Mechanical release of homogenous proteins from supramolecular gels.

A long-standing challenge is how to formulate proteins and vaccines to retain function during storage and transport and to remove the burdens of cold-chain management. Any solution must be practical to use, with the protein being released or applied using clinically relevant triggers. Advanced biologic therapies are distributed cold, using substantial energy, limiting equitable distribution in low-resource countries and placing responsibility on the user for correct storage and handling. Cold-chain management is the best solution at present for protein transport but requires substantial infrastructure and energy. For example, in research laboratories, a single freezer at -80 °C consumes as much energy per day as a small household1. Of biological (protein or cell) therapies and all vaccines, 75% require cold-chain management; the cost of cold-chain management in clinical trials has increased by about 20% since 2015, reflecting this complexity. Bespoke formulations and excipients are now required, with trehalose2, sucrose or polymers3 widely used, which stabilize proteins by replacing surface water molecules and thereby make denaturation thermodynamically less likely; this has enabled both freeze-dried proteins and frozen proteins. For example, the human papilloma virus vaccine requires aluminium salt adjuvants to function, but these render it unstable against freeze-thaw4, leading to a very complex and expensive supply chain. Other ideas involve ensilication5 and chemical modification of proteins6. In short, protein stabilization is a challenge with no universal solution7,8. Here we designed a stiff hydrogel that stabilizes proteins against thermal denaturation even at 50 °C, and that can, unlike present technologies, deliver pure, excipient-free protein by mechanically releasing it from a syringe. Macromolecules can be loaded at up to 10 wt% without affecting the mechanism of release. This unique stabilization and excipient-free release synergy offers a practical, scalable and versatile solution to enable the low-cost, cold-chain-free and equitable delivery of therapies worldwide.

Multidimensional screening of pancreatic cancer spheroids reveals vulnerabilities in mitotic and cell-matrix adhesion signaling that associate with metastatic progression and decreased patient survival.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, with a median survival of less than 12 months and a 5-year survival of less than 10 %. Here, we have established an image-based screening pipeline for quantifying single PDAC spheroid dynamics in genetically and phenotypically diverse PDAC cell models. Wild-type KRas PDAC cells formed tight/compact spheroids - compaction of these structures was completely blocked by cytoplasmic dynein and focal adhesion kinase (FAK) inhibitors. In contrast, PDAC cells containing mutant KRas formed loosely aggregated spheroids that grew significantly slower following inhibition of polo-like kinase 1 (PLK1) or focal adhesion kinase (FAK). Independent of genetic background, multicellular PDAC-mesenchymal stromal cell (MSC) spheroids self-organized into structures with an MSC-dominant core. The inclusion of MSCs into wild-type KRas PDAC spheroids modestly affected their compaction; however, MSCs significantly increased the compaction and growth of mutant KRas PDAC spheroids. Notably, exogenous collagen 1 potentiated PANC1 spheroid compaction while ITGA1 knockdown in PANC1 cells blocked MSC-induced PANC1 spheroid compaction. In agreement with a role for collagen-based integrin adhesion complexes in stromal cell-induced PDAC phenotypes, we also discovered that MSC-induced PANC1 spheroid growth was completely blocked by the ITGB1 immunoneutralizing antibody mAb13. Finally, multiplexed single-cell immunohistochemical analysis of a 25 patient PDAC tissue microarray revealed a relationship between decreased variance in Spearman r correlation for ITGA1 and PLK1 expression within the tumor cell compartment of PDAC in patients with advanced disease stage, and elevated expression of both ITGA1 and PLK1 in PDAC was found to be associated with decreased patient survival. Taken together, this work uncovers new therapeutic vulnerabilities in PDAC that are relevant to the progression of this stromal cell-rich malignancy and which may reveal strategies for improving patient outcomes.

Understanding the intergenerational impact of migration: An adult mortality advantage for the children of forced migrants?

