Characterizing the gut microbiota in adults with bipolar disorder: a pilot study.

Background: Convergent evidence implicates gut microbiota in human health and disease. Hitherto, relatively few studies have evaluated the gut microbiota profile in individuals with bipolar disorder (BD) relative to healthy controls (HC). Methods: Fecal samples were collected from subjects (aged 18-65) meeting DSM-5-defined criteria for BD and age- and sex-matched HC without current or past history of mental or major medical disorders. Samples were sequenced using Illumina sequencing and association of specific taxa and co-occurrence of taxa with sample groups including the effect of diet was assessed using cluster analysis and analysis of communities of microorganisms (ANCOM). Nutritional composition was evaluated using the Dietary Questionnaire for Epidemiological Studies (DQES v2) Food Frequency Questionnaire. Results: Forty-six subjects were enrolled (n=23 BD, n=23 HC). Cluster analyses did not identify any significant differences between BD and HC (p=0.38). Lower microbiota diversity was observed among BD subjects relative to HC (p=0.04). A greater abundance of a Clostridiaceae OTU was observed among BD subjects when compared to HC and of Collinsella among BD-II subjects relative to BD-I. Cluster analysis revealed that neither diagnosis (p=0.38) nor diet (p=0.43) had a significant effect on overall gut microbiota composition. Limitations: This study has a small sample size and insufficient control for some potential moderating factors (e.g. psychotropic medication and smoking). Conclusion: This study suggests that individuals with BD may have a distinct gut microbiota profile compared to healthy controls, with a greater abundance of Clostridiaceae and Collinsella. These findings need to be replicated in future studies with larger sample sizes.

NCL Inhibition Exerts Antineoplastic Effects against Prostate Cancer Cells by Modulating Oncogenic MicroRNAs.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men and second most common cause of cancer-related deaths in the United States. Androgen deprivation therapy (ADT) is only temporarily effective for advanced-stage PCa, as the disease inevitably progresses to castration-resistant prostate cancer (CRPC). The protein nucleolin (NCL) is overexpressed in several types of human tumors where it is also mislocalized to the cell surface. We previously reported the identification of a single-chain fragment variable (scFv) immuno-agent that is able to bind NCL on the surface of breast cancer cells and inhibit proliferation both in vitro and in vivo. In the present study, we evaluated whether NCL could be a valid therapeutic target for PCa, utilizing DU145, PC3 (CRPC), and LNCaP (androgen-sensitive) cell lines. First, we interrogated the publicly available databases and noted that higher NCL mRNA levels are associated with higher Gleason Scores as well as with recurrent and metastatic tumors. Then, using our anti-NCL scFv, we demonstrated that NCL is expressed on the surface of all three tested cell lines and that NCL inhibition results in reduced proliferation and migration. We also measured the inhibitory effect of NCL targeting on the biogenesis of oncogenic microRNAs such as miR-21, -221 and -222, which was cell context dependent. Taken together, our data provide evidence that NCL targeting inhibits the key hallmarks of malignancy in PCa cells and may provide a novel therapeutic option for patients with advanced-stage PCa.

Insulin receptor signaling regulates renal collecting duct and intercalated cell antibacterial defenses.

People with diabetes mellitus have increased infection risk. With diabetes, urinary tract infection (UTI) is more common and has worse outcomes. Here, we investigate how diabetes and insulin resistance impact the kidney's innate defenses and urine sterility. We report that type 2 diabetic mice have increased UTI risk. Moreover, insulin-resistant prediabetic mice have increased UTI susceptibility, independent of hyperglycemia or glucosuria. To identify how insulin resistance affects renal antimicrobial defenses, we genetically deleted the insulin receptor in the kidney's collecting tubules and intercalated cells. Intercalated cells, located within collecting tubules, contribute to epithelial defenses by acidifying the urine and secreting antimicrobial peptides (AMPs) into the urinary stream. Collecting duct and intercalated cell-specific insulin receptor deletion did not impact urine acidification, suppressed downstream insulin-mediated targets and AMP expression, and increased UTI susceptibility. Specifically, insulin receptor-mediated signaling regulates AMPs, including lipocalin 2 and ribonuclease 4, via phosphatidylinositol-3-kinase signaling. These data suggest that insulin signaling plays a critical role in renal antibacterial defenses.

