Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one-pot/two-step biocatalytic whole-cell system

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer–Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L ⁻¹·h ⁻¹) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L ⁻¹·h ⁻¹).     

Uptake of infant and preschool immunisations in Scotland and England during the COVID-19 pandemic: An observational study of routinely collected data

BackgroundIn 2020, the SARS-CoV-2 (COVID-19) pandemic and lockdown control measures threatened to disrupt routine childhood immunisation programmes with early reports suggesting uptake would fall. In response, public health bodies in Scotland and England collected national data for childhood immunisations on a weekly or monthly basis to allow for rapid analysis of trends. The aim of this study was to use these data to assess the impact of different phases of the pandemic on infant and preschool immunisation uptake rates.Methods and findingsWe conducted an observational study using routinely collected data for the year prior to the pandemic (2019) and immediately before (22 January to March 2020), during (23 March to 26 July), and after (27 July to 4 October) the first UK “lockdown”. Data were obtained for Scotland from the Public Health Scotland “COVID19 wider impacts on the health care system” dashboard and for England from ImmForm.Five vaccinations delivered at different ages were evaluated; 3 doses of “6-in-1” diphtheria, tetanus, pertussis, polio, Haemophilus influenzae type b, and hepatitis B vaccine (DTaP/IPV/Hib/HepB) and 2 doses of measles, mumps, and rubella (MMR) vaccine. This represented 439,754 invitations to be vaccinated in Scotland and 4.1 million for England. Uptake during the 2020 periods was compared to the previous year (2019) using binary logistic regression analysis. For Scotland, uptake within 4 weeks of a child becoming eligible by age was analysed along with geographical region and indices of deprivation. For Scotland and England, we assessed whether immunisations were up-to-date at approximately 6 months (all doses 6-in-1) and 16 to 18 months (first MMR) of age.We found that uptake within 4 weeks of eligibility in Scotland for all the 5 vaccines was higher during lockdown than in 2019. Differences ranged from 1.3% for first dose 6-in-1 vaccine (95.3 versus 94%, odds ratio [OR] compared to 2019 1.28, 95% confidence intervals [CIs] 1.18 to 1.39) to 14.3% for second MMR dose (66.1 versus 51.8%, OR compared to 2019 1.8, 95% CI 1.74 to 1.87). Significant increases in uptake were seen across all deprivation levels.In England, fewer children due to receive their immunisations during the lockdown period were up to date at 6 months (6-in-1) or 18 months (first dose MMR). The fall in percentage uptake ranged from 0.5% for first 6-in-1 (95.8 versus 96.3%, OR compared to 2019 0.89, 95% CI 0.86– to 0.91) to 2.1% for third 6-in-1 (86.6 versus 88.7%, OR compared to 2019 0.82, 95% CI 0.81 to 0.83).The use of routinely collected data used in this study was a limiting factor as detailed information on potential confounding factors were not available and we were unable to eliminate the possibility of seasonal trends in immunisation uptake.ConclusionsIn this study, we observed that the national lockdown in Scotland was associated with an increase in timely childhood immunisation uptake; however, in England, uptake fell slightly. Reasons for the improved uptake in Scotland may include active measures taken to promote immunisation at local and national levels during this period and should be explored further. Promoting immunisation uptake and addressing potential vaccine hesitancy is particularly important given the ongoing pandemic and COVID-19 vaccination campaigns.

Fiona McQuaid and colleagues assess the uptake of infant and pre-school immunisations in Scotland and England during the COVID-19 pandemic.     

Perceived and Physiological Indicators of Relaxation: As Different as Mozart and Alice in Chains

The effects of listening to different types of music on perceived and physiological indicators of relaxation were evaluated. Fifty-six undergraduate students, 24 males and 32 females, mean age of 21, were randomly assigned to listen to classical, hard rock, self-selected relaxing music, or no music. Participants' relaxation level, skin temperature, muscle tension and heart rate were evaluated before and after exposure to a music condition. Analyses of variance using baseline measures as covariates indicated that skin temperature decreased for all conditions (p = 0.001) and the classical, self-selected relaxing music and no music groups reported significant increases in feelings of relaxation (p = 0.004). These results partially support the hypothesis that classical and self-selected relaxing music can increase perceptions of relaxation to a greater degree than listening to hard rock music. However, no differences were found between different types of music on physiological indicators of arousal. Implications for using music to reduce stress were discussed.     

Ecological study of social fragmentation,poverty, and suicide

ObjectivesTo investigate the association between suicide and area based measures of deprivation and social fragmentation.DesignEcological study.Setting633 parliamentary constituencies of Great Britain as defined in 1991.ResultsMortality from suicide and all other causes increased with increasing Townsend deprivation score, social fragmentation score, and abstention from voting in all age and sex groups. Suicide mortality was most strongly related to social fragmentation, whereas deaths from other causes were more closely associated with Townsend score. Constituencies with absolute increases in social fragmentation and Townsend scores between 1981 and 1991 tended to have greater increases in suicide rates over the same period. The relation between change in social fragmentation and suicide was largely independent of Townsend score, whereas the association with Townsend score was generally reduced after adjustment for social fragmentation.ConclusionsSuicide rates are more strongly associated with measures of social fragmentation than with poverty at a constituency level.

