An insight into the commercial piglet's microbial gut colonization: from birth towards weaning.

BACKGROUND: The establishment of the gut microbiota can be influenced by several perinatal factors, including, most importantly, the maternal microbiota. Moreover, early-life environmental variation affects gut microbial colonization and the intestinal health of offspring throughout life. The present study aimed to explore the development of piglet gut microbiota from birth to weaning in the commercial practice and also to assess how different farm environments could condition this process. Although it is possible to find in the literature other studies with similar objectives this work probably represents one of the few studies that make a systematic evaluation of such differential factors under a real scenario. To achieve this objective, we performed two trials. In a first Trial, we selected 2 farms in which we performed an intensive sampling (5 samples /animal) to characterize the gut colonization pattern during the first days of life and to identify the time window with the greatest impact. Both farms differed in their health status and the use of antimicrobials in the piglets. In a second Trial, we selected 4 additional farms with variable rearing conditions and a distinctive use of antimicrobials in the sows with a simplified sampling pattern (2 samples/animal). Faecal samples were obtained with swabs and DNA was extracted by using the PSP® Spin Stool DNA Kit and sequencing of the 16S rRNA gene (V3-V4 region) performed by Illumina MiSeq Platform. RESULTS: The present study contributes to a better understanding of microbiome development during the transition from birth to weaning in commercial conditions. Alpha diversity was strongly affected by age, with an increased richness of species through time. Beta diversity decreased after weaning, suggesting a convergent evolvement among individuals. We pinpointed the early intestinal colonizers belonging to Bacteroides, Escherichia-Shigella, Clostridium sensu stricto 1, and Fusobacterium genera. During lactation(d7-d21 of life), the higher relative abundances of Bacteroides and Lactobacillus genera were correlated with a milk-oriented microbiome. As the piglets aged and after weaning (d36 of life), increasing abundances of genera such as Prevotella, Butyricimonas, Christensenellaceae R-7 group, Dorea, Phascolarctobacterium, Rikenellaceae RC9 gut group, Subdoligranulum, and Ruminococcaceae UCG-002 were observed. These changes indicate the adaptation of the piglets to a cereal-based diet rich in oligosaccharides and starch. Our results also show that the farm can have a significant impact in such a process, evidencing the influence of different environments and rearing systems on the gut microbiota development of the young piglet. Differences between farms were more noticeable after weaning than during lactation with changes in alpha and beta biodiversity and specific taxa. The analysis of such differences suggests that piglets receiving intramuscular amoxicillin (days 2-5 of life) and being offered an acidifying rehydrating solution (Alpha farm in Trial 1) have a greater alpha diversity and more abundant Lactobacillus population. Moreover, the only farm that did not offer any rehydrating solution (Foxtrot farm in Trial 2) showed a lower alpha diversity (day 2 of life) and increased abundance of Enterobacteriaceae (both at 2 and 21 days). The use of in-feed antibiotics in the sows was also associated with structural changes in the piglets' gut ecosystem although without changes in richness or diversity. Significant shifts could be registered in different microbial groups, particularly lower abundances of Fusobacterium in those piglets from medicated sows. CONCLUSIONS: In conclusion, during the first weeks of life, the pig microbiota showed a relevant succession of microbial groups towards a more homogeneous and stable ecosystem better adapted to the solid dry feed. In this relevant early-age process, the rearing conditions, the farm environment, and particularly the antimicrobial use in piglets and mothers determine changes that could have a relevant impact on gut microbiota maturation. More research is needed to elucidate the relative impact of these farm-induced early life-long changes in the growing pig.

Potential effect of two Bacillus probiotic strains on performance and fecal microbiota of breeding sows and their piglets.

