Site specific insertion of a transgene into the murine α-casein (CSN1S1) gene results in the predictable expression of a recombinant protein in milk.

Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. The α-casein (CSN1S1) gene has assessed as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA, and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression.

Physical activity in childhood and adolescence and future depressive symptoms: an 11-year prospective cohort study.

BACKGROUND: Physical activity (PA) can reduce young peoples' risk of depressive symptoms. Associations between PA and depressive symptoms are often investigated over timeframes spanning minutes to weeks. Less is known about whether childhood/adolescent PA can predict depressive symptoms in early adulthood. METHODS: Using a nationally representative sample from Ireland, latent growth mixture modelling was performed to investigate the extent to which different PA trajectories existed from ages 9-17, whether gender, weight status, and socio-economic deprivation at age 9, predicted PA trajectories from ages 9-17, and whether trajectory class membership predicted depressive symptoms at age 20. RESULTS: A 4-class solution was the best fit to the data (AIC = 52 175.69; BIC = 52 302.69; ssaBIC = 52 245.49; entropy = 1.00). Classes were labelled according to their baseline PA and slope of their trajectory: 'High-Decreasers'; 'Moderate-Decreasers'; 'Moderate-Stable'; and 'Low-Increasers'. A negative linear association existed between activity trajectory and the likelihood class members were female, overweight or socioeconomically deprived at age 9. The most active class (High-Decreasers) were significantly less likely to report depressive symptoms at age 20 than other classes. CONCLUSIONS: Multiple PA trajectories exist throughout childhood and adolescence although differences in PA levels reduced over time. The most/least active children continued to be the most/least active throughout adolescence. Those most active were least at risk of depressive symptoms in early adulthood. Being female, overweight or experiencing deprivation at age 9 were all risk factors for inactivity throughout adolescence. Findings have implications for public health and PA promotion in young people.

Comparing Mental Health of Athletes and Non-athletes as They Emerge From a COVID-19 Pandemic Lockdown.

Athletes going through transition periods such as injury or retirement have previously reported feelings of depression and anxiety, especially when feeling unsupported. Cessation of competitive sport during the pandemic has forced athletes through a non-normative transition and has reduced many opportunities to satisfy their basic psychological needs increasing the risk of poor wellbeing and loneliness. Whilst athletes are often praised for their resilience-a trait that serves to support them during tough times-the inability to play sport can be particularly challenging for those with strong athletic identities. An online cross-sectional survey (n = 744) was conducted to capture adult athlete and non-athlete mental health factors (specifically wellbeing, depression, anxiety, loneliness) during emergence from a COVID-19 lockdown. Results showed that resilience was positively correlated with mental health but was no higher in athletes than non-athletes. Furthermore, athletes reported greater anxiety than non-athletes, a difference mediated by negative affectivity-a subfactor of athletic identity. We present evidence that after a temporary transition away from sport, athletes' resilience is comparable to non-athletes leaving them just as likely to suffer poor mental health. Moreover, athletes with strong athletic identities are likely to experience anxiety symptoms above and beyond those reported by non-athletes. Findings have implications for the development of self-management guidance for athletes as the COVID-19 pandemic and restrictions on sport participation continue.

A study on episodic memory reconsolidation that tells us more about consolidation.

Awake quiescence immediately after encoding is conducive to episodic memory consolidation. Retrieval can render episodic memories labile again, but reconsolidation can modify and restrengthen them. It remained unknown whether awake quiescence after retrieval supports episodic memory reconsolidation. We sought to examine this question via an object-location memory paradigm. We failed to probe the effect of quiescence on reconsolidation, but we did observe an unforeseen "delayed" effect of quiescence on consolidation. Our findings reveal that the beneficial effect of quiescence on episodic memory consolidation is not restricted to immediately following encoding but can be achieved at a delayed stage and even following a period of task engagement.

Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors.

Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

Using the CRISPR/Cas9 system to understand neuropeptide biology and regulation.