BACKGROUND: Children of immigrants often have excess mortality rates, in contrast to the low mortality typically exhibited by their parents' generation. However, prior research has studied children of immigrants who were selected into migration, thereby rendering it difficult to isolate the intergenerational impact of migration on adult mortality. METHODS: We use semi-parametric survival analysis to carry out a total population cohort study estimating all-cause and cause-specific mortality among all adult men and women from age 17 among all men and women born in 1953-1972 and resident in Finland in 1970-2020. We compare children of forced migrants from ceded Karelia-an area of Finland that was ceded to Russia during the Second World War-with the children of parents born in present-day Finland. RESULTS: Children with two parents who were forced migrants have higher mortality than children with two parents born in Northern, Southern and Western Finland, but similar or lower mortality than the subpopulation of children whose parents were born in the more comparable areas of Eastern Finland. For women and men, a mortality advantage is largest for external causes and persists after controlling for socio-economic factors. CONCLUSIONS: Our findings suggest that forced migration can have a beneficial impact on the mortality of later generations, at least in the case where forced migrants are able to move to contextually similar locations that offer opportunities for rapid integration and social mobility. The findings also highlight the importance of making appropriate comparisons when evaluating the impact of forced migration.

COVID-19 mortality among immigrants by duration of residence in Sweden: a population-based cohort study.

BACKGROUND: Explanations for the disproportional COVID-19 burden among immigrants relative to host-country natives include differential exposure to the virus and susceptibility due to poor health conditions. Prior to the pandemic, immigrants displayed deteriorating health with duration of residence that may be associated with increased susceptibility over time. The aim of this study was to compare immigrant-native COVID-19 mortality by immigrants' duration of residence to examine the role of differential susceptibility. METHODS: A population-based cohort study was conducted with individuals between 18 and 100 years old registered in Sweden between 1 January 2015 and 15 June 2022. Cox regression models were run to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: Inequalities in COVID-19 mortality between immigrants and the Swedish-born population in the working-age group were concentrated among those of non-Western origins and from Finland with more than 15 years in Sweden, while for those of retirement age, these groups showed higher COVID-19 mortality HRs regardless of duration of residence. Both age groups of immigrants from Africa and the Middle East showed consistently higher COVID-19 mortality HRs. For the working-age population: Africa: HR<15: 2.46, 95%CI: 1.78, 3.38; HR≥15: 1.49, 95%CI: 1.01, 2.19; and from the Middle East: HR<15: 1.20, 95%CI: 0.90, 1.60; HR≥15: 1.65, 95%CI: 1.32, 2.05. For the retirement-age population: Africa: HR<15: 3.94, 95%CI: 2.85, 5.44; HR≥15: 1.66, 95%CI: 1.32, 2.09; Middle East: HR<15: 3.27, 95%CI: 2.70, 3.97; HR≥15: 2.12, 95%CI: 1.91, 2.34. CONCLUSIONS: Differential exposure, as opposed to differential susceptibility, likely accounted for the higher COVID-19 mortality observed among those origins who were disproportionately affected by the pandemic in Sweden.

The Teledermatology Experience: Cost Savings and Image Quality Control.

Introduction: Teledermatology adoption continues to increase, in part, spurred by the COVID-19 pandemic. This study analyzes the utility and cost savings of a store-and-forward teledermatology consultative system within the Veterans Health Administration (VA). Methods: Retrospective cohort of 4,493 patients across 14 remote sites in Tennessee and Kentucky from May 2017 through August 2019. The study measured the agreement between the teledermatology diagnoses and follow-up face-to-face clinic evaluations as well as the cost effectiveness of the teledermatology program over the study period. Results: Fifty-four percent of patients were recommended for face-to-face appointment for biopsy or further evaluation. Most patients, 80.5% received their face-to-face care by a VA dermatologist. There was a high level of concordance between teledermatologist and clinic dermatologist for pre-malignant and malignant cutaneous conditions. Veterans were seen faster at a VA clinic compared with a community dermatology site. Image quality improved as photographers incorporated teledermatologist feedback. From a cost perspective, teledermatology saved the VA system $1,076,000 in community care costs. Discussion: Teledermatology is a useful diagnostic tool within the VA system providing Veteran care at a cost savings.

Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein-Ligand Interactions from a Single NMR Sample.

