Mechanistic insights into complement pathway inhibition by CR1 domain duplication.

Complement receptor 1 (CR1) is a membrane glycoprotein with a highly duplicated domain structure able to bind multiple ligands such as C3b and C4b, the activated fragments of complement components C3 and C4, respectively. We have previously used our knowledge of this domain structure to identify CSL040, a soluble extracellular fragment of CR1 containing the long homologous repeat (LHR) domains A, B, and C. CSL040 retains the ability to bind both C3b and C4b but is also a more potent complement inhibitor than other recombinant CR1-based therapeutics. To generate soluble CR1 variants with increased inhibitory potential across all three complement pathways, or variants with activity skewed to specific pathways, we exploited the domain structure of CR1 further by generating LHR domain duplications. We identified LHR-ABCC, a soluble CR1 variant containing a duplicated C3b binding C-terminal LHR-C domain that exhibited significantly enhanced alternative pathway inhibitory activity in vitro compared to CSL040. Another variant, LHR-BBCC, containing duplications of both LHR-B and LHR-C with four C3b binding sites, was shown to have reduced classical/lectin pathway inhibitory activity compared to CSL040, but comparable alternative pathway activity. Interestingly, multiplication of the C4b-binding LHR-A domain resulted in only minor increases in classical/lectin pathway inhibitory activity. The CR1 duplication variants characterized in these in vitro potency assays, as well as in affinity in solution C3b and C4b binding assays, not only provides an opportunity to identify new therapeutic molecules, but also additional mechanistic insights to the multiple interactions between CR1 and C3b/C4b.

A framework for the biophysical screening of antibody mutations targeting solvent-accessible hydrophobic and electrostatic patches for enhanced viscosity profiles.

The formulation of high-concentration monoclonal antibody (mAb) solutions in low dose volumes for autoinjector devices poses challenges in manufacturability and patient administration due to elevated solution viscosity. Often many therapeutically potent mAbs are discovered, but their commercial development is stalled by unfavourable developability challenges. In this work, we present a systematic experimental framework for the computational screening of molecular descriptors to guide the design of 24 mutants with modified viscosity profiles accompanied by experimental evaluation. Our experimental observations using a model anti-IL8 mAb and eight engineered mutant variants reveal that viscosity reduction is influenced by the location of hydrophobic interactions, while targeting positively charged patches significantly increases viscosity in comparison to wild-type anti-IL-8 mAb. We conclude that most predicted in silico physicochemical properties exhibit poor correlation with measured experimental parameters for antibodies with suboptimal developability characteristics, emphasizing the need for comprehensive case-by-case evaluation of mAbs. This framework combining molecular design and triage via computational predictions with experimental evaluation aids the agile and rational design of mAbs with tailored solution viscosities, ensuring improved manufacturability and patient convenience in self-administration scenarios.

STEAP2 promotes hepatocellular carcinoma progression via increased copper levels and stress-activated MAP kinase activity.

Six Transmembrane Epithelial Antigen of Prostate 2 (STEAP2) belongs to a family of metalloreductases, which indirectly aid in uptake of iron and copper ions. Its role in hepatocellular carcinoma (HCC) remains to be characterized. Here, we report that STEAP2 expression was upregulated in HCC tumors compared with paired adjacent non-tumor tissues by RNA sequencing, RT-qPCR, Western blotting, and immunostaining. Public HCC datasets demonstrated upregulated STEAP2 expression in HCC and positive association with tumor grade. Transient and stable knockdown (KD) of STEAP2 in HCC cell lines abrogated their malignant phenotypes in vitro and in vivo, while STEAP2 overexpression showed opposite effects. STEAP2 KD in HCC cells led to significant alteration of genes associated with extracellular matrix organization, cell adhesion/chemotaxis, negative enrichment of an invasiveness signature gene set, and inhibition of cell migration/invasion. STEAP2 KD reduced intracellular copper levels and activation of stress-activated MAP kinases including p38 and JNK. Treatment with copper rescued the reduced HCC cell migration due to STEAP2 KD and activated p38 and JNK. Furthermore, treatment with p38 or JNK inhibitors significantly inhibited copper-mediated cell migration. Thus, STEAP2 plays a malignant-promoting role in HCC cells by driving migration/invasion via increased copper levels and MAP kinase activities. Our study uncovered a novel molecular mechanism contributing to HCC malignancy and a potential therapeutic target for HCC treatment.

