Zinc mediates control of nitrogen fixation via transcription factor filamentation.

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

CD5L is a canonical component of circulatory IgM.

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass spectrometry.

Cross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over-length cross-links compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can provide new insights into the initial stages of the terminal complement pathway.

Longitudinal Fluctuations in Protein Concentrations and Higher-Order Structures in the Plasma Proteome of Kidney Failure Patients Subjected to a Kidney Transplant.

Using proteomics and complexome profiling, we evaluated in a year-long study longitudinal variations in the plasma proteome of kidney failure patients, prior to and after a kidney transplantation. The post-transplant period was complicated by bacterial infections, resulting in dramatic changes in the proteome, attributed to an acute phase response (APR). As positive acute phase proteins (APPs), being elevated upon inflammation, we observed the well-described C-reactive protein and Serum Amyloid A (SAA), but also Fibrinogen, Haptoglobin, Leucine-rich alpha-2-glycoprotein, Lipopolysaccharide-binding protein, Alpha-1-antitrypsin, Alpha-1-antichymotrypsin, S100, and CD14. As negative APPs, being downregulated upon inflammation, we identified the well-documented Serotransferrin and Transthyretin, but added Kallistatin, Heparin cofactor 2, and interalpha-trypsin inhibitor heavy chain H1 and H2 (ITIH1, ITIH2). For the patient with the most severe APR, we performed plasma complexome profiling by SEC-LC-MS on all longitudinal samples. We observed that several plasma proteins displaying alike concentration patterns coelute and form macromolecular complexes. By complexome profiling, we expose how SAA1 and SAA2 become incorporated into high-density lipid particles, replacing largely Apolipoprotein (APO)A1 and APOA4. Overall, our data highlight that the combination of in-depth longitudinal plasma proteome and complexome profiling can shed further light on correlated variations in the abundance of several plasma proteins upon inflammatory events.

The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2.

Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, that is, induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF-7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.

Phase II study of vemurafenib in children and young adults with tumors harboring BRAF V600 mutations: NCI-COG pediatric MATCH trial (APEC1621) Arm G.

BACKGROUND: This is a phase II subprotocol of the NCI-COG Pediatric MATCH study evaluating vemurafenib, a selective oral inhibitor of BRAF V600 mutated kinase, in patients with relapsed or refractory solid tumors harboring BRAF V600 mutations. METHODS: Patients received vemurafenib at 550 mg/m2 (maximum 960 mg/dose) orally twice daily for 28-day cycles until progression or intolerable toxicity. The primary aim was to determine the objective response rate and secondary objectives included estimating progression-free survival and assessing the tolerability of vemurafenib. RESULTS: Twenty-two patients matched to the subprotocol and 4 patients (18%) enrolled. Primary reasons for non-enrollment were ineligibility due to exclusions of low-grade glioma (nâ=â7) and prior BRAF inhibitor therapy (nâ=â7). Enrolled diagnoses were one each of histiocytosis, ameloblastoma, Ewing sarcoma, and high-grade glioma, all with BRAF V600E mutations. Treatment was overall tolerable with mostly expected grade 1/2 adverse events (AE). Grade 3 or 4 AE on treatment were acute kidney injury, hyperglycemia, and maculopapular rash. One patient came off therapy due to AE. One patient (glioma) had an objective partial response and remained on protocol therapy for 15 cycles. CONCLUSION: There was a low accrual rate on this MATCH subprotocol, with only 18% of those who matched with BRAFV600 mutations enrolling, resulting in early termination, and limiting study results (ClinicalTrials.gov Identifier: NCT03220035).

Tranexamic Acid for the Prevention of Blood Loss after Cesarean Delivery.

