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Clinical infection and death caused by bluetongue virus infection has been reported in the Eurasian lynx. Bluetongue virus surveillance in the Iberian lynx revealed widespread and repeated exposure to serotypes 1 and 4 in wild and captive populations of this species. This exposure is possibly from a spillover event from sympatric ruminants.

Abstract Background Apoptosis, a form of programmed cell death, is critical for the development and homeostasis of the immune system. Chimeric antigen receptor T (CAR-T) cell therapy, approved for hematologic cancers, retains several limitations and challenges associated with ex vivo manipulation, including CAR T-cell susceptibility to apoptosis. Therefore, strategies to improve T-cell survival and persistence are required. Mesenchymal stem/stromal cells (MSCs) exhibit immunoregulatory and tissue-restoring potential. We have previously shown that the transfer of umbilical cord MSC (UC-MSC)-derived mitochondrial (MitoT) prompts the genetic reprogramming of CD3+ T cells towards a Treg cell lineage. The potency of T cells plays an important role in effective immunotherapy, underscoring the need for improving their metabolic fitness. In the present work, we evaluate the effect of MitoT on apoptotis of native T lymphocytes and engineered CAR-T cells. Methods We used a cell-free approach using artificial MitoT (Mitoception) of UC-MSC derived MT to peripheral blood mononuclear cells (PBMCs) followed by RNA-seq analysis of CD3+ MitoTpos and MitoTneg sorted cells. Target cell apoptosis was induced with Staurosporine (STS), and cell viability was evaluated with Annexin V/7AAD and TUNEL assays. Changes in apoptotic regulators were assessed by flow cytometry, western blot, and qRT-PCR. The effect of MitoT on 19BBz CAR T-cell apoptosis in response to electroporation with a non-viral transposon-based vector was assessed with Annexin V/7AAD. Results Gene expression related to apoptosis, cell death and/or responses to different stimuli was modified in CD3+ T cells after Mitoception. CD3+MitoTpos cells were resistant to STS-induced apoptosis compared to MitoTneg cells, showing a decreased percentage in apoptotic T cells as well as in TUNEL+ cells. Additionally, MitoT prevented the STS-induced collapse of the mitochondrial membrane potential (MMP) levels, decreased caspase-3 cleavage, increased BCL2 transcript levels and BCL-2-related BARD1 expression in FACS-sorted CD3+ T cells. Furthermore, UC-MSC-derived MitoT reduced both early and late apoptosis in CAR-T cells following electroporation, and exhibited an increasing trend in cytotoxic activity levels. Conclusions Artificial MitoT prevents STS-induced apoptosis of human CD3+ T cells by interfering with the caspase pathway. Furthermore, we observed that MitoT confers protection to apoptosis induced by electroporation in MitoTpos CAR T-engineered cells, potentially improving their metabolic fitness and resistance to environmental stress. These results widen the physiological perspective of organelle-based therapies in immune conditions while offering potential avenues to enhance CAR-T treatment outcomes where their viability is compromised.

Abstract This systematic review and meta-analysis addresses the effects of minimally invasive surgical techniques, specifically the Reverdin Isham osteotomy, on functional and radiological outcomes in patients with moderate Hallux Valgus, a common foot deformity. The review included randomized and non-randomized controlled trials, as well as case reports, assessing the osteotomy in adults with moderate to severe Hallux Valgus. Searches were conducted in electronic databases such as MEDLINE and Web of Science up until July 2023, and the Joanna Briggs Institute’s critical appraisal tool was used to assess the risk of bias. Meta-analytical analyses employed a random-effects model with small-sample correction, presenting results as standardized mean differences and mean differences with 95% confidence intervals. Seven studies involving 554 patients and 643 operated feet were included, showing significant improvements in AOFAS scores (an average improvement of 36 points from 28.61 to 45.16) and reductions in radiological angles such as the distal metatarsal angle and hallux valgus angle post-surgery (IMA improved by − 3.07° from − 4.68 to − 1.46, DMAA by − 6.12° from − 9.52 to − 2.71, and HVangle by − 15.27° from − 17.98 to − 12.57). Despite these positive outcomes, most studies exhibited risks of bias and other methodological limitations, impacting the generalizability of the results. Overall, the findings highlight the efficacy of the Reverdin Isham osteotomy in improving both functional and radiological parameters in patients with moderate Hallux Valgus, although further research is warranted to solidify these results. No specific funding was received for this study, and the protocol was registered on PROSPERO with the number CRD-42023445886.

