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Hephaestin is a vertebrate multicopper ferroxidase important for the transfer of dietary iron from intestinal cells to the blood. Hephaestin is mutated in the sex-linked anemia mouse, resulting in iron deficiency. However, sex-linked anemia mice still retain some hephaestin ferroxidase activity. They survive, breed, and their anemia improves with age. To gain a better understanding of the role of hephaestin in iron homeostasis, we used the Cre-lox system to generate knockout mouse models with whole body or intestine-specific (Villin promoter) ablation of hephaestin. Both types of mice were viable, indicating that hephaestin is not essential and that other mechanisms, multicopper ferroxidase-dependent or not, must compensate for hephaestin deficiency. The knockout strains, however, both developed a microcytic, hypochromic anemia, suggesting severe iron deficiency and confirming that hephaestin plays an important role in body iron acquisition. Consistent with this, the knockout mice accumulated iron in duodenal enterocytes and had reduced intestinal iron absorption. In addition, the similarities of the phenotypes of the whole body and intestine-specific hephaestin knockout mice clarify the important role of hephaestin specifically in intestinal enterocytes in maintaining whole body iron homeostasis. These mouse models will serve as valuable tools to study the role of hephaestin and associated proteins in iron transport in the small intestine and other tissues.

IntroductionOptimized health system approaches to improving guideline-congruent care require evaluation of multilevel factors associated with prescribing practices and outcomes after total knee and hip arthroplasty.Materials and methodsElectronic health data from patients who underwent a total knee or hip arthroplasty between January 2016–January 2020 in the Military Health System Data were retrospectively analyzed. A generalized linear mixed-effects model (GLMM) examined the relationship between fixed covariates, random effects, and the primary outcome (30-day opioid prescription refill).ResultsIn the sample (N = 9151, 65% knee, 35% hip), the median discharge morphine equivalent dose was 660 mg [450, 892] and varied across hospitals and several factors (e.g., joint, race and ethnicity, mental and chronic pain conditions, etc.). Probability of an opioid refill was higher in patients who underwent total knee arthroplasty, were white, had a chronic pain or mental health condition, had a lower age, and received a presurgical opioid prescription (all p < 0.01). Sex assigned in the medical record, hospital duration, discharge non-opioid prescription receipt, discharge morphine equivalent dose, and receipt of an opioid-only discharge prescription were not significantly associated with opioid refill.ConclusionIn the present study, several patient-, care-, and hospital-level factors were associated with an increased probability of an opioid prescription refill within 30 days after arthroplasty. Future work is needed to identify optimal approaches to reduce unwarranted and inequitable healthcare variation within a patient-centered framework.

ANH could provide coagulation factors to the circulating blood; shortening clot initiation until these factors are consumed at sites of surgical injury. The identification of specific products or scientific instrumentation is considered an integral part of the scientific endeavor and does not constitute endorsement or implied endorsement on the part of the author, DoD, or any component agency. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Army/Navy/Air Force, Department of Defense, or U.S. Government.Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.CRediT authorship contribution statement Benjamin M. Kristobak:Conceptualization, Data curation, Investigation, Methodology, Writing - original draft, Writing - review & editing.Daniel I. Brooks:Conceptualization, Formal analysis.Austin G. Bell:Data curation.Peter B. Willett:Data curation.Derek N. Lodico:Conceptualization, Data curation, Investigation, Methodology, Writing - review & editing.Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Mitochondria are dynamic organelles that undergo rounds of fission and fusion and exhibit a wide range of morphologies that contribute to the regulation of different signaling pathways and various cellular functions. It is important to understand the differences between mitochondrial structure in health and disease so that therapies can be developed to maintain the homeostatic balance of mitochondrial dynamics. Mitochondrial disorders are multisystemic and characterized by complex and variable clinical pathologies. The dynamics of mitochondria in mitochondrial disorders is thus worthy of investigation. Therefore, in this study, we performed a comprehensive analysis of mitochondrial dynamics in ten patient-derived fibroblasts containing different mutations and deletions associated with various mitochondrial disorders. Our results suggest that the most predominant morphological signature for mitochondria in the diseased state is fragmentation, with eight out of the ten cell lines exhibiting characteristics consistent with fragmented mitochondria. To our knowledge, this is the first comprehensive study that quantifies mitochondrial dynamics in cell lines with a wide array of developmental and mitochondrial disorders. A more thorough analysis of the correlations between mitochondrial dynamics, mitochondrial genome perturbations, and bioenergetic dysfunction will aid in identifying unique morphological signatures of various mitochondrial disorders in the future.

Using population-based surveillance data, we quantified the secondary invasive group A Streptococcus disease risk among household contacts. The disease risk in the 30 days postexposure to an index-case patient was highest among individuals aged ≥65 years, versus the annual background incidence of all ages.

