Differential sensitivity to Wnt signaling gradients in human gastric organoids derived from corpus and antrum.

Wnt signaling regulates gastric stem cell proliferation and differentiation. Although similar Wnt gradients exist within the corpus and antrum of the human stomach, there are striking differences in gland architecture and disease manifestation that suggest Wnt may differentially regulate progenitor cell function in each compartment. In this study, we tested sensitivities to Wnt activation in human gastric corpus and antral organoids to determine whether progenitor cells have region-specific differences in Wnt responsiveness. Human patient-matched corpus and antral organoids were grown in the presence of varying concentrations of the Wnt pathway activator CHIR99021 to assess regional sensitivity to Wnt signaling on growth and proliferation. Corpus organoids were further studied to understand how high Wnt affected cellular differentiation and progenitor cell function. A lower concentration of CHIR99021 stimulated peak growth in corpus organoids compared with patient-matched antral organoids. Supramaximal Wnt signaling levels in corpus organoids suppressed proliferation, altered morphology, reduced surface cell differentiation, and increased differentiation of deep glandular neck and chief cells. Surprisingly, corpus organoids grown in high CHIR99021 had enhanced organoid forming potential, indicating that progenitor cell function was maintained in these nonproliferative, deep glandular cell-enriched organoids. Passaging high-Wnt quiescent organoids into low Wnt rescued normal growth, morphology, and surface cell differentiation. Our findings suggest that human corpus progenitor cells have a lower threshold for optimal Wnt signaling than antral progenitor cells. We demonstrate that Wnt signaling in the corpus regulates a bimodal axis of differentiation, with high Wnt promoting deep glandular cell differentiation and suppressing proliferation while simultaneously promoting progenitor cell function.NEW & NOTEWORTHY This study demonstrates that human gastric corpus organoids have a lower Wnt signaling threshold to drive optimal growth relative to patient-matched antral organoids. Paradoxically, supramaximal Wnt levels suppress corpus organoid proliferation, yet promote differentiation toward deep glandular cell types while simultaneously enhancing progenitor cell function. These findings provide novel insights into how Wnt signaling differentially regulates homeostasis in the human gastric corpus and antrum and contextualizes patterns of Wnt activation diseases.

Rutin: Family Farming Products' Extraction Sources, Industrial Applications and Current Trends in Biological Activity Protection.

In vitro and in vivo studies have demonstrated the bioactivity of rutin, a dietary flavonol naturally found in several plant species. Despite widespread knowledge of its numerous health benefits, such as anti-inflammatory, antidiabetic, hepatoprotective and cardiovascular effects, industrial use of rutin is still limited due to its low solubility in aqueous media, the characteristic bitter and astringent taste of phenolic compounds and its susceptibility to degradation during processing. To expand its applications and preserve its biological activity, novel encapsulation systems have been developed. This review presents updated research on the extraction sources and methodologies of rutin from fruit and vegetable products commonly found in a regular diet and grown using family farming approaches. Additionally, this review covers quantitative analysis techniques, encapsulation methods utilizing nanoparticles, colloidal and heterodisperse systems, as well as industrial applications of rutin.

A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels.

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.

Enhancing the physicochemical stability and antioxidant activity of cape gooseberry calyx extract through nanoencapsulation in soy lecithin liposomes.

The focus of this study was on the development, physicochemical characterisation and evaluation of the antioxidant activity of cape gooseberry calyx extract loaded into nanoliposomal systems. Various nanoliposomes were prepared and optimised using the ethanol injection method and characterised based on particle size, polydispersity and zeta potential measurements. Subsequently, the encapsulation efficiency and in vitro release profile of the natural antioxidant extract (NAE) were evaluated, and its antioxidant activity was assessed using the oxygen radical absorbance capacity assay. The results revealed that NAE-loaded nanoliposomes described desired quality features (e.g., particle size of < 200 nm, polydispersity index of < 0.3, zeta potential of > -40 mV and encapsulation efficiency of ∼70%). Furthermore, it was found that NAE release is controlled by various stages, and its antioxidant activity improves by around 30% when loaded into the nanoliposomes, suggesting that it could be a promising antioxidant functional raw material.

Region-specific Wnt signaling responses promote gastric polyp formation in patients with familial adenomatous polyposis.

