Enzyme engineering in biomimetic compartments.

The success of a directed evolution approach to creating custom-made enzymes relies in no small part on screening as many clones as possible. The miniaturisation of assays into pico to femtoliter compartments (emulsion droplets, vesicles or gel-shell beads) makes directed evolution campaigns practically more straightforward than current large scale industrial screening that requires liquid handling equipment and much manpower. Several recent experimental formats have established protocols to screen more than 10 million compartments per day, representing unprecedented throughput at low cost. This review introduces the emerging approaches towards making biomimetic man-made compartments that are poised to be adapted by a wider circle of researchers. In addition to cost and time saving, control of selection pressures and conditions, the quantitative readout that reports on every library members and the ability to develop strategies based on these data will increase the degrees of freedom in designing and testing strategies for directed evolution experiments.Our research was funded by the Biological and Biotechnological Research Council (BBSRC) and the Engineering and Physical Sciences Research Council (EPSRC). FH is an ERC Starting Investigator, PYC and AZ were supported by the EU Marie-Curie training network PhosChemRec. AZ received further support from the BBSRC and the Cambridge European Trust.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.sbi.2015.06.00

One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution.

Directed evolution relies on iterative cycles of randomization and selection. The outcome of an artificial evolution experiment is crucially dependent on (i) the numbers of variants that can be screened and (ii) the quality of the assessment of each clone that forms the basis for selection. Compartmentalization of screening assays in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with good signal quality. Microfluidic systems have been developed to make and sort droplets, but the operator skill required precludes their ready implementation in nonspecialist settings. We now establish a protocol for the creation of monodisperse double-emulsion droplets in two steps in microfluidic devices with different surface characteristics (first hydrophobic, then hydrophilic). The resulting double-emulsion droplets are suitable for quantitative analysis and sorting in a commercial flow cytometer. The power of this approach is demonstrated in a series of enrichment experiments, culminating in the successful recovery of catalytically active clones from a sea of 1 000 000-fold as many low-activity variants. The modular workflow allows integration of additional steps: the encapsulated lysate assay reactions can be stopped by heat inactivation (enabling ready control of selection stringency), the droplet size can be contracted (to concentrate its contents), and storage (at -80 °C) is possible for discontinuous workflows. The control that can be thus exerted on screening conditions will facilitate exploitation of the potential of protein libraries compartmentalized in droplets in a straightforward protocol that can be readily implemented and used by protein engineers

Whole exome sequencing links dental tumor to an autosomal-dominant mutation in ANO5 gene associated with gnathodiaphyseal dysplasia and muscle dystrophies

Tumors of the jaws may represent different human disorders and frequently associate with pathologic bone fractures. In this report, we analyzed two affected siblings from a family of Russian origin, with a history of dental tumors of the jaws, in correspondence to original clinical diagnosis of cementoma consistent with gigantiform cementoma (GC, OMIM: 137575). Whole exome sequencing revealed the heterozygous missense mutation c.1067G \u3e A (p.Cys356Tyr) in ANO5 gene in these patients. To date, autosomal-dominant mutations have been described in the ANO5 gene for gnathodiaphyseal dysplasia (GDD, OMIM: 166260), and multiple recessive mutations have been described in the gene for muscle dystrophies (OMIM: 613319, 611307); the same amino acid (Cys) at the position 356 is mutated in GDD. These genetic data and similar clinical phenotypes demonstrate that the GC and GDD likely represent the same type of bone pathology. Our data illustrate the significance of mutations in single amino-acid position for particular bone tissue pathology. Modifying role of genetic variations in another gene on the severity of the monogenic trait pathology is also suggested. Finally, we propose the model explaining the tissue-specific manifestation of clinically distant bone and muscle diseases linked to mutations in one gene

The short-term effect of high versus moderate protein intake on recovery after strength training in resistance-trained individuals