An Anti-VEGF-B Antibody Reduces Abnormal Tumor Vasculature and Enhances the Effects of Chemotherapy.

The vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key regulators of blood vessel formation, including in tumors, where their deregulated function can promote the production of aberrant, leaky blood vessels, supporting tumor development. Here we investigated the VEGFR1 ligand VEGF-B, which we demonstrate to be expressed in tumor cells and in tumor stroma and vasculature across a range of tumor types. We examined the anti-VEGF-B-specific monoclonal antibody 2H10 in preclinical xenograft models of breast and colorectal cancer, in comparison with the anti-VEGF-A antibody bevacizumab. Similar to bevacizumab, 2H10 therapy was associated with changes in tumor blood vessels and intra-tumoral diffusion consistent with normalization of the tumor vasculature. Accordingly, treatment resulted in partial inhibition of tumor growth, and significantly improved the response to chemotherapy. Our studies indicate the importance of VEGF-B in tumor growth, and the potential of specific anti-VEGF-B treatment to inhibit tumor development, alone or in combination with established chemotherapies.

Variants of Unknown Significance in Maturity-Onset Diabetes of the Young: High Rate of Conundrum Resolution via Variants of Unknown Significance Reanalysis.

INTRODUCTION: In the era of next-generation sequencing, clinicians frequently encounter variants of unknown significance (VUS) in genetic testing. VUS may be reclassified over time as genetic knowledge grows. We know little about how best to approach VUS in the maturity-onset diabetes of the young (MODY). Therefore, our study aimed to determine the utility of reanalysis of previous VUS results in genetic confirmation of MODY. METHODS: A single-center retrospective chart review identified 85 subjects with a MODY clinical diagnosis. We reanalyzed genetic testing in 10 subjects with 14 unique VUS on MODY genes that was performed >3 years before the study. Demographic, clinical, and biochemical data was collected for those individuals. RESULTS: After reanalysis, 43% (6/14) of the gene variants were reclassified to a different category: 7% (1/14) were "likely pathogenic" and 36% (5/14) were "benign" or "likely benign." The reclassified pathogenic variant was in HNF1A and all reclassified benign variants were in HNF1A, HNF1B and PDX1. The median time between MODY testing and reclassification was 8 years (range: 4-10 years). CONCLUSION: In sum, iterative reanalyzing the genetic data from VUS found during MODY testing may provide high-yield diagnostic information. Further studies are warranted to identify the optimal time and frequency for such analyses.

Integrative multi-omics characterization of hepatocellular carcinoma in Hispanic patients.

Background: The incidence and mortality rates of hepatocellular carcinoma (HCC) among Hispanics in the United States are much higher than those of non-Hispanic whites. We conducted comprehensive multi-omics analyses to understand molecular alterations in HCC among Hispanic patients. Methods: Paired tumor and adjacent non-tumor samples were collected from 31 Hispanic HCC in South Texas (STX-Hispanic) for genomic, transcriptomic, proteomic, and metabolomic profiling. Additionally, serum lipids were profiled in 40 Hispanic and non-Hispanic patients with or without clinically diagnosed HCC. Results: Exome sequencing revealed high mutation frequencies of AXIN2 and CTNNB1 in STX Hispanic HCCs, suggesting a predominant activation of the Wnt/ß-catenin pathway. The TERT promoter mutation frequency was also remarkably high in the Hispanic cohort. Cell cycles and liver functions were identified as positively- and negatively-enriched, respectively, with gene set enrichment analysis. Gene sets representing specific liver metabolic pathways were associated with dysregulation of corresponding metabolites. Negative enrichment of liver adipogenesis and lipid metabolism corroborated with a significant reduction in most lipids in the serum samples of HCC patients. Two HCC subtypes from our Hispanic cohort were identified and validated with the TCGA liver cancer cohort. The subtype with better overall survival showed higher activity of immune and angiogenesis signatures, and lower activity of liver function-related gene signatures. It also had higher levels of immune checkpoint and immune exhaustion markers. Conclusions: Our study revealed some specific molecular features of Hispanic HCC and potential biomarkers for therapeutic management of HCC and provides a unique resource for studying Hispanic HCC.