Abstract Recently, Lactobacillus johnsonii N6.2-derived extracellular vesicles (EVs) were shown to reduce apoptosis in human beta cell lines and stimulate insulin secretion in human islets. Our goal was to identify a physiologically relevant environmental condition that induces a hypervesiculation phenotype in L. johnsonii N6.2 and to evaluate if transcriptional changes are involved in this process. Culturing this strain in the presence of 0.2% bovine bile, which mimics a stressor encountered by the bacterium in the small intestine, resulted in approximately a 100-fold increase in EVs relative to cells grown in media without bile. Whole transcriptome analysis of cells grown with bile revealed upregulation of several peptidoglycan hydrolases as well as several genes involved in fatty acid utilization. These results suggest that the hypervesiculation phenotype may be the result of increased cell wall turnover combined with increased accumulation of phospholipids, in agreement with our previous proteomic and lipidomics results. Additionally, EVs isolated from L. johnsonii N6.2 grown in presence of bile maintained their immunomodulatory properties in host-derived βlox5 pancreatic and THP-1 macrophage cell lines. Our findings suggest that in L. johnsonii N6.2 vesiculogenesis is significantly impacted by the expression of cell wall modifying enzymes and proteins utilized for exogenous fatty acid uptake that are regulated at the transcriptional level. Furthermore, this data suggests that vesiculogenesis could be stimulated in vivo using small molecules thereby maximizing the beneficial interactions between bacteria and their hosts.

Introduction Therapeutic exercise has an important role in the population living with cancer as it improves function and quality of life and reduces the symptoms of cancer treatment. There is little clinical evidence on the effects of hypopressive exercise in women with gynecological cancer. Objective Evaluate the effects of 4 weeks of hypopressive exercise associated with muscle strength training and aerobic exercises on fatigue, urinary incontinence symptoms, sexual function, and quality of life in women treated for gynecological cancer compared to a group that will perform conventional training. Methods This randomized, single-blinded clinical trial study is set in the Clinical Research Laboratory, Department of Kinesiotherapy, at a Chilean University. Patients will be randomly assigned to an experimental group of hypopressive exercises associated with muscle strength training and aerobic exercises or a control group of muscle strength training and aerobic exercises. Twelve tele-rehabilitation sessions will be performed. Women over 18 years of age with gynecologic cancer who have been prescribed radiotherapy or chemotherapy will participate. Fatigue, quality of life, urinary incontinence symptoms, and sexual function will be assessed before and after the intervention. Expected results The results of this clinical trial have important implications for specific treatment for the cancer population and generate new techniques in the practice of oncology-specialized kinesiologists. Hypopressive exercise is expected to reduce incontinence symptoms due to neuromuscular activation of the pelvic floor muscles. However, more studies are needed to confirm the beneficial effects of hypopressive exercises in face-to-face or remote rehabilitation.

Malaria is an infectious disease caused by Plasmodium spp. parasites, with widespread drug resistance to most antimalarial drugs. We report the development of two 3D-QSAR models based on comparative molecular field analysis (CoMFA), comparative molecular similarity index analysis (CoMSIA), and a 2D-QSAR model, using a database of 349 compounds with activity against the P. falciparum 3D7 strain. The models were validated internally and externally, complying with all metrics (q2 > 0.5, r2test > 0.6, r2m > 0.5, etc.). The final models have shown the following statistical values: r2test CoMFA = 0.878, r2test CoMSIA = 0.876, and r2test 2D-QSAR = 0.845. The models were experimentally tested through the synthesis and biological evaluation of ten quinoline derivatives against P. falciparum 3D7. The CoMSIA and 2D-QSAR models outperformed CoMFA in terms of better predictive capacity (MAE = 0.7006, 0.4849, and 1.2803, respectively). The physicochemical and pharmacokinetic properties of three selected quinoline derivatives were similar to chloroquine. Finally, the compounds showed low cytotoxicity (IC50 > 100 µM) on human HepG2 cells. These results suggest that the QSAR models accurately predict the toxicological profile, correlating well with experimental in vivo data.