The effect of long-term administration of two Bacillus strains was tested on 98 breeding sows and their litters allotted into three treatments: a control group (CON); supplemented with 5 × 108 cfu/kg B. subtilis - 541 (BSU); or with 5 × 108 cfu/kg B. amyloliquefaciens - 516 (BAM). Reproductive and performance variables were recorded over three cycles with 56 dams remaining through the third lactation. Blood and fecal samples were taken longitudinally from 12 sows per treatment on days 8 and 21 of the third lactation and milk samples were taken on day 21. Feces from one piglet per litter was sampled on days 21 and 33 and jejunal gene expression was assessed in two piglets on day 21. Changes in fecal microbiota were assessed by 16S rRNA gene sequencing (Illumina MiSeq) and gene expression by Open-Array technology. Metabolomic responses were analyzed in milk by NMR and Ig-G and Ig-A specific antibodies were determined by ELISA. No significant differences were observed on feed intake, body weight, or fat mobilization of the sows. However, a significant increase in the total number of piglets born was observed in supplemented sows. Although the increase was seen from the first cycle with BAM, improvements were not seen with BSU until the third cycle. BAM also increased the number of born-alive and weaned piglets. NMR analysis showed an impact of BAM on milk composition. No differences were found in milk or blood immunoglobulins. A different structure of the fecal microbiota was found in supplemented sows, with changes across phylum, family, and genus. These changes were greater at day 8, suggesting a relevant role of probiotics establishing a new intestinal balance after labor. Shifts in the microbiota were also seen in the piglets, with a clearer impact post-weaning than in suckling. In this regard, correlations between microbial groups of sows and piglets showed a higher link with weaned (d33) than with suckling pigs (d21), reinforcing the idea of an early maternal carry-over. No changes due to treatment in jejunal gene expression were detected; however, piglet size had a clear impact on different genes. In summary, the addition of both probiotics, and particularly Bacillus amyloliquefaciens, demonstrated potential benefits on the prolificacy of sows. Daily feeding of Bacillus amyloliquefaciens resulted in an increase in the number of weaned piglets. The high correlations between the compositions of the microbiota of sows and their piglets are evidence of maternal imprinting, with effects lasting beyond weaning.

The aim of the present study was to determine if the inclusion of probiotic microorganisms in the mother's diet during gestation and the lactation period is capable of modifying the performance of mothers and piglets and the possible effect on the intestinal health of piglets after separation from the mother. For this, 98 females were distributed in three experimental treatments: a control diet, or the same diet in which one of two probiotic strains to be tested (Bacillus subtilis or Bacillus amyloliquefaciens) were incorporated. The experimental diets were administered during pregnancy and the lactation phase for three consecutive productive cycles. Among the most striking results, it is worth highlighting the impact of probiotic treatments on the reproductive performance of sows. Both supplemented groups showed a higher number of total piglets per sow. Furthermore, sows that received the Bacillus amyloliquefaciens diet showed a significant increase in the number of live-born piglets. Probiotic supplementation also showed effects on the fecal microbiota composition of the mothers and their piglets. Changes in the composition of sow milk were also observed. In summary, results demonstrated the potential benefits of supplementing probiotics, and particularly a strain of Bacillus amyloliquefaciens, to improve prolificacy, modulate the intestinal microbial composition, and improve the performance of piglets during lactation.

Effectiveness of Two Plant-Based In-Feed Additives against an Escherichia coli F4 Oral Challenge in Weaned Piglets.

This study evaluates the efficacy of two plant-based feed supplementations to fight colibacillosis in weanlings. A total of 96 piglets (32 pens) were assigned to four diets: a control diet (T1) or supplemented with ZnO (2500 ppm Zn) (T2) or two different plant supplements, T3 (1 kg/t; based on essential oils) and T4 (T3 + 1.5 kg/t based on non-volatile compounds). After one week, animals were challenged with ETEC F4, and 8 days after, one animal per pen was euthanized. Performance, clinical signs, microbial analysis, inflammatory response, intestinal morphology, and ileal gene expression were assessed. ZnO improved daily gains 4 days after challenge, T3 and T4 showing intermediate values (96, 249, 170, and 157 g/d for T1, T2, T3, and T4, p = 0.035). Fecal lactobacilli were higher with T3 and T4 compared to ZnO (7.55, 6.26, 8.71, and 8.27 cfu/gFM; p = 0.0007) and T3 increased the lactobacilli/coliforms ratio (p = 0.002). T4 was associated with lower levels of Pig-MAP (p = 0.07) and increases in villus/crypt ratio (1.49, 1.90, 1.73, and 1.84; p = 0.009). Moreover, T4 was associated with an upregulation of the REG3G gene (p = 0.013; pFDR = 0.228) involved in the immune response induced by enteric pathogens. In conclusion, both plant supplements enhanced animal response in front of an ETEC F4 challenge probably based on different modes of action.