Key messages

• Place of residence may affect health, and mortality from most common diseases tends to be higher in areas characterised by low socioeconomic position
• Research dating back over 100 years suggests that social fragmentation may influencesuicide
• In the 1980s and 1990s, parliamentary constituencies with high levels of social fragmentation had high rates of suicide, independent of deprivation
• Constituencies with the greatest increases in social fragmentation between 1981 and 1991 also had the greatest increases in suicide rates over the same period
• Any targeting of suicide prevention may be more effective if aimed at socially fragmented rather than deprived areas

     

Oxidative stress inactivates cobalamin-independent methionine synthase (MetE) in Escherichia coli

In nature, Escherichia coli are exposed to harsh and non-ideal growth environments—nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper (Leichert and Jakob 2004), which provide evidence that MetE is one of the proteins in E. coli most susceptible to oxidation. In eukaryotes, glutathionylation of key proteins involved in protein synthesis leads to inhibition of translation. Our studies suggest a simpler mechanism is employed by E. coli to achieve the same effect.     

Trabecular bone modulus-density relationships depend on anatomic site

One outstanding issue regarding the relationship between elastic modulus and density for trabecular bone is whether the relationship depends on anatomic site. To address this, on-axis elastic moduli and apparent densities were measured for 142 specimens of human trabecular bone from the vertebra (n=61), proximal tibia (n=31), femoral greater trochanter (n=23), and femoral neck (n=27). Specimens were obtained from 61 cadavers (mean+/-SD age=67+/-15 years). Experimental protocols were used that minimized end-artifact errors and controlled for specimen orientation. Tissue moduli were computed for a subset of 18 specimens using high-resolution linear finite element analyses and also using two previously developed theoretical relationships (Bone 25 (1999) 481; J. Elasticity 53 (1999) 125). Resultant power law regressions between modulus and density did depend on anatomic site, as determined via an analysis of covariance. The inter-site differences were among the leading coefficients (p<0.02), but not the exponents (p>0.08), which ranged 1.49-2.18. At a given density, specimens from the tibia had higher moduli than those from the vertebra (p=0.01) and femoral neck (p=0.002); those from the trochanter had higher moduli than the vertebra (p=0.02). These differences could be as large as almost 50%, and errors in predicted values of modulus increased by up to 65% when site-dependence was ignored. These results indicate that there is no universal modulus-density relationship for on-axis loading. Tissue moduli computed using methods that account for inter-site architectural variations did not differ across site (p>0.15), suggesting that the site-specificity in apparent modulus-density relationships may be attributed to differences in architecture.     

Rapid upregulation of endothelial P-selectin expression via reactive oxygen species generation

Endothelial cell ICAM-1 upregulation in response to TNF-alpha is mediated in part by reactive oxygen species (ROS) generated by the endothelial membrane-associated NADPH oxidase and occurs maximally after 4 h as the synthesis of new protein is required. However, thrombin-stimulated P-selectin upregulation is bimodal, the first peak occurring within minutes. We hypothesize that this early peak, which results from the release of preformed P-selectin from within Weibel-Palade bodies, is mediated in part by ROS generated from the endothelial membrane-associated xanthine oxidase. We found that this rapid expression of P-selectin on the surface of endothelial cells was accompanied by qualitatively parallel increases in ROS generation. Both P-selectin expression and ROS generation were inhibited, dose dependently, by the exogenous administration of disparate cell-permeable antioxidants and also by the inhibition of either of the known membrane-associated ROS-generating enzymes NADPH oxidase or xanthine oxidase. This rapid, posttranslational cell signaling response, mediated by ROS generated not only by the classical NADPH oxidase but also by xanthine oxidase, may well represent an important physiological trigger of the microvascular inflammatory response.     

Pain as a counterpoint to culture: toward an analysis of pain associated with infibulation among Somali immigrants in Norway

This article focuses on how some Somali women experience and reflect on the pain of infibulation as a lived bodily experience within shifting social and cultural frameworks. Women interviewed for this study describe such pain as intolerable, as an experience that has made them question the cultural values in which the operation is embedded. Whereas this view has gone largely unvoiced in their natal communities, the Norwegian exile situation in which the present study's informants live has brought about dramatic changes. In Norway, where female circumcision is both condemned and illegal, most of the women have come to reconsider the practice--not merely as a theoretical topic or as a "cultural tradition" to be maintained or abolished but, rather, as part of their embodied and lived experience.