Purpose: To evaluate the association between ejaculation frequency (EF) during four stages of life and prostate cancer (PCa) according to tumor aggressiveness, PCa stage, and urinary symptomatology. Materials and Methods: A total of 456 incident PCa cases histologically confirmed, and 427 controls aged 40–80 years from the CAPLIFE study were analyzed. This study is a population-based case-control study carried out in the south of Spain. Average EF was measured for: (1) 20s, (2) 30s, (3) 40s, and (4) one year before the interview. EF was categorized into: (1) 0–3, (2) 4, and (3) >4 ejaculations/month. Sociodemographic, lifestyle, and medical information were also collected. To estimate the association between EF and PCa, adjusted ORs (aORs) and 95% CIs were calculated by logistic regression models. Results: A year before the interview, PCa cases ejaculated less frequently than the controls. An inverse association was observed between the EF a year before and PCa, aOR=1.64 (95% CI 1.03–2.61) for men with 4 ejaculations/month, and aOR=2.38 (95% CI 1.57–3.60) for men with 0–3 ejaculations/month, compared to men with >4. The association was higher for cases with ISUP 3–5 (aOR=2.76 [95% CI 1.34–5.67] for men with 0–3 ejaculations/month) or with a locally advancedmetastatic tumor (aOR=4.70 [95% CI 1.55–14.29]). Moreover, men with moderate urinary symptoms and 0–3 ejaculations/ month had the highest risk, aOR=3.83 (95% CI 1.84–7.95). Conclusions: A low EF could be associated with a higher risk of PCa, especially for cases with ISUP 3–5 or with a locally advanced-metastatic tumor.

Abstract In this report, we evaluated the effect of the pasteurization (P) process of mother’s own milk (MOM) on the miRNA content of extracellular vesicles (EVs) and its impact on innate immune responses. Differences in size or particle number were not observed upon pasteurization of MOM (PMOM). However, significant differences were observed in the EV membrane marker CD63 and miRNA profiles. miRNA sequencing identified 33 differentially enriched miRNAs between MOMEV and PMOMEV. These changes correlated with significant decreases in the ability of PMOMEV to modulate IL-8 secretion in intestinal Caco2 cells where only MOMEV were able to decrease IL-8 secretion in presence of TNFα. While EVs from MOMEV and PMOMEV were both able to induce a tolerogenic M2-like phenotype in THP-1 macrophages, a significant decrease in the transcript levels of IL-10 and RNA sensing genes was observed with PMOMEV. Together, our data indicates that pasteurization of MOM impacts the integrity and functionality of MOMEV, decreasing its EVs-mediated immunomodulatory activity. This data provides biomarkers that may be utilized during the optimization of milk processing to preserve its bioactivity.

Abstract Growth alterations have been described in patients operated on for oral clefts. The purpose of this work was to analyze the craniofacial and palate morphology and dimensions of young adults operated on for oral clefts in early childhood in Spain. Eighty-three patients from eight different hospitals were divided into four groups based on their type of cleft: cleft lip (CL, n = 6), unilateral cleft lip and palate (UCLP, n = 37), bilateral cleft lip and palate (BCLP, n = 16), and cleft palate only (CPO, n = 24). A control group was formed of 71 individuals. Three-dimensional (3D) digital models were obtained from all groups with an intraoral scanner, together with cephalometries and frontal, lateral, and submental facial photographs. Measurements were obtained and analyzed statistically. Our results showed craniofacial alterations in the BCLP, UCLP, and CPO groups with an influence on the palate, maxilla, and mandible and a direct impact on facial appearance. This effect was more severe in the BCLP group. Measurements in the CL group were similar to those in the control group. Cleft characteristics and cleft type seem to be the main determining factors of long-term craniofacial growth alterations in these patients. Prospective research is needed to clearly delineate the effects of different treatments on the craniofacial appearance of adult cleft patients.