Association between knowledge and attitudes towards advance directives in emergency services.

BACKGROUND: Implementing the routine consultation of patient advance directives in hospital emergency departments and emergency medical services has become essential, given that advance directives constitute the frame of reference for care personalisation and respect for patients' values and preferences related to healthcare. The aim of this study was to assess the levels and relationship of knowledge and attitudes of nursing and medical professionals towards advance directives in hospital emergency departments and emergency medical services, and to determine the correlated and predictor variables of favourable attitudes towards advance directives. METHODS: Observational, descriptive, and cross-sectional study. The study was conducted in the emergency department of a second-level hospital and in the emergency medical service. Data collection was performed from January 2019 to February 2020. The STROBE guidelines were followed for the preparation of the study. RESULTS: A total of 173 healthcare professionals responded to the questionnaire. Among them, 91.3% considered that they were not sufficiently informed about advance directives, and 74% acknowledged not having incorporated them into their usual practice. Multinomial analysis indicated a statistically significant relationship between the variable emergency medical service and having more favourable attitudes towards consulting the advance directives in their practical application (OR 2.49 [95% CI 1.06-5.88]; p = 0.037) and compliance in complex scenarios (OR 3.65 [95% CI 1.58 - 8.41]; p = 0.002). Working the afternoon and night shift was a predictor variable for obtaining a higher score with respect to attitudes in complex scenarios. CONCLUSION: There is an association between the level of knowledge that nursing and medical professionals have about advance directives and the scores obtained on the attitude scales at the time of practical implementation and in complex scenarios. This shows that the more knowledge professionals have, the more likely they are to consult patients' advance directives and to respect their wishes and preferences for care and/or treatment.

EBUS-TBNA Cytological Samples for Comprehensive Molecular Testing in Non-Small Cell Lung Cancer.

Clinical guidelines promote the identification of several targetable biomarkers to drive treatment decisions in advanced non-small cell lung cancer (NSCLC), but half of all patients do not have a viable biopsy. Specimens from endobronchial-ultrasound transbronchial needle aspiration (EBUS-TBNA) are an alternative source of material for the initial diagnosis of NSCLC, however their usefulness for a complete molecular characterization remains controversial. EBUS-TBNA samples were prospectively tested for several biomarkers by next-generation sequencing (NGS), nCounter, and immunohistochemistry (PD-L1). The primary objectives were to assess the sensitivity of EBUS-TBNA samples for a comprehensive molecular characterization and to compare its performance to the reference standard of biopsy samples. Seventy-two EBUS-TBNA procedures were performed, and 42 NSCLC patients were diagnosed. Among all cytological samples, 92.9% were successfully genotyped by NGS, 95.2% by nCounter, and 100% by immunohistochemistry. There were 29 paired biopsy samples; 79.3% samples had enough tumor material for genomic genotyping, and 96.6% for PD-L1 immunohistochemistry. A good concordance was found between both sources of material: 88.9% for PD-L1, 100% for NGS and nCounter. EBUS-TBNA is a feasible alternative source of material for NSCLC genotyping and allows the identification of patient candidates for personalized therapies with high concordance when compared with biopsy.

Validation and psychometric properties of the Spanish version of the questionnaire for assessing the childbirth experience (QACE).

OBJECTIVE: When evaluating childbirth experience, some of the factors considered by women include their previous births experience, pain management during birth, and companion and healthcare professional support received. The objective of this paper is to validate the Questionnaire for Assessing the Childbirth Experience (QACE) into the Spanish population by assessing its psychometric properties. METHODS: Due to the differences between the Spanish and English languages, a careful translation process was the first step to making the QACE useable to Spanish speaking cohorts, once thoroughly translated their conceptual equivalence was evaluated by a group of experts and tested later via interviews with postpartum women for comprehensibility evaluation. Secondly, the validation process was obtained throughout the factorial analysis, internal consistency, test-retest evaluation and convergent and discriminant validity. RESULTS: A total of 268 postpartum women participated in the validity study. The KMO (0.84) and Bartlett test (p < 0.001) confirmed the adequacy of factor analysis and the Screen plot showed four factors with the predictive power of 52.63%, which supported total variance. Confirmatory factor analysis indicated an adequate/good fitness for the new model (χ2/df = 1.47, GFI = 0.979, RMSEA = 0.052, CFI = 0.889, NFI = 0.727, NNFI = 0.873, and SRMR = 0.155). Internal consistency was confirmed with McDonal's Omega level of 0.818. Test-retest evaluation supported test stability (r = 0.79, p < 0.01). Convergent and discriminant validity were obtained with 0.803 and 0.475 Pearson coefficients respectively. CONCLUSIONS: The Spanish version of QACE is a relevant tool for measuring childbirth experience into the Spanish context with acceptable validity and stability.

