Integrating molecular microbial methods to improve faecal pollution management in rivers with designated bathing waters.

Rivers are at risk from a variety of pollution sources. Faecal pollution is of particular concern since it disperses pathogenic microorganisms in the aquatic environment. Currently, faecal pollution levels in rivers is monitored using faecal indicator bacteria (FIB) that do not offer information about pollution sources and associated risks. This study used a combined molecular approach, along with measurements of water quality, to gain information on pollution sources, and risk levels, in a newly designated recreational bathing site in the River Wharfe (UK). Physico-chemical parameters were monitored in situ, with water quality multiparameter monitoring sondes installed during the 2021 bathing season. The molecular approach was based on quantitative PCR (qPCR)-aided Microbial Source Tracking (MST) and 16S rRNA gene metabarcoding to obtain a fingerprint of bacterial communities and identify potential bioindicators. The analysis from the water quality sondes showed that ammonium was the main parameter determining the distribution of FIB values. Lower faecal pollution levels were detected in the main river when compared to tributaries, except for samples in the river located downstream of a wastewater treatment plant. The faecal pollution type (anthropogenic vs. zoogenic) changed the diversity and the structure of bacterial communities, giving a distinctive fingerprint that can be used to inform source. DNA-based methods showed that the presence of human-derived bacteria was associated with Escherichia coli spikes, coinciding with higher bacterial diversity and the presence of potential pathogenic bacteria mainly of the genus Mycobacterium, Aeromonas and Clostridium. Samples collected after a heavy rainfall event were associated with an increase in Bacteroidales, which are markers of faecal pollution, including Bacteroides graminisolvens, a ruminant marker associated with surface run-off from agricultural sources. The combined use of qPCR and 16S rRNA sequencing was able to identify pollution sources, and novel bacterial indicators, thereby aiding decision-making and management strategies in recreational bathing rivers.

An Efficient Transformation Method for the Bioplastic-Producing "Knallgas" Bacterium Ralstonia eutropha H16.

Ralstonia eutropha H16 (also known as Cupriavidus necator H16) is a Gram-negative lithoautotrophic ß-proteobacterium with increasing biotechnological applications, including carbon capture and utilization, biopolymer synthesis, and biofuel production. Engineering of this organism is supported by the availability of its genome sequence and suitable plasmid systems. However, the lack of a simple and robust transformation method remains a challenge as it limits both the pace and ease of engineering this organism. To overcome this limitation, a systematic study is performed to evaluate the effects of different parameters on the transformation efficiency of R. eutropha H16. The optimized electroporation protocol uses R. eutropha H16 cells grown to OD600 0.6. These cells are made competent by a 15-min incubation in 50 mM CaCl2 , followed by two cell washes and final resuspension in 0.2 M sucrose prior to electroporation using 2.3 kV. This protocol achieves a transformation efficiency of (3.86 ± 0.29) × 105 cfu µg-1 DNA, a 103 -fold improvement compared to a previously published value for the same plasmid. This transformation method is a valuable tool for R. eutropha H16 research and will further enable the development of other advanced molecular biology methods for this industrially relevant microorganism.

Validation of energy requirement references for exclusively breast-fed infants.

In paediatric practice, mean reference energy requirements for groups are often used to predict individual infant energy requirements. References from the FAO/WHO/United Nations University are based on infants not fed according to the current infant feeding recommendations. The objective of the present study was to measure total energy expenditure (TEE) and determine energy requirements using criterion methods, and validate the use of TEE prediction equation and mean energy requirement references for predicting individual TEE and energy requirements, respectively, in infants who were exclusively breast-fed (EBF) to 6 months of age. EBF infants were included from Greater Glasgow for measurements at 3·5 (n 36) and 6 (n 33) months of age. TEE was measured using doubly labelled water and energy requirements were determined using the factorial approach. TEE and energy requirements were also predicted using equations based on body weight. Relationships between criterion methods and predictions were assessed using correlations. Paired t tests and Bland-Altman plots were used to assess agreement. At the population level, predicted and measured TEE were similar. The energy requirement reference significantly underestimated energy requirements by 7·2% at 3·5 months at the population level, but there was no bias at 6 months. Errors at individual levels were large and energy requirements were underestimated to a larger extent for infants with higher energy requirements. This indicates that references presently used in clinical practice to estimate energy requirements may not fully account for the different growth pattern of EBF infants. More studies in infants EBF to 6 months of age are needed to understand how growth of EBF infants influences energy requirements.

Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

Adequacy of milk intake during exclusive breastfeeding: a longitudinal study.

OBJECTIVE: To test whether and how human lactation and breastfeeding practices can adapt to fulfill infant energy requirements during exclusive breastfeeding for 6 months. METHODS: The First-Feed study was a longitudinal, observational field study to measure milk intake, energy intake (from the doubly labeled water method), anthropometry, and breastfeeding practices at 2 time points around 15 and 25 weeks of age. Fifty healthy exclusively breastfeeding mother-infant dyads were included from breastfeeding support groups in greater Glasgow, Scotland. Forty-seven completed (23 boys), and 41 were exclusively breastfed to 25 weeks of age. RESULTS: Milk intakes were higher than literature values (923 [SD: 122] g/day, n = 36; and 999 [SD: 146] g/day, n = 33) at both 15 and 25 weeks of age (both P < .001) and increased significantly between time points (mean increase: 61 g/day [95% confidence interval: 23-99]; P = .003). Infant growth was normal compared with World Health Organization Child Growth Standards, and energy intakes were adequate compared with references for energy requirements. Behavioral data indicated no evidence of strain on breastfeeding practices. CONCLUSIONS: Results of this prospective study reveal that when mothers are well supported and follow the World Health Organization recommendation on breastfeeding, milk intakes are high and increase over time, and there is adequate energy intake, normal infant growth, and no marked changes in breastfeeding practices. This new empirical evidence on adaptations during exclusive breastfeeding should help health professionals promote the initiation, duration, and exclusivity of breastfeeding.

A genome annotation-driven approach to cloning the human ORFeome.

We have developed a systematic approach to generating cDNA clones containing full-length open reading frames (ORFs), exploiting knowledge of gene structure from genomic sequence. Each ORF was amplified by PCR from a pool of primary cDNAs, cloned and confirmed by sequencing. We obtained clones representing 70% of genes on human chromosome 22, whereas searching available cDNA clone collections found at best 48% from a single collection and 60% for all collections combined.