We have combined saturation transfer difference NMR (STD NMR) with chemical shift imaging (CSI) and controlled concentration gradients of small molecule ligands to develop imaging STD NMR, a new tool for the assessment of protein-ligand interactions. Our methodology allows the determination of protein-ligand dissociation constants (KD) and assessment of the binding specificity in a single NMR tube, avoiding time-consuming titrations. We demonstrate the formation of suitable and reproducible concentration gradients of ligand along the vertical axis of the tube, against homogeneous protein concentration, and present a CSI pulse sequence for the acquisition of STD NMR experiments at different positions along the sample tube. Compared to the conventional methodology in which the [ligand]/[protein] ratio is increased manually, we can perform STD NMR experiments at a greater number of ratios and construct binding epitopes in a fraction (∼20%) of the experimental time. Second, imaging STD NMR also allows us to screen for non-specific binders, by monitoring any variation of the binding epitope map at increasing [ligand]/[protein] ratios. Hence, the proposed method does carry the potential to speed up and smooth out the drug discovery process.

Measurement of the pKa Values of Organic Molecules in Aqueous-Organic Solvent Mixtures by 1H NMR without External Calibrants.

Aqueous-organic solvent mixtures are commonly used for reactions or analyses, where the components of a system are insoluble in pure water. The acid dissociation constant is an important property to measure in these media as it determines the charge state, solubility, and reactivity of a molecule. While NMR spectroscopy is an established tool for the measurement of pKa in water, its use in aqueous-organic solvents is greatly hindered by the requirement for external calibrants on which a working pH scale is set. Such calibrants include buffer solutions, "anchor" molecules with known pKa values, and pH electrodes that have undergone lengthy calibration procedures in the solvent mixture of interest. However, such calibrations are often inconvenient to perform, while literature pKa data covering the required range may not be available at the solvent composition or the temperature of interest. Here, we present a method to determine pKa in aqueous-organic solvents directly by NMR. We first determine pKa of an organic acid such as 2,6-dihydroxybenzoic acid (2,6-DHB) by measuring its 1H chemical shift as a function of concentration along a concentration gradient using chemical shift imaging (CSI). Using 2,6-DHB as a reference, we then determine pKa of less acidic molecules in single CSI experiments via the variation of their 1H chemical shifts along pH gradients. As proof of concept, we determine the pKa values of organic acids and bases up to pKa 10 in 50% (v/v) 1-propanol/water, 50% (v/v) dimethyl sulfoxide/water, and 30% (v/v) acetonitrile/water and obtain good agreement with the literature values.

Elevated mortality among the second-generation (children of migrants) in Europe: what is going wrong? A review.

INTRODUCTION: The 'second-generation' (i.e. the children of migrants) represent one of the fastest growing subpopulations of the child and young adult populations in Europe today. The research so far appears to indicate that their mortality risk is elevated relative to people with non-migrant backgrounds. SOURCES OF DATA: Peer-reviewed publications. AREAS OF AGREEMENT: Second-generation status is a clear marker of elevated mortality risk in Europe in early life (including stillbirth, perinatal, neonatal and infant mortality) and adulthood, particularly if the parent(s) were born outside of Europe. Socioeconomic inequality plays an important, albeit rarely defining, role in these elevated risks. AREAS OF CONTROVERSY: It remains unclear what causes-of-death are driving these elevated mortality risks. The exact influence of (non-socioeconomic) explanatory factors (e.g. health care, racism & discrimination, and factors related to integration) on the elevated mortality risks of the second-generation also remains unclear. GROWING POINTS: The second-generation will continue to grow and diversify in Europe; we must intervene to address these inequalities now. AREAS TIMELY FOR DEVELOPING RESEARCH: Place more emphasis on the complexity of migration background, specific causes-of-death, and understanding the roles of explanatory factors beyond socioeconomic background.

Novel Biomaterial Containing Gelatin, Manuka Honey, and Hydroxyapatite Enhanced Secondary Intention Healing Versus Standard Secondary Intention Healing in Mohs Surgical Defects on the Head and Distal Lower Extremities-A Randomized Controlled Trial: Pilot Study.