Managing ethical aspects of advance directives in emergency care services.

BACKGROUND: In Hospital Emergency Department and Emergency Medical Services professionals experience situations in which they face difficulties or barriers to know patient's advance directives and implement them. OBJECTIVES: To analyse the barriers, facilitators, and ethical conflicts perceived by health professionals derived from the management of advance directives in emergency services. RESEARCH DESIGN, PARTICIPANTS, AND CONTEXT: This is a qualitative phenomenological study conducted with purposive sampling including a population of nursing and medical professionals linked to Hospital Emergency Department and Emergency Medical Services. Three focus groups were formed, totalling 24 participants. We performed an inductive-type thematic discourse analysis. ETHICAL CONSIDERATIONS: This study was approved by ethical committees of Ethical Commitee of Clínic Hospital (Barcelona) and Comittee of Emergency Medical Services (Barcelona). The participants received information about the purpose of the study. Patients' anonymity and willingness to participate in the study were guaranteed. FINDINGS: There were four types of barriers that hindered the proper management of patients' advance directives in Hospital Emergency Department and Emergency Medical Services: personal and professional, family members, organisational and structural, and those derived from the health system. These barriers caused ethical conflicts and hindered professionals' decision-making. DISCUSSION: These results are in line with those of previous studies and indicate that factors such as gender, professional category, and years of experience, in addition to professionals' beliefs and the opinions of colleagues and family members, can also influence the professionals' final decisions. CONCLUSION: The different strategies described in this study can contribute to the development of health policies and action protocols to help reduce both the barriers that hinder the correct management and implementation of advance directives and the ethical conflicts generated.

Should Overnight Orthokeratology Patients Wear Their Lenses During Their Afternoon Nap?

PURPOSE: The purpose of this study was to evaluate changes in visual acuity, corneal curvature, elevation, pachymetry, and objective quality of vision of experienced orthokeratology patients using their contact lenses during a simulated 30-min afternoon nap. METHOD: Twelve patients aged 30.8±8.3 years were recruited for the study, with a history of overnight orthokeratology of 27.4±23.0 months. Patients were instructed to close their eyes for 30 min while wearing their contact lenses or without lenses. Anterior corneal curvature, elevation, and corneal pachymetry were assessed with the Pentacam Scheimpflug System at 17 predefined corneal locations, and the HD Analyzer (Terrassa, Spain) was used to measure objective quality of vision. Measurements were conducted before eye closure (baseline), immediately after eye opening/lens removal (M1), and 30 min later (M2). RESULTS: No statistically significant differences were found in anterior corneal curvature and elevation between baseline values and M1 or M2, with and without contact lenses. Corneal swelling at M1 was greater without contact lenses (change in central corneal thickness of 2.3%±3.1%, P=0.001) than with contact lenses (1.7%±1.3%, P<0.001). Recovery at M2 was slower when lenses were worn. A statistically significant improvement in objective quality of vision and visual acuity was found only when patients napped with their lenses. CONCLUSIONS: Even if no significant changes were found in corneal curvature and elevation, patients of overnight orthokeratology may benefit from using their contact lenses during their afternoon nap in terms of objective quality of vision and visual acuity.

TP53 mutation and tumoral PD-L1 expression are associated with depth of invasion in desmoplastic melanomas.

