Characterization of a Stemness-Optimized Purification Method for Human Dental-Pulp Stem Cells: An Approach to Standardization.

Human dental pulp stem cells (hDPSCs) are promising for oral/craniofacial regeneration, but their purification and characterization is not yet standardized. hDPSCs from three donors were purified by magnetic activated cell sorting (MACS)-assisted STRO-1-positive cell enrichment (+), colony derivation (c), or a combination of both (c/+). Immunophenotype, clonogenicity, stemness marker expression, senescence, and proliferation were analyzed. Multilineage differentiation was assessed by qPCR, immunohistochemistry, and extracellular matrix mineralization. To confirm the credibility of the results, repeated measures analysis and post hoc p-value adjustment were applied. All hDPSC fractions expressed STRO-1 and were similar for several surface markers, while their clonogenicity and expression of CD10/44/105/146, and 166 varied with the purification method. (+) cells proliferated significantly faster than (c/+), while (c) showed the highest increase in metabolic activity. Colony formation was most efficient in (+) cells, which also exhibited the lowest cellular senescence. All hDPSCs produced mineralized extracellular matrix. Regarding osteogenic induction, (c/+) revealed a significant increase in mRNA expression of COL5A1 and COL6A1, while osteogenic marker genes were detected at varying levels. (c/+) were the only population missing BDNF gene transcription increase during neurogenic induction. All hDPSCs were able to differentiate into chondrocytes. In summary, the three hDPSCs populations showed differences in phenotype, stemness, proliferation, and differentiation capacity. The data suggest that STRO-1-positive cell enrichment is the optimal choice for hDPSCs purification to maintain hDPSCs stemness. Furthermore, an (immuno) phenotypic characterization is the minimum requirement for quality control in hDPSCs studies.

The Effects of Feeding Waste Milk Containing Antimicrobial Residues on Dairy Calf Health.

A number of studies have reported that there is a high prevalence of antimicrobial-resistant faecal bacteria excreted by dairy calves. Although faecal shedding is influenced by a variety of factors, such as the environment and calf age, feeding milk with antimicrobial residues contributes significantly to an increased prevalence of antimicrobial-resistant (AMR) bacteria, such as extended spectrum beta-lactamase (ESBL)-producing E.coli. As a follow-up to the European Food Safety Authority (EFSA) Scientific Opinion on the risk of AMR development in dairy calves published in January 2017, this review aims to illustrate more recent research in this area, focusing on the period 2016 to 2020. A total of 19 papers are reviewed here. The vast majority assess the commensal faecal bacteria, E.coli, isolated from dairy calves, in particular its antimicrobial-resistant forms such as ESBL-producing E.coli and AmpC-producing E.coli. The effect of waste milk feeding on the prevalence of pathogens such as Salmonella spp. has also been investigated. Current research findings include positive effects on daily liveweight gain and other advantages for calf health from feeding waste milk compared to milk replacer. However, the negative effects, such as the demonstrable selection for antimicrobial-resistant bacteria, the shift in the intestinal microbiome and the possible negative consequences that these could have on global public health, should always be taken into consideration.

Rapid spread of OXA-244-producing Escherichia coli ST38 in Germany: insights from an integrated molecular surveillance approach; 2017 to January 2020.

Annually, increasing numbers of OXA-244-producing Escherichia coli in 13 German federal states prompted us to initiate an outbreak investigation. Whole genome sequencing revealed that among 148 isolates analysed, most belonged to sequence type 38 with 62 isolates forming a genetically distinct cluster. Although no epidemiological link could be identified between cases, ongoing investigations suggest non-healthcare associated transmission. A screening-PCR was developed facilitating early detection of ST38 cluster isolates to identify the source and transmission route.

An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes.

The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.