Bipolar disorder in youth is associated with increased levels of vitamin D-binding protein.

Genetic, dietary, and inflammatory factors contribute to the etiology of major mood disorders (MMD), thus impeding the identification of specific biomarkers to assist in diagnosis and treatment. We tested association of vitamin D and inflammatory markers in 36 adolescents with bipolar disorder (BD) and major depressive disorder (MDD) forms of MMD and without MMD (non-mood control). We also assessed the overall level of inflammation using a cell-based reporter assay for nuclear factor kappa-B (NFκB) activation and measuring antibodies to oxidized LDL. We found that these factors were similar between non-mood and MMD youth. To identify potential biomarkers, we developed a screening immunoprecipitation-sequencing approach based on inflammatory brain glia maturation factor beta (GMFß). We discovered that a homolog of GMFß in human plasma is vitamin D-binding protein (DBP) and validated this finding using immunoprecipitation with anti-DBP antibodies and mass spectrometry/sequencing analysis. We quantified DBP levels in participants by western blot. DBP levels in BD participants were significantly higher (136%) than in participants without MMD (100%). The increase in DBP levels in MDD participants (121.1%) was not statistically different from these groups. The DBP responds early to cellular damage by binding of structural proteins and activating inflammatory cells. A product of enzymatic cleavage of DBP has been described as macrophage-activating factor. Circulating DBP is comprised of heterogenous high and low molecular fractions that are only partially recognized by mono- and polyclonal ELISA and are not suitable for the quantitative comparison of DBP in non-mood and MDD participants. Our data suggest DBP as a marker candidate of BD warranting its validation in a larger cohort of adolescent and adult MMD patients.

A novel fully human anti-NCL immunoRNase for triple-negative breast cancer therapy.

Breast cancer is the most common cancer in women worldwide. A new promising anti-cancer therapy involves the use of monoclonal antibodies specific for target tumor-associated antigens (TAAs). A TAA of interest for immunotherapy of Triple Negative Breast Cancer (TNBC) is nucleolin (NCL), a multifunctional protein, selectively expressed on the surface of cancer cells, which regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and drug-resistance. We previously isolated a novel human anti-NCL scFv, called 4LB5, that is endowed with selective anti-tumor effects. Here we report the construction and characterization of a novel immunoRNase constituted by 4LB5 and a human pancreatic RNase (HP-RNase) called "4LB5-HP-RNase". This immunoRNase retains both the enzymatic activity of human pancreatic RNase and the specific binding of the parental scFv to a panel of surface NCL-positive breast cancer cells. Notably, 4LB5-HP-RNase dramatically and selectively reduced the viability and proliferation of NCL-positive tumor cells in vitro and in vivo. Specifically, it induced apoptosis and reduced the levels of the tumorigenic miRNAs miR-21, -221 and -222. Thus, this novel immunoagent could be a valuable tool for the treatment of TNBC patients ineligible for currently available targeted treatments.

Human anti-nucleolin recombinant immunoagent for cancer therapy.

Nucleolin (NCL) is a nucleocytoplasmic protein involved in many biological processes, such as ribosomal assembly, rRNA processing, and mRNA stabilization. NCL also regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and aggressiveness. Interestingly, NCL is expressed on the surface of actively proliferating cancer cells, but not on their normal counterparts. Therefore, NCL is an attractive target for antineoplastic treatments. Taking advantage of phage-display technology, we engineered a fully human single-chain fragment variable, named 4LB5. This immunoagent binds NCL on the cell surface, it is translocated into the cytoplasm of target cells, and it abrogates the biogenesis of NCL-dependent miRNAs. Binding of 4LB5 to NCL on the cell surface of a variety of breast cancer and hepatocellular carcinoma cell lines, but not to normal-like MCF-10a breast cells, dramatically reduces cancer cell viability and proliferation. Finally, in orthotopic breast cancer mouse models, 4LB5 administration results in a significant reduction of the tumor volume without evident side effects. In summary, here we describe, to our knowledge, the first anti-NCL single-chain fragment variable displaying antineoplastic activity against established solid tumors, which could represent the prototype of novel immune-based NCL-targeting drugs with clinical potential as diagnostic and therapeutic tools in a wide variety of human cancers.