BACKGROUND: Desmoplastic melanoma (DM) is a rare subtype of spindle cell malignant melanoma characterized by frequent local recurrences and hematogenous spread, but without molecular classification. The aim of the study was to investigate in a DM series the incidence of relevant gene alterations in cancer, the programmed death-ligand 1 (PD-L1) expression status and the association with clinicopathological features and melanoma progression. METHODS: A total of 38 patients were included. Clinical follow-up and the histopathological features of all cases were retrospectively collected. PD-L1 expression by immunohistochemistry (IHC) and BRAF genomic alterations by real-time PCR were determined in 34 samples. Additionally, a molecular analysis by next-generation sequencing was performed in 25 DMs. RESULTS: Tumors occurred predominantly in men (76%) and in the head and neck region (50%). Most tumors were pure DMs (66%), containing less than 10% of conventional melanoma. Overall, 48% of our cohort harbored TP53 mutations, most of them showing a molecular signature associated with ultraviolet (UV)-oncogenesis, and 29%, BRAF mutations. A positive correlation between TP53 with depth of invasion (P=0.005) and presence of elastosis (P=0.002) was found. High-expression of PD-L1 in tumor cells was observed in 38% of cases and correlated with depth of tumoral infiltration (P=0.003), TP53 (P=0.016), PD-1 (P<0.001) and tumor-infiltrating lymphocytes (TILS) (P<0.001). PD-L1 expression in immune cells correlated with PD-1 (P=0.006), tumoral PD-L1 expression (P=0.029) and TP53 mutation (P=0.002). Survival correlated with depth of invasion (P=0.003), stage of tumors (P=0.015), positive sentinel lymph node (P=0.004), lymph node metastasis (P=0.024) and distant metastasis (P<0.001). CONCLUSIONS: Our results suggest that progressed DMs with deep tumoral infiltration frequently harbor TP53 mutations, PD-L1 expression and present a high inflammatory response, probably related to adaptive immune resistance in this tumor-type.

Usefulness of Two Independent DNA and RNA Tissue-Based Multiplex Assays for the Routine Care of Advanced NSCLC Patients.

Personalized medicine is nowadays a paradigm in lung cancer management, offering important benefits to patients. This study aimed to test the feasibility and utility of embedding two multiplexed genomic platforms as the routine workup of advanced non-squamous non-small cell lung cancer (NSCLC) patients. Two parallel multiplexed approaches were performed based on DNA sequencing and direct digital detection of RNA with nCounter® technology to evaluate gene mutations and fusions. The results were used to guide genotype-directed therapies and patient outcomes were collected. A total of 224 advanced non-squamous NSCLC patients were prospectively included in the study. Overall, 85% of samples were successfully characterized at DNA and RNA levels and oncogenic drivers were found in 68% of patients, with KRAS, EGFR, METΔex14, BRAF, and ALK being the most frequent (31%, 19%, 5%, 4%, and 4%, respectively). Among all patients with complete genotyping results and follow-up data (n = 156), the median overall survival (OS) was 1.90 years (confidence interval (CI) 95% 1.69-2.10) for individuals harbouring an actionable driver treated with a matched therapy, compared with 0.59 years (CI 95% 0.39-0.79) in those not eligible for any targeted therapy and 0.61 years (CI 95% 0.12-1.10) in patients with no drivers identified (p < 0.001). Integrating DNA and RNA multiplexing technologies into the routine molecular testing of advanced NSCLC patients is feasible and useful and highlights the necessity of widespread integrating comprehensive molecular diagnosis into lung cancer care.

Implementation of an NGS panel for clinical practice in paraffin-embedded tissue samples from locally advanced and metastatic melanoma patients.

