Myeloid vitamin D receptor regulates Paneth cells and microbial homeostasis.

Cross talk between immune cells and the intestinal crypt is critical in maintaining intestinal homeostasis. Recent studies highlight the direct impact of vitamin D receptor (VDR) signaling on intestinal and microbial homeostasis. However, the tissue-specific role of immune VDR signaling is not fully understood. Here, we generated a myeloid-specific VDR knockout (VDRΔLyz ) mouse model and used a macrophage/enteroids coculture system to examine tissue-specific VDR signaling in intestinal homeostasis. VDRΔLyz mice exhibited small intestine elongation and impaired Paneth cell in maturation and localization. Coculture of enteroids with VDR-/- macrophages increased the delocalization of Paneth cells. VDRΔLyz mice exhibited significant changes in the microbiota taxonomic and functional files, and susceptibility to Salmonella infection. Interestingly, loss of myeloid VDR impaired Wnt secretion in macrophages, thus inhibiting crypt ß-catenin signaling and disrupting Paneth cell differentiation in the epithelium. Taken together, our data have demonstrated that myeloid cells regulate crypt differentiation and the microbiota in a VDR-dependent mechanism. Dysregulation of myeloid VDR led to high risks of colitis-associated diseases. Our study provided insight into the mechanism of immune/Paneth cell cross talk in regulating intestinal homeostasis.

OUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression.

MicroRNAs (miRNAs or miRs) are single-stranded, ∼22-nucleotide noncoding RNAs that regulate many cellular processes. While numerous miRNA quantification technologies are available, a recent analysis of 12 commercial platforms revealed high variations in reproducibility, sensitivity, accuracy, specificity and concordance within and/or between platforms. Here, we developed a universal hairpin primer (UHP) system that negates the use of miRNA-specific hairpin primers (MsHPs) for quantitative reverse transcription PCR (RT-qPCR)-based miRNA quantification. Specifically, we analyzed four UHPs that share the same hairpin structure but are anchored with two, three, four and six degenerate nucleotides at 3'-ends (namely UHP2, UHP3, UHP4 and UHP6), and found that the four UHPs yielded robust RT products and quantified miRNAs with high efficiency. UHP-based RT-qPCR miRNA quantification was not affected by long transcripts. By analyzing 14 miRNAs, we demonstrated that UHP4 closely mimicked MsHPs in miRNA quantification. Fine-tuning experiments identified an optimized UHP (OUHP) mix with a molar composition of UHP2:UHP4:UHP6 = 8:1:1, which closely recapitulated MsHPs in miRNA quantification. Using synthetic LET7 isomiRs, we demonstrated that the OUHP-based qPCR system exhibited high specificity and sensitivity. Collectively, our results demonstrate that the OUHP system can serve as a reliable and cost-effective surrogate of MsHPs for RT-qPCR-based miRNA quantification for basic research and precision medicine.

Mediterranean Diet Adherence and Depressive Symptoms among a Nationally Representative Sample of Pregnant Women in the United States.