Ribonucleases 6 and 7 have antimicrobial function in the human and murine urinary tract.

Recent evidence suggests antimicrobial peptides protect the urinary tract from infection. Ribonuclease 7 (RNase 7), a member of the RNase A superfamily, is a potent epithelial-derived protein that maintains human urinary tract sterility. RNase 7 expression is restricted to primates, limiting evaluation of its antimicrobial activity in vivo. Here we identified ribonuclease 6 (RNase 6) as the RNase A superfamily member present in humans and mice that is most conserved at the amino acid level relative to RNase 7. Like RNase 7, recombinant human and murine RNase 6 has potent antimicrobial activity against uropathogens. Quantitative real-time PCR and immunoblot analysis indicate that RNase 6 mRNA and protein are upregulated in the human and murine urinary tract during infection. Immunostaining located RNase 6 to resident and infiltrating monocytes, macrophages, and neutrophils. Uropathogenic E. coli induces RNase 6 peptide expression in human CD14(+) monocytes and murine bone marrow-derived macrophages. Thus, RNase 6 is an inducible, myeloid-derived protein with markedly different expression from the epithelial-derived RNase 7 but with equally potent antimicrobial activity. Our studies suggest RNase 6 serves as an evolutionarily conserved antimicrobial peptide that participates in the maintenance of urinary tract sterility.

Bacterial overgrowth syndrome in myotonic muscular dystrophy is potentially treatable.

Over one third of patients with myotonic muscular dystrophy type 1 (DM1) have gastrointestinal complaints. The cause is multifactorial, and treatment options are limited. Twenty DM1 patients with gastrointestinal symptoms were screened over a 2-year period using glucose breath hydrogen testing (GBHT) to evaluate the prevalence of small intestinal bacterial overgrowth (SIBO). Sixty-five percent of patients had a positive GBHT, and diarrhea was the most common presenting symptom. Ciprofloxacin was the most common antibiotic used for treatment, and 70% of patients reported a good response to the initial course of treatment. Although the causes of gastrointestinal symptoms in patients with DM1 are multifactorial, small intestinal bacterial overgrowth is an important diagnostic consideration that is easily diagnosed using glucose breath hydrogen testing and often shows a good response to treatment with common antibiotics.

Postinfectious irritable bowel syndrome after a food-borne outbreak of acute gastroenteritis attributed to a viral pathogen.

BACKGROUND & AIMS: A large outbreak of acute gastroenteritis at the annual meeting of the Canadian Society of Gastroenterology Nurses and Associates (CSGNA) was attributed to food-borne norovirus. A prospective study was undertaken to determine the incidence and natural history of postinfectious irritable bowel syndrome (PI-IBS). METHODS: Questionnaires addressing demographics, medical history, acute illness, prior bowel function, and current symptoms were mailed to all delegates within 1 month of the outbreak. Follow-up questionnaires were mailed at 3, 6, 12, and 24 months. The prevalence of new Rome I IBS among participants with and without acute enteric illness during the outbreak was calculated for each time point. Risk factors were assessed by multiple logistic regression. RESULTS: Baseline surveys were returned by 139 of 197 delegates (70.6%; mean age, 48 +/- 6 years; 95.0% female), of whom 135 (97.1%), 133 (95.7%), 128 (92.1%), and 116 (83.4%) returned the 3-, 6-, 12-, and 24-month surveys, respectively. One hundred seven respondents (76.9%) reported an acute enteric illness during the outbreak. Eighteen subjects reported premorbid IBS. Among the remainder, 21 of 89 who experienced gastroenteritis (23.6%) reported symptoms consistent with PI-IBS at 3 months versus 1 of 29 (3.4%) who remained well (odds ratio, 6.9; 95% confidence interval, 1.0-48.7; P = .014). At 6, 12, and 24 months, the prevalence of IBS was similar among exposed versus nonexposed individuals. In multiple logistic regression, vomiting during the acute illness independently predicted risk of PI-IBS at 3 months (odds ratio, 10.5; 95% confidence interval, 1.3-85.5; P = .028). CONCLUSIONS: PI-IBS is common after presumptive viral gastroenteritis but might be more transient than after bacterial dysentery.

