Single-cell microglial transcriptomics during demyelination defines a microglial state required for lytic carcass clearance.

BACKGROUND: Microglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood. METHODS: To understand how microglia respond during demyelination, we fed mice cuprizone-a potent demyelinating agent-and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response. RESULTS: The cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination. CONCLUSIONS: Microglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state.

Seal, replacement or monitoring amalgam restorations with occlusal marginal defects? Results of a 10-year clinical trial.

The aim of this prospective and blind clinical trial was to assess the effectiveness of sealing localized marginal defects of amalgam restoration that were initially scheduled to be replaced. A cohort of twenty six patients with 60 amalgam restorations (n=44Class I and n=16Class II), that presented marginal defects deviating from ideal (Bravo) according to USPHS criteria, were assigned to either sealing or replacement groups: A: sealing n=20, Replacement n=20, and no treatment (n=20). Two blind examiners evaluated the restorations at baseline (K=0.74) and after ten years (K=0.84) according with USPHS criteria, in four parameters: marginal adaptation (MA), secondary caries (SC), marginal staining (MS) and teeth sensitivity (TS). Multiple comparison of restorations degradation/upgrade was analyzed by Friedman test and the comparisons within groups were performed by Wilcoxon test. After 10 years, 44 restorations were assessed (73.3%), Group A: n=14 and Group B: n=16; and Group C: n=14 sealing and replacement amalgam restorations presented similar level of quality in MA (p=0.76), SC (p=0.25) and TS (p=0.52), while in MS (p=0.007) presented better performance in replacement group after 10-years. Most of the occlusal amalgam restorations with marginal gaps showed similar long term outcomes than the restorations were sealed, replaced, or not treated over a 10-year period. Most of the restorations of the three groups were clinically acceptable, under the studied parameters. All restorations had the tendency to present downgrade/deterioration over time.

Detection of pathogenic micro-organisms on children's hands and toys during play.

AIMS: This study aimed to determine if the children's leisure activities impact the presence of pathogens on their hands and toys. METHODS & RESULTS: To assess the microbiological hazard in playground areas, a pilot study that included 12 children was conducted. We then conducted an intervention study; children's hands and toys were washed before playing. Faecal coliforms, pathogenic bacteria and Giardia lamblia were quantified by membrane filtration, selective media and flotation techniques, respectively; rotavirus, hepatitis A and rhinovirus by RT-PCR. Pilot study results revealed faecal contamination on children's hands and toys after playing on sidewalks and in public parks. Pathogenic bacteria, hepatitis A and G. lamblia on children's hands were also found. In the intervention study, Staphylococcus aureus and Klebsiella pneumoniae were found on children's hands at concentrations up to 2·5 × 10(4) and 1 × 10(4) CFU hands(-1), respectively. E. coli and Kl. pneumoniae were detected on toys (2·4 × 10(3) and 2·7 × 10(4) CFU toy(-1), respectively). Salmonella spp, Serratia spp and G. lamblia cysts were also present on toys. CONCLUSION: Children's play activities influence microbial presence on hands and toys; the transfer seems to occur in both ways. SIGNIFICANCE AND IMPACT OF THE STUDY: Control strategy needs to be implemented to protect children from infectious diseases.

Increased plasma citrulline in mice marks diet-induced obesity and may predict the development of the metabolic syndrome.