BACKGROUND: Prophylactic administration of tranexamic acid has been associated with reduced postpartum blood loss after cesarean delivery in several small trials, but evidence of its benefit in this clinical context remains inconclusive. METHODS: In a multicenter, double-blind, randomized, controlled trial, we assigned women undergoing cesarean delivery before or during labor at 34 or more gestational weeks to receive an intravenously administered prophylactic uterotonic agent and either tranexamic acid (1 g) or placebo. The primary outcome was postpartum hemorrhage, defined as a calculated estimated blood loss greater than 1000 ml or receipt of a red-cell transfusion within 2 days after delivery. Secondary outcomes included gravimetrically estimated blood loss, provider-assessed clinically significant postpartum hemorrhage, use of additional uterotonic agents, and postpartum blood transfusion. RESULTS: Of the 4551 women who underwent randomization, 4431 underwent cesarean delivery, 4153 (93.7%) of whom had primary outcome data available. The primary outcome occurred in 556 of 2086 women (26.7%) in the tranexamic acid group and in 653 of 2067 (31.6%) in the placebo group (adjusted risk ratio, 0.84; 95% confidence interval [CI], 0.75 to 0.94; P = 0.003). There were no significant between-group differences in mean gravimetrically estimated blood loss or in the percentage of women with provider-assessed clinically significant postpartum hemorrhage, use of additional uterotonic agents, or postpartum blood transfusion. Thromboembolic events in the 3 months after delivery occurred in 0.4% of women (8 of 2049) who received tranexamic acid and in 0.1% of women (2 of 2056) who received placebo (adjusted risk ratio, 4.01; 95% CI, 0.85 to 18.92; P = 0.08). CONCLUSIONS: Among women who underwent cesarean delivery and received prophylactic uterotonic agents, tranexamic acid treatment resulted in a significantly lower incidence of calculated estimated blood loss greater than 1000 ml or red-cell transfusion by day 2 than placebo, but it did not result in a lower incidence of hemorrhage-related secondary clinical outcomes. (Funded by the French Ministry of Health; TRAAP2 ClinicalTrials.gov number, NCT03431805.).

Race as a Risk Marker, Not a Risk Factor: Revising Race-Based Algorithms to Protect Racially Oppressed Patients.

Emerging consensus in the medical and public health spheres encourages removing race and ethnicity from algorithms used in clinical decision-making. Although clinical algorithms remain appealing given their promise to lighten the cognitive load of medical practice and save time for providers, they risk exacerbating existing health disparities. Race is a strong risk marker of health outcomes, yet it is not a risk factor. The use of race as a factor in medical algorithms suggests that the effect of race is intrinsic to the patient or that its effects can be distinct or separated from other social and environmental variables. By contrast, incisive public health analysis coupled with a race-conscious perspective recognizes that race serves as a marker of countless other dynamic variables and that structural racism, rather than race, compromises the health of racially oppressed individuals. This perspective offers a historical and theoretical context for the current debates regarding the use of race in clinical algorithms, clinical and epidemiologic perspectives on "risk," and future directions for research and policy interventions that combat color-evasive racism and follow the principles of race-conscious medicine.

Volume expansion mitigates Shiga toxin-producing E. coli-hemolytic uremic syndrome in children.

BACKGROUND: Shiga toxin-producing E. coli-hemolytic uremic syndrome (STEC-HUS) is associated with high morbidity and relevant mortality. Previous small studies showed that volume expansion could improve the course and outcome of STEC-HUS. The aim of this single-center study was to evaluate the effect of volume expansion on the clinical course and outcome in STEC-HUS. METHODS: Data of pediatric patients with STEC-HUS were analyzed retrospectively. Course and outcome of patients treated with volume expansion (VE) from 2019 to 2022 (n = 38) were compared to historical controls (HC) from 2009 to 2018 (n = 111). RESULTS: Patients in the VE group had a significant relative median weight gain compared to HC (7.8% (3.4-11.3) vs. 1.2% (- 0.7-3.9), p < 0.0001) 48 h after admission. The need for dialysis was not reduced by VE (VE 21/38 (55.3%) vs. HC 64/111 (57.7%), p = 0.8). However, central nervous system involvement (impairment of consciousness, seizures, focal neurological deficits, and/or visual disturbances) was significantly reduced (VE 6/38 (15.8%) vs. HC 38/111 (34.2%), p = 0.039). None of the patients in the VE group died or developed chronic kidney disease (CKD) stage 5, whereas in the HC group, three patients died and three patients had CKD stage 5 at discharge. CONCLUSIONS: This study suggests that volume expansion may be associated with the mitigation of the acute course of STEC-HUS, especially severe neurological involvement and the development of CKD. Prospective trials should lead to standardized protocols for volume expansion in children with STEC-HUS.

The Utility of AI in Writing a Scientific Review Article on the Impacts of COVID-19 on Musculoskeletal Health.