BACKGROUND: Prenatal depression affects ∼12% of pregnant women in the United States and is associated with an increased risk of adverse birth outcomes and maternal mortality. Adherence to a healthy dietary pattern may reduce and/or protect against depressive symptoms. OBJECTIVES: To investigate the relationship between adherence to a Mediterranean diet and depressive symptoms among pregnant women in the United States. METHODS: We used data from the National Health and Nutrition Examination Survey (2005-2018, N = 540) and included pregnant women aged 18-44 y with a positive urine pregnancy test. The Mediterranean diet score (aMED) was calculated from 1 24-h recall; aMED typically ranges from 0-9, but in these analyses, it ranged from 0-8 because alcohol was not included. The aMED score was dichotomized as high (>3) compared with low (≤3). The Patient Health Questionnaire-9 (PHQ-9), which measures depressive symptoms, was dichotomized as lower compared with higher (PHQ-9 score ≥10), based on the clinical cutoff for patient referral. Our primary model employed logistic regression to investigate the association between aMED adherence and high depressive symptoms when controlling for socio-demographics (age, racial/ethnicity, education, poverty, and relationship status), total calories, and prepregnancy body mass index (kg/m2). We also modeled the PHQ-9 score as a continuous variable using a random-effects model. RESULTS: About 5% of pregnant women had moderate to severe depressive symptoms, and 45% were highly adherent to a Mediterranean diet. Higher adherence to a Mediterranean diet was associated with lower odds of depressive symptoms (odds ratio: 0.31, 95% confidence interval: 0.10, 0.98). Results were not significant for the continuous PHQ-9 score (ß: -0.30; 95% confidence interval: -0.90, 0.30). CONCLUSIONS: Adherence to a Mediterranean diet may have the potential to lower depressive symptoms among pregnant women; however, these results should be interpreted with caution. Nevertheless, considering the public health significance of promoting mental wellness among pregnant women, this relationship merits further examination using experimental designs.

Paneth Cell Alertness to Pathogens Maintained by Vitamin D Receptors.

BACKGROUND AND AIMS: Vitamin D exerts a regulatory role over mucosal immunity via the vitamin D receptor (VDR). Although Paneth cells and their products are known to regulate the commensal and pathogenic microbiota, the role that VDRs in Paneth cells play in these responses is unknown. METHODS: We identified the decreased intestinal VDR significantly correlated with reduction of an inflammatory bowel disease risk gene ATG16L1 and Paneth cell lysozymes in patients with Crohn's disease. We generated Paneth cell-specific VDR knockout (VDRΔPC) mice to investigate the molecular mechanisms. RESULTS: Lysozymes in the Paneth cells were significantly decreased in the VDRΔPC mice. Isolated VDRΔPC Paneth cells exhibited weakened inhibition of pathogenic bacterial growth and displayed reduced autophagic responses. VDRΔPC mice had significantly higher inflammation after Salmonella infections. VDRΔPC mice also showed high susceptibility to small intestinal injury induced by indomethacin, a nonsteroidal anti-inflammatory drug. Co-housing of VDRΔPC and VDRlox mice made the VDRΔPC less vulnerable to dextran sulfate sodium colitis, suggesting the transmission of protective bacterial from the VDRlox mice. Thus, a lack of VDR in Paneth cells leads to impaired antibacterial activities and consequently increased inflammatory responses. Genetically and environmentally regulated VDRs in the Paneth cells may set the threshold for the development of chronic inflammation, as observed in inflammatory bowel diseases. CONCLUSIONS: We provide new insights into the tissue-specific functions of VDRs in maintaining Paneth cell alertness to pathogens in intestinal disorders. Targeting the VDR affects multiple downstream events within Paneth cells that inhibit intestinal inflammation and establish host defense against enteropathogens.

Do Gastroenterologists Have a "Weak" Day? Impact of Day of the Week on the Quality of Outpatient Screening Colonoscopies.

GOAL: The goal of this study is to determine the significance of day of the work week and its contribution to endoscopist performance using adenoma detection rate (ADR) and proposed surrogate quality measures. BACKGROUND: Nearly a quarter of adenomas are missed on routine screening colonoscopy which contributes to between 50% and 60% of interval colorectal cancer. MATERIALS AND METHODS: Adult patients who underwent outpatient screening colonoscopy between January 2015 and April 2020 were included. Measurement of ADR and proposed quality metrics were analyzed for each day of the work week. Secondary outcomes included rates of good or excellent bowel preparation, trainee fellow participation, performance quartile of individual endoscopists, and patient demographic data. A generalized linear mixed model was used to analyze predictors of ADR. RESULTS: A total of 1884 screening procedures were included in our analysis. ADR on Friday (35.6%) was significantly lower than all other days of the work week ( P <0.001). When compared with Friday, all days were found to be independent predictors of increased ADR. Male gender [95% confidence interval (CI): 1.12-1.65, P =0.002], good rather than excellent bowel preparation (95% CI: 1.22-2.28, P =0.001) and colonoscopy withdrawal time (CWT) (95% CI: 1.02-1.03, P <0.001) were all found to be predictors of increased ADR. Proposed quality indicators were all well correlated with ADR ( r >0.811, P ≤0.001) apart from CWT ( r =0.28, P =0.379). CONCLUSIONS: The data suggests there is a decline in endoscopist performance on Friday when compared with all other days of the work week. ADR correlates well with many proposed quality parameters, however, CWT may be of additional value as a quality metric.