Aim: Single biomarker diagnostic test of BRAFV600 locus in metastatic melanoma is mandatory for treatment decision; however, multiple-gene based techniques, such as targeted next-generation sequencing (NGS) are being used to maximize the number of patients that can benefit from a targeted therapy. The main objective of this study is to investigate whether an NGS panel could be adopted in routine clinical care for advanced melanoma. Methods: Patients diagnosed with advanced melanoma at our center from 2017 to 2019 were included. Presence of genetic alterations was performed using two methods: real-time polymerase chain reaction-based Idylla test (Biocartis) and NGS with the oncomine solid tumor DNA kit (Thermo Fisher Scientific). Total genomic DNA was extracted from formalin-fixed and paraffin embedded samples for sequencing. Results: A total of 155 samples were evaluated for molecular analysis but 40 samples (25.8%) were inadequate for sequencing. The clinical utility of BRAFV600 real-time polymerase chain reaction and targeted-NGS was compared in 29 samples and a very good concordance was observed (Kappa = 0.89, 95% confidence interval 0.68 ± 1.05). An oncogenic mutation by NGS was found in 75 samples (65%)-53% of whom were candidates for personalized therapies. The most prevalent mutated genes were BRAF (39%), TP53 (23%), and NRAS (14%). Other genes identified at lower incidence (< 5%) were: PIK3CA, ERBB4, CTNNB1, STK11, FGFR1, SMAD4, KRAS, FGFR3, PTEN and AKT. Co-occurrence of oncogenic mutations was detected in 40% of the samples. Among the mutations identified, TP53 was significantly more prevalent in men (men 31.8% versus women 12.2%, P = 0.03) and NRAS in women (men 9.1% versus women 24.4%, P = 0.03). Conclusions: Targeted-NGS testing is a feasible technique to implement in the routine clinical practice. Based on our results, NGS has provided more information on target-genes than RT-PCR technique, maximizing the benefit for patients with advanced melanoma.

Avoidable hospitalizations due to adverse drug reactions in an acute geriatric unit. Analysis of 3,292 patients. / Hospitalizaciones evitables por reacciones adversas a medicamentos en una unidad geriátrica de agudos. Análisis de 3.292 pacientes.

OBJECTIVE: To determine prevalence of admissions due to an adverse drug reaction (ADR) and determine whether or not admission was avoidable, and what drugs and risk factors were implicated. DESIGN: Cross-sectional observational study. STUDY SAMPLE: All patients hospitalized in an acute geriatric unit during the period January 2001 to December 2010 were studied. MEASUREMENT: To determine whether admissions were due toADR, we used the World Health Organization-Uppsala Monitoring Centre criteria and the Naranjo scale. Beers criteria were used to detect potentially inappropriate medication. RESULTS: A total of 3,292 patients (mean age 84.7 years, 60.1% women) were studied. Of these, 197 (6%) were admissions for ADR and nearly three quarters (76.4%, 152 cases) were considered avoidable admissions. The 5 most frequent drugs associated with admissions for ADR were digoxin, nonsteroidal anti-inflammatory drugs, benzodiazepines, diuretics and antibiotics. Independent risk factors for admissions for ADR were being female (OR 1.84; 95% CI 1.30-2.61), inappropriate medication according to Beers criteria (OR 4.20; 95% CI 2.90-6.03), polypharmacy (>5 drugs) (OR 1.50; 95% CI 1.04-2.13), glomerular filtration rate<30mL/min (OR 3; 95% CI 2.12-4.23) and sedative use (OR 1.40; 95% CI 1-1.91). CONCLUSION: ADR were responsible for 6% of admissions to an acute geriatric unit, and over 75% of these admissions were considered avoidable. Associated risk factors were being female, inappropriate medication, polypharmacy, renal insufficiency and sedative use.

Topical use of 5% acyclovir cream for the treatment of occult and verrucous equine sarcoids: a double-blinded placebo-controlled study.

BACKGROUND: Previous studies mention the use of topical acyclovir for the treatment of equine sarcoids. Success rates vary and since the bovine papillomavirus (BPV) lacks the presence of a kinase necessary to activate acyclovir, there is no proof of its activity against equine sarcoids. RESULTS: Twenty-four equine sarcoids were topically treated with acyclovir cream and 25 with a placebo. Both creams were applied twice daily during 6 months. Before the start of the treatment and further on a monthly basis, photographs and swabs were obtained. On the photographs, sarcoid diameter and surface area were measured and verrucosity of the tumours was quantified using a visual analog scale (VAS). The swabs were analysed by PCR for the presence of BPV DNA and positivity rates were calculated as the number of positive swabs divided by the total number of swabs for each treatment group at each time point. Success rates were not significantly different between both treatment groups. There was also no significant effect of treatment on sarcoid diameter, surface area or VAS score. For the swabs, a significantly higher BPV positivity rate was found for acyclovir treated tumours compared to placebo treated sarcoids only after 1 month of treatment and not at other time points. CONCLUSIONS: None of the results indicate that treatment with acyclovir yields any better results compared to placebo treatment.