Mammalian immunology has been studied in great detail in laboratory animals, but few of the tools and less of the insight derived from these studies have been placed in the context of natural, outbred wildlife populations subject to variable environments. We investigated patterns of innate immunity in free-ranging African buffalo in relation to host traits (age, reproductive status, body condition, white blood cell counts) and disease status (bovine tuberculosis [BTB], gastrointestinal nematodes, coccidia, ticks). We evaluated and used an in vitro assay measuring bactericidal competence of blood to assess a component of innate immunity in 200 female buffalo captured at Kruger National Park, South Africa, in June/July and October 2008. Animals with BTB had higher bactericidal competence of blood. Animals with higher neutrophil counts had higher bactericidal competence, whereas animals with lower lymphocyte counts had higher bactericidal competence. This pattern was driven by animals captured at the end of the dry season (October) and may be evidence of immune polarization, whereby individuals are unable to upregulate multiple components of immunity simultaneously. Bactericidal competence did not vary with host pregnancy status, body condition, age, lactation, tick infestation, nematode egg count, or coccidia oocyst count. Overall, we demonstrate that the bactericidal competence assay is practical and informative for field-based studies in wild bovids. Our results also show a correlation between bactericidal competence and bovine tuberculosis infection and reveal possible functional polarizations between different types of immune response in a free-ranging mammal.

Background Outbreaks of influenza can result in significant morbidity, including secondary bacterial infections. Invasive group A streptococcal (iGAS) infections are associated with a 12% case fatality rate. We used surveillance data to examine if there was a correlation between hospitalized influenza and GAS cases. Methods Minnesota Department of Health conducts population-based surveillance for hospitalized lab-confirmed influenza and iGAS (sterile site isolation) cases in the Minneapolis–St. Paul area as part of the CDC Emerging Infections Program. Cases were categorized by week during October–April of each year for 2010–2016, based on specimen collection date. Using STATA (v15), the correlation between the number of influenza (N = 11,768), and overall iGAS (N = 687), iGAS septic shock (n = 104), and iGAS pneumonia cases (n = 59) was assessed in weekly time periods using the Granger causality test. Results The number of hospitalized influenza cases was associated with an increase in the overall number of iGAS cases (Wald χ2 = 10.22, P = 0.04). Hospitalized influenza cases were associated with an increase in iGAS septic shock cases; every 1,000 increase in case counts were associated with one case of iGAS septic shock 1 week later (P = 0.02). Similarly, every 1,000 increase in hospitalized influenza cases were associated with one case of iGAS pneumonia 1 week later (P < 0.01). While the effect of Granger causality is cumulative when describing the causal relationship between hospitalized influenza and total iGAS, the correlation between influenza and the iGAS subgroups is best described with a 1-week lag. Conclusion Granger causality tests are commonly used in economic modeling but have not been routinely applied to infectious diseases. Using this test, we found a strong correlation between weekly cases of hospitalized influenza and iGAS cases, with a 1-week lag between influenza and iGAS septic shock or pneumonia. This approach can provide insight into the potential impact of developing prevention interventions for infections with strong correlation. Further exploration of Granger tests in infectious disease modeling should be considered. Disclosures All authors: No reported disclosures.

As part of the response to the highly pathogenic avian influenza A(H5N1) virus outbreak in U.S. cattle and poultry and the associated human cases, CDC and partners are monitoring influenza A virus levels and detection of the H5 subtype in wastewater. Among 48 states and the District of Columbia that performed influenza A testing of wastewater during May 12-July 13, 2024, a weekly average of 309 sites in 38 states had sufficient data for analysis, and 11 sites in four states reported high levels of influenza A virus. H5 subtype testing was conducted at 203 sites in 41 states, with H5 detections at 24 sites in nine states. For each detection or high level, CDC and state and local health departments evaluated data from other influenza surveillance systems and partnered with wastewater utilities and agriculture departments to investigate potential sources. Among the four states with high influenza A virus levels detected in wastewater, three states had corresponding evidence of human influenza activity from other influenza surveillance systems. Among the 24 sites with H5 detections, 15 identified animal sources within the sewershed or adjacent county, including eight milk-processing inputs. Data from these early investigations can help health officials optimize the use of wastewater surveillance during the upcoming respiratory illness season.