Effect of the Weekend on Bowel Preparation Quality in Outpatient Colonoscopies.

INTRODUCTION: Inadequate bowel preparation has been associated with a higher likelihood of missing adenomatous polyps. Colonoscopies immediately following a weekend may be prone to inadequate bowel preparation. This study aims to evaluate if day of the week is a predictor of bowel preparation adequacy, while assessing other patient and procedural variables and their effect on the Boston Bowel Preparation Scale (BBPS). METHODS: A retrospective review was conducted of all adult patients undergoing outpatient colonoscopy between January 2015 and April 2020. Adequacy of bowel preparation was compared among all days of the week and days following federal holidays. Secondary outcomes included patient demographics, indication and timing of the procedure. RESULTS: Of 4,279 colonoscopies, Monday had the highest rate of inadequate preparation (BBPS < 6) (16.5%) compared to other days of the week (p < .001). Post-holiday procedures were not associated with poor bowel preparation (p = .901). Similarly, on multivariate analysis, we found that procedures on Monday (OR 1.67 95%CI 1.33-2.10, p < .001) and African-American race (OR 1.34 95%CI 1.11-1.62, p = .003) were associated with inadequate bowel preparation. Females were more likely to have adequate bowel preparation (OR 0.71 95%CI 0.59-0.86, p < .001). DISCUSSION: Bowel preparation on Mondays is more likely to be inadequate than other days of the week. Additionally, gender and ethnicity appear to be associated with quality of bowel preparation. A better characterization of procedural and patient variables can lead to a more personalized approach to bowel preparation.

The olfactory G protein-coupled receptor (Olfr-78/OR51E2) modulates the intestinal response to colitis.

Olfactory receptor-78 (Olfr-78) is a recently identified G protein-coupled receptor activated by short-chain fatty acids acetate and propionate. A suggested role for this receptor exists in the prostate where it may influence chronic inflammatory response leading to intraepithelial neoplasia. Olfr-78 has also been shown to be expressed in mouse colon. Short-chain fatty acids and their receptors are well known to modulate inflammation in the gut. Considering this possibility, we first explored if colitis regulated Olfr-78 expression in the gut, where we observed a significant reduction in the expression of Olfr-78 transcript in mouse models of dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. To more directly test this, mice deficient in Olfr-78 were administered with DSS in water for 7 days and were found to have increased expression of IL-1ß and inflammatory signs in colon compared with control mice. Next, we explored the expression of its human counterpart olfactory receptor family 51, subfamily E, member 2 (OR51E2) in human intestinal samples and observed that it was in fact also expressed in human colon samples. RNA sequence analysis revealed significant changes in the genes involved in infection, immunity, inflammation, and colorectal cancer between wild-type and Olfr-78 knockout mice. Collectively, our findings show that Olfr-78 is highly expressed in colon and downregulated in DSS- and TNBS-induced colitis, and DSS-treated Olfr-78 null mice had increased colonic expression of cytokine RNA levels, suggesting a potential role for this receptor in intestinal inflammation. Future investigations are needed to understand how Olfr-78/OR51E2 in both mouse and human intestine modulates gastrointestinal pathophysiology.

Imbalance of autophagy and apoptosis in intestinal epithelium lacking the vitamin D receptor.