Neuropeptides and their receptors play a role in physiological responses such as appetite, stress and inflammatory pain. With neuropeptides having such diverse and important physiological roles, knocking-out the genes encoding them, their receptors, parts of their regulatory sequences, or reproducing disease associated polymorphic variants are important steps in studying neuropeptides and how they may contribute to disease. Previously, knock-outs were generated using methods such as targeted homologous recombination in embryonic stem cells but this method is costly and time-consuming. The CRISPR/Cas9 system has rapidly taken over the genome editing field and will advance our understanding of neuropeptide genes and their regulation. With CRISPR/Cas9 technology, the time and costs involved in producing transgenic animal models, is greatly reduced. In this review, we describe how the system can be used to manipulate genomic sequences by "knock-out" or "knock-in" mutations in cell lines or in animal models. We also discuss the specificity of the system and methods to limit off-target effects. When combined with the availability of genome sequences, CRISPR/Cas9 directed genome editing in vitro and in vivo, promises to provide a deeper understanding of the biology of the neuropeptides in health and disease than has ever been available before.

The effect of whole cell immobilisation on the biotransformation of benzonitrile and the use of direct electric current for enhanced product removal.

The nitrilase of Rhodococcus rhodochrous performs a one-step biotransformation of nitriles to their corresponding carboxylic acids. Application of a direct electric current moves the charged carboxylic acid towards an anode, across an anion exchange membrane, into a separate compartment. Cells encapsulated within alginate beads (2.9 mm diameter) for protection against the current biotransformed benzonitrile to benzoic acid with a 26% reduction in the biotransformation rate, from 0.054 mmol/min/g dcw with free cells to 0.040 mmol/min/g dcw with immobilised cells. When the electric current was applied, the biotransformation rate increased to 0.047 mmol/min/g dcw and product recovery increased from 19% to 79%.

Enhanced biotransformations and product recovery in a membrane bioreactor through application of a direct electric current.

The simultaneous enhancement of biotransformation coupled to product recovery, purification and concentration is presented. The nitrilase of Rhodococcus rhodochrous LL100-21 catalyses the single-step hydrolytic biotransformation of benzonitrile to benzoic acid and ammonia. When a direct electric current is applied across a bioreactor containing the bacterium and benzonitrile, the charged product (benzoic acid) can be removed in situ across an anion exchange membrane and recovered in a separate compartment. Over the course of a 24-hour biotransformation, benzonitrile was converted to benzoic acid which was completely removed from the bioreactor chamber and concentrated 3-fold in a separate chamber. The rate of production of benzoic acid increased by 42% when the current was applied (0.044 mmol/min/g dry cell weight in the presence of current as compared to 0.03 mmol/min/g dry cell weight in its absence). The enhanced reaction rate was achieved irrespective of product separation and therefore appears to be a direct effect upon the bacterial cells. This process has potential for enhanced productivity from biotransformations through a simultaneous increase in metabolic activity and in situ product recovery.

Bioaugmentation strategies for remediating mixed chemical effluents.

Operationally exhausted metal working fluids are chemically mixed, produced in large quantities (400 000 tonnes year in the U.K.), and potentially environmentally toxic. It is essential to develop more reliable and economical approaches for their disposal. We investigated the effectiveness of a defined bacterial consortium, constructed specifically for treating metal-working fluid (MWF), and contrasted its performance to that of undefined inocula from activated sludge. Construction of the consortium was based on knowledge of the diversity of bacterial communities that naturally colonize MWF and determination of their catabolic abilities and tolerance to the chemical constituents. Chemical analysis of the inoculated MWF bioreactor revealed that, after 100 h at 28 degrees C, the defined inoculum reduced the pollution load by over 80% from an initial chemical oxygen demand of approximately 48 000 mg L(-)(1). The inocula performance was approximately 50% more effective than that of the undefined microbial community from the activated sludge. Furthermore, the performance of the constructed consortium was more reproducible than that of an undefined community, an essential feature for bioaugmentation treatment of industrial wastes.

Bacterial community structure and function in a metal-working fluid.

The diversity of bacterial populations colonizing spatially and temporally separated samples of the same metal-working fluid (MWF) formulation was investigated. Analyses were performed with a view to improve strategies for bioaugmentation of waste MWF in bioreactor systems and prevention of in-use MWF biodeterioration in engineering workshops. Significantly, complementary phenotypic, genotypic and in situ methods revealed that the bacterial communities in operationally exhausted MWFs had low diversity and were similar in species composition from different locations and uses. Of the 179 bacterial isolates studied, only 11 genera and 15 species were identified using fatty acid methyl ester (FAME) analysis, with culture independent analyses by 16S rDNA denaturing gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization being congruent with these FAME data. In order to gain some insight into functional role of detected populations, we correlated the MWF chemical composition and potential pollution load with bacterial abundance and community composition detected within samples.