT system is clinically feasible in classifying Chinese patients with CP/CPPS and directing therapy.

Preparation of monoclonal antibodies against Mycobacterium tuberculosis TB10.4 antigen.

TB10.4 protein is a member of the ESX family that is necessary for Mycobacterium tuberculosis survival and plays a vital role in mycobacterial pathogenesis. In this study, the gene encoding TB10.4 was cloned into prokaryotic expression vecters pET-30(a) and pGEX-6p-1. The two recombinant proteins His-TB10.4 and GST-TB10.4 were then expressed in vitro in prokaryotic expression systems to develop monoclonal antibodies (MAbs) against TB10.4 protein. The purified rHis-TB10.4 protein was used to immunize BALB/c mice, and eight MAbs were produced. An immunoblotting analysis indicated that all these MAbs specifically recognize the TB10.4 protein. These new MAbs provide powerful reagents for further functional research into TB10.4 protein.

Retroperitoneoscopic dismembered pyeloplasty for ureteropelvic junction obstruction: modification of the procedure and our experience.

PURPOSE: To report a modified retroperitoneoscopic dismembered pyeloplasty technique and its application in the treatment of ureteropelvic junction obstruction (UPJO). MATERIALS AND METHODS: From June 2010 to March 2012, retroperitoneoscopic dismembered pyeloplasty was performed in 46 patients with UPJO. Briefly, the renal pelvis was incised in the anterior aspect instead of the lateral aspect, and proximal ureter was spatulated with incision on its posterior wall. After adequately trim­ming, two layers of ureteropelvic anastomosis respectively lay on left and right side of one laparoscopic plane other than two different planes. In our refined procedure, the difficulty of intracorporeal suturing was greatly decreased. Data from 19 months mean follow-up were analyzed to evaluate the surgical outcomes. RESULTS: All operations were completed without open conversion. The mean operative time, estimated blood loss, and postoperative hospitalization stay were 108 min (75 to 155 min), 30 mL (15 to 60 mL) and 4 days (2 to 9 days), respectively. No intraoperative complications were occurred. Postoperative complications included 2 cases of minor abdominal wall hematoma and 1 case of transient postoperative anastomotic leakage for 8 days, which all were successfully treated by conservative management. A mean follow-up of 19 months (12 to 36 months) was performed which showed a success rate of 97.8%. One case (2.2%) underwent open surgery for persistence UPJO two months later. CONCLUSION: Our modification to the retroperitoneoscopic dismembered pyeloplasty procedure is technically feasible and reliable with low complications. It could be implemented as a useful alternative technique to great­ly decrease the difficulty of this procedure.

The antibiofilm activity of lingonberry flavonoids against oral pathogens is a case connected to residual complexity.

The antimicrobial activity of lingonberry (Vaccinium vitis-idaea L.) was evaluated against two oral pathogens, Streptococcus mutans and Fusobacterium nucleatum. Long-bed gel permeation chromatography (GPC; Sephadex LH-20) yielded purified flavonoids, with the most efficient minimum inhibitory concentrations (MICs) against planktonic cells in the anthocyanin and procyanidin primary fractions against F. nucleatum (63-125 µg/ml) and in the procyanidin rich fraction against S. mutans (16-31 µg/ml). The purified flavonol glycosides and procyanidins inhibited biofilm formation of S. mutans (MICs 16-31 µg/ml), while the corresponding reference compounds showed no activity. Secondary GPC purification yielded flavonol glycosides devoid of antibiofilm activity in the 50% MeOH fraction, while elution with 70% acetone recovered a brownish material with activity against S. mutans biofilm (MIC 8 µg/ml). Even after HPLC-PDA, NMR, and MALDI-TOF analyses, the structural identity of this material remained unknown, while its color and analytical characteristics appear to be consistent with flavonoid oxidation products.

Human coronaviruses are uncommon in patients with gastrointestinal illness.