Apoptosis and autophagy are dynamic processes that determine the fate of cells. Vitamin D receptor (VDR) deficiency in the intestine leads to abnormal Paneth cells and impaired autophagy function. Here, we will elucidate the mechanisms of the intestinal epithelial VDR regulation of autophagy and apoptosis. We used in vivo VDRlox and VDR∆IEC mice and ex vivo organoids generated from small intestine and colon tissues. We found that VDR deficiency induced more apoptotic cells and significantly increased cell death in the small intestine and colon of VDR∆IEC mice. The proapoptotic protein B-cell lymphoma 2 (BCL-2) associated X protein (Bax) was enhanced, whereas autophagy related 16 like 1 (ATG16L1) and Beclin-1 were decreased in the intestines of VDRΔIEC mice. Apoptosis induced by Bax reduced autophagy by decreasing Beclin-1. Physical interactions between Beclin-1 and Bcl-2 were increased in the VDR-deficient epithelia from mice. The growth of VDR∆IEC organoids was significantly slower with fewer Paneth cells than that of VDR+/+ organoids. The expression levels of Beclin-1 and lysozyme were decreased in VDR∆IEC organoids. Bacterial endotoxin levels were high in the serum from VDR∆IEC mice and made mice susceptible to colitis. In the organoids and colitis IL-10-/- mice, vitamin D3 treatment increased VDR and ATG16L1 protein expression levels, which activated autophagic responses. In summary, intestinal epithelial VDR regulates autophagy and apoptosis through ATG16L1 and Beclin-1. Our studies provide fundamental insights into the tissue-specific function of VDR in modulating the balance between autophagy and apoptosis.-Lu, R., Zhang, Y.-G., Xia, Y., Sun, J. Imbalance of autophagy and apoptosis in intestinal epithelium lacking the vitamin D receptor.

Deletion of sorting nexin 27 suppresses proliferation in highly aggressive breast cancer MDA-MB-231 cells in vitro and in vivo.

BACKGROUND: Sorting Nexin 27 (SNX27) belongs to a family of sortin nexins and possesses a unique binding domain at the C-terminus which mediates protein-protein interaction in intracellular trafficking, membrane remodeling, organelle motility, and tight junctions. However, its role in cancer development, especially in vivo, remains largely unknown. METHODS: We have generated a stable SNX27 knockdown clone in a highly aggressive breast cancer cell line MDA-MB-231 using an inducible lentiviral shRNA system. Cell migration and proliferation of SNX27 knockdown (KD) cells were compared with wild-type (WT) cells by MTT and wound healing assay, respectively. The differences in colony formation between SNX27-KD and WT cells were detected by soft agar culture and matrigel 3D culture. Furthermore, tumor growth was examined in a xenograft nude mouse model using SNX27-KD and WT MDA-MB-231 cells. The critical EMT (epithelial-mesenchymal transition) regulators were examined in vitro and in vivo. RESULTS: The wound healing assay showed that SNX27 knockdown significantly decreased cell motility and proliferation. Colony formation in soft agar showed that the SNX27 knockdown cells formed significantly fewer and smaller colonies than the parental MDA-MB-231 cells. Western blots and immunostaining showed that knockdown of SNX27 led to increased expression of E-cadherin and ß-catenin proteins, which facilitate adhesion formation and reverse EMT. EMT is a cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal cells, promoting carcinoma invasion. The expression levels of Vimentin, the transcription factor of EMT, and tight junction protein Claudin-5, were significantly diminished in the SNX27 knockdown cells. The expression of PCNA, the cell proliferation marker, was increased in SNX27-KD cells transfected with E-cadherin siRNA. In a xenograft nude mouse model, we found that knockdown of SNX27 significantly inhibited tumor growth. The tumors from mice with SNX27-KD cells showed less proliferation compared to tumors from mice injected with wildtype cells. The increase in E-cadherin and ß-catenin and decrease in Vimentin and Claudin-5 were observed in tumors of mice injected with SNX27-KD cells. CONCLUSIONS: Our data have demonstrated that SNX27 plays a crucial role in tumor growth in vitro and in vivo.