Objetivo. Evaluar la eficacia de un protocolo estandarizado y simplificado basado en los pilares técnicos de la Iniciativa HEARTS para el control de pacientes hipertensos del Programa de Salud Cardiovascular en el primer nivel de atención de salud en Chile. Métodos. Estudio observacional longitudinal (cohorte histórica), en 2 centros de salud familiar del primer nivel de atención en Santiago, que comparó el control de presión arterial en adultos hipertensos logrado con el protocolo estandarizado y simplificado, frente al protocolo habitual, según las guías nacionales. Las innovaciones del protocolo estandarizado incluyeron cambios en la coordinación del equipo de salud, inicio de tratamiento farmacológico inmediatamente después de confirmación diagnóstica, tratamiento farmacológico estandarizado con combinación de al menos 2 o 3 fármacos antihipertensivos en un sólo comprimido, en una toma diaria. Se realizó seguimiento por 1 año para evaluar el porcentaje de adherencia al tratamiento y cumplimiento de metas de control de presión arterial (menor a 140/90 mmHg). Resultados. Se evaluaron 1 490 pacientes: 562 que utilizaron el protocolo estandarizado y simplificado y 928 tratados con el protocolo habitual (centros de salud familiar-1: 650, centros de salud familiar -2: 278). A 1 año de seguimiento, los pacientes del grupo del protocolo estandarizado y simplificado tuvieron mayor proporción de cumplimiento de metas de control de presión arterial (65% versus 37% y 41%, p

La infección por SARS-CoV-2 podría gatillar la ocurrencia de numerosas patologías, algunas de ellas de tipo endocrinológico. Se presenta una revisión narrativa sobre la asociación entre COVID-19 y tiroiditis subaguda, en la que se describen aspectos epidemiológicos, fisiopatológicos, manifestaciones clínicas y de laboratorio, tratamiento y pronóstico. La prevalencia de alguna disfunción tiroidea en pacientes COVID-19 fluctúa entre 13 y 64%, específicamente en relación a tiroiditis subaguda se describe un cambio en la prevalencia de 0,5% a 10% entre los años 2019 y 2020. La tiroiditis subaguda se produce por la unión del virus SARS-CoV-2 con la enzima convertidora de angiotensina 2 (ACE-2) ampliamente expresada en la glándula tiroides, generando manifestaciones clínicas tipo tirotoxicosis, aunque la presentación de la tiroiditis subaguda asociada a COVID-19 puede ser atípica. Los elementos centrales del tratamiento son similares a los empleados ante cualquier tiroiditis subaguda. Se concluye la necesidad de considerar esta entidad en el diagnóstico diferencial de pacientes actual o previamente infectados por SARS-CoV-2 que presenten síndrome febril, elevación de parámetros inflamatorios o taquicardia persistente, sin etiología precisada.

Objective To describe the experiences of nephrologists on caring for patients undergoing in-centre haemodialysis during the COVID-19 pandemic in Latin America.Design Twenty-five semistructured interviews were conducted by Zoom videoconference in English and Spanish languages during 2020 until data saturation. Using thematic analysis, we conducted line-by-line coding to inductively identify themes.Setting 25 centres across nine countries in Latin America.Participants Nephrologists (17 male and 8 female) were purposively sampled to include diverse demographic characteristics and clinical experience.Results We identified five themes: shock and immediate mobilisation for preparedness (overwhelmed and distressed, expanding responsibilities to manage COVID-19 infection and united for workforce resilience); personal vulnerability (being infected with COVID-19 and fear of transmitting COVID-19 to family); infrastructural susceptibility of dialysis units (lacking resources and facilities for quarantine, struggling to prevent cross-contamination, and depletion of personal protective equipment and cleaning supplies); helplessness and moral distress (being forced to ration life-sustaining equipment and care, being concerned about delayed and shortened dialysis sessions, patient hesitancy to attend to dialysis sessions, being grieved by socioeconomic disparities, deterioration of patients with COVID-19, harms of isolation and inability to provide kidney replacement therapy); and fostering innovative delivery of care (expanding use of telehealth, increasing uptake of PD and shifting focus on preventing syndemics).Conclusion Nephrologists felt personally and professionally vulnerable and reported feeling helpless and morally distressed because they doubted their capacity to provide safe care for patients undergoing dialysis. Better availability and mobilisation of resources and capacities to adapt models of care, including telehealth and home-based dialysis, are urgently needed.