Mechanistic Basis of the Cu(OAc)2 Catalyzed Azide-Ynamine (3 + 2) Cycloaddition Reaction.

The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is used as a ligation tool throughout chemical and biological sciences. Despite the pervasiveness of CuAAC, there is a need to develop more efficient methods to form 1,4-triazole ligated products with low loadings of Cu. In this paper, we disclose a mechanistic model for the ynamine-azide (3 + 2) cycloadditions catalyzed by copper(II) acetate. Using multinuclear nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy, and high-performance liquid chromatography analyses, a dual catalytic cycle is identified. First, the formation of a diyne species via Glaser-Hay coupling of a terminal ynamine forms a Cu(I) species competent to catalyze an ynamine-azide (3 + 2) cycloaddition. Second, the benzimidazole unit of the ynamine structure has multiple roles: assisting C-H activation, Cu coordination, and the formation of a postreaction resting state Cu complex after completion of the (3 + 2) cycloaddition. Finally, reactivation of the Cu resting state complex is shown by the addition of isotopically labeled ynamine and azide substrates to form a labeled 1,4-triazole product. This work provides a mechanistic basis for the use of mixed valency binuclear catalytic Cu species in conjunction with Cu-coordinating alkynes to afford superior reactivity in CuAAC reactions. Additionally, these data show how the CuAAC reaction kinetics can be modulated by changes to the alkyne substrate, which then has a predictable effect on the reaction mechanism.

Treadmill walking economy is not affected by body fat and body mass index in adults.

To determine whether body fat and body mass index (BMI) affect the energy cost of walking (Cw; J/kg/m), ventilation, and gas exchange data from 205 adults (115 females; percent body fat range = 3.0%-52.8%; BMI range = 17.5-43.2 kg/m2) were obtained at rest and during treadmill walking at 1.34 m/s to calculate gross and net Cw. Linear regression was used to assess relationships between body composition indices, Cw, and standing metabolic rate (SMR). Unpaired t-tests were used to assess differences between sex, and one-way ANOVA was used to assess differences by BMI categories: normal weight, <25.0 kg/m2; overweight, 25.0-29.9 km/m2; and obese, ≥30 kg/m2. Net Cw was not related to body fat percent, fat mass, or BMI (all R2 ≤ 0.011). Furthermore, mean net Cw was similar by sex (male: 2.19 ± 0.30 J/kg/m; female: 2.24 ± 0.37 J/kg/m, p = 0.35) and across BMI categories (normal weight: 2.23 ± 0.36 J/kg/m; overweight: 2.18 ± 0.33 J/kg/m; obese: 2.26 ± 0.31, p = 0.54). Gross Cw and SMR were inversely associated with percent body fat, fat mass, and BMI (all R2 between 0.033 and 0.270; all p ≤ 0.008). In conclusion, Net Cw is not influenced by body fat percentage, total body fat, and BMI and does not differ by sex.

Liver Transplant Costs and Activity After United Network for Organ Sharing Allocation Policy Changes.

