Fecal indicator organisms are measured to indicate the presence of fecal pollution, yet the association between indicators and pathogens varies by context. The goal of this study was to empirically evaluate the relationships between indicator Escherichia coli , microbial source tracking markers, select enteric pathogen genes, and potential sources of enteric pathogens in 600 rural Bangladeshi households. We measured indicators and pathogen genes in stored drinking water, soil, and on mother and child hands. Additionally, survey and observational data on sanitation and domestic hygiene practices were collected. Log 10 concentrations of indicator E. coli were positively associated with the prevalence of pathogenic E. coli genes in all sample types. Given the current need to rely on indicators to assess fecal contamination in the field, it is significant that in this study context indicator E. coli concentrations, measured by IDEXX Colilert-18, provided quantitative information on the presence of pathogenic E. coli in different sample types. There were no significant associations between the human fecal marker (HumM2) and human-specific pathogens in any environmental sample type. There was an increase in the prevalence of Giardia lamblia genes, any E. coli virulence gene, and the specific E. coli virulence genes stx1/2 with every log 10 increase in the concentration of the animal fecal marker (BacCow) on mothers’ hands. Thus, domestic animals were important contributors to enteric pathogens in these households.

Converting adult cells from one cell type to another is a particularly interesting idea for regenerative medicine. Terminally differentiated cells can be induced to de-differentiate in vitro to become multipotent progenitors. In mammals these changes do not occur naturally, however exposing differentiated adult cells to synthetic molecules capable of selectively reverting cells from their lineage commitment to a more plastic state makes it possible to re-address their fate. Only scattered information are available on the morphological changes and ultrastructural remodeling taking place when cells convert into a different and specific type. To better clarify these aspects, we derived human granulosa cell (GC) primary cultures and analyzed the morphological changes taking place in response to the exposure to the epigenetic modifier 5-azacytidine (5-aza-CR) and to the treatment with VEGF, as a stimulus for inducing differentiation into muscle cells. Ultrastructural modifications and molecular marker expression were analyzed at different intervals during the treatments. Our results indicate that the temporary up regulation of pluripotency markers is accompanied by the loss of GC-specific ultrastructural features, mainly through autophagocitosis, and is associated with a temporary chromatin decondensation. After exposure to VEGF the induction of muscle specific genes was combined with the appearance of multinucleated cells with a considerable quantity of non-spatially organized filaments. The detailed analysis of the morphological changes occurring in cells undergoing lineage re-addressing allows a better understanding of these process and may prove useful for refining the use of somatic cells in regenerative medicine and tissue replacement therapies.

Large animal models provide useful data for pre-clinical research including regenerative medicine. However whereas the derivation of tissue specific stem cells has been successful. pluripotent stem cells so far have been difficult to obtain in these species. A possible alternative could be direct reprogramming but this has only been described in mouse and human. We have recently described an alternative method for reprogramming human somatic cells based on a brief demethylation step immediately followed by an induction protocol. Aim of the present paper was to determine whether this method is applicable to pig in the attempt to achieve cell reprogramming in a large animal model for the first time. Pig dermal fibroblasts were exposed to DNA methyltransferase inhibitor 5-aza-cytidine (5-aza-CR) for 18 h. After a brief recovery period, fibroblast were subjected to a three-step protocol for the induction of endocrine pancreatic differentiation that was completed after 42 days. During the process pig fibroblast rapidly lost their typical elongated form and gradually became organized in a reticular pattern that evolved into distinct cell aggregates. After a brief expression of some pluripotency genes, cells expression pattern mimicked the transition from primitive endoderm to endocrine pancreas. Not only converted cells expressed insulin but were able to release it in response to a physiological glucose challenge in vitro. Finally they were able to protect recipient mice against streptozotocin-induced diabetes. This work shows, that the conversion of a somatic cell into another, even if belonging to a different germ layer, is possible also in pig.

Supporting caregivers’ mental wellbeing and ability to provide psychosocial stimulation may promote early childhood development. This paper describes the systematic approach of developing an integrated stimulation intervention, identifying the feasibility and challenges faced throughout the period. We developed an integrated curriculum by culturally adapting three interventions (Reach Up, Thinking Healthy, and general nutrition advice) and piloted this curriculum (Mar–April 2017) in courtyard groups sessions and individual home visits with pregnant women (n = 11) and lactating mothers (of children <24 months) (n = 29). We conducted qualitative interviews with the participants (n = 8) and the community health workers who delivered the intervention (n = 2). Most participants reported willingness to attend the sessions if extended for 1 year, and recommended additional visual cues and interactive role-play activities to make the sessions more engaging. Participants and community health workers found it difficult to understand the concept of “unhealthy thoughts” in the curriculum. This component was then revised to include a simplified behavior-focused story. Community health workers reported difficulty balancing the required content of the integrated curriculum but were able to manage after the contents were reduced. The revised intervention is likely feasible to deliver to a group of pregnant and lactating mothers in a low-resource setting.