PURPOSE OF REVIEW: There were two primary purposes to our reviews. First, to provide an update to the scientific community about the impacts of COVID-19 on musculoskeletal health. Second, was to determine the value of using a large language model, ChatGPT 4.0, in the process of writing a scientific review article. To accomplish these objectives, we originally set out to write three review articles on the topic using different methods to produce the initial drafts of the review articles. The first review article was written in the traditional manner by humans, the second was to be written exclusively using ChatGPT (AI-only or AIO), and the third approach was to input the outline and references selected by humans from approach 1 into ChatGPT, using the AI to assist in completing the writing (AI-assisted or AIA). All review articles were extensively fact-checked and edited by all co-authors leading to the final drafts of the manuscripts, which were significantly different from the initial drafts. RECENT FINDINGS: Unfortunately, during this process, it became clear that approach 2 was not feasible for a very recent topic like COVID-19 as at the time, ChatGPT 4.0 had a cutoff date of September 2021 and all articles published after this date had to be provided to ChatGPT, making approaches 2 and 3 virtually identical. Therefore, only two approaches and two review articles were written (human and AI-assisted). Here we found that the human-only approach took less time to complete than the AI-assisted approach. This was largely due to the number of hours required to fact-check and edit the AI-assisted manuscript. Of note, the AI-assisted approach resulted in inaccurate attributions of references (about 20%) and had a higher similarity index suggesting an increased risk of plagiarism. The main aim of this project was to determine whether the use of AI could improve the process of writing a scientific review article. Based on our experience, with the current state of technology, it would not be advised to solely use AI to write a scientific review article, especially on a recent topic.

SARS-CoV-2 and its Multifaceted Impact on Bone Health: Mechanisms and Clinical Evidence.

PURPOSE OF REVIEW: SARS-CoV-2 infection, the culprit of the COVID-19 pandemic, has been associated with significant long-term effects on various organ systems, including bone health. This review explores the current understanding of the impacts of SARS-CoV-2 infection on bone health and its potential long-term consequences. RECENT FINDINGS: As part of the post-acute sequelae of SARS-CoV-2 infection, bone health changes are affected by COVID-19 both directly and indirectly, with multiple potential mechanisms and risk factors involved. In vitro and preclinical studies suggest that SARS-CoV-2 may directly infect bone marrow cells, leading to alterations in bone structure and osteoclast numbers. The virus can also trigger a robust inflammatory response, often referred to as a "cytokine storm", which can stimulate osteoclast activity and contribute to bone loss. Clinical evidence suggests that SARS-CoV-2 may lead to hypocalcemia, altered bone turnover markers, and a high prevalence of vertebral fractures. Furthermore, disease severity has been correlated with a decrease in bone mineral density. Indirect effects of SARS-CoV-2 on bone health, mediated through muscle weakness, mechanical unloading, nutritional deficiencies, and corticosteroid use, also contribute to the long-term consequences. The interplay of concurrent conditions such as diabetes, obesity, and kidney dysfunction with SARS-CoV-2 infection further complicates the disease's impact on bone health. SARS-CoV-2 infection directly and indirectly affects bone health, leading to potential long-term consequences. This review article is part of a series of multiple manuscripts designed to determine the utility of using artificial intelligence for writing scientific reviews.

Invited Commentary: Undiagnosed and Undertreated-the Suffocating Consequences of the Use of Racially Biased Medical Devices During the COVID-19 Pandemic.

While medical technology is typically considered neutral, many devices rely upon racially biased algorithms that prioritize care for White patients over Black patients, who may require more urgent medical attention. In their accompanying article, Sudat et al. (Am J Epidemiol. 2023;XXX(XX):XXX-XXX) document striking inaccuracies in pulse oximeter readings among Black patients, with significant clinical implications. Their findings suggest that this resulted in racial differences in delivery of evidence-based care during the coronavirus disease 2019 (COVID-19) pandemic, affecting admissions and treatment protocols. Despite the medical community's growing awareness of the pulse oximeter's significant design flaw, the device is still in use. In this article, I contextualize Sudat et al.'s study results within the larger history of racial bias in medical devices by highlighting the consequences of the continued underrepresentation of diverse populations in clinical trials. I probe the implications of racially biased assessments within clinical practice and research and illustrate the disproportionate impact on patients of color by examining 2 medical tools, the pulse oximeter and pulmonary function tests. Both cases result in the undertreatment and underdiagnosis of Black patients. I also demonstrate how the social underpinnings of racial bias in medical technology contribute to poor health outcomes and reproduce health disparities, and propose several recommendations for the field to rectify the harms of racial bias in medical technology.