Validation of telephone-based behavioral assessments in aging services clients.

BACKGROUND: The Behavioral Health Laboratory (BHL), a telephone-based mental health assessment, is a cost-effective approach that can improve mental illness identification and management. The individual BHL instruments, which were originally designed to be administered in-person, have not yet been validated with an in-person BHL assessment. This study therefore aims to characterize the concordance between the BHL data gathered by telephone and in-person interviews. METHODS: A cross-sectional study was conducted with English-speaking aging services network (ASN) clients aged 60 years and older in Monroe County, NY who were randomized to a BHL interview either in-person (n = 55) or by telephone (n = 53). RESULTS: There was strong evidence of equivalence between telephone and in-person interviews for depressive disorders, generalized anxiety, panic disorder, drug misuse, psychosis, PTSD, mental illness symptom severity, and five of the six questions assessing suicidality. There was marginal equivalence in PHQ-9 total scores and one of the six questions assessing suicidal ideation, and no evidence of equivalence between interview modalities for assessing cognitive impairment. CONCLUSIONS: With a few exceptions, the BHL gathered nearly equivalent information via telephone as compared to in-person interviews. This suggests that the BHL may be a cost-effective approach appropriate for dissemination in a wide variety of settings including the ASN. Dissemination of the BHL has the potential to strengthen the linkages between primary care, mental healthcare, and social service providers and improve identification and management of those with late-life mental illness.

Salmonella enteritidis Effector AvrA Stabilizes Intestinal Tight Junctions via the JNK Pathway.

Salmonella pathogenesis studies to date have focused on Salmonella typhimurium, and the pathogenesis of a second major serotype, Salmonella enteritidis, is poorly understood. Salmonella spp. possess effector proteins that display biochemical activities and modulate host functions. Here, we generated a deletion mutant of the effector AvrA, S.E-AvrA-, and a plasmid-mediated complementary strain, S.E-AvrA-/pAvrA+ (S.E-AvrA+), in S. Enteritidis. Using in vitro and in vivo infection models, we showed that AvrA stabilizes epithelial tight junction (TJ) proteins, such as ZO-1, in human intestinal epithelial cells. Transepithelial electrical resistance was significantly higher in cells infected with S.E-AvrA+ than in cells infected with S.E-AvrA- Inhibition of the JNK pathway suppresses the disassembly of TJ proteins; we found that enteritidis AvrA inhibited JNK activity in cells infected with wild type or S.E-AvrA+ strains. Therefore, Enteritidis AvrA-induced ZO-1 stability is achieved via suppression of the JNK pathway. Furthermore, the S.E-AvrA- strain led to enhanced bacterial invasion, both in vitro and in vivo Taken together, our data reveal a novel role for AvrA in S. Enteritidis: Enteritidis AvrA stabilizes intestinal TJs and attenuates bacterial invasion. The manipulation of JNK activity and TJs in microbial-epithelial interactions may be a novel therapeutic approach for the treatment of infectious diseases.

Alcohol Injury Damages Intestinal Stem Cells.