Germline adenomatous polyposis coli (APC) mutation in patients with familial adenomatous polyposis (FAP) promotes gastrointestinal polyposis, including the formation of frequent gastric fundic gland polyps (FGPs). In this study, we investigated how dysregulated Wnt signaling promotes FGPs and why they localize to the corpus region of the stomach. We developed a biobank of FGP and surrounding nonpolyp corpus biopsies and organoids from patients with FAP for comparative studies. Polyp biopsies and polyp-derived organoids exhibited enhanced Wnt target gene expression. Polyp-derived organoids with intrinsically upregulated Wnt signaling showed poor tolerance to further induction, suggesting that high Wnt restricts growth. Targeted genomic sequencing revealed that most gastric polyps did not arise via APC loss of heterozygosity. Studies in genetic mouse models demonstrated that heterozygous Apc loss increased epithelial cell proliferation in the corpus but not the antrum, while homozygous Apc loss was not maintained in the corpus yet induced hyperproliferation in the antrum. Our findings suggest that heterozygous APC mutation in patients with FAP may be sufficient to drive polyp formation in the corpus region while subsequent loss of heterozygosity to further enhance Wnt signaling is not tolerated. This finding contextualizes the abundant yet benign nature of gastric polyps in FAP patient corpus compared with the rare, yet adenomatous polyps in the antrum.

Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1.

Malaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1) variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with the pfcrt1 K76T variant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ resistance but at a cost of reduced fitness, while pfaat1 F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.

Delta-like 1-Expressing Cells at the Gland Base Promote Proliferation of Gastric Antral Stem Cells in Mouse.

BACKGROUND & AIMS: Notch pathway signaling maintains gastric epithelial cell homeostasis by regulating stem cell proliferation and differentiation. We previously identified NOTCH1 and NOTCH2 as the key Notch receptors controlling gastric stem cell function. Here, we identify the niche cells and critical Notch ligand responsible for regulating stem cell proliferation in the distal mouse stomach. METHODS: Expression of Notch ligands in the gastric antrum was determined by quantitative reverse-transcriptase polymerase chain reaction and cellular localization was determined by in situ hybridization and immunostaining. The contribution of specific Notch ligands to regulate epithelial cell proliferation in adult mice was determined by inducible gene deletion, or by pharmacologic inhibition using antibodies directed against specific Notch ligands. Mouse gastric organoid cultures were used to confirm that Notch ligand signaling was epithelial specific. RESULTS: Delta-like 1 (DLL1) and Jagged 1 (JAG1) were the most abundantly expressed Notch ligands in the adult mouse stomach, with DLL1 restricted to the antral gland base and JAG1 localized to the upper gland region. Inhibition of DLL1 alone or in combination with other Notch ligands significantly reduced epithelial cell proliferation and the growth of gastric antral organoids, while inhibition of the other Notch ligands, DLL4, JAG1, and JAG2, did not affect proliferation or organoid growth. Similarly, DLL1, and not DLL4, regulated proliferation of LGR5+ antral stem cells, which express the NOTCH1 receptor. CONCLUSIONS: DLL1 is the key Notch ligand regulating epithelial cell proliferation in the gastric antrum. We propose that DLL1-expressing cells at the gland base are Notch niche cells that signal to adjacent LGR5+ antral stem cells to regulate stem cell proliferation and epithelial homeostasis.

A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media.

What genes determine in vitro growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between NF54, clone 3D7, a lab-adapted African parasite, and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: 3D7 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes 3D7 in media containing AlbuMAX, a commercial lipid-rich bovine serum formulation. To determine the basis for this polymorphism, we conducted parasite genetic crosses using humanized mice and compared genome-wide allele frequency changes in three independent progeny populations cultured in media containing human serum or AlbuMAX. This bulk segregant analysis detected three quantitative trait loci (QTL) regions [on chromosome (chr) 2 containing aspartate transaminase AST; chr 13 containing EBA-140; and chr 14 containing cysteine protease ATG4] linked with differential growth in serum or AlbuMAX in each of the three independent progeny pools. Selection driving differential growth was strong (s = 0.10 - 0.23 per 48-hour lifecycle). We conducted validation experiments for the strongest QTL on chr 13: competition experiments between ΔEBA-140 and 3D7 wildtype parasites showed fitness reversals in the two medium types as seen in the parental parasites, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of bulk segregant analysis for dissecting fitness traits in P. falciparum genetic crosses, and (ii) reveal intimate links between red blood cell invasion and nutrient composition of growth media. Use of parasite crosses combined with bulk segregant analysis will allow systematic dissection of key nutrient acquisition/metabolism and red blood cell invasion pathways in P. falciparum.

Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice.

Classical malaria parasite genetic crosses involve isolation, genotyping, and phenotyping of progeny parasites, which is time consuming and laborious. We tested a rapid alternative approach-bulk segregant analysis (BSA)-that utilizes sequencing of bulk progeny populations with and without drug selection for rapid identification of drug resistance loci. We used dihydroartemisinin (DHA) selection in two genetic crosses and investigated how synchronization, cryopreservation, and the drug selection regimen impacted BSA success. We detected a robust quantitative trait locus (QTL) at kelch13 in both crosses but did not detect QTLs at four other candidate loci. QTLs were detected using synchronized, but not unsynchronized progeny pools, consistent with the stage-specific action of DHA. We also successfully applied BSA to cryopreserved progeny pools, expanding the utility of this approach. We conclude that BSA provides a powerful approach for investigating the genetic architecture of drug resistance in Plasmodium falciparum.

CRISPR-Based Synthetic Transcription Factors In Vivo: The Future of Therapeutic Cellular Programming.

Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the CRISPR bacterial immune system, this dream is becoming a powerful reality. Engineered CRISPR/Cas9-based transcriptional regulators and epigenome editors have enabled researchers to perturb endogenous gene expression in vivo, allowing for the therapeutic reprogramming of cell and tissue behavior. For this technology to be of maximal use, a variety of technological hurdles still need to be addressed. Better understanding of the design principle controlling gene expression together with technologies that enable spatiotemporal control of transcriptional engineering are fundamental for rational design, improved efficacy, and ultimately safe translation to humans. In this review, we will discuss recent advances and integrative strategies that can help pave the path toward a new class of transcriptional therapeutics.

Eficiencia de la terapia periodontal no quirúrgica en la actividad clínica de la artritis reumatoide

Introducción: El efecto del tratamiento no quirúrgico periodontal en los pacientes con artritis reumatoide es escaso y controversial. Objetivo: Evaluar el efecto de la terapia periodontal no quirúrgica en la actividad de la artritis reumatoide. Métodos: Se realizó un estudio cuasi experimental de intervención terapéutica en 30 pacientes de ambos sexos entre 35 y 70 años de edad con diagnóstico clínico de artritis reumatoide y periodontitis. Fueron incluidos en este estudio los pacientes con más de ocho dientes presentes y aprobación escrita para participar en la investigación y excluidos las embarazadas, fumadores y los que recibieron terapia periodontal o antibiótica. Los parámetros clínicos-serológicos y periodontales fueron evaluados antes y 30 días después de la terapia periodontal. Las variables estudiadas fueron: formas de periodontitis y actividad de la enfermedad reumática (DAS-28/VSG). Se utilizaron frecuencias absolutas, relativas, chi-cuadrado y correlación de Spearman al 95 % de confianza. Resultados: El grupo de edad de 45-54 años y el sexo femenino fueron predominantes. La periodontitis incipiente y moderada fue la más prevalente antes del tratamiento periodontal y estuvo asociada a los niveles de actividad moderada de la AR. Tras el tratamiento periodontal se confirmó disminuciones en el estado periodontal y en la actividad de la afección reumática en el 73.3 % de los pacientes. Conclusiones: La terapia periodontal no quirúrgica mejoró el estado periodontal y redujo la actividad de la artritis reumatoide.