Establishing a global regulatory floor for children's decisions about participation in clinical research.

BACKGROUND: Enrolling children in clinical trials typically requires parental or guardian permission and, when appropriate, child assent. Aligning requirements across jurisdictions would facilitate multisite pediatric trials. Guidance from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) is the best candidate for a global standard but would benefit from additional specification. METHODS: Ethical analysis of ICH guidance for permission and assent for pediatric trials, with recommendations for clarification. RESULTS: ICH guidance regarding permission and assent would be enhanced by additional detail in the following areas: (1) what information should be provided to parents, guardians, and children considering a trial, and how that information should be provided; (2) the definition of "assent," the criteria for when assent should be required, and the need to include children in discussions even when assent is not mandated; (3) criteria for requiring children's signatures indicating agreement; (4) greater specificity regarding children's right to decline or withdraw; and (5) clarification of when children's wish to decline or withdraw from participation may be overridden and of what the overriding process should entail. CONCLUSION: ICH guidance provides a global standard for decision making regarding children's participation in trials. Several clarifications would facilitate the conduct of multinational pediatric research. IMPACT: Enrolling children in clinical trials requires the permission of a parent/guardian ± the assent of the minor. Differing global regulatory requirements for enrolling children complicate the conduct of multicenter and multinational trials. The authors identify points of ambiguity and/or contradiction in the International Council for Harmonization guidelines and offer recommendations for a common ethical platform for conducting global pediatric research.

Impacts of multiple anthropogenic stressors on the transcriptional response of Gammarus fossarum in a mesocosm field experiment.

BACKGROUND: Freshwaters are exposed to multiple anthropogenic stressors, leading to habitat degradation and biodiversity decline. In particular, agricultural stressors are known to result in decreased abundances and community shifts towards more tolerant taxa. However, the combined effects of stressors are difficult to predict as they can interact in complex ways, leading to enhanced (synergistic) or decreased (antagonistic) response patterns. Furthermore, stress responses may remain undetected if only the abundance changes in ecological experiments are considered, as organisms may have physiological protective pathways to counteract stressor effects. Therefore, we here used transcriptome-wide sequencing data to quantify single and combined effects of elevated fine sediment deposition, increased salinity and reduced flow velocity on the gene expression of the amphipod Gammarus fossarum in a mesocosm field experiment. RESULTS: Stressor exposure resulted in a strong transcriptional suppression of genes involved in metabolic and energy consuming cellular processes, indicating that G. fossarum responds to stressor exposure by directing energy to vitally essential processes. Treatments involving increased salinity induced by far the strongest transcriptional response, contrasting the observed abundance patterns where no effect was detected. Specifically, increased salinity induced the expression of detoxification enzymes and ion transporter genes, which control the membrane permeability of sodium, potassium or chloride. Stressor interactions at the physiological level were mainly antagonistic, such as the combined effect of increased fine sediment and reduced flow velocity. The compensation of the fine sediment induced effect by reduced flow velocity is in line with observations based on specimen abundance data. CONCLUSIONS: Our findings show that gene expression data provide new mechanistic insights in responses of freshwater organisms to multiple anthropogenic stressors. The assessment of stressor effects at the transcriptomic level and its integration with stressor effects at the level of specimen abundances significantly contribute to our understanding of multiple stressor effects in freshwater ecosystems.

Race, Ethnicity, and Immigration Status in a Medical Licensing Educational Resource: a Systematic, Mixed-Methods Analysis.