Background: Dietary protein intakes up to 2.9 g.kg-1.d-1 and protein consumption before and after resistance training may enhance recovery, resulting in hypertrophy and strength gains. However, it remains unclear whether protein quantity or nutrient timing is central to positive adaptations. This study investigated the effect of total dietary protein content, whilst controlling for protein timing, on recovery in resistance trainees. Methods: Fourteen resistance-trained individuals underwent two 10-day isocaloric dietary regimes with a protein content of 1.8 g.kg-1.d-1 (PROMOD) or 2.9 g.kg-1.d-1 (PROHIGH) in a randomised, counterbalanced, crossover design. On days 8-10 (T1-T3), participants undertook resistance exercise under controlled conditions, performing 3 sets of squat, bench press and bent-over rows at 80% 1 repetition maximum until volitional exhaustion. Additionally, participants consumed a 0.4 g.kg-1 whey protein concentrate/isolate mix 30 minutes before and after exercise sessions to standardise protein timing specific to training. Recovery was assessed via daily repetition performance, muscle soreness, bioelectrical impedance phase angle, plasma creatine kinase (CK) and tumor necrosis factor-α (TNF-α). Results: No significant differences were reported between conditions for any of the performance repetition count variables (p>0.05). However, within PROMOD only, squat performance total repetition count was significantly lower at T3 (19.7 ± 6.8) compared to T1 (23.0 ± 7.5; p=0.006). Pre and post-exercise CK concentrations significantly increased across test days (p≤0.003), although no differences were reported between conditions. No differences for TNF-α or muscle soreness were reported between dietary conditions. Phase angle was significantly greater at T3 for PROHIGH (8.26 ± 0.82°) compared with PROMOD (8.08 ± 0.80°; p=0.012). Conclusions: When energy intake and peri-exercise protein intake was controlled for, a short term PROHIGH diet did not improve markers of muscle damage or soreness in comparison to a PROMOD approach following repeated days of intensive training. Whilst it is therefore likely that protein intakes (1.8g.kg-1.d-1) may be sufficient for resistance-trained individuals, it is noteworthy that both lower body exercise performance and bioelectrical phase angle were maintained with PROHIGH. Longer term interventions are warranted to determine whether PROMOD intakes are sufficient during prolonged training periods or when extensive exercise (e.g. training twice daily) is undertaken

The impact of consumer cooperatives on the living standards of the population in Russia

The socio-economic development of Russia in recent years has significantly slowed down, which is caused by both external factors (a difficult geopolitical situation, sanctions wars, the economic confrontation between leading world economic powers) and internal factors (poverty, low efficiency of the economy, a weak innovation environment). In these conditions, it is necessary to find various ways to stimulate the economic and social development of the country and regions. This paper discusses the development of cooperation as a way to develop economic relations and improve living standards. The aim of the study is to reveal the relationship between consumer co-operation and the standard of living of the population, and to determine its impact on the economy. The main research methods were analysis and synthesis, generalization, and the case method. This study used data from the Federal State Statistics Service of the Russian Federation, analytical reviews on the development of cooperation in Russia, and data from the Central Union of Russia. As a result of the study, the influence of cooperation on the growth of living standards of the population has been identified, some recommendations have been made on the development of cooperation in Russia

Tuberculosis in the Russian Federation: Dynamics of the Epidemic Indicators before and after COVID-19 Pandemic

The measures taken against tuberculosis (TB) in recent years in the Russian Federation have been highly effective. Unfortunately, the COVID-19 pandemic may seriously undermine the progress that has been made in the fight against TB. The aim of this study was to assess changes in the epidemiological rates of tuberculosis in the Russian Federation before and after the COVID-19 pandemic. Materials and methods. The analysis was conducted by considering the main epidemiological indicators of tuberculosis, according to the federal statistics for the period from 2017 to 2021. The parameters were estimated according to the data received from 11 areas in the North-Western region. Statistical analysis was carried out using the free software computing environment R (v.3.5.1) and the commercial software package Statistical Package for the Social Sciences (SPSS Statistics for Windows, version 24.0, IBM Corp., 2016). Research results. We found a positive correlation between the incidence among the overall population and the incidence among children aged 0–17, inclusively (r = 0.55 in 2017, r = 0.60 in 2020, and r = 0.53 in 2021). Along with the received regularities, a different trend is shown in the data analysis of general incidence and health X-ray examination for tuberculosis among the general population. The correlation has decreased threefold from 2017 (r = 0.72) to 2020 (r = 0.32); this negative trend might be the result of factors such as the quality of X-ray screening examinations among the general population, and the reduced assessment objectivity of the tuberculosis incidence rate. Conclusions. In assessing the correlation between general incidence and incidence in children under 17 years of age, as well as between incidence and mortality in the Russian Federation, a positive correlation was found with an increasing trend. Such a discrepancy might be due to decreases in the occupational health examination coverage among the general population. Therefore, in the years ahead, we can expect epidemiological indicators to increase incidence and mortality, including child mortality, associated with the insufficient detection of tuberculosis among the population during the COVID-19 pandemic

Satiating Effect of High Protein Diets on Resistance-Trained Subjects in Energy Deficit.

