Trends in Medicare Costs of Hyperbaric Oxygen Therapy, 2013 through 2022.

Objective: To analyze Hyperbaric Oxygen Therapy Registry (HBOTR) data to estimate the Medicare costs of hyperbaric oxygen therapy (HBO2) based on standard treatment protocols and the annual mean number of treatments per patient reported by the registry. Methods: We performed a secondary analysis of deidentified data for all payers from 53 centers registered in the HBOTR from 2013 to 2022. We estimated the mean annual per-patient costs of HBO2 based on Medicare (outpatient facility + physician) reimbursement fees adjusted to 2022 inflation using the Medicare Economic Index. Costs were calculated for the annual average number of treatments patients received each year and for a standard 40-treatment series. We estimated the 2022 costs of standard treatment protocols for HBO2 indications treated in the outpatient setting. Results: Generally, all costs decreased from 2013 to 2022. The facility cost per patient per 40 HBO2 treatments decreased by 10.7% from $21,568.58 in 2013 to $19,488.00 in 2022. The physician cost per patient per 40 treatments substantially decreased by -37.8%, from $5,993.16 to $4,346.40. The total cost per patient per 40 treatments decreased by 15.6% from $27,561.74 to $23,834.40. In 2022, a single HBO2 session cost $595.86. For different indications, estimated costs ranged from $2,383.4-$8,342.04 for crush injuries to $17,875.80-$35,751.60 for diabetic foot ulcers and delayed radiation injuries. Conclusions: This real-world analysis of registry data demonstrates that the actual cost of HBO2 is not nearly as costly as the literature has insinuated, and the per-patient cost to Medicare is decreasing, largely due to decreased physician costs.

Efficient and reproducible generation of human iPSC-derived cardiomyocytes and cardiac organoids in stirred suspension systems.

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality, inter-batch consistency, cryopreservation and scale remain, reducing experimental reproducibility and clinical translation. Here, we report a robust stirred suspension cardiac differentiation protocol, and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines, the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs, bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks, which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible, scalable, and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications, and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols.

Mechanisms of inclusion of thallium-201 into Prussian blue nanoparticles for nuclear medicine applications.

Prussian blue is known for its high affinity for thallium and other univalent metal cations and has been used as a treatment for radiocaesium and thallium/radiothallium poisoning. While Prussian blue nanoparticles (PBNPs) show potential for binding radioactive thallium for further use in nuclear medicine applications, the inclusion mechanism remains elusive. Understanding the interaction between PBNPs and 201Tl is essential for identifying the physicochemical and radiochemical properties required for optimal in vivo performance. In this work, we evaluated the binding mechanism between Tl and PBNPs with different coatings and core shapes. Combining PBNPs with [201Tl] thallium(I) chloride provided high radiolabelling yields and radiochemical stabilities under physiological conditions. Comprehensive characterisation by different X-ray techniques confirmed that Tl ions are located in the interstitial sites within the crystal structure, maintaining the integrity of the iron (Fe) 4p electronic distribution and inducing local modifications in the nearby C-N ligands. Additionally, this inclusion does not impact the core or the shell of the nanoparticles but does alter their ionic composition. The PB ionic network undergoes significant changes, with a substantial drop in K+ content, confirming that Tl+ ions replace K+ and occupy additional spaces within the crystal structure. These results open new opportunities in nuclear medicine applications with 201Tl-PBNPs where the size, shape and composition of the particles can be specifically tuned depending on the desired biological properties without affecting the radiochemical performance as a vehicle for 201Tl.

Rocky Mountain spotted fever is a neglected tropical disease in Latin America.

Rocky Mountain spotted fever (RMSF), a severe and extraordinarily lethal infectious disease, has emerged as a widespread public health crisis among predominantly vulnerable populations in several countries of Latin America, particularly evident in northern Mexico. Historically, RMSF has gained less attention than many other tropical infectious diseases, resulting in insufficient allocations of resources and development of capabilities for its prevention and control in endemic regions. We argue that RMSF fulfills accepted criteria for a neglected tropical disease (NTD). The relative neglect of RMSF in most Latin American countries contributes to disparities in morbidity and mortality witnessed in this region. By recognizing RMSF as an NTD, an increased public policy interest, equitable and more appropriate allocation of resources, scientific interest, and social participation can ameliorate the impact of this potentially treatable disease, particularly in vulnerable populations.

Redundancy of p75NTR neurotrophin receptor function in development, growth and fertility in the rat.

The p75NTR neurotrophin receptor has positive and negative roles regulating cell survival in the nervous system. Unambiguous interpretation of p75NTR function in vivo has been complicated, however, by residual expression of alternate forms of p75NTR protein in initial p75NTR knock-out mouse models. As rats are the preferred rodent for studying brain and behaviour, and to simplify interpretation of the knock-out phenotype, we report here the generation of a mutant rat devoid of the p75NTR protein. TALEN-mediated recombination in embryonic stem cells (ESCs) was used to flank exon 2 of p75NTR with Lox P sites and produce transgenic rats carrying either un-recombined floxed p75NTREx2-fl, or recombined, exon-2 deleted p75NTREx2-Δ alleles. Crossing p75NTREx2-fl rats with a Cre-deleter strain efficiently removed exon 2 in vivo. Excision of exon 2 causes a frameshift after p75NTR Gly23 and eliminated p75NTR protein expression. Rats lacking p75NTR were healthy, fertile, and histological analysis did not reveal significant changes in cellular density or overall structure in their brains. p75NTR function is therefore largely dispensable for normal development, growth and basal homeostasis in the rat. However, the availability of constitutive and conditional p75NTREx2-Δ rats provides new opportunities to investigate specific roles of p75NTR upon injury and during tissue repair.