BACKGROUND: Randomized, comparative studies evaluating augmented secondary intention healing (SIH) compared with conventional SIH in dermatologic surgery are limited. This study aimed to evaluate whether the use of a novel biomaterial enhances SIH, particularly in shortening time to complete re-epithelialization. OBJECTIVE: The purpose of this study was to elucidate whether a novel biomaterial containing gelatin, manuka honey, and hydroxyapatite enhances SIH when compared with conventional SIH for surgical defects after Mohs micrographic surgery (MMS) on the head and distal lower extremities. MATERIALS AND METHODS: Thirty-seven patients were enrolled in this randomized controlled trial. Patients undergoing MMS on the head or distal lower extremities were eligible for recruitment. After clear surgical margins were obtained post-MMS, patients were randomized to receive standard SIH or biomaterial enhanced SIH. Patients had regularly scheduled follow-ups with questionnaires at each visit until complete re-epithelialization was achieved. RESULTS: Overall, there was no significant difference in time to re-epithelialization between standard SIH and biomaterial-enhanced SIH. However, there was a significant decrease in pain scores and skin thickness in the biomaterial-enhanced SIH group. CONCLUSION: Biomaterial-enhanced SIH is noninferior to standard SIH and produces less pain and favorable skin thickness compared with standard SIH. ClinicalTrials.gov listing: NCT04545476.

Controlled Annealing in Adaptive Multicomponent Gels.

We use a pH-driven annealing process to convert between co-assembled and self-sorted networks in multicomponent gels. The initially formed gels at low pH are co-assembled, with the two components coexisting within the same self-assembled structures. We use an enzymatic approach to increase the pH, resulting in a gel-to-sol transition, followed by a hydrolysis to lower the pH once again. As the pH decreases, a self-sorted network is formed by a two-stage gelation process determined by the pKa of each component. This approach can be expanded to layered systems to generate many varied systems by changing composition and rates of pH change, adapting their microstructure and so allowing access to a far greater range of morphologies and complexity than can be achieved in single component systems.

Does It Bind? A Method to Determine the Affinity of Calcium and Magnesium Ions for Polymers Using 1H NMR Spectroscopy.

The binding of calcium and magnesium ions (M2+) by polymers and other macromolecules in aqueous solution is ubiquitous across chemistry and biology. At present, it is difficult to assess the binding affinity of macromolecules for M2+ without recourse to potentiometric titrations and/or isothermal titration calorimetry. Both of these techniques require specialized equipment, and the measurements can be difficult to perform and interpret. Here, we present a new method based on 1H NMR chemical shift imaging (CSI) that enables the binding affinity of polymers to be assessed in a single experiment on standard high-field NMR equipment. In our method, M2+ acetate salt is weighed into a standard 5 mm NMR tube and a solution of polymer layered on top. Dissolution and diffusion of the salt carry the M2+ and acetate ions up through the solution. The concentrations of acetate, [Ac], and free (unbound) M2+, [M2+]f, are measured at different positions along the sample by CSI. Binding of M2+ to the polymer reduces [M2+]f and hinders the upward diffusion of M2+. A discrepancy is thus observed between [Ac] and [M2+]f from which the binding affinity of the polymer can be assessed. For systems which form insoluble complexes with M2+, such as sodium polyacrylate or carboxylate-functionalized nanocellulose (CNC), we can determine the concentration of M2+ at which the polymer will precipitate. We can also predict [M2+]f when a solution of polymer is mixed homogeneously with M2+ salt. We assess the binding properties of sodium polyacrylate, alginate, polystyrene sulfonate, CNC, polyethyleneimine, ethylenediamenetetraacetic acid, and maleate.

Efficient pKa Determination in a Nonaqueous Solvent Using Chemical Shift Imaging.

pKa is an important property of a molecule which impacts many fields, such as drug design, catalysis, reactivity, and environmental toxicity. It is often necessary to measure pKa in nonaqueous media due to the poor solubility of an analyte in water, for example, many compounds of pharmaceutical interest. Although NMR methods to measure pKa in water are well established, determining pKa in organic solvents is laborious and problematic. We present an efficient one-shot method to determine the pKa of an analyte in an organic solvent in a single measurement. Diffusion of an acid into a basic solution of the analyte and a set of pH indicators establishes a pH gradient in the NMR tube. The chemical shift of a pH sensitive resonance of the analyte and the pH of the solution are then determined simultaneously as a function of position along the pH gradient by recording a chemical shift image of the NMR tube. The pKa of the analyte is then determined using the Henderson-Hasselbalch equation. The method can be implemented in any laboratory with a gradient equipped NMR high-field spectrometer and is demonstrated for a range of pharmaceutical compounds and inorganic phosphazene bases.