Introduction: The effect of nonsurgical periodontal treatment in patients with rheumatoid arthritis is limited and controversial. Objective: To evaluate the effect of non-surgical periodontal therapy on the activity of rheumatoid arthritis. Methods: a quasi-experimental study of therapeutic intervention was carried out in 30 patients of both sexes between 35 and 70 years of age with a clinical diagnosis of rheumatoid arthritis and periodontitis. Patients with more than eight teeth present and written approval to participate in the research and excluded were included in this study: pregnant women, smokers and those who received periodontal or antibiotic therapy. The clinical - serological rheumatoid and periodontal parameters were evaluated: before and 30 days after periodontal therapy. The variables studied were: forms of periodontitis and activity of the arthritic disease. Absolute and relative frequencies, chi-square and Spearman's correlation at 95 % confidence were used. Results: The age group of 45-54 years and the female sex were predominant. Early and moderate periodontitis was the most prevalent before periodontal treatment and was associated with moderate levels of rheumatoid activity. After periodontal treatment, decreases in periodontal status and rheumatic disease activity were confirmed in 73.3 % of patients. Conclusions: Non-surgical periodontal therapy improved the periodontal status and activity levels of rheumatoid arthritis.

Improving learning about familial risks using a multicomponent approach: the GRACE program.

AIM: To enhance learning (knowledge, attitudes and practices) about the importance of family health history (FHH) information and familial risks. METHODS: A pre-post design with one group was employed in this study. Five learning sessions were conducted with a community-based sample (n = 75) recruited from five counties in Texas, USA. Each learning session included: a short online video; enactive instructions on how to use the online Surgeon General FHH tool; and a presentation on how to assess familial risks. Participants completed the pre-post knowledge, attitudes and practices questionnaires and the study's satisfaction survey, and participated in a short focus group interview. RESULTS: Participants' average age was 48.1 ± 13.3 years. Over half of the participants (79%) were female, and 55% described themselves as non-Hispanic White. Our findings showed significant changes (p < 0.05) in participants' specific knowledge about factors that affect their familial risks. Similarly, significant changes (p < 0.05) in participants' attitudes toward familial risks assessment for common disease complications and confidence in controlling these risks have been documented. Participants' reported a high level of satisfaction in using online FHH tools, yet no significant change (p > 0.05) was detected in their reported practices regarding sharing FHH information with their providers or relatives. Focus group interviews revealed that participants were uncertain about providers' or relatives' reactions to sharing FHH information. CONCLUSION: Using different learning styles may have a significant impact on improving knowledge and attitudes about familial risks.

Abordaje infraclavicular en pediatría: concordancia del abordaje de Wilson modificado y el ultrasonido para la localización del sitio de punción ideal / Infraclavicular block in paediatric anaesthesia: Concordance between the modified Wilson approach and ultrasound in determining the ideal puncture site

Introduction: The modified Wilson infraclavicular approach (MWIA) was described in our institution for brachial plexus blocks in paediatric patients. However, concordance studies between this approach and ultrasound for the identification of ideal puncture site have no been reported. Objective: To determine the concordance between MWIA and ultrasound for localization of the ideal puncture site. Materials and methods: Descriptive observational study; we included 100 healthy patients between 1 and 16 years of age, with parental consent, over a 5-month period. Continuous variables were described and kappa statistics were used for concordance evaluation. We also conducted a multivariate analysis to confirm the relationship between the measured distances and weight and height. Results: The distance fromthe skin to the brachial plexus, aswell as the distance between the coracoid process and the brachial plexus, and the distance from the coracoid process to the pleura were all smaller in abduction, with no statistically significant difference. Height and weight are independent factors that determine the distance between the coracoid process and the posterior cord, both in adduction and abduction. The concordance of MWIA vs. ultrasound for determining the ideal puncture site was 47% in both positions. Conclusions: Concordance between MWIA and ultrasound for the determination of the ideal puncture site is low when it comes to anatomic localization; however, this technique must be evaluated in randomized clinical studies in order to determine its efficacy and usefulness. Height and weight are independent factors that determine the distance between the coracoid process and the posterior cord in adduction and abduction.

