Bacterial sensors define intracellular free energies for correct enzyme metalation.

There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: the less competitive the metal, the less favorable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt chelatase for vitamin B12.

Biomarkers of neonatal skin barrier adaptation reveal substantial differences compared to adult skin.

BACKGROUND: The objective of this study was to measure skin characteristics in premature (PT), late preterm (LPT), and full-term (FT) neonates compared with adults at two times (T1, T2). METHODS: Skin samples of 61 neonates and 34 adults were analyzed for protein biomarkers, natural moisturizing factor (NMF), and biophysical parameters. Infant groups were: <34 weeks (PT), 34-<37 weeks (LPT), and ≥37 weeks (FT). RESULTS: Forty proteins were differentially expressed in FT infant skin, 38 in LPT infant skin, and 12 in PT infant skin compared with adult skin at T1. At T2, 40 proteins were differentially expressed in FT infants, 38 in LPT infants, and 54 in PT infants compared with adults. All proteins were increased at both times, except TMG3, S100A7, and PEBP1, and decreased in PTs at T1. The proteins are involved in filaggrin processing, protease inhibition/enzyme regulation, and antimicrobial function. Eight proteins were decreased in PT skin compared with FT skin at T1. LPT and FT proteins were generally comparable at both times. Total NMF was lower in infants than adults at T1, but higher in infants at T2. CONCLUSIONS: Neonates respond to the physiological transitions at birth by upregulating processes that drive the production of lower pH of the skin and water-binding NMF components, prevent protease activity leading to desquamation, and increase the barrier antimicrobial properties. IMPACT: Neonates respond to the transitions at birth by upregulating processes that drive the production of lower pH of the skin and NMF, prevent protease activity leading to desquamation, and increase the antimicrobial properties of the barrier. The neonatal epidermal barrier exhibits a markedly different array of protein biomarkers both shortly after birth and 2-3 months later, which are differentially expressed versus adults. The major biomarker-functional classes included filaggrin processing, protease inhibitor/enzyme regulators, antimicrobials, keratins, lipids, and cathepsins. The findings will guide improvement of infant skin care practices, particularly for the most premature infants with the ultimate goals mitigating nosocomial infection.

The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant.

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmR(E64H) gains responsiveness to Zn(II) and cobalt in vivo Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro Sensitivity to formaldehyde requires a cysteine (Cys(35) in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmR(E64H) reveals that an FrmR-specific amino-terminal Pro(2) is proximal to Cys(35), and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmR(P2S) and RcnR(S2P), respectively, impair and enhance formaldehyde reactivity in vitro Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.

Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells.

FrmR from Salmonella enterica serovar typhimurium (a CsoR/RcnR-like transcriptional de-repressor) is shown to repress the frmRA operator-promoter, and repression is alleviated by formaldehyde but not manganese, iron, cobalt, nickel, copper, or Zn(II) within cells. In contrast, repression by a mutant FrmRE64H (which gains an RcnR metal ligand) is alleviated by cobalt and Zn(II). Unexpectedly, FrmR was found to already bind Co(II), Zn(II), and Cu(I), and moreover metals, as well as formaldehyde, trigger an allosteric response that weakens DNA affinity. However, the sensory metal sites of the cells' endogenous metal sensors (RcnR, ZntR, Zur, and CueR) are all tighter than FrmR for their cognate metals. Furthermore, the endogenous metal sensors are shown to out-compete FrmR. The metal-sensing FrmRE64H mutant has tighter metal affinities than FrmR by approximately 1 order of magnitude. Gain of cobalt sensing by FrmRE64H remains enigmatic because the cobalt affinity of FrmRE64H is substantially weaker than that of the endogenous cobalt sensor. Cobalt sensing requires glutathione, which may assist cobalt access, conferring a kinetic advantage. For Zn(II), the metal affinity of FrmRE64H approaches the metal affinities of cognate Zn(II) sensors. Counter-intuitively, the allosteric coupling free energy for Zn(II) is smaller in metal-sensing FrmRE64H compared with nonsensing FrmR. By determining the copies of FrmR and FrmRE64H tetramers per cell, then estimating promoter occupancy as a function of intracellular Zn(II) concentration, we show how a modest tightening of Zn(II) affinity, plus weakened DNA affinity of the apoprotein, conspires to make the relative properties of FrmRE64H (compared with ZntR and Zur) sufficient to sense Zn(II) inside cells.

Perceived feedback and school belonging: the mediating role of subjective well-being.