BACKGROUND: Medical students preparing for the United States Medical Licensing Exam (USMLE) Step 2 Clinical Knowledge (CK) Exam frequently use the UWorld Step 2 CK Question Bank (QBank). Over 90% of medical students use UWorld QBanks to prepare for at least one USMLE. Although several questions in the QBank mention race, ethnicity, or immigration status, their contributions to the QBank remain underexamined. OBJECTIVE: We conducted a systematic, mixed-methods content analysis to assess whether and how disease conditions might be racialized throughout this popular medical education resource. DESIGN: We screened 3537 questions in the QBank between May 28 and August 11, 2020, for mentions of race, ethnicity, or immigration status. We performed multinomial logistic regression to assess the likelihood of each racial/ethnic category occurring in either the question stem, answer explanation, or both. We used an inductive technique for codebook development and determined code frequencies. MAIN MEASURES: We reviewed the frequency and distribution of race or ethnicity in question stems, answer choices, and answer explanations; assessed associations between disease conditions and racial and ethnic categories; and identified whether and how these associations correspond to race-, ethnicity-, or migration-based care. RESULTS: References to Black race occurred most frequently, followed by Asian, White, and Latinx groups. Mentions of race/ethnicity varied significantly by location in the question: Asian race had 6.40 times greater odds of occurring in the answer explanation only (95% CI 1.19-34.49; p < 0.031) and White race had 9.88 times greater odds of occurring only in the question stem (95% CI 2.56-38.08; p < 0.001). Qualitative analyses suggest frequent associations between disease conditions and racial, ethnic, and immigration categories, which often carry implicit or explicit biological and genetic explanations. CONCLUSIONS: Our analysis reveals patterns of race-based disease associations that have potential for systematic harm, including promoting incorrect race-based associations and upholding cultural conventions of White bodies as normative.

A multi-site method to capture turnover in rare to common interactions in bipartite species networks.

Ecological network structure is maintained by a generalist core of common species. However, rare species contribute substantially to both the species and functional diversity of networks. Capturing changes in species composition and interactions, measured as turnover, is central to understanding the contribution of rare and common species and their interactions. Due to a large contribution of rare interactions, the pairwise metrics used to quantify interaction turnover are, however, sensitive to compositional change in the interactions of, often rare, peripheral specialists rather than common generalists in the network. Here we expand on pairwise interaction turnover using a multi-site metric that enables quantifying turnover in rare to common interactions (in terms of occurrence of interactions). The metric further separates this turnover into interaction turnover due to species turnover and interaction rewiring. We demonstrate the application and value of this method using a host-parasitoid system sampled along gradients of environmental modification. In the study system, both the type and amount of habitat needed to maintain interaction composition depended on the properties of the interactions considered, that is, from rare to common. The analyses further revealed the potential of host switching to prevent or delay species loss, and thereby buffer the system from perturbation. Multi-site interaction turnover provides a comprehensive measure of network change that can, for example, detect ecological thresholds to habitat loss for rare to common interactions. Accurate description of turnover in common, in addition to rare, species and their interactions is particularly relevant for understanding how network structure and function can be maintained.

TRAPing Ghrelin-Activated Circuits: A Novel Tool to Identify, Target and Control Hormone-Responsive Populations in TRAP2 Mice.

The availability of Cre-based mouse lines for visualizing and targeting populations of hormone-sensitive cells has helped identify the neural circuitry driving hormone effects. However, these mice have limitations and may not even be available. For instance, the development of the first ghrelin receptor (Ghsr)-IRES-Cre model paved the way for using the Cre-lox system to identify and selectively manipulate ghrelin-responsive populations. The insertion of the IRES-Cre cassette, however, interfered with Ghsr expression, resulting in defective GHSR signaling and a pronounced phenotype in the homozygotes. As an alternative strategy to target ghrelin-responsive cells, we hereby utilize TRAP2 (targeted recombination in active populations) mice in which it is possible to gain genetic access to ghrelin-activated populations. In TRAP2 mice crossed with a reporter strain, we visualized ghrelin-activated cells and found, as expected, much activation in the arcuate nucleus (Arc). We then stimulated this population using a chemogenetic approach and found that this was sufficient to induce an orexigenic response of similar magnitude to that induced by peripheral ghrelin injection. The stimulation of this population also impacted food choice. Thus, the TRAPing of hormone-activated neurons (here exemplified by ghrelin-activated pathways) provides a complimentary/alternative technique to visualize, access and control discrete pathways, linking hormone action to circuit function.

Similarities and differences in the structures and proteoform profiles of the complement proteins C6 and C7.