TFB1 or TFB2 is sufficient for Thermococcus kodakaraensis viability and for basal transcription in vitro.

Archaeal RNA polymerases (RNAPs) are most similar to eukaryotic RNAP II (Pol II) but require the support of only two archaeal general transcription factors, TBP (TATA-box binding protein) and TFB (archaeal homologue of the eukaryotic general transcription factor TFIIB) to initiate basal transcription. However, many archaeal genomes encode more than one TFB and/or TBP leading to the hypothesis that different TFB/TBP combinations may be employed to direct initiation from different promoters in Archaea. As a first test of this hypothesis, we have determined the ability of RNAP purified from Thermococcus kodakaraensis (T.k.) to initiate transcription from a variety of T.k. promoters in vitro when provided with T.k. TBP and either TFB1 or TFB2, the two TFBs encoded in the T.k. genome. With every promoter active in vitro, transcription initiation occurred with either TFB1 or TFB2 although the optimum salt concentration for initiation was generally higher for TFB2 (approximately 250 mM K(+)) than for TFB1 (approximately 200 mM K(+)). Consistent with this functional redundancy in vitro, T.k. strains have been constructed with the TFB1- (tfb1; TK1280) or TFB2- (tfb2; TK2287) encoding gene deleted. These mutants exhibit no detectable growth defects under laboratory conditions. Domain swapping between TFB1 and TFB2 has identified a central region that contributes to the salt sensitivity of TFB activity, and deleting residues predicted to form the tip of the B-finger region of TFB2 had no detectable effects on promoter recognition or transcription initiation but did eliminate the production of very short (< or =5 nt) abortive transcripts.

Randomized active and placebo-controlled endoscopy study of a novel protected formulation of oral alendronate.

Although generally well tolerated, oral aminobisphosphonates have been associated with sporadic cases of severe esophageal injury attributed to pill contact. A novel protected formulation of oral alendronate uses an inert cylindrical shell to prevent mucosal contact with intact tablets. An active and placebo-controlled endoscopy study was undertaken to assess mucosal injury associated with this protected formulation. Healthy volunteers with normal baseline endoscopy were randomly assigned to receive protected alendronate 70 mg/day, standard alendronate 70 mg/day, or placebo for 14 days. Endoscopy was repeated on days 8 and 15. Of 78 subjects, 30 received protected alendronate, 28 received standard alendronate, and 20 received placebo. Mean gastric injury scores did not differ significantly among treatment groups. However, subjects on standard alendronate were more likely than those on protected alendronate to develop severe gastric injury, defined as Lanza score 3 or 4 (67.9% versus 33.3%, P=.009), and more likely to develop a gastric ulcer (21.4% versus 3.3%, P=.015). No differences in symptoms or adverse events were observed. In conclusion, a novel protected formulation of oral alendronate is less likely than standard alendronate to induce severe mucosal injury to the upper gastrointestinal tract.

Time course and pattern of blood loss with ibuprofen treatment in healthy subjects.