Introduction: This study examined the interplay between perceived feedback (PF), subjective wellbeing (SWB), and students' sense of belonging to school (SBS). School belonging is a key factor for decisions regarding academic studies, and is usually impacted by PF. The current study explored whether SWB mediates the established relationship between PF and SBS. Method: This was achieved by applying a mediation model to PISA 2018 data from 12,058 students in four areas of China. Results: Perceived feedback positively affected students' SBS (ß = 0.26, p < 0.01); and that SWB partially mediated this relationship between PF and SBS (ß = 0.47, p < 0.01). Discussion: The findings of this study have the potential to contribute to the existing literature on educational psychology and well-being. By shedding light on the mediating role of SWB, the research offers practical implications for educators and policymakers seeking to enhance students' school belonging and other implications for their academic success. Further research can contribute to this promising area, by focusing on subjective wellbeing and its impact on a range of decisions being made by students during a critical phase of their personal and academic development.

Assessing the Dynamic Outcomes of Containment Strategies against COVID-19 under Different Public Health Governance Structures: A Comparison between Pakistan and Bangladesh.

COVID-19 scenarios were run using an epidemiological mathematical model (system dynamics model) and counterfactual analysis to simulate the impacts of different control and containment measures on cumulative infections and deaths in Bangladesh and Pakistan. The simulations were based on national-level data concerning vaccination level, hospital capacity, and other factors, from the World Health Organization, the World Bank, and the Our World in Data web portal. These data were added to cumulative infections and death data from government agencies covering the period from 18 March 2020 to 28 February 2022. Baseline curves for Pakistan and Bangladesh were obtained using piecewise fitting with a consideration of different events against the reported data and allowing for less than 5% random errors in cumulative infections and deaths. The results indicate that Bangladesh could have achieved more reductions in each key outcome measure by shifting its initial lockdown at least five days backward, while Pakistan would have needed to extend its lockdown to achieve comparable improvements. Bangladesh's second lockdown appears to have been better timed than Pakistan's. There were potential benefits from starting the third lockdown two weeks earlier for Bangladesh and from combining this with the fourth lockdown or canceling the fourth lockdown altogether. Adding a two-week lockdown at the beginning of the upward slope of the second wave could have led to a more than 40 percent reduction in cumulative infections and a 35 percent reduction in cumulative deaths for both countries. However, Bangladesh's reductions were more sensitive to the duration of the lockdown. Pakistan's response was more constrained by medical resources, while Bangladesh's outcomes were more sensitive to both vaccination timing and capacities. More benefits were lost when combining multiple scenarios for Bangladesh compared to the same combinations in Pakistan. Clearly, cumulative infections and deaths could have been highly impacted by adjusting the control and containment measures in both national settings. However, COVID-19 outcomes were more sensitive to adjustment interventions for the Bangladesh context. Disaggregated analyses, using a wider range of factors, may reveal several sub-national dynamics. Nonetheless, the current research demonstrates the relevance of lockdown timing adjustments and discrete adjustments to several other control and containment measures.

Introduction to the Special Issue on Cascading Disaster Modelling and Prevention.

The 2019 Global Assessment Report (GAR2019) on Disaster Risk Reduction [...].

Infrastructural Aspects of Rain-Related Cascading Disasters: A Systematic Literature Review.

Cascading disasters progress from one hazard event to a range of interconnected events and impacts, with often devastating consequences. Rain-related cascading disasters are a particularly frequent form of cascading disasters in many parts of the world, and they are likely to become even more frequent due to climate change and accelerating coastal development, among other issues. (1) Background: The current literature review extended previous reviews of documented progressions from one natural hazard event to another, by focusing on linkages between rain-related natural hazard triggers and infrastructural impacts. (2) Methods: A wide range of case studies were reviewed using a systematic literature review protocol. The review quality was enhanced by only including case studies that detailed mechanisms that have led to infrastructural impacts, and which had been published in high-quality academic journals. (3) Results: A sum of 71 articles, concerning 99 case studies of rain-related disasters, were fully reviewed. Twenty-five distinct mechanisms were identified, as the foundation for a matrix running between five different natural hazards and eight types of infrastructural impacts. (4) Conclusion: Relatively complex quantitative methods are needed to generate locality-specific, cascading disaster likelihoods and scenarios. Appropriate methods can leverage the current matrix to structure both Delphi-based approaches and network analysis using longitudinal data.

The Inverse Response Law: Theory and Relevance to the Aftermath of Disasters.

The Inverse Care Law is principally concerned with the effect of market forces on health care which create inequities in access to health services through privileging individuals who possess the forms of social capital that are valued within health care settings. The fields of disaster risk reduction need to consider the ways in which inequities, driven by economic and social policy as well as institutional decision-making, create vulnerabilities prior to a disaster, which are then magnified post disaster through entrenched structural differences in access to resources. Drawing on key principles within the Inverse Care Law, the Inverse Response Law refers to the idea that people in lower socio-economic groups are more likely to be impacted and to experience disparities in service provision during the disaster response and recovery phase. In a market model of recovery, vulnerable groups struggle to compete for necessary services creating inequities in adaptive capacity as well as in social and wellbeing outcomes over time. Both the Inverse Care Law and the Inverse Response Law focus on the structural organisation of services at a macro level. In this article, the Inverse Care Law is outlined, its application to medical treatment following disasters considered and an explanation of the Inverse Response Law provided. Case studies from recent disasters, in London, New Zealand, Puerto Rico and Mexico City are examined in order to illustrate themes at work relating to the Inverse Response Law.