Treatment of disorders that disrupt homeostasis of skeletal tissues, such as osteoarthritis, and developmental skeletal diseases, such as overgrowth syndromes, requires a precise understanding of skeletal development. Osteoarthritis is a whole joint disorder that can be caused by articular cartilage defects that illicit an inflammatory immune response. Articular cartilage does not have intrinsic processes for repair or regeneration, so it is necessary to develop tissue constructs to repair the native tissue.Since articular cartilage is not capable of repair, it is difficult to engraft cartilage tissue constructs. As such, it is necessary to develop a layered osteochondral construct that can integrate with the bone layer just beneath the articular cartilage. Generation of a layered tissue construct presents a major challenge because bone and cartilage differentiation require very different stimulating cues. Here, we attempted to develop a multi-compartmental “bi-culture” system to promote spatially restricted differentiation of an osteochondral tissue construct. We use a progenitor cell derived from articular cartilage called a cartilage progenitor cell which is capable of differentiating into articular-like chondrocytes, hypertrophic-like chondrocytes, or osteoblasts, all of which play important roles in osteochondral tissue development. We use a demineralized human cancellous bone scaffold which has demonstrated its propensity to support bone and cartilage formation and success in engrafting with the host tissues. However, we find that the osteogenic and chondrogenic cues we use to develop the osteochondral tissue construct are insufficient, and we need to apply more native-like stimulation of tissue development to obtain an osteochondral construct.Epigenetic regulation has steadily gained attention in the field of orthopaedics. It has become clear that a variety of epigenetic factors play important roles in the development and homeostasis of skeletal tissues. Among these epigenetic factors are microRNAs. MicroRNA profiling studies have identified many differentially expressed miRNAs in chondrocytes at distinct sites of developing limb growth plate. Among the top differentially expressed microRNAs are miR-181a-1 and miR-138.miR-181a-1 is part of the miR-181a/b-1 cluster and has a reported pro-differentiation function in multiple in vitro differentiation assays. Here, we used lentiviral approaches to over-express this microRNA cluster during chondrogenesis of cartilage progenitor cells in micromass pellet cultures and report a pro-differentiation role. Using bulk RNA-sequencing, we identify a number of pro-anabolic genes and pathways upregulated by miR-181a/b-1 over-expression during cartilage progenitor cell chondrogenesis. The gene most downregulated by miR-181a/b-1 over-expression was aquaporin-9 which has not been studied extensively in cartilage development or homeostasis. We report that aquaporin-9 is expressed intracellularly during cartilage progenitor cell chondrogenesis and that it is not a direct target of miR-181a/b-1. The miR-181a/b-1 cluster appears to positively regulate chondrogenesis and chondrocyte anabolism, and one of the mechanisms for this effect may be indirect downregulation of aquaporin-9.miR-138 has been identified as an anti-proliferation, anti-adhesion, anti-motility agent, primarily in the context of cancers. Here, we used lentiviral approaches to over-express miR-138 during cartilage progenitor cell micromass pellet chondrogenesis and report that it has limited impact on differentiation. Bulk RNA-sequencing analysis detected only one significantly differentially expressed gene and minimal perturbation of chondrogenic-related signaling pathways. Our report adds to the ambiguous literature on the role of miR-138 in chondrogenesis.Another class of epigenetic regulators that are known to regulate skeletal development is DNA methyltransferases. Mutations in one of member of this class, DNMT3A, are responsible for a skeletal overgrowth syndrome called Tatton-Brown-Rahman syndrome. Interestingly, Tatton-Brown-Rahman syndrome patients also have intellectual and behavioral disorders, so it falls under the umbrella of overgrowth and intellectual disability syndromes.Recently, two different mouse models were generated with two distinct mutations in Dnmt3a, both of which are homologous to mutations found in human Tatton-Brown-Rahman syndrome patients. Here, we perform a thorough skeletal characterization on mice with either of the Dnmt3a mutations to assess the potential of these mice as an animal model for Tatton-Brown-Rahman syndrome. We report that these mice mimic some of the skeletal overgrowth phenotypes found in patients with these mutations. Investigating the cellular mechanism responsible for this overgrowth, we find that the growth plates in both Dnmt3a mutant mice are significantly thicker than their wild-type littermates during skeletal development. These findings indicate that both Dnmt3a mutant mice are good animal models for Tatton-Brown-Rahman syndrome.Given that DNMT3A has been reported to have a pro-osteogenic and anti-osteoclastogenic function, we hypothesized that there may be additional skeletal phenotypes present in the Dnmt3a mutant mice. We expanded our skeletal characterization to include other skeletal parameters such as trabecular and cortical bone indices, bone mechanical properties, and bone marrow adipose tissue accumulation. We found reduced cortical bone thickness and impaired bone mechanical properties in both of the Dnmt3a mutants. We investigated osteoblast function and osteoclast number in the mice during development but did not identify a clear cellular mechanism. We also report a sex-specific and mutation-specific upregulation of bone marrow adipose tissue in mice with one of the Dnmt3a mutations. These additional skeletal phenotypes represent opportunity to improve our understanding of Tatton-Brown-Rahman syndrome and, more broadly, overgrowth and intellectual disability syndromes.These investigations of microRNAs and DNA methyltransferases during in vitro chondrogenesis and in vivo skeletal overgrowth add to the rapidly expanding body of literature on epigenetic regulators in skeletal development. As we learn more about the functional roles of epigenetic regulators, we can better use them as tools to treat diseases and improve human health.