BACKGROUND: Alcohol consumption is associated with intestinal injury including intestinal leakiness and the risk of developing progressive gastrointestinal cancer. Alcoholics have disruption of intestinal barrier dysfunction that persists weeks after stopping alcohol intake, and this occurs in spite of the fact that intestinal epithelial cells turn over every 3 to 5 days. The renewal and functional regulation of the intestinal epithelium largely relies on intestinal stem cells (ISCs). Chronic inflammation and tissue damage in the intestine can injure stem cells including accumulation of mutations that may result in ISC dysfunction and transformation. ISCs are a key element in intestinal function and pathology; however, very little is known about the effects of alcohol on ISCs. We hypothesize that dysregulation of ISCs is one mechanism by which alcohol induces long-lasting intestinal damage. METHODS: In Vivo: Small intestinal samples from alcohol- and control-fed mice were assessed for ISC markers (Lgr5 and Bmi1) and the changes of the ß-catenin signaling using immunofluorescent microscopy, Western blotting, and RT-PCR. Ex Vivo: Organoids were generated from small intestine tissue and subsequently exposed to alcohol and analyzed for ISC markers, ß-catenin signaling. RESULTS: Chronic alcohol consumption significantly decreased the expression of stem cell markers, Bmi1 in the small intestine of the alcohol-fed mice and also resulted in dysregulation of the ß-catenin signaling-an essential regulator of its target gene Lgr5 and ISC function. Exposure of small intestine-derived organoids to 0.2% alcohol significantly reduced the growth of the organoids, including budding, and total surface area of the organoid cultures. Alcohol also significantly decreased the expression of Lgr5, p-ß-catenin (ser552), and Bmi1 in the organoid model. CONCLUSIONS: Both chronic alcohol feeding and acute exposure of alcohol resulted in ISC dysregulation which might be one mechanism for alcohol-induced long-lasting intestinal damage.

Diabetes Screening in US Women With a History of Gestational Diabetes, National Health and Nutrition Examination Survey, 2007-2012.

INTRODUCTION: Women with a history of gestational diabetes mellitus (GDM) are at increased risk for developing type 2 diabetes mellitus. We examined individual, socioeconomic, and health care use characteristics of women with a history of GDM and the association of those characteristics with diabetes screening, and we estimated their rates of undiagnosed prediabetes and diabetes. METHODS: Using 3 cycles of the National Health and Nutrition Examination Survey (2007-2008, 2009-2010, and 2011-2012), we identified 284 women with a history of GDM who were eligible for diabetes screening. Screening status was defined by self-report of having had a blood test for diabetes within the prior 3 years. Undiagnosed prediabetes and diabetes were assessed by hemoglobin A1c measurement. RESULTS: Among women with a history of GDM, 67% reported diabetes screening within the prior 3 years. Weighted bivariate analyses showed screened women differed from unscreened women in measured body mass index (BMI) category (P = .01) and number of health visits in the prior year (P = .001). In multivariable analysis, screening was associated with a greater number of health visits in the prior year (1 visit vs 0 visits, adjusted odds ratio [AOR], 1.91; 95% confidence interval [CI], 0.71-5.18; 2 or 3 visits, AOR, 7.05; and ≥4 visits, AOR, 5.83). Overall, 24.4% (95% CI, 18.3%-31.7%) of women had undiagnosed prediabetes and 6.5% (95% CI, 3.7%-11.3%) had undiagnosed diabetes. CONCLUSION: More health visits in the prior year was associated with receiving diabetes screening. Fewer opportunities for screening may delay early detection, clinical management, and prevention of diabetes. Prediabetes in women with a history of GDM may be underrecognized and inadequately treated.

Intestinal epithelial vitamin D receptor deletion leads to defective autophagy in colitis.

OBJECTIVE: Vitamin D and the vitamin D receptor (VDR) appear to be important immunological regulators of inflammatory bowel diseases (IBD). Defective autophagy has also been implicated in IBD, where interestingly, polymorphisms of genes such as ATG16L1 have been associated with increased risk. Although vitamin D, the microbiome and autophagy are all involved in pathogenesis of IBD, it remains unclear whether these processes are related or function independently. DESIGN: We investigated the effects and mechanisms of intestinal epithelial VDR in healthy and inflamed states using cell culture models, a conditional VDR knockout mouse model (VDR(ΔIEC)), colitis models and human samples. RESULTS: Absence of intestinal epithelial VDR affects microbial assemblage and increases susceptibility to dextran sulfate sodium-induced colitis. Intestinal epithelial VDR downregulates expressions of ATG16L1 and lysozyme, and impairs antimicrobial function of Paneth cells. Gain and loss-of-function assays showed that VDR levels regulate ATG16L1 and lysozyme at the transcriptional and translational levels. Moreover, low levels of intestinal epithelial VDR correlated with reduced ATG16L1 and representation by intestinal Bacteroides in patients with IBD. Administration of the butyrate (a fermentation product of gut microbes) increases intestinal VDR expression and suppresses inflammation in a colitis model. CONCLUSIONS: Our study demonstrates fundamental relationship between VDR, autophagy and gut microbial assemblage that is essential for maintaining intestinal homeostasis, but also in contributing to the pathophysiology of IBD. These insights can be leveraged to define therapeutic targets for restoring VDR expression and function.