Importance: A new liver allocation policy was implemented by United Network for Organ Sharing (UNOS) in February 2020 with the stated intent of improving access to liver transplant (LT). There are growing concerns nationally regarding the implications this new system may have on LT costs, as well as access to a chance for LT, which have not been captured at a multicenter level. Objective: To characterize LT volume and cost changes across the US and within specific center groups and demographics after the policy implementation. Design, Setting, and Participants: This cross-sectional study collected and reviewed LT volume from multiple centers across the US and cost data with attention to 8 specific center demographics. Two separate 12-month eras were compared, before and after the new UNOS allocation policy: March 4, 2019, to March 4, 2020, and March 5, 2020, to March 5, 2021. Data analysis was performed from May to December 2022. Main Outcomes and Measures: Center volume, changes in cost. Results: A total of 22 of 68 centers responded comparing 1948 LTs before the policy change and 1837 LTs postpolicy, resulting in a 6% volume decrease. Transplants using local donations after brain death decreased 54% (P < .001) while imported donations after brain death increased 133% (P = .003). Imported fly-outs and dry runs increased 163% (median, 19; range, 1-75, vs 50, range, 2-91; P = .009) and 33% (median, 3; range, 0-16, vs 7, range, 0-24; P = .02). Overall hospital costs increased 10.9% to a total of $46 360 176 (P = .94) for participating centers. There was a 77% fly-out cost increase postpolicy ($10 600 234; P = .03). On subanalysis, centers with decreased LT volume postpolicy observed higher overall hospital costs ($41 720 365; P = .048), and specifically, a 122% cost increase for liver imports ($6 508 480; P = .002). Transplant centers from low-income states showed a significant increase in hospital (12%) and import (94%) costs. Centers serving populations with larger proportions of racial and ethnic minority candidates and specifically Black candidates significantly increased costs by more than 90% for imported livers, fly-outs, and dry runs despite lower LT volume. Similarly, costs increased significantly (>100%) for fly-outs and dry runs in centers from worse-performing health systems. Conclusions and Relevance: Based on this large multicenter effort and contrary to current assumptions, the new liver distribution system appears to place a disproportionate burden on populations of the current LT community who already experience disparities in health care. The continuous allocation policies being promoted by UNOS could make the situation even worse.

Five multivariate Duchenne muscular dystrophy progression models bridging six-minute walk distance and MRI relaxometry of leg muscles.

The study aimed to provide quantitative information on the utilization of MRI transverse relaxation time constant (MRI-T2) of leg muscles in DMD clinical trials by developing multivariate disease progression models of Duchenne muscular dystrophy (DMD) using 6-min walk distance (6MWD) and MRI-T2. Clinical data were collected from the prospective and longitudinal ImagingNMD study. Disease progression models were developed by a nonlinear mixed-effect modeling approach. Univariate models of 6MWD and MRI-T2 of five muscles were developed separately. Age at assessment was the time metric. Multivariate models were developed by estimating the correlation of 6MWD and MRI-T2 model variables. Full model estimation approach for covariate analysis and five-fold cross validation were conducted. Simulations were performed to compare the models and predict the covariate effects on the trajectories of 6MWD and MRI-T2. Sigmoid Imax and Emax models best captured the profiles of 6MWD and MRI-T2 over age. Steroid use, baseline 6MWD, and baseline MRI-T2 were significant covariates. The median age at which 6MWD is half of its maximum decrease in the five models was similar, while the median age at which MRI-T2 is half of its maximum increase varied depending on the type of muscle. The models connecting 6MWD and MRI-T2 successfully quantified how individual characteristics alter disease trajectories. The models demonstrate a plausible correlation between 6MWD and MRI-T2, supporting the use of MRI-T2. The developed models will guide drug developers in using the MRI-T2 to most efficient use in DMD clinical trials.

Taking High-Fidelity Simulation to the Next Level: Nursing in Nontraditional Environments.

ABSTRACT: Nurse educators must prepare nursing students to be competent first responders and providers in nontraditional situations. We developed a course that provides in-depth experiential instruction in disaster nursing, remote/austere nursing, and global health. The Nursing in Nontraditional Environments course provides nursing students with the knowledge and skills to provide quality care to patients in environments outside traditional hospitals and clinics. The course merges survival skills with austere care using evidence-based practice derived from the US Military and the Wilderness Medical Society.

High Modulus, Strut-like poly(ether ether ketone) Aerogels Produced from a Benign Solvent.