Fra-2 regulates B cell development by enhancing IRF4 and Foxo1 transcription.

The role of AP-1 transcription factors in early B cell development and function is still incompletely characterized. Here we address the role of Fra-2 in B cell differentiation. Deletion of Fra-2 leads to impaired B cell proliferation in the bone marrow. In addition, IL-7-stimulated pro-B cell cultures revealed a reduced differentiation from large pre-B cells to small B cells and immature B cells. Gene profiling and chromatin immunoprecipitation sequencing analyses unraveled a transcriptional reduction of the transcription factors Foxo1, Irf4, Ikaros, and Aiolos in Fra-2-deficient B cells. Moreover, expression of IL7Rα and Rag 1/2, downstream targets of Irf4 and Foxo1, were also reduced in the absence of Fra-2. Pro-B cell proliferation and small pre-B cell differentiation were fully rescued by expression of Foxo1 and Irf4 in Fra-2-deficient pro-B cells. Hence, Fra-2 is a key upstream regulator of Foxo1 and Irf4 expression and influences proliferation and differentiation of B cells at multiple stages.

Relationship between Resilience and Self-regulation: A Study of Spanish Youth at Risk of Social Exclusion.

The ability to self-regulate behavior is one of the most important protective factors in relation with resilience and should be fostered especially in at-risk youth. Previous research has characterized these students as having behaviors indicating lack of foresight. The aim of the present study was to test the hypothetical relationship between these personal variables. It was hypothesized that self-regulation would be associated with and would be a good predictor of resilience, and that low-medium-high levels of self-regulation would lead to similar levels of resilience. The participants were 365 students -aged 15 and 21- from Navarre (Spain) who were enrolled in Initial Vocational Qualification Programs (IVQP). For the assessment, the Connor Davidson Resilience Scale (CD-RISC) and the Short Self-Regulation Questionnaire (SSRQ) were applied. We carried out linear association analyses (correlational and structural) and non-linear interdependence analyses (MANOVA) between the two constructs. Relationships between them were significant and positive. Learning from mistakes (self-regulation) was a significant predictor of coping and confidence, tenacity and adaptation, and tolerance to negative situations (resilience). Likewise, low-medium-high levels of self-regulation correlated with scores on resilience factors. Implications of these results for educational practice and for future research are discussed.

Inhibition of Osteoarthritis by Adipose-Derived Stromal Cells Overexpressing Fra-1 in Mice.

OBJECTIVE: To determine whether overexpression of the activator protein 1 (AP-1) transcription factor Fra-1 in adipose-derived stromal cells (ADSCs) is an effective treatment of collagenase-induced osteoarthritis (OA). METHODS: OA was induced by injection of collagenase into the knee joints of male C57BL/6 mice. ADSCs were isolated from the inguinal fat pads of 8-week-old wild-type or Fra-1-transgenic mice and injected into the knee joints of mice with collagenase-induced OA 7 days after OA induction. Histologic analyses of cartilage destruction and chondrocyte cell death were performed. Adipogenic differentiation capacity was evaluated, gene expression was analyzed, and cytokine profiling was performed in stromal vascular fractions (SVFs) and ADSCs. RESULTS: OA-related cartilage destruction and chondrocyte cell death were significantly reduced in mouse knee joints treated with ADSCs from Fra-1-transgenic mice compared to mouse knee joints treated with ADSCs from wild-type mice. This effect did not result from the higher number of adipogenic progenitors observed in SVFs from Fra-1-transgenic compared to wild-type mouse fat pads, since injection of wild-type mouse ADSCs enriched for adipogenic progenitors did not show any additional chondroprotective effects compared to nonsorted ADSCs. However, Fra-1-transgenic mouse ADSCs showed decreased adipogenic differentiation capacity. Moreover, Fra-1 significantly inhibited proinflammatory interleukin-6 and pentraxin 3 expression, while increasing the expression of extracellular matrix proteins, such as periostin and spondin 1. These findings suggest that Fra-1 overexpression leads to an increased chondroprotective effect of ADSCs in OA. CONCLUSION: ADSCs overexpressing Fra-1 effectively protect against OA. Our data indicate that genetic modifications of ADSCs, such as Fra-1 overexpression, may improve their potential to protect articular cartilage against OA-mediated damage.