BACKGROUND: Coronaviruses infect numerous animal species causing a variety of illnesses including respiratory, neurologic and enteric disease. Human coronaviruses (HCoV) are mainly associated with respiratory tract disease but have been implicated in enteric disease. OBJECTIVES: To investigate the frequency of coronaviruses in stool samples from children and adults with gastrointestinal illness by RT-PCR. STUDY DESIGN: Clinical samples submitted for infectious diarrhea testing were collected from December 2007 through March 2008. RNA extraction and RT-PCR was performed for stools negative for Clostridium difficile using primer sets against HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. Clinical data from samples positive for coronaviruses were reviewed and recorded. RESULTS: Samples from 479 patients were collected including 151 pediatric (< or = 18 years), and 328 adults (>18 years). Of these samples, 4 patients (1.3%, 2 adult; 2 pediatric) screened positive for the presence of a coronavirus. All detected coronaviruses were identified as HCoV-HKU1. No stools screened positive for either HCoV-229E, HCoV-NL63 or HCoV-OC43. All HCoV-HKU1 positive samples occurred between mid-January to mid-February. Clinical manifestations from HCoV-HKU1 positive patients included diarrhea, emesis and respiratory complaints. Three (75%) patients were admitted to the hospital with a median length of stay of 6 days. CONCLUSIONS: Coronaviruses as a group are not commonly identified in stool samples of patients presenting with gastrointestinal illness. HCoV-HKU1 can be identified in stool samples from children and adults with gastrointestinal disease, with most individuals having respiratory findings as well. No stool samples screened positive for HCoV-NL63, HCoV-229E, or HCoV-OC43.

Newly recognized bocaviruses (HBoV, HBoV2) in children and adults with gastrointestinal illness in the United States.

BACKGROUND: The human bocavirus (HBoV) is a newly recognized parvovirus associated with respiratory and gastrointestinal disease. Recently, two new members of the parvovirus family have been recognized, HBoV2 and HBoV3. OBJECTIVES: Here we investigate stool and respiratory samples for the presence of HBoV, HBoV2 and HBoV3. STUDY DESIGN: Stool samples collected from 12/1/2007 to 3/31/2008 were screened by PCR for the presence of HBoV, HBoV2, and HBoV3. Extracted DNA from respiratory specimens archived between 10/17/2005 and 3/29/2006 were screened by PCR for HBoV2 and HBoV3. Medical records for all bocavirus positive patients were reviewed. RESULTS: Of 479 stool samples screened, 328 (68.5%) were from adults, and 151 (31.5%) were from children. Sixteen (3.4%) patients were positive for the presence of a bocavirus, including 10 (2.1%) HBoV and 6 (1.3%) HBoV2. No HBoV3 was detected in stool samples. Frequency of HBoV and HBoV2 in stool samples from children was 3.3% and 0.7%, and from adults was 1.5% and 1.5% respectively. Clinical findings in patients with HBoV and HBoV2 in stool include diarrhea (50% and 83.3%), abdominal pain (40%, 33.3%), and cough (10%, 50%). Of 868 respiratory samples screened, none were positive for either HBoV2 or HBoV3. CONCLUSIONS: The newly recognized parvovirus HBoV2 circulates in the United States. Patients with bocaviruses in stool have evidence of gastrointestinal illness. HBoV2 was not detected in respiratory samples. HBoV3 was not detected in either stool or respiratory samples.

[Effect of human beta defensin 2 on Staphylococcus aureus infection: in vivo study with transgenic mice].

OBJECTIVE: To investigate the effect of human beta defensin 2 (HBD-2) on Staphylococcus aureus infection. METHODS: A minigene of HBD-2 containing pCMV promoter, full length of HBD-2 cDNA, and BGH polyA tail was generated by PCR amplification and introduced into the fertilized oocytes of C57/ICR hybridized mouse by microinjection. After gestation of 3 - 4 weeks, immunohistochemistry was used to detect the expression of HBD-2 peptide in different tissues of the transgenic young mice. Staphylococcus aureus was cultured and injected intraperitoneally to wild type mice and transgenic mice to observe their surviving status. RESULTS: PCR showed that the HBD-2 fragment had been successfully integrated into the chromosome of the mice. A widespread expression of HBD-2 gene was found in many tissues of the transgenic mice: trachea, lung, intestine, esophagus, testis, spleen, skin, endothelium, brain, etc. Four of the 7 transgenic mice survived the Staphylococcus aureus infection, and 10 wild type mice all died within 24 hours. CONCLUSION: HBD-2 may play an important role ion the host defense against Staphylococcus aureus infection.