Poly(ether ether ketone) (PEEK) was found to form gels in the benign solvent 1,3-diphenylacetone (DPA). Gelation of PEEK in DPA was found to form an interconnected, strut-like morphology composed of polymer axialites. To our knowledge, this is the first report of a strut-like morphology for PEEK aerogels. PEEK/DPA gels were prepared by first dissolving PEEK in DPA at 320 °C. Upon cooling to 50 °C, PEEK crystallizes and forms a gel in DPA. The PEEK/DPA phase diagram indicated that phase separation occurs by solid-liquid phase separation, implying that DPA is a good solvent for PEEK. The Flory-Huggins interaction parameter, calculated as χ12 = 0.093 for the PEEK/DPA system, confirmed that DPA is a good solvent for PEEK. PEEK aerogels were prepared by solvent exchanging DPA to water then freeze-drying. PEEK aerogels were found to have densities between 0.09 and 0.25 g/cm3, porosities between 80 and 93%, and surface areas between 200 and 225 m2/g, depending on the initial gel concentration. Using nitrogen adsorption analyses, PEEK aerogels were found to be mesoporous adsorbents, with mesopore sizes of about 8 nm, which formed between stacks of platelike crystalline lamellae. Scanning electron microscopy and X-ray scattering were utilized to elucidate the hierarchical structure of the PEEK aerogels. Morphological analysis found that the PEEK/DPA gels were composed of a highly nucleated network of PEEK axialites (i.e., aggregates of stacked crystalline lamellae). The highly connected axialite network imparted robust mechanical properties on PEEK aerogels, which were found to densify less upon freeze-drying than globular PEEK aerogel counterparts gelled from dichloroacetic acid (DCA) or 4-chlorphenol (4CP). PEEK aerogels formed from DPA were also found to have a modulus-density scaling that was far more efficient in supporting loads than the poorly connected aerogels formed from PEEK/DCA or PEEK/4CP solutions. The strut-like morphology in these new PEEK aerogels also significantly improved the modulus to a degree that is comparable to high-performance crosslinked aerogels based on polyimide and polyurea of comparable densities.

Safety plan use and suicide-related coping in a sample of Australian online help-seekers.

BACKGROUND: Suicide safety plans can improve suicide-related coping skills and reduce suicidal thoughts and behaviours (STBs). However, little is known about their use and impact outside of treatment settings, where most suicidal crises will occur. The current study explored the prevalence of safety plan use among an online sample of help-seekers with lifetime STBs, and whether STBs and suicide-related coping differed between those with and without safety plans. An exploratory aim was to investigate barriers to safety plan use. METHOD: Participants (N = 1251) completed an online, anonymous survey at a mental health support website (Beyond Blue). The survey measured lifetime STBs, past-month suicidal ideation, suicide-related coping, help-seeking intentions and behaviour. RESULTS: Despite high levels of past-month suicidal ideation and past-year help-seeking, most participants (89.5 %) did not have a safety plan, and most of those were not familiar with the concept (70.5 %). Participants with safety plans reported a higher rate of past suicide attempts, but higher suicide-related coping and help-seeking behaviour. Among participants without safety plans, negative attitudes toward safety planning were positively associated with suicidal ideation and negatively associated with suicide-related coping. LIMITATIONS: Participants were primarily female, English-speaking visitors to a mental health support website. Cross-sectional design precludes conclusions being drawn about safety planning effectiveness over time. CONCLUSION: This study highlights the low prevalence of safety plan use among online help-seekers with lifetime STBs and the need to better promote safety planning as an intervention with autonomous benefits, including crisis preparedness and improved suicide-related coping skills.

Innovative, combinatorial and high-throughput approaches to degrader synthesis.

Targeted protein degraders such as PROTACs and molecular glues are a rapidly emerging therapeutic modality within industry and academia. Degraders possess unique mechanisms of action that lead to the removal of specific proteins by co-opting the cell's natural degradation mechanisms via induced proximity. Their optimisation thus far has often been largely empirical, requiring the synthesis and screening of a large number of analogues. In addition, the synthesis and development of degraders is often challenging, leading to lengthy optimisation campaigns to deliver candidate-quality compounds. This review highlights how the synthesis of degraders has evolved in recent years, in particular focusing on means of applying high-throughput chemistry and screening approaches to expedite these timelines, which we anticipate to be valuable in shaping the future of degrader optimisation campaigns.

Recognizing and coping with suicidal thoughts: A mixed-methods investigation of digital safety plan content.