In humans, plasma amino acid concentrations of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) increase in states of obesity, insulin resistance and diabetes. We here assessed whether these putative biomarkers can also be identified in two different obesity and diabetic mouse models. C57BL/6 mice with diet-induced obesity (DIO) mimic the metabolic impairments of obesity in humans characterized by hyperglycemia, hyperinsulinemia and hepatic triglyceride accumulation. Mice treated with streptozotocin (STZ) to induce insulin deficiency were used as a type 1 diabetes model. Plasma amino acid profiling of two high fat (HF) feeding trials revealed that citrulline and ornithine concentrations are elevated in obese mice, while systemic arginine bioavailability (ratio of plasma arginine to ornithine + citrulline) is reduced. In skeletal muscle, HF feeding induced a reduction of arginine levels while citrulline levels were elevated. However, arginine or citrulline remained unchanged in their key metabolic organs, intestine and kidney. Moreover, the intestinal conversion of labeled arginine to ornithine and citrulline in vitro remained unaffected by HF feeding excluding the intestine as prime site of these alterations. In liver, citrulline is mainly derived from ornithine in the urea cycle and DIO mice displayed reduced hepatic ornithine levels. Since both amino acids share an antiport mechanism for mitochondrial import and export, elevated plasma citrulline may indicate impaired hepatic amino acid handling in DIO mice. In the insulin deficient mice, plasma citrulline and ornithine levels also increased and additionally these animals displayed elevated BCAA and AAA levels like insulin resistant and diabetic patients. Therefore, type 1 diabetic mice but not DIO mice show the "diabetic fingerprint" of plasma amino acid changes observed in humans. Additionally, citrulline may serve as an early indicator of the obesity-dependent metabolic impairments.

Occurrence of faecal contamination in households along the US-Mexico border.

AIMS: The study aim was to determine the presence of total and faecal coliforms on kitchen surfaces, in tap water and on the hands of caregivers in households on both sides of the US-Mexico border. METHODS AND RESULTS: Samples were collected in 135 randomly selected households in Ciudad Juarez, Mexico, and El Paso, Texas. Different surfaces throughout the kitchen and head of households' hands were sampled using sterile cotton swabs moistened in D/E neutralizing solution. Sponge/dishcloth and drinking water samples were also obtained. Total and faecal coliforms were enumerated on m-Endo LES and mFC respectively. Total coliforms and Escherichia coli in drinking water samples were enumerated in accordance with the Quanti-Tray method. Sponge/dishcloth samples were the most commonly contaminated kitchen sites, followed by countertops and cutting boards. We recovered faecal coliforms from 14% of the hands of child caregivers, and this indicator was moderately associated with self-reported failure to wash hands after using the toilet (OR = 3.2; 95% CI: 0.9, 11.1). CONCLUSIONS: Hand washing should continue to be emphasized, and additional interventions should be directed to specific kitchen areas, such as sponges/dishcloths, tables/countertops and cutting boards. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for additional interventions regarding kitchen sanitation.

Population-based survey of taeniasis along the United States-Mexico border.

Taenia solium and T. saginata are zoonotic tapeworms of substantial medical and economic importance. Although human taeniasis is widely recognised as an endemic problem in Mexico, its presence in the United States is poorly understood. The first population-based study to estimate the prevalence of human infection with Taenia tapeworms along the Texas-Mexico border has recently been conducted. Households were interviewed in the Texan city of El Paso and in the neighbouring Ciudad Juárez, in Mexico. Faecal samples from household members were then checked for Taenia eggs by flotation and/or for Taenia copro-antigens in an ELISA. The overall prevalence of taeniasis in this border region was found to be 3% but, compared with the residents of Juárez, El Paso residents were 8.6-fold more likely to be tapeworm carriers. The interviews revealed some important differences between the two study sites, particularly the more frequent use of anthelminthic drugs on the Mexican side of the border. These findings have implications in terms of the planning of effective health-education campaigns to decrease the prevalence of taeniasis in the human populations along the Texas-Mexico border.

Assessment of the risk of infection by Cryptosporidium and Giardia in non-potable reclaimed water.

Quantitative risk assessment for Cryptosporidium oocysts and Giardia cysts was performed to determine the public health significance of non-potable use of tertiary treated reclaimed water. Seven reclaimed water treatment plants in the southwestern United States participated in this study. The average public exposure to oocysts and cysts was estimated, based on concentrations, recovery efficiency, viability and three exposure scenarios. The exponential dose-response model was chosen to determine the probability of infection from ingestion of various numbers of oocysts and cysts. The risks of infection for Giardia were approximately one or two orders of magnitude higher than those for Cryptosporidium. The combined risks of infection from oocysts and cysts at sites using a combination of chlorination and UV disinfection would meet the annual acceptable risk of 1.00E-04, whereas those at the other utilities using only chlorination indicated higher probability of infection than the 1.00E-04 resulting from accidental consumption of a small amount of non-potable reclaimed water.