The human complement system provides a first line of defence against pathogens. It requires a well-orchestrated sequential assembly of an array of terminal complement components (C5, C6, C7, C8, and C9), ultimately forming the membrane attack complex (MAC). Although much information about MAC assembly is available, the structure of the soluble C7 has remained elusive. The complement proteins C7 and C6 share very high sequence homology and exhibit several conserved domains, disulphide bridges, and C-mannosylation sites. Here, we used an integrative structural MS-based approach combining native MS, glycopeptide-centric MS, in-gel cross-linking MS (IGX-MS) and structural modelling to describe structural features, including glycosylation, of human serum soluble C7. We compare this data with structural and glycosylation data for human serum C6. The new structural model for C7 shows that it adopts a compact conformation in solution. Although C6 and C7 share many similarities, our data reveals distinct O-, and N-linked glycosylation patterns in terms of location and glycan composition. Cumulatively, our data provide valuable new insight into the structure and proteoforms of C7, solving an essential piece of the puzzle in our understanding of MAC assembly.

Complement activation is a crucial driver of acute kidney injury in rhabdomyolysis.

Rhabdomyolysis is a life-threatening condition caused by skeletal muscle damage with acute kidney injury being the main complication dramatically worsening the prognosis. Specific treatment for rhabdomyolysis-induced acute kidney injury is lacking and the mechanisms of the injury are unclear. To clarify this, we studied intra-kidney complement activation (C3d and C5b-9 deposits) in tubules and vessels of patients and mice with rhabdomyolysis-induced acute kidney injury. The lectin complement pathway was found to be activated in the kidney, likely via an abnormal pattern of Fut2-dependent cell fucosylation, recognized by the pattern recognition molecule collectin-11 and this proceeded in a C4-independent, bypass manner. Concomitantly, myoglobin-derived heme activated the alternative pathway. Complement deposition and acute kidney injury were attenuated by pre-treatment with the heme scavenger hemopexin. This indicates that complement was activated in a unique double-trigger mechanism, via the alternative and lectin pathways. The direct pathological role of complement was demonstrated by the preservation of kidney function in C3 knockout mice after the induction of rhabdomyolysis. The transcriptomic signature for rhabdomyolysis-induced acute kidney injury included a strong inflammatory and apoptotic component, which were C3/complement-dependent, as they were normalized in C3 knockout mice. The intra-kidney macrophage population expressed a complement-sensitive phenotype, overexpressing CD11b and C5aR1. Thus, our results demonstrate a direct pathological role of heme and complement in rhabdomyolysis-induced acute kidney injury. Hence, heme scavenging and complement inhibition represent promising therapeutic strategies.

Translation of experimental cardioprotective capability of P2Y12 inhibitors into clinical outcome in patients with ST-elevation myocardial infarction.

We studied the translational cardioprotective potential of P2Y12 inhibitors against acute myocardial ischemia/reperfusion injury (IRI) in an animal model of acute myocardial infarction and in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). P2Y12 inhibitors may have pleiotropic effects to induce cardioprotection against acute myocardial IRI beyond their inhibitory effects on platelet aggregation. We compared the cardioprotective effects of clopidogrel, prasugrel, and ticagrelor on infarct size in an in vivo rat model of acute myocardial IRI, and investigated the effects of the P2Y12 inhibitors on enzymatic infarct size (48-h area-under-the-curve (AUC) troponin T release) and clinical outcomes in a retrospective study of STEMI patients from the CONDI-2/ERIC-PPCI trial using propensity score analyses. Loading with ticagrelor in rats reduced infarct size after acute myocardial IRI compared to controls (37 ± 11% vs 52 ± 8%, p < 0.01), whereas clopidogrel and prasugrel did not (50 ± 11%, p > 0.99 and 49 ± 9%, p > 0.99, respectively). Correspondingly, troponin release was reduced in STEMI patients treated with ticagrelor compared to clopidogrel (adjusted 48-h AUC ratio: 0.67, 95% CI 0.47-0.94). Compared to clopidogrel, the composite endpoint of cardiac death or hospitalization for heart failure within 12 months was reduced in STEMI patients loaded with ticagrelor (HR 0.63; 95% CI 0.42-0.94) but not prasugrel (HR 0.84, 95% CI 0.43-1.63), prior to PPCI. Major adverse cardiovascular events did not differ between clopidogrel, ticagrelor, or prasugrel. The cardioprotective effects of ticagrelor in reducing infarct size may contribute to the clinical benefit observed in STEMI patients undergoing PPCI.