Introducción: En pacientes pediátricos, el sitio óptimo de inyección para el abordaje infraclavicular sigue siendo sujeto de debate; no hay estudios de concordancia entre el abordaje infraclavicular de Wilson modificado para bloqueo del plexo braquial (AIWM) y el ultrasonido para la localización del sitio ideal de punción. Objetivo: Determinar la concordancia entre AIWM y ultrasonido para localizar el sitio ideal de punción. Materiales y métodos: estudio observacional descriptivo; se incluyeron 100 niños sanos entre 1-16 años, en un periodo de 5 meses. Se describieron variables continuas y se evaluó la concordancia mediante la estadística kappa. Se realizó análisis multivariado para confirmar la correlación entre las distancias, el peso y la talla. Resultados: Se encontró una menor profundidad del plexo braquial y menor distancia entre el plexo braquial y la apófisis coracoides y entre la coracoides y la pleura en abducción, sin significancia estadística. El peso y la talla son factores independientes que determinan la distancia entre la apófisis coracoides y el cordón posterior en abducción y en aducción. La concordancia del sitio ideal de punción entre AIWM y ultrasonido fue 0,47 en ambas posiciones. Conclusiones: La concordancia entre el AIWM y el ultrasonido para determinar el sitio ideal de punción es sorprendentemente baja. Sin embargo, desde el punto de vista de utilidad y eficacia clínica, estas técnicas deben ser comparadas y evaluadas con estudios clínicos aleatorizados. El peso y la talla determinan de forma independiente la distancia entre el cordón posterior y la apófisis coracoides en aducción y abducción del brazo.

Prevalencia de Hipertensión Arterial y factores asociados: municipio Matanzas 2009-2010 / Arterial hypertension prevalence and associated factors: municipality of Matanzas 2009-2010

Se realizó un estudio observacional analítico de tipo transversal de base poblacional para estimar la prevalencia de la hipertensión arterial en la población matancera y determinar la presencia de los factores de riesgo atribuibles a esta enfermedad durante el año 2009-2010. El universo estuvo constituido por la población del municipio Matanzas entre 15 y 74 años de edad que corresponde a 112 348 habitantes. La muestra quedó conformada por 2 640 personas. En la recolección de la información se aplicó un cuestionario validado por la Organización Panamericana de la Salud para la vigilancia de factores de riesgos de las enfermedades no transmisibles. Los datos fueron procesados en el programa EpiInfo 3.4.3 2007. Para el análisis de los datos se emplearon medidas de frecuencias absolutas y relativas, medidas de tendencia central como la media, mediana, y moda; se utilizó el enfoque de riesgo en análisis bivariado, con cálculo de Odds Ratio con sus intervalos de confianza. Se utilizó la prueba de Chi Cuadrado. También Fracción Atribuible de riesgo con sus Intervalos de Confianza del 95 por ciento. Se consideraron diferencias significativas cuando los valores p fueron menores que 0.,05 y los intervalos de confianza del Odds Ratio no incluyera el valor de 1. Se obtuvo una prevalencia de hipertensión arterial de 31,3 por ciento, siendo los controlados de un 67,4 por ciento. De las nueve variables que resultaron significativas en el análisis bivariado, ocho de ellas se mostraron como verdaderos factores asociados al hipertenso al ser analizadas mediante la regresión logística: el colesterol alto, la edad mayor de 50 años, la diabetes mellitus, el hábito de fumar (tabaquismo), la obesidad, los antecedentes familiares de hipertensión arterial, el sobre peso y el bajo nivel escolar.

We carried out a cross-sectional analytic observational research on the population basis to estimate the arterial hypertension prevalence in the Matanzasan population and determine the presence of risk factors that could be attributed to this disease during the year 2009-2010. The universe was formed by the population of the municipality of Matanzas whose age ranged from 15 to 70 years, corresponding to 112 348 inhabitants. The sample was formed by 2 640 persons. In collecting information we applied a questionnaire validated by the Pan-American Health Organization for the surveillance of the non- transmissible disease risk factors. Data were processed in the program EpiInfo 3.4.3 2007. For the data analysis there were used measures of absolute and relative frequencies, central tendency measures like average, medium, and fashion; we used the risk approach in bi-variable analysis, calculating the odds ratio with their confidence intervals. In this case we used the Chi square test. We also used the attributed risk fraction with its confidence intervals 95 per cent. We considered significant differences when the value p were less than 0.05 and the confidence interval did not included the value 1. From the nine variables that were significant in the bi-variable study, 8 were shown true factors associated to the hypertensive persons when they were analyzed through the logistic regression: high levels of cholesterol, people aged more than 50 years, Diabetes Mellitus, the habit of smoking, obesity, arterial hypertension familiar antecedents, overweight, and low level of scholarship.