Vitamin D receptor pathway is required for probiotic protection in colitis.

Low expression of vitamin D receptor (VDR) and dysfunction of vitamin D/VDR signaling are reported in patients with inflammatory bowel disease (IBD); therefore, restoration of VDR function to control inflammation in IBD is desirable. Probiotics have been used in the treatment of IBD. However, the role of probiotics in the modulation of VDR signaling to effectively reduce inflammation is unknown. We identified a novel role of probiotics in activating VDR activity, thus inhibiting inflammation, using cell models and VDR knockout mice. We found that the probiotics Lactobacillus rhamnosus strain GG (LGG) and Lactobacillus plantarum (LP) increased VDR protein expression in both mouse and human intestinal epithelial cells. Using the VDR luciferase reporter vector, we detected increased transcriptional activity of VDR after probiotic treatment. Probiotics increased the expression of the VDR target genes, such as antimicrobial peptide cathelicidin, at the transcriptional level. Furthermore, the role of probiotics in regulating VDR signaling was tested in vivo using a Salmonella-colitis model in VDR knockout mice. Probiotic treatment conferred physiological and histologic protection from Salmonella-induced colitis in VDR(+/+) mice, whereas probiotics had no effects in the VDR(-/-) mice. Probiotic treatment also enhanced numbers of Paneth cells, which secrete AMPs for host defense. These data indicate that the VDR pathway is required for probiotic protection in colitis. Understanding how probiotics enhance VDR signaling and inhibit inflammation will allow probiotics to be used effectively, resulting in innovative approaches to the prevention and treatment of chronic inflammation.

Observational measures of implementer fidelity for a school-based preventive intervention: development, reliability, and validity.

Current measures of implementer fidelity often fail to adequately measure core constructs of adherence and competence, and their relationship to outcomes can be mixed. To address these limitations, we used observational methods to assess these constructs and their relationships to proximal outcomes in a randomized trial of a school-based preventive intervention (Rochester Resilience Project) designed to strengthen emotion self-regulation skills in first-third graders with elevated aggressive-disruptive behaviors. Within the intervention group (n = 203), a subsample (n = 76) of students was selected to reflect the overall sample. Implementers were 10 paraprofessionals. Videotaped observations of three lessons from year 1 of the intervention (14 lessons) were coded for each implementer-child dyad on adherence (content) and competence (quality). Using multilevel modeling, we examined how much of the variance in the fidelity measures was attributed to implementer and to the child within implementer. Both measures had large and significant variance accounted for by implementer (competence, 68 %; adherence, 41 %); child within implementer did not account for significant variance indicating that ratings reflected stable qualities of the implementer rather than the child. Raw adherence and competence scores shared 46 % of variance (r = .68). Controlling for baseline differences and age, the amount (adherence) and quality (competence) of program delivered predicted children's enhanced response to the intervention on both child and parent reports after 6 months, but not on teacher report of externalizing behavior. Our findings support the use of multiple observations for measuring fidelity and that adherence and competence are important components of fidelity which could be assessed by many programs using these methods.

Patterns of visit attendance in the nurse-family partnership program.