BACKGROUND & AIMS: Nonselective nonsteroidal anti-inflammatory drug (NSAID) users are at increased risk of gastrointestinal bleeding. We aimed to assess the pattern and extent of fecal blood loss (FBL) with ibuprofen, which is considered to have a favorable gastrointestinal safety profile. METHODS: We conducted a post hoc analysis of 2 separate randomized, parallel-group, double-blind studies, in which ibuprofen was used as a positive control. FBL was measured by radioactive analysis of chromium-51 labeled red cells in stools during baseline and then followed by 4 weeks of treatment with ibuprofen (800 mg 3 times daily) or placebo in 68 healthy volunteers. FBL was considered significant when blood loss was >2 mL daily. RESULTS: The baseline period was identical for all subjects, with an average FBL of 0.36 mL (standard deviation, +/-0.075) per day. During the study period, all subjects receiving ibuprofen had a daily mean FBL >2 mL, with a group daily mean loss 3.64-fold greater than in the placebo group (2.55 mL [+/-3.2] vs 0.7 mL [+/-0.37], P < .001). In the ibuprofen group (n = 31), 26 subjects had between 1 and 7 random episodes of microbleeding with FBL >3 mL. Nine had a maximum FBL >10 mL (29.35 +/- 23.32 mL), and in 2 subjects blood loss reached 73 mL and 66 mL, respectively. CONCLUSIONS: Treatment with a therapeutic dose of ibuprofen, a commonly used nonselective NSAID, in healthy subjects is associated with significant FBL, which occurs randomly with spikes of bleeding, sometimes exceeding 66 mL in a single day. Chronic anemia or gastrointestinal bleeding in patients taking nonselective NSAIDs should be thoroughly investigated.

Effect on intragastric pH of a PPI with a prolonged plasma half-life: comparison between tenatoprazole and esomeprazole on the duration of acid suppression in healthy male volunteers.

OBJECTIVE: To compare the inhibitory effect of a novel proton pump inhibitor (PPI), tenatoprazole 40 mg once daily, with esomeprazole 40 mg once daily on intragastric acidity. METHODS: A randomized, investigator-blind, two-way, crossover study was conducted in 30 healthy Helicobacter pylori negative male volunteers. Tenatoprazole 40 mg or esomeprazole 40 mg was administered once daily for 7 consecutive days with a 4-wk washout period between treatments. Ambulatory 24-h intragastric pH was recorded at baseline, after 7 days' treatment, and 3 and 5 days after treatment was stopped. RESULTS: At presumed steady-state (day 7), median 24-h pH values were 5.02 and 4.79 for tenatoprazole and esomeprazole, respectively. There was a significant difference between tenatoprazole and esomeprazole during the nocturnal period when mean pH was 4.64 +/- 0.67 versus 3.61 +/- 0.90, respectively (p < 0.0001), as well as a significantly higher mean percentage of time with pH >4 on tenatoprazole (72.5 +/- 14.9 vs 62.2 +/- 13.6, p < 0.0001). The effect of tenatoprazole was still present 5 days after treatment withdrawal especially during the night-time. The mean area under the plasma concentration-time curve and elimination half-time was significantly higher in the tenatoprazole group as compared with the esomeprazole group. CONCLUSION: Tenatoprazole 40 mg daily provides a prolonged duration of acid suppression and a shorter nocturnal acid breakthrough in healthy volunteers, even after stopping the drug. Thus, tenatoprazole may provide greater clinical efficacy for patients in whom a once daily PPI is ineffective. Further studies are indicated.

Electrocardiographic findings over time in arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Thirty-five patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy underwent serial electrocardiographic (ECG) testing and were evaluated for evidence of ECG progression. Over a median of 43 months, 89% of the patients had evidence of progression, with S-wave prolongation being the most prevalent marker of ECG progression.

Proteinase activity and stability of natural bromelain preparations.