Fine control of metal concentrations is necessary for cells to discern zinc from cobalt.

Bacteria possess transcription factors whose DNA-binding activity is altered upon binding to specific metals, but metal binding is not specific in vitro. Here we show that tight regulation of buffered intracellular metal concentrations is a prerequisite for metal specificity of Zur, ZntR, RcnR and FrmR in Salmonella Typhimurium. In cells, at non-inhibitory elevated concentrations, Zur and ZntR, only respond to Zn(II), RcnR to cobalt and FrmR to formaldehyde. However, in vitro all these sensors bind non-cognate metals, which alters DNA binding. We model the responses of these sensors to intracellular-buffered concentrations of Co(II) and Zn(II) based upon determined abundances, metal affinities and DNA affinities of each apo- and metalated sensor. The cognate sensors are modelled to respond at the lowest concentrations of their cognate metal, explaining specificity. However, other sensors are modelled to respond at concentrations only slightly higher, and cobalt or Zn(II) shock triggers mal-responses that match these predictions. Thus, perfect metal specificity is fine-tuned to a narrow range of buffered intracellular metal concentrations.

Bridging the gap: providing health care to newly released men.

Men who are incarcerated have higher morbidity rates than the general U.S. population and upon release most rely on the public sector to get their medical needs met. However, little is known about the health care needs, service utilization patterns, and costs of providing primary care to released inmates in an ambulatory setting. Using data from a primary care health center for uninsured men, health profiles and service use over a 12-month period is described for men newly released from prison (n=221). Health care needs and utilization were measured by medical diagnosis and primary care consultation rates. The results show that the newly released men studied had different service use patterns from that of the general population. The average cost of providing care was slightly higher than the center's average cost per individual for a man newly released from prison. The findings demonstrate the need for future research to support program planning of organizations seeking to meet the needs of people newly released from prison.

Unique Responses are Observed in Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and TRPV1) Co-Expressing Cells.

Transient receptor potential (TRP) ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are implicated in modulation of cough and nociception. In vivo, TRPA1 and TRPV1 are often co-expressed in neurons and TRPA1V1 hetero-tetramer formation is noted in cells co-transfected with the respective expression plasmids. In order to understand the impact of TRP receptor interaction on activity, we created stable cell lines expressing the TRPA1, TRPV1 and co-expressing the TRPA1 and TRPV1 (TRPA1V1) receptors. Among the 600 compounds screened against these receptors, we observed a number of compounds that activated the TRPA1, TRPV1 and TRPA1V1 receptors; compounds that activated TRPA1 and TRPA1V1; compounds that activated TRPV1 and TRPA1V1; compounds in which TRPA1V1 response was modulated by either TRPA1 or TRPV1; and compounds that activated only TRPV1 or TRPA1 or TRPA1V1; and one compound that activated TRPA1 and TRPV1, but not TRPA1V1. These results suggest that co-expression of TRPA1 and TRPV1 receptors imparts unique activation profiles different from that of cells expressing only TRPA1 or TRPV1.

Trace analysis of bromate in potato snacks using high-performance liquid chromatography-tandem mass spectrometry.

A simple, sensitive, and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method in the negative-ion electrospray ionization (ESI(-)) mode was validated for the quantitation of bromate (BrO(3)(-)) in potato snacks. Ground snack specimens ( approximately 0.5 g/sample) are spiked with Br(18)O(3)(-), stable-isotope labeled bromate internal standard (IS), and vortexed with a mixture of distilled/deionized water (dd water) and heptane. Subsequently, the specimens are centrifuged, and a small portion of the aqueous extract is isolated, diluted with dd water (1:4), and analyzed by HPLC-MS/MS. The methodology has a quantitation range of 10-1000 ppb, an accuracy of 1.5-7.5%, and a precision of 5.2-13.4% across the concentration range.

10th Annual University of Wisconsin Land O'Lakes Bioanalytical Conference.

This conference was arranged by the Extension Services in Pharmacy at the University of Wisconsin School of Pharmacy. The purpose of this annual 4-day conference is to provide an educational forum to discuss issues and applications associated with the analysis of xenobiotics and metabolites in biological matrices. The conference is designed to include and encourage an open exchange of scientific and methodological applications for bioanalysis. To increase the interactive nature of the conference, the program will be a mixture of lectures, poster sessions, round table discussions and workshops. This paper summarizes the presentations at the Tenth Annual Conference.