OBJECTIVES: We examined visit attendance patterns in the Memphis trial of the Nurse-Family Partnership and associations between these patterns and family characteristics, outcomes, and treatment-control differences in outcomes. METHODS: We employed repeated measures latent class analysis to identify attendance patterns among the 228 mothers assigned to receive home nurse visits during pregnancy and until the child was aged 2 years, associated background characteristics, outcomes, and treatment-control differences by visit class. Home visits were conducted from June 1990 to March 1994. We collected outcome data from May 1992 to April 1994 and July 2003 to December 2006. RESULTS: We identified 3 visit attendance patterns. High attenders (48%) had the most visits and good outcomes. Low attenders (33%) had the most education and the best outcomes. Increasing attenders (18%) had the fewest completed visits during pregnancy, the poorest intake characteristics, and the poorest outcomes. Treatment-control group differences varied by class, with high and low attenders having better outcomes on some measures than did their control group counterparts. CONCLUSIONS: Three patterns were associated with distinct groups of mothers with different long-term outcomes. Further examination and use of patterns to classify mothers and prioritize resources may improve efficiency in the Nurse-Family Partnership.

Cyclin E involved in early stage carcinogenesis of esophageal adenocarcinoma by SNP DNA microarray and immunohistochemical studies.

BACKGROUND: Cyclin E is a cell cycle regulator which is critical for driving G1/S transition. Abnormal levels of cyclin E have been found in many cancers. However, the level changes of cyclin E in esophageal adenocarcinoma and its precancerous lesion have not been well studied. Here, we focus on the gene amplification and expression of cyclin E in these lesions, and aim to ascertain the relationship with clinicopathological characteristics. METHODS: Genomic DNA was analyzed from 116 esophageal adenocarcinoma and 26 precancerous lesion patients using Affymetrix SNP 6.0 arrays. The protein overexpression of cyclin E was also detected using immunohistochemistry from tissue microarrays containing esophageal adenocarcinoma and precancerous lesions. Patient survival and other clinical data were collected and analyzed. The intensity and percentage of the cyclin E expressing cells in tissue microarrays were scored by two pathologists. Fisher exact tests and Kaplan-Meier methods were used to analyze data. RESULTS: By genomic analysis, cyclin E was amplified in 19.0% of the EAC samples. By immunohistochemistry, high expression of cyclin E was observed in 2.3% of squamous mucosa tissues, 3.7% in columnar cell metaplasia, 5.8% in Barrett's esophagus, 19.0% in low grade dysplasia, 35.7% in high grade dysplasia, and 16.7% in esophageal adenocarcinoma. The differences in cyclin E high expression between neoplastic groups and non-dysplasia groups are statistically significant (p < 0.05). The prognosis for patients with high cyclin E expression appeared slightly better than for those with low cyclin E expression although this was not statistically significant (p = 0.13). CONCLUSIONS: The expression of cyclin E significantly increases from non-dysplasia esophageal lesion to low and high grade dysplasia, suggesting that cyclin E plays an important role in the early stage of carcinogenesis. Importantly, cyclin E is also amplified and highly expressed in a subset of esophageal adenocarcinoma patients, but this increase is not associated with worse prognosis.

Pretreating and normalizing metabolomics data for statistical analysis.

Metabolomics as a research field and a set of techniques is to study the entire small molecules in biological samples. Metabolomics is emerging as a powerful tool generally for precision medicine. Particularly, integration of microbiome and metabolome has revealed the mechanism and functionality of microbiome in human health and disease. However, metabolomics data are very complicated. Preprocessing/pretreating and normalizing procedures on metabolomics data are usually required before statistical analysis. In this review article, we comprehensively review various methods that are used to preprocess and pretreat metabolomics data, including MS-based data and NMR -based data preprocessing, dealing with zero and/or missing values and detecting outliers, data normalization, data centering and scaling, data transformation. We discuss the advantages and limitations of each method. The choice for a suitable preprocessing method is determined by the biological hypothesis, the characteristics of the data set, and the selected statistical data analysis method. We then provide the perspective of their applications in the microbiome and metabolome research.