Dialysis treatment has improved the survival of patients with kidney failure. However, the hospitalization and mortality rates remain alarmingly high, primarily due to incomplete uremic toxin elimination. High-volume hemodiafiltration (HDF) has emerged as a promising approach that significantly improves patient outcomes by effectively eliminating medium and large uremic toxins, which explains its increasing adoption, particularly in Europe and Japan. Interest in this therapy has grown following the findings of the recently published CONVINCE study, as well as the need to understand the mechanisms behind the benefits. This comprehensive review aims to enhance the scientific understanding by explaining the underlying physiological mechanisms that contribute to the positive effects of HDF in terms of short-term benefits, like hemodynamic tolerance and cardiovascular disease. Additionally, it explores the rationale behind the medium-term clinical benefits, including phosphorus removal, the modulation of inflammation and oxidative stress, anemia management, immune response modulation, nutritional effects, the mitigation of bone disorders, neuropathy relief, and amyloidosis reduction. This review also analyzes the impact of HDF on patient-reported outcomes and mortality. Considering the importance of applying personalized uremic toxin removal strategies tailored to the unique needs of each patient, high-volume HDF appears to be the most effective treatment to date for patients with renal failure. This justifies the need to prioritize its application in clinical practice, initially focusing on the groups with the greatest potential benefits and subsequently extending its use to a larger number of patients.

Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an instrumental role in driving CME initiation. The F-BAR domain-only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted in vitro and cellular systems. Our results indicate that PI(4,5)P2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like-shaped protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P2-interacting proteins at endocytic sites.

La prevalencia de enfermedad diverticular en Chile se estima en 28% y cada vez es más frecuente su presentación como diverticulitis complicada. Durante los últimos años ha surgido evidencia que ha hecho replantear el manejo de la diverticulitis aguda. Históricamente los antibióticos han sido pilar fundamental del tratamiento de la diverticulitis aguda, sin embargo, evidencia reciente muestra que pacientes con un episodio de diverticulitis aguda no complicada pueden ser tratados sin ellos. Con respecto al manejo quirúrgico, la cirugía de emergencia está indicada en peritonitis difusa, absceso no puncionable asociado a sepsis y fracaso de tratamiento; tanto la cirugía de Hartmann como resección y anastomosis primaria son opciones válidas según el escenario. La cirugía electiva debe indicarse caso a caso y se debe optar por abordaje laparoscópico dentro de lo posible. Al enfrentarse a pacientes inmunosuprimidos se debe ser más agresivo en el manejo. La indicación de colonoscopía posterior a un episodio de diverticulitis aguda se reserva para pacientes en los que se haya presentado como un episodio de diverticulitis complicada, en aquellos que corresponda realizar tamizaje para cáncer colorrectal y en los que presenten síntomas o signos sugerentes de neoplasia colorrectal.

Brain aquaporin 1 (AQP1) and AQP4 are involved in cerebrospinal fluid (CSF) homeostasis and might participate in the origin of hydrocephalus. Studies have shown alterations of perivascular AQP4 expression in idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer's disease (AD). Due to the overlapping of clinical signs between iNPH and certain neurological conditions, mainly AD, specific biomarkers might improve the diagnostic accuracy for iNPH. The goal of the present study was to analyze and quantify the presence of AQP1 and AQP4 in the CSF of patients with iNPH and AD to determine whether these proteins can be used as biomarkers of iNPH. We examined AQP1 and AQP4 protein levels in the CSF of 179 participants (88 women) classified into 5 groups: possible iNPH (81 participants), hydrocephalus associated with other neurological disorders (13 participants), AD (41 participants), non-AD dementia (32 participants) and healthy controls (12 participants). We recorded each participant's demographic and clinical variables and indicated, when available in the clinical history, the record of cardiovascular and respiratory complications. An ELISA showed virtually no AQP content in the CSF. Information on the vascular risk factors (available for 61 patients) confirmed some type of vascular risk factor in 86% of the patients with possible iNPH and 58% of the patients with AD. In conclusion, the ELISA analysis showed insufficient sensitivity to detect the presence of AQP1 and AQP4 in CSF, ruling out the possible use of these proteins as biomarkers for diagnosing iNPH.

Una de las explosiones más notables que ha ocurrido en el último año, el 2020 del siglo XXI, es la preocupación y debate sobre las vacunas para controlar la pandemia del nuevo virus SARS-CoV-2 al cumplirse un año de iniciada. Sobre esta pandemia ya se han escrito miles de comunicaciones científicas y de divulgación, incluyendo las de carácter puramente políticas o apocalípticas. A los millones de casos diagnosticados en el planeta, con sus dos millones de fallecidos, se une al cumplirse doce meses de este breve y devastador fenómeno la iniciación del proceso vacunatorio, que abre la esperanza de salir de la inmensa enfermedad cuanto antes.