Patient preferences pertaining to treatment options for drug-resistant focal epilepsy.

OBJECTIVE: To determine patient acceptability of benefit-risk trade-offs in selecting treatment options for drug-resistant mesial temporal lobe epilepsy, including open brain surgery, laser ablation (laser interstitial thermal therapy [LITT]), and continued medications. METHODS: A discrete-choice experiment survey was developed, consisting of 20 versions that were randomly assigned to respondents. Each version had 8 sets of constructed treatment alternatives, representing open brain surgery, LITT, or continued medical management. For each set, respondents indicated the treatment alternative they would choose first. Treatment alternatives were characterized by varying levels of chance of seizure freedom for at least 2 years (20-70%), risk of 30-day mortality (0-10%), and risk of neurological deficits (0-40%). Respondents' choices were analyzed using random-parameters logit models to quantify acceptable benefit-risk trade-offs. Preference heterogeneity was evaluated using latent-class analysis. RESULTS: The survey was administered to 2 cohorts of adult patients with drug-resistant epilepsy: a Duke cohort identified using diagnostic codes (n = 106) and a web-recruited panel with a self-reported physician diagnosis of drug-resistant epilepsy (n = 300). Based on mean preference weights, respondents who indicated a willingness to consider surgical intervention would accept a reduction in chance of seizure freedom from 70% to a minimum-acceptable benefit (MAB) of 23% if they could undergo LITT rather than open brain surgery. For a reduction in 30-day mortality from 1% to 0%, MAB was 52%. For a reduction in risk of long-term deficits from 10% to 0%, MAB was 39%. Latent-class analysis revealed additional choice patterns identifying respondent groups that more strongly favored continuing medications or undergoing surgery. CONCLUSION: Patients who are receptive to surgery would accept significantly lower treatment effectiveness to undergo a minimally invasive procedure relative to open brain surgery. They also were willing to accept lower treatment benefit to reduce risks of mortality or neurological deficits.

What can the UK learn from the impact of migrant populations on national life expectancy?

Improvements in life expectancy at birth in the UK had stalled prior to 2020 and have fallen during the COVID-19 pandemic. The stagnation took place at a time of relatively high net migration, yet we know that migrants to Australia, the USA and some Nordic countries have positively impacted national life expectancy trends, outperforming native-born populations in terms of life expectancy. It is important to ascertain whether migrants have contributed positively to life expectancy in the UK, concealing worsening trends in the UK-born population, or whether relying on national life expectancy calculations alone may have masked excess mortality in migrant populations. We need a better understanding of the role and contribution of migrant populations to national life expectancy trends in the UK.

Age variations and population over-coverage: Is low mortality among migrants merely a data artefact?

The migrant mortality advantage has been observed extensively, but its authenticity is debated. In particular, concerns persist that the advantage is an artefact of the data, generated by the problems of recording mobility among foreign-born populations. Here, we build on the intersection of two recent developments: the first showing substantial age variation in the advantage-a deep U-shaped advantage at peak migration ages-and the second showing high levels of population over-coverage, the principal source of data artefact, at the same ages. We use event history analysis of Sweden's population registers (2010-15) to test whether this over-coverage can explain age variation in the migrant mortality advantage. We document its U-shape in Sweden and, crucially, demonstrate that large mortality differentials persist after adjusting for estimated over-coverage. Our findings contribute to ongoing debate by demonstrating that the migrant mortality advantage is real and by ruling out one of its primary mechanisms.

Disparities in Coronavirus Disease 2019 Mortality by Country of Birth in Stockholm, Sweden: A Total-Population-Based Cohort Study.