Short-term energy deficit strategies are practiced by weight class and physique athletes, often involving high protein intakes to maximize satiety and maintain lean mass despite a paucity of research. This study compared the satiating effect of two protein diets on resistance-trained individuals during short-term energy deficit. Following ethical approval, 16 participants (age: 28 ± 2 years; height: 1.72 ± 0.03 m; body-mass: 88.83 ± 5.54 kg; body-fat: 21.85 ± 1.82%) were randomly assigned to 7-days moderate (PROMOD: 1.8 g·kg-1·d-1) or high protein (PROHIGH: 2.9 g·kg-1·d-1) matched calorie-deficit diets in a cross-over design. Daily satiety responses were recorded throughout interventions. Pre-post diet, plasma ghrelin and peptide tyrosine tyrosine (PYY), and satiety ratings were assessed in response to a protein-rich meal. Only perceived satisfaction was significantly greater following PROHIGH (67.29 ± 4.28 v 58.96 ± 4.51 mm, p = 0.04). Perceived cravings increased following PROMOD only (46.25 ± 4.96 to 57.60 ± 4.41 mm, p = 0.01). Absolute ghrelin concentration significantly reduced post-meal following PROMOD (972.8 ± 130.4 to 613.6 ± 114.3 pg·mL-1; p = 0.003), remaining lower than PROHIGH at 2 h (-0.40 ± 0.06 v -0.26 ± 0.06 pg·mL-1 normalized relative change; p = 0.015). Absolute PYY concentration increased to a similar extent post-meal (PROMOD: 84.9 ± 8.9 to 147.1 ± 11.9 pg·mL-1, PROHIGH: 100.6 ± 9.5 to 143.3 ± 12.0 pg·mL-1; p 0.05). However, desire to eat remained significantly blunted for PROMOD (p = 0.048). PROHIGH does not confer additional satiating benefits in resistance-trained individuals during short-term energy deficit. Ghrelin and PYY responses to a test-meal support the contention that satiety was maintained following PROMOD, although athletes experiencing negative symptoms (i.e., cravings) may benefit from protein-rich meals as opposed to over-consumption of protein

GPR55 Receptor Activation by the N-Acyl Dopamine Family Lipids Induces Apoptosis in Cancer Cells via the Nitric Oxide Synthase (nNOS) Over-Stimulation

GPR55 is a GPCR of the non-CB1/CB2 cannabinoid receptor family, which is activated by lysophosphatidylinositol (LPI) and stimulates the proliferation of cancer cells. Anandamide, a bioactive lipid endocannabinoid, acts as a biased agonist of GPR55 and induces cancer cell death, but is unstable and psychoactive. We hypothesized that other endocannabinoids and structurally similar compounds, which are more hydrolytically stable, could also induce cancer cell death via GPR55 activation. We chemically synthesized and tested a set of fatty acid amides and esters for cell death induction via GPR55 activation. The most active compounds appeared to be N-acyl dopamines, especially N-docosahexaenoyl dopamine (DHA-DA). Using a panel of cancer cell lines and a set of receptor and intracellular signal transduction machinery inhibitors together with cell viability, Ca2+, NO, ROS (reactive oxygen species) and gene expression measurement, we showed for the first time that for these compounds, the mechanism of cell death induction differed from that published for anandamide and included neuronal nitric oxide synthase (nNOS) overstimulation with concomitant oxidative stress induction. The combination of DHA-DA with LPI, which normally stimulates cancer proliferation and is increased in cancer setting, had an increased cytotoxicity for the cancer cells indicating a therapeutic potential

GPR55 Receptor Activation by the <i>N</i>-Acyl Dopamine Family Lipids Induces Apoptosis in Cancer Cells via the Nitric Oxide Synthase (nNOS) Over-Stimulation

GPR55 is a GPCR of the non-CB1/CB2 cannabinoid receptor family, which is activated by lysophosphatidylinositol (LPI) and stimulates the proliferation of cancer cells. Anandamide, a bioactive lipid endocannabinoid, acts as a biased agonist of GPR55 and induces cancer cell death, but is unstable and psychoactive. We hypothesized that other endocannabinoids and structurally similar compounds, which are more hydrolytically stable, could also induce cancer cell death via GPR55 activation. We chemically synthesized and tested a set of fatty acid amides and esters for cell death induction via GPR55 activation. The most active compounds appeared to be N-acyl dopamines, especially N-docosahexaenoyl dopamine (DHA-DA). Using a panel of cancer cell lines and a set of receptor and intracellular signal transduction machinery inhibitors together with cell viability, Ca2+, NO, ROS (reactive oxygen species) and gene expression measurement, we showed for the first time that for these compounds, the mechanism of cell death induction differed from that published for anandamide and included neuronal nitric oxide synthase (nNOS) overstimulation with concomitant oxidative stress induction. The combination of DHA-DA with LPI, which normally stimulates cancer proliferation and is increased in cancer setting, had an increased cytotoxicity for the cancer cells indicating a therapeutic potential