Objetivo: explorar la siguiente pregunta de investigación: ¿De qué manera el nivel socioeconómico de una persona, específicamente el nivel de pobreza se asocia a las características de las exposiciones agudas a sustancias con potencial tóxico en Chile? Métodos: estudio epidemiológico, de diseño transversal y semi-ecológico. Se recopiló información proveniente de una muestra de casos atendidos por el Centro de Información Toxicológica y de Medicamentos de la Pontificia Universidad Católica de Chile (CITUC). La variable independiente corresponde al índice de pobreza comunal de la Encuesta de Caracterización Socioeconómica Nacional (CASEN). Las variables dependientes fueron las características de las exposiciones registradas por el centro toxicológico de acuerdo con el nivel de pobreza de los casos observados. Se analizó el fenómeno a través de tablas cruzadas, cálculo de Odds Ratio con análisis estratificado. Resultados: se observan diferencias significativas en las circunstancias de las exposiciones: las personas que viven en comunas con mayor porcentaje de pobreza muestran más exposiciones intencionales versus aquellas con menor porcentaje de pobreza. Otro hallazgo significativo es el uso del sistema de salud en casos de exposiciones dependiente de la situación socioeconómica. Para las otras variables no se encuentran diferencias significativas entre los grupos comparados. Conclusiones: se observa una relación entre el nivel de pobreza y las exposiciones a sustancias potencialmente tóxicas. Los resultados abren una línea investigativa y nuevas interrogantes que podrían generar medidas de control de las intoxicaciones en Chile.

Sin resumen

Aims: To determine the lifetime and monthly prevalence of people with mental disorders and its association with sociodemographic factors and criminal risk in three Spanish prisons (Ocaña, Madrid I, II and VI). Method: Cross-sectional epidemiological study of a sample of 184 inmates. Socio-demographic and criminal data were collected by an ad hoc interview. Mental disorders were assessed with the clinical version of the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders Axis I Disorders (SCID-I). Results: Life prevalence of mental disorders was 90.2%. The most common mental disorders and substance abuse or dependence was 72.3%, followed by mood disorder (38.5%) and psychotic disorders (34.2%). Moreover, the prevalence of any mental disorder in the last month was 52.2%. The main psychotic disorder (20.7%) was followed by substance abuse or dependence (18.5%), and mood disorder state (13%). A socio-demographic profile as a risk for each disorder was found. Discussion: The prevalence of people with mental disorders is very high in Spanish prisons, and is associated with a distinct demographic profile. It is essential to continue researching this reality, translating the results into therapeutic and preventive action adapted to the status of inmates to reduce social inequalities in this high priority public health situation.

Sin resumen

OBJECTIVES/GOALS: Feeding preterm infants with mother’s own milk (MOM) lowers rates of sepsis, decreases necrotizing enterocolitis, and shortens hospital stay. Our objective is to determine whether a similar microbial diversity to MOM can be obtained when fresh or frozen MOM is inoculated in donor human milk (DHM). METHODS/STUDY POPULATION: Subjects included 12 mothers of infants born 100ml of MOM per day and were excluded if they had taken antibiotics within 3 days of the 1-time pumped MOM sample collection. MOM sample was divided into fresh (processed immediately) and frozen (−20°C) for 24h fractions. MOM was inoculated in DHM [referred to as refaunated milk (RM)] at 10% (RM10) and 30% (RM30) dilutions, then incubated at timepoints: 0h, 2h, 4h at 37°C. At each timepoint, total viable microbial cell counts were performed in differential or selective media along with future 16S rRNA sequencing. RESULTS/ANTICIPATED RESULTS: Microbiota expansion was detected in MOM, RM10 and RM30 over time whether fresh or frozen milk was used as the inoculum. Incubated fresh and frozen MOM had similar bacterial loads when tested on nutrient agar (10^5-10^6 CFU/mL), mannitol salt (10^6 CFU/mL), MacConkey (10^2-10^5 CFU/mL), blood agar (10^6 CFU/mL) and MRS (10^4 CFU/mL) plates. Based on these CFU counts, RM30 incubated for 2h and RM10 at 4h showed similar counts to that of MOM at 0h. DISCUSSION/SIGNIFICANCE OF IMPACT: RM, inoculated with fresh or frozen MOM, obtained a similar microbial count compared to MOM at 0h indicates that fresh or frozen MOM can inoculate DHM. 16s rRNA sequencing is ongoing. Future studies are needed to support an inoculation protocol to be used in clinical practice and human milk banking.