Bromelain is a complex mixture of proteinases typically derived from pineapple stem. Similar proteinases are also present in pineapple fruit. Beneficial therapeutic effects of bromelain have been suggested or proven in several human inflammatory diseases and animal models of inflammation, including arthritis and inflammatory bowel disease. However, it is not clear how each of the proteinases within bromelain contributes to its anti-inflammatory effects in vivo. Previous in vivo studies using bromelain have been limited by the lack of assays to control for potential differences in the composition and proteolytic activity of this naturally derived proteinase mixture. In this study, we present model substrate assays and assays for cleavage of bromelain-sensitive cell surface molecules can be used to assess the activity of constituent proteinases within bromelain without the need for biochemical separation of individual components. Commercially available chemical and nutraceutical preparations of bromelain contain predominately stem bromelain. In contrast, the proteinase activity of pineapple fruit reflects its composition of fruit bromelain>ananain approximately stem bromelain. Concentrated bromelain solutions (>50 mg/ml) are more resistant to spontaneous inactivation of their proteolytic activity than are dilute solutions, with the proteinase stability in the order of stem bromelain>fruit bromelain approximately ananain. The proteolytic activity of concentrated bromelain solutions remains relatively stable for at least 1 week at room temperature, with minimal inactivation by multiple freeze-thaw cycles or exposure to the digestive enzyme trypsin. The relative stability of concentrated versus dilute bromelain solutions to inactivation under physiologically relevant conditions suggests that delivery of bromelain as a concentrated bolus would be the preferred method to maximize its proteolytic activity in vivo.

Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway.

The bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli catalyzes non-consecutive reactions in the aspartate pathway of amino acid biosynthesis. Both catalytic activities are subject to allosteric regulation by the end product amino acid L-threonine. To examine the kinetics and regulation of the enzymes in this pathway, each of these catalytic domains were separately expressed and purified. The separated catalytic domains remain active, with each of their catalytic activities enhanced in comparison to the native enzyme. The allosteric regulation of the kinase activity is lost, and regulation of the dehydrogenase activity is dramatically decreased in these separate domains. To create a new bifunctional enzyme that can catalyze consecutive metabolic reactions, the aspartokinase I domain was fused to the enzyme that catalyzes the intervening reaction in the pathway, aspartate semialdehyde dehydrogenase. A hybrid bifunctional enzyme was also created between the native monofunctional aspartokinase III, an allosteric enzyme regulated by lysine, and the catalytic domain of homoserine dehydrogenase I with its regulatory interface domain still attached. In this hybrid the kinase activity remains sensitive to lysine, while the dehydrogenase activity is now regulated by both threonine and lysine. The dehydrogenase domain is less thermally stable than the kinase domain and becomes further destabilized upon removal of the regulatory domain. The more stable aspartokinase III is further stabilized against thermal denaturation in the hybrid bifunctional enzyme and was found to retain some catalytic activity even at temperatures approaching 100 degrees C.

Production and characterization of bifunctional enzymes. Substrate channeling in the aspartate pathway.

The direct channeling of an intermediate between enzymes that catalyze consecutive reactions in a pathway offers the possibility of an efficient, exclusive, and protected means of metabolite delivery. Aspartokinase-homoserine dehydrogenase I (AK-HDH I) from Escherichia coli is an unusual bifunctional enzyme in that it does not catalyze consecutive reactions. The potential channeling of the intermediate beta-aspartyl phosphate between the aspartokinase of this bifunctional enzyme and aspartate semialdehyde dehydrogenase (ASADH), the enzyme that catalyzes the intervening reaction, has been examined. The introduction of increasing levels of inactivated ASADH has been shown to compete against enzyme-enzyme interactions and direct intermediate channeling, leading to a decrease in the overall reaction flux through these consecutive enzymes. These same results are obtained whether these experiments are conducted with aspartokinase III, a naturally occurring monofunctional isozyme, with an artificially produced monofunctional aspartokinase I, or with a fusion construct of AK I-ASADH. These results provide definitive evidence for the channeling of beta-aspartyl phosphate between aspartokinase and aspartate semialdehyde dehydrogenase in E. coli and suggest that ASADH may provide a bridge to channel the intermediates between the non-consecutive reactions of AK-HDH I.