There is growing interest in local elimination of soil-transmitted helminth (STH) infection in endemic settings. In such settings, highly sensitive diagnostics are needed to detect STH infection. We compared double-slide Kato-Katz, the most commonly used copromicroscopic detection method, to multi-parallel quantitative polymerase chain reaction (qPCR) in 2,799 stool samples from children aged 2-12 years in a setting in rural Bangladesh with predominantly low STH infection intensity. We estimated the sensitivity and specificity of each diagnostic using Bayesian latent class analysis. Compared to double-slide Kato-Katz, STH prevalence using qPCR was almost 3-fold higher for hookworm species and nearly 2-fold higher for Trichuris trichiura. Ascaris lumbricoides prevalence was lower using qPCR, and 26% of samples classified as A. lumbricoides positive by Kato-Katz were negative by qPCR. Amplicon sequencing of the 18S rDNA from 10 samples confirmed that A. lumbricoides was absent in samples classified as positive by Kato-Katz and negative by qPCR. The sensitivity of Kato-Katz was 49% for A. lumbricoides, 32% for hookworm, and 52% for T. trichiura; the sensitivity of qPCR was 79% for A. lumbricoides, 93% for hookworm, and 90% for T. trichiura. Specificity was � 97% for both tests for all STH except for Kato-Katz for A. lumbricoides (specificity = 68%). There were moderate negative, monotonic correlations between qPCR cycle quantification values and eggs per gram quantified by Kato-Katz. While it is widely assumed that double-slide Kato-Katz has few false positives, our results indicate otherwise and highlight inherent limitations of the Kato-Katz technique. qPCR had higher sensitivity than Kato-Katz in this low intensity infection setting.

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Reduced oocyte competence causes the lower fertility reported in domestic sows during the warm months of the year. Somatic cells express heat shock proteins (HSPs) to protect themselves from damage caused by thermal stress. HSPs are classified as molecular chaperones and control the correct folding of newly synthesized or damaged proteins. The present work performed a comprehensive survey of the different components of the heat shock chaperone machinery in the pig ovary, which included the HSP40, HSP70, HSP90, and HSP110 families, as well as heat shock factors (HSF) 1 and 2. Pig ovarian follicles constitutively expressed different members of these families; therefore, we examined their ability to respond to heat stress. In order to take into account the role of the complex follicular architecture, whole pig ovaries were exposed to 41.5°C for 1 h. This exposure significantly disrupted oocyte maturation and determined the upregulation of the HSP70, HSP40, HSPH1, HSPA4, HSPA4L, HSF1, and HFS2 genes, whereas expression levels of HSP90A and HSP90B, as well as those of genes unrelated to heat stress were not altered. Unexpectedly HSP and HSF expression levels changed only in oocytes but not in cumulus cells. Cumulus-oocyte complexes isolated from ovaries collected in summer showed the same pattern as those collected in winter. We conclude that the HSP chaperone machinery is constitutively fully operational in the pig ovary. However, following thermal stimuli or seasonal variations, cumulus cell HS-related gene expression remains unchanged, and only oocytes activate a response, suggesting why this mechanism is insufficient to preserve their competence both in vitro and in vivo.

Connection between mastitis and fertility is multifaceted; therefore, several aspects need more elucidation. In particular, the aim was to investigate if naturally occurring chronic mastitis has an effect on ovarian function. At the time of slaughter, a milk sample and both ovaries were collected from 68 cows. The presence and intensity of chronic mastitis was diagnosed by the combined evaluation of bacteriological examination and somatic cell count of the milk of each individual quarter according to the measures of the National Mastitis Council. Animals were divided into 4 groups characterized by a low (n=15), mild (n=14), intense (n=19), or severe (n=16) degree of infection. A count of visible follicles on each ovary was followed by a quantitative analysis of microscopic traits on a selected group of animals (n=16). The latter included the classification and count of the entire preantral follicle population, and the morphometric analysis of the vascular bed extension and connective stroma in the cortical region. Finally, the expression of growth and differentiation factor-9 (GDF-9) was studied. The number of follicles with diameters ranging from 1 to 3 mm and 4 to 7 mm was not affected by the degree of infection. A significant effect of the degree of udder infection was observed on the number of follicles with a diameter larger than 8 mm. Furthermore, the intensity of mastitis had no effect on the number of primordial and primary follicles, but severely affected cows showed a lower number of secondary follicles (0.5±0.1 vs. 0.2±0.03). Quantitative analysis demonstrated a decrease in the density of blood vessels (6.30±1.08 vs. 4.68±0.28) expressed as ratio of vascular bed/total area) and a higher incidence of fibrous stroma (1.60±0.99 vs. 6.04±3.08 expressed as ratio of connective tissue/total area) in the cortical area of the most affected animals. Finally, the level of GDF-9 protein within the oocytes of different follicle size was lower in the animals with the severe form of chronic mastitis (1.34±0.05 vs. 0.78±0.21 expressed as arbitrary units). In conclusion, decreased fertility of cows with chronic mastitis takes place through an effect on the ovary altering the dynamics of folliculogenesis. Within the ovary, this implies a reduction of the vascular bed and an increase in the fibrotic tissue together with a direct effect on oocyte-specific factors as GDF-9, all of which are essential regulatory elements of folliculogenesis.