Half of the high-risk colorectal cancer families that fulfill the clinical criteria for Lynch syndrome lack germline mutations in the mismatch repair (MMR) genes and remain unexplained. Genetic testing for hereditary cancers is rapidly evolving due to the introduction of multigene panels, which may identify more mutations than the old screening methods. The aim of this study is the use of a Next Generation Sequencing panel in order to find the genes involved in the cancer predisposition of these families. For this study, 98 patients from these unexplained families were tested with a multigene panel targeting 94 genes involved in cancer predisposition. The mutations found were validated by Sanger sequencing and the segregation was studied when possible. We identified 19 likely pathogenic variants in 18 patients. Out of these, 8 were found in MMR genes (5 in MLH1, 1 in MSH6 and 2 in PMS2). In addition, 11 mutations were detected in other genes, including high penetrance genes (APC, SMAD4 and TP53) and moderate penetrance genes (BRIP1, CHEK2, MUTYH, HNF1A and XPC). Mutations c.1194G>A in SMAD4, c.714_720dup in PMS2, c.2050T>G in MLH1 and c.1635_1636del in MSH6 were novel. In conclusion, the detection of new pathogenic mutations in high and moderate penetrance genes could contribute to the explanation of the heritability of colorectal cancer, changing the individual clinical management. Multigene panel testing is a more effective method to identify germline variants in cancer patients compared to single-gene approaches and should be therefore included in clinical laboratories.

LdtR is a master regulator of gene expression in Liberibacter asiaticus, one of the causative agents of citrus greening disease. LdtR belongs to the MarR-family of transcriptional regulators and it has been linked to the regulation of more than 180 genes in Liberibacter species, most of them gathered in the following Clusters of Orthologous Groups: cell motility, cell wall envelope, energy production, and transcription. Our previous transcriptomic evidence suggested that LdtR is directly involved in the modulation of the zinc uptake system genes (znu) in the closely related L. crescens. In this report, we show that LdtR is involved in the regulation of one of the two encoded zinc uptake mechanisms in L. asiaticus, named znu2. We also show that LdtR binds zinc with higher affinity than benzbromarone, a synthetic effector inhibitory molecule, resulting in the disruption of the LdtR:promoter interactions. Using site-directed mutagenesis, electrophoretic mobility shift assays (EMSAs), and isothermal titration calorimetry, we identified that residues C28 and T43 in LdtR, located in close proximity to the Benz1 pocket, are involved in the interaction with zinc. These results provided new evidence of a high-affinity effector molecule targeting a key player in L. asiaticus' physiology and complemented our previous findings about the mechanisms of signal transduction in members of the MarR-family.