Development and Validation of a Stability Indicating RP-HPLC Method for Hydrocortisone Acetate Active Ingredient, Propyl Parahydroxybenzoate and Methyl Parahydroxybenzoate Preservatives, Butylhydroxyanisole Antioxidant, and Their Degradation Products in a Rectal Gel Formulation.

A stability indicating method was established through a stress study, wherein different methods of degradation (oxidation, hydrolysis, photolysis, and temperature) were studied simultaneously to determine the active ingredient hydrocortisone acetate, preservatives propyl parahydroxybenzoate, and methyl parahydroxybenzoate, antioxidant butylhydroxyanisole (BHA), and their degradation products in a semisolid dosage gel form. The proposed method was suitably validated using a Zorbax SB-Phenyl column and gradient elution. The mobile phase consisted of a mixture of methanol, acetonitrile, and water in different proportions according to a planned program at a flow rate of 1.5 mL/min. The diode array detector was set at 240 nm for the active substance and two preservatives, and 290 nm for BHA. The validation study was conducted according to International Conference on Harmonization guidelines for specificity, linearity, repeatability, precision, and accuracy. The method was used for QC of hydrocortisone acetate gel and for the stability studies with the aim of quantifying the active substance, preservatives, antioxidant, and degradation products. It has proved to be suitable as a fast and reliable method for QC.

NF-κB-direct activation of microRNAs with repressive effects on monocyte-specific genes is critical for osteoclast differentiation.

BACKGROUND: Monocyte-to-osteoclast conversion is a unique terminal differentiation process that is exacerbated in rheumatoid arthritis and bone metastasis. The mechanisms implicated in upregulating osteoclast-specific genes involve transcription factors, epigenetic regulators and microRNAs (miRNAs). It is less well known how downregulation of osteoclast-inappropriate genes is achieved. RESULTS: In this study, analysis of miRNA expression changes in osteoclast differentiation from human primary monocytes revealed the rapid upregulation of two miRNA clusters, miR-212/132 and miR-99b/let-7e/125a. We demonstrate that they negatively target monocyte-specific and immunomodulatory genes like TNFAIP3, IGF1R and IL15. Depletion of these miRNAs inhibits osteoclast differentiation and upregulates their targets. These miRNAs are also upregulated in other inflammatory monocytic differentiation processes. Most importantly, we demonstrate for the first time the direct involvement of Nuclear Factor kappa B (NF-κB) in the regulation of these miRNAs, as well as with their targets, whereby NF-κB p65 binds the promoters of these two miRNA clusters and NF-κB inhibition or depletion results in impaired upregulation of their expression. CONCLUSIONS: Our results reveal the direct involvement of NF-κB in shutting down certain monocyte-specific genes, including some anti-inflammatory activities, through a miRNA-dependent mechanism for proper osteoclast differentiation.

PU.1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation.

BACKGROUND: DNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Local deposition and removal of DNA methylation are tightly coupled with transcription factor binding, although the relationship varies with the specific differentiation process. Conversion of monocytes to osteoclasts is a unique terminal differentiation process within the hematopoietic system. This differentiation model is relevant to autoimmune disease and cancer, and there is abundant knowledge on the sets of transcription factors involved. RESULTS: Here we focused on DNA methylation changes during osteoclastogenesis. Hypermethylation and hypomethylation changes took place in several thousand genes, including all relevant osteoclast differentiation and function categories. Hypomethylation occurred in association with changes in 5-hydroxymethylcytosine, a proposed intermediate toward demethylation. Transcription factor binding motif analysis revealed an over-representation of PU.1, NF-κB, and AP-1 (Jun/Fos) binding motifs in genes undergoing DNA methylation changes. Among these, only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes; ChIP-seq data analysis confirmed its association to both gene sets. Moreover, PU.1 interacts with both DNMT3b and TET2, suggesting its participation in driving hypermethylation and hydroxymethylation-mediated hypomethylation. Consistent with this, siRNA-mediated PU.1 knockdown in primary monocytes impaired the acquisition of DNA methylation and expression changes, and reduced the association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation. CONCLUSIONS: The work described here identifies key changes in DNA methylation during monocyte-to-osteoclast differentiation and reveals novel roles for PU.1 in this process.