Preliminary evidence points to higher morbidity and mortality from coronavirus disease 2019 (COVID-19) in certain racial and ethnic groups, but population-based studies using microlevel data are lacking so far. We used register-based cohort data including all adults living in Stockholm, Sweden, between January 31, 2020 (the date of the first confirmed case of COVID-19) and May 4, 2020 (n = 1,778,670) to conduct Poisson regression analyses with region/country of birth as the exposure and underlying cause of COVID-19 death as the outcome, estimating relative risks and 95% confidence intervals. Migrants from Middle Eastern countries (relative risk (RR) = 3.2, 95% confidence interval (CI): 2.6, 3.8), Africa (RR = 3.0, 95% CI: 2.2, 4.3), and non-Sweden Nordic countries (RR = 1.5, 95% CI: 1.2, 1.8) had higher mortality from COVID-19 than persons born in Sweden. Especially high mortality risks from COVID-19 were found among persons born in Somalia, Lebanon, Syria, Turkey, Iran, and Iraq. Socioeconomic status, number of working-age household members, and neighborhood population density attenuated up to half of the increased COVID-19 mortality risks among the foreign-born. Disadvantaged socioeconomic and living conditions may increase infection rates in migrants and contribute to their higher risk of COVID-19 mortality.

Infant mortality among native-born children of immigrants in France, 2008-17: results from a socio-demographic panel survey.

BACKGROUND: Within Europe, France stands out as a major country that lacks recent and reliable evidence on how infant mortality levels vary among the native-born children of immigrants compared with the native-born children of two parents born in France. METHODS: We used a nationally representative socio-demographic panel consisting of 296 400 births and 980 infant deaths for the period 2008-17. Children of immigrants were defined as being born to at least one parent born abroad and their infant mortality was compared with that of children born to two parents born in France. We first calculated infant mortality rates per 1000 live births. Then, using multi-level logit models, we calculated odds ratios of infant mortality in a series of models adjusting progressively for parental origins (M1), core demographic factors (M2), father's socio-professional category (M3) and area-level urbanicity and deprivation score (M4). RESULTS: We documented a substantial amount of excess infant mortality among those children born to at least one parent from Eastern Europe, Northern Africa, Western Africa, Other Sub-Saharan Africa and the Americas, with variation among specific origin countries belonging to these groups. In most of these cases, the excess infant mortality levels persisted after adjusting for all individual-level and area-level factors. CONCLUSIONS: Our findings, which can directly inform national public health policy, reaffirm the persistence of longstanding inequality in infant mortality according to parental origins in France and add to a growing body of evidence documenting excess infant mortality among the children of immigrants in Europe.

Rapid Determination of the Acidity, Alkalinity and Carboxyl Content of Aqueous Samples by 1H NMR with Minimal Sample Quantity.

The titratable acidity, alkalinity, and carboxylate content are fundamental properties required for the understanding of aqueous chemical systems. Here, we present a set of new methods that allow these properties to be determined directly by 1H NMR without the labor, cost, and sample quantity associated with running separate potentiometric or conductometric titrations. Our methods require only the measurement of the pH-sensitive 1H chemical shifts of indicator molecules and do not require the tedious titration of reagents into a sample. To determine the titratable acidity, an excess of 2-methylimidazole (2MI) is added to a sample and the quantity of protons absorbed by 2MI is determined from its 1H chemical shifts. The titratable alkalinity of a sample can be similarly determined using acetic acid. To determine the concentration of deprotonated carboxylates, a sample is acidified with HCl, and the quantity of H+ absorbed is determined from the 1H chemical shift of methylphosphonic acid. We validate our methods by demonstrating the measurement of the acidity of fruit-flavored drinks, the alkalinity of tap water, and the carboxylate content of nanocellulose dispersions.

Mechanoresponsive Self-Assembled Perylene Bisimide Films.

In this work, self-assembled amino-acid appended perylene bisimides (PBIs) have been studied that when processed into thin films change their resistivity in response to being bent. The PBIs assemble into structures in water and form thin films upon drying. These normally delicate thin films can be tolerant to bending, depending on the aggregates they form. Furthermore, the films then reversibly change their resistivity in response to this mechanical stimulus. This change is proportional to the degree of bending of the film giving them the potential to be used quantitatively to measure mechanical movement, such as in wearable devices.

A unique variant of juvenile papular-purpuric gloves and socks syndrome.

Parvovirus B19 is the most common causative agent of papular-purpuric gloves and socks syndrome (PPGSS), an often-underreported condition in the pediatric population. Classically, PPGSS presents with a papular-purpuric and at times petechial eruption of the hands and feet. (Dermatology. 1994;188:85; Int J Dermatol. 1996;35:626) We report a unique variant of juvenile PPGSS with prominent involvement of the flexural and extensor elbows, wrists, and knees.