OBJECTIVES/SPECIFIC AIMS: Aim 1: To compare frozen MOM to fresh MOM over time as an agent to inoculate DHM and measure the enrichment of commensal microbes and their beneficial bioactive components similar to MOM. Hypothesis: Frozen or fresh MOM inoculated in DHM will produce similar microbial content to MOM over time allowing for the production of beneficial bacterial compounds that may contribute to host immune response. Aim 2: To determine the effect of MOM storage (fresh vs frozen) on the expansion of bioactive components from live microbiota in DHM. Hypothesis: Both fresh and frozen MOM will produce similar results when inoculated into DHM to restore the microbial content (including their bioactive components) similar to each MOM sample. Aim 3: To compare the microbiome found in a mother’s MOM to the microbiome in her infant’s stool. Hypothesis: The mother/infant pair will share a common microbiome between the mother’s MOM and her infant’s stool. METHODS/STUDY POPULATION: Subjects will include 12 pump-dependent mothers of infants born < 34 weeks gestation admitted to the University of Florida Health Shands Hospital, Neonatal Intensive Care Unit (NICU). Inclusion criteria consists of mothers expressing over 100 ml of MOM per day, producing at least 45 ml of MOM at an expression session, at least 18 years of age, and speak English. Mothers are excluded if they have taken antibiotics within 3 days of sample collection, are HIV+, or delivered an infant who has a chromosomal abnormality or is severely ill. An expressed MOM sample will be collected and divided into two fractions: (A) fresh and (B) frozen at -20C for 24 h. The fresh fraction (A) will be processed immediately while the frozen fraction (B) will be processed after 24 h. Each MOM will be inoculated in DHM at dilutions of 10% and 30% and incubated at different time points: 0 h (T0), 2 h (T2), 4 h (T4) at 37°C. At each time point, total viable cell counts as well as microbiome analysis through 16S ribosomal shotgun sequencing will be performed and compared for differences. Bacteria isolated from each MOM will be saved, identified through 16S ribosomal sequencing and grown in culture for future studies. Fecal samples from each corresponding infant will be collected within 48 h after collection of the MOM sample. Stools will be homogenized and subjected to DNA extraction to perform 16S ribosomal shotgun sequencing. Microbiome analysis will be conducted, compared between fecal samples as well as with the microbiome of the MOM. RESULTS/ANTICIPATED RESULTS: Study is ongoing. We anticipate similar results with fresh or frozen MOM to that of a previous pilot study, where enriched microbiota similar to MOM was found when fresh MOM was inoculated and incubated in DHM. The microbiome analysis of the infant fecal samples may illustrate the influence that the microbiome of the MOM may have on the development of the infants’ gastrointestinal microbiota. DISCUSSION/SIGNIFICANCE OF IMPACT: The purpose of this study is to provide evidence on the ability, timing, and efficacy of inoculating DHM with fresh and frozen MOM. Study results will inform future studies to support the implementation of an inoculation procedural protocol to be used in clinical practice and human milk banking. The description of the MOM microbiome, as well as the gastrointestinal microbiome, will expand scientific knowledge on the role breast milk has on the origins of health and disease.

The involvement of GALNT12 in colorectal carcinogenesis has been demonstrated but it is not clear to what extent it is implicated in familial CRC susceptibility. Partially inactivating variant, NM_024642.4:c.907G>A, p.(D303N), has been previously detected in familial CRC and proposed as the causative risk allele. Since phenotypes of the described carrier families showed not only CRC but also a polyp history, we hypothesized that GALNT12 could be involved in adenoma predisposition and consequently, in hereditary polyposis CRC syndromes. For that purpose, we have screened the GALNT12 gene in germline DNA from 183 unrelated attenuated polyposis patients. c.907G>A, p.(D303N) was detected in 4 cases (MAF = 1.1%) and no other candidate variants were found. After segregation studies, LOH analyses, glycosylation pattern tests and case-control studies, our results did not support the role of c.907G>A, p.(D303N) as a high-penetrance risk allele for polyposis CRC.

Large conductance voltage- and Ca2+-activated K+ (BKCa) channels are essential regulators of membrane excitability in a wide variety of cells and tissues. An important mechanism of modulation of BKCa channel activity is its association with auxiliary subunits. In smooth muscle cells, the most predominant regulatory subunit of BKCa channels is the β1-subunit. We have previously described that BKCa channels with distinctive N-terminal ends (starting with the amino acid sequence MDAL, MSSN or MANG) are differentially modulated by the β1-subunit, but not by the β2. Here we extended our studies to understand how the distinct N-terminal regions differentially modulate channel activity by β-subunits. We recorded inside-out single-channel currents from HEK293T cells co-expressing the BKCa containing three N-terminal sequences with two β1-β2 chimeric constructs containing the extracellular loop of β1 or β2, and the transmembrane and cytoplasmic domains of β2 or β1, respectively. Both β chimeric constructs induced leftward shifts of voltage-activation curves of channels starting with MANG and MDAL, in the presence of 10 or 100 μM intracellular Ca2+. However, MSSN showed no shift of the voltage-activation, at the same Ca2+ concentrations. The presence of the extracellular loop of β1 in the chimera resembled results seen with the full β1 subunit, suggesting that the extracellular region of β1 might be responsible for the lack of modulation observed in MSSN. We further studied a poly-serine stretch present in the N-terminal region of MSSN and observed that the voltage-activation curves of BKCa channels either containing or lacking this poly-serine stretch were leftward shifted by β1-subunit in a similar way. Overall, our results provide further insights into the mechanism of modulation of the different N-terminal regions of the BKCa channel by β-subunits and highlight the extension of this region of the channel as a form of modulation of channel activity.