OBJECTIVES: Suicide safety plans are a personalized means of documenting how a person at risk of suicide recognizes and intends to cope with emerging suicidal thoughts. This study aimed to understand how users of digital suicide safety plans describe their warning signs, methods of coping and any relationships between these that may emerge. METHODS: A sample comprising 150 users of the Australian suicide safety planning smartphone app Beyond Now consented to share the content of their safety plans. Reflexive thematic analysis was used to identify themes in overall plan content. Most participants identified as women (61%), had a history of at least one suicide attempt (61%) and completed their plans by themselves (84%). RESULTS: Three major themes emerged: (1) interpersonal challenges and complexity; (2) matching coping strategies to warning signs; and (3) helpful and harmful digital technology use. Most plans appeared to demonstrate high self-awareness of warning signs and available supports. CONCLUSIONS: Safety plan content provides a window into the thought process underlying the recognition of suicidal thoughts and the attempts to manage them. An opportunity exists for practitioners and support persons to use this content when collaboratively supporting a safety plan user to improve their coping strategies and support networks.

Trauma Center Hospitals Charged Higher Prices For Some Nontrauma Care Than Non-Trauma Center Hospitals, 2012-18.

Rising prices are a major cause of increased health care spending and health insurance premiums in the US. Hospital prices, specifically-for both inpatient and outpatient care-are the largest driver of rising health care spending in the commercial insurance market. As a result, policy makers and employers are increasingly interested in understanding the determinants of hospital prices. Hospitals serving as trauma centers are often endowed by regulators with monopoly power over trauma services in their geographic areas, and this monopoly power may spill over to nontrauma services. This study focused on the growing number of designated trauma centers and how trauma center status affects hospital prices for other, nontrauma services. We found that hospitals designated as trauma centers charged higher prices for nontrauma inpatient admissions and nontrauma emergency department visits when compared with hospitals that were not designated as trauma centers, even after controlling for potential confounders.

Tectonic trigger to the first major extinction of the Phanerozoic: The early Cambrian Sinsk event.

The Cambrian explosion, one of the most consequential biological revolutions in Earth history, occurred in two phases separated by the Sinsk event, the first major extinction of the Phanerozoic. Trilobite fossil data show that Series 2 strata in the Ross Orogen, Antarctica, and Delamerian Orogen, Australia, record nearly identical and synchronous tectono-sedimentary shifts marking the Sinsk event. These resulted from an abrupt pulse of contractional supracrustal deformation on both continents during the Pararaia janeae trilobite Zone. The Sinsk event extinction was triggered by initial Ross/Delamerian supracrustal contraction along the edge of Gondwana, which caused a cascading series of geodynamic, paleoenvironmental, and biotic changes, including (i) loss of shallow marine carbonate habitats along the Gondwanan margin; (ii) tectonic transformation to extensional tectonics within the Gondwanan interior; (iii) extrusion of the Kalkarindji large igneous province; (iv) release of large volumes of volcanic gasses; and (v) rapid climatic change, including incursions of marine anoxic waters and collapse of shallow marine ecosystems.

Perceived Usefulness of Self-Guided Versus Collaborative Suicide Safety Plans in Online Help-Seekers.

Background: Suicide safety plans were originally devised to be paper-based and clinician-guided, but digital self-guided plans are now common. Aim: This study explored whether plan format (paper vs. digital), assistance (self-authored vs. collaboration), and suicide attempt history were associated with differences in suicidal ideation, suicide-related coping, and perceived usefulness. Method: An online sample of safety plan users (N = 131) completed a survey assessing suicidal ideation, suicide-related coping, and perceived usefulness of their plan. t tests compared outcomes by plan format, collaboration, and suicide attempt history. Pearson correlations explored associations between reasons for plan use, suicidal ideation, and suicide-related coping. Results: Suicidal ideation was significantly higher, and perceived usefulness significantly lower in participants with a past suicide attempt (vs. none) and in those who had collaborated to make their safety plan (vs. self-authored). Collaborators were largely health professionals. No significant differences were found between plan formats. Suicide-related coping was associated with higher perceived usefulness overall. Limitations: Our study design was cross-sectional, utilizing a largely young, female, English-speaking, online help-seeking sample. Conclusions: For clients with prior suicide attempts and higher levels of suicidal ideation, meaningful collaboration may be needed to find safety plan coping strategies that are perceived as useful.