Establishment of a multisite umbrella cohort study protocol to describe the epidemiology and aetiologies of acute undifferentiated febrile illness in Latin America.

INTRODUCTION: Acute undifferentiated febrile illnesses (AUFIs) impose a large burden in the tropics. Understanding of AUFI's epidemiology is limited. Insufficient diagnostic capacity hinders the detection of outbreaks. The lack of interconnection in healthcare systems hinders timely response. We describe a protocol to study the epidemiology and aetiologies of AUFI and pathogen discovery in strategic areas of Latin America (LA). METHODS AND ANALYSIS: Global Infectious Diseases Network investigators comprising institutions in Colombia, Dominican Republic, México, Perú and the USA, developed a common cohort study protocol. The primary objective is to determine the aetiologies of AUFI at healthcare facilities in high-risk areas. Data collection and laboratory testing for viral, bacterial and parasitic agents are performed in rural and urban healthcare facilities and partner laboratories. Centralised laboratory and data management cores deploy diagnostic tests and data management tools. Subjects >6 years with fever for <8 days without localised infection are included in the cohort. They are evaluated during the acute and convalescent phases of illness. Study personnel collect clinical and epidemiological information. Blood, urine, nasal or pharyngeal swabs and saliva are collected in the acute phase and blood in convalescent phase. Specimens are banked at -80°C. Malaria, dengue and COVID-19 are tested onsite in the acute phase. The acute-phase serum is PCR tested for dengue, chikungunya, Venezuelan equine encephalitis, Mayaro, Oropouche, Zika, and yellow fever viruses. Paired convalescent and acute serum antibody titters are tested for arbovirus, Leptospira spp, and Rickettsia spp. Serum is used for viral cultures and next-generation sequencing for pathogen discovery. Analysis includes variable distributions, risk factors and regression models. Laboratory results are shared with health authorities and network members. ETHICS AND DISSEMINATION: The protocol was approved by local ethics committees and health authorities. The results will be published in peer-reviewed journals. All study results are shared with local and regional health authorities.

Optimising parameters for pilot scale steam explosion and continuous pressurised disc refining of Miscanthus and sugarcane bagasse for xylose and xylo-oligosaccharide release.

The first comparative pre-treatment study of Miscanthus (Mxg) and sugarcane bagasse (SCB) using steam explosion (SE) and pressurised disc refining (PDR) pretreatment to optimise xylose and xylo-oligosaccharide release is described. The current investigation aimed to 1) Develop optimised batch-wise steam explosion parameters for Mxg and SCB, 2) Scale from static batch steam explosion to dynamic continuous pressurised disc refining, 3) Identify, understand, and circumvent scale-up production hurdles. Optimised SE parameters released 82% (Mxg) and 100% (SCB) of the available xylan. Scaling to PDR, Miscanthus yielded 85% xylan, highlighting how robust scouting assessments for boundary process parameters can result in successful technical transfer. In contrast, SCB technical transfer was not straightforward, with significant differences observed between the two processes, 100% (SE) and 58% (PDR). This report underlines the importance of feedstock-specific pretreatment strategies to underpin process development, scale-up, and optimisation of carbohydrate release from biomass.

Realising a global One Health disease surveillance approach: insights from wastewater and beyond.

One Health is a recognition of the shared environment inhabited by humans, animals and plants, and the impact of their interactions on the health of all organisms. The COVID-19 pandemic highlighted the need for a framework of pathogen surveillance in a tractable One Health paradigm to allow timely detection and response to threats to human and animal health. We present case studies centered around the recent global approach to tackle antimicrobial resistance and the current interest in wastewater testing, with the concept of "one sample many analyses" to be further explored as the most appropriate means of initiating this endeavor.

Deletion of CD44 promotes adipogenesis by regulating PPARγ and cell cycle-related pathways.

CD44, a cell surface adhesion receptor and stem cell biomarker, is recently implicated in chronic metabolic diseases. Ablation of CD44 ameliorates adipose tissue inflammation and insulin resistance in obesity. Here, we investigated cell type-specific CD44 expression in human and mouse adipose tissue and further studied how CD44 in preadipocytes regulates adipocyte function. Using Crispr Cas9-mdediated gene deletion and lentivirus-mediated gene re-expression, we discovered that deletion of CD44 promotes adipocyte differentiation and adipogenesis, whereas re-expression of CD44 abolishes this effect and decreases insulin responsiveness and adiponectin secretion in 3T3-L1 cells. Mechanistically, CD44 does so via suppressing Pparg expression. Using quantitative proteomics analysis, we further discovered that cell cycle-regulated pathways were mostly decreased by deletion of CD44. Indeed, re-expression of CD44 moderately restored expression of proteins involved in all phases of the cell cycle. These data were further supported by increased preadipocyte proliferation rates in CD44-deficient cells and re-expression of CD44 diminished this effect. Our data suggest that CD44 plays a crucial role in regulating adipogenesis and adipocyte function possibly through regulating PPARγ and cell cycle-related pathways. This study provides evidence for the first time that CD44 expressed in preadipocytes plays key roles in regulating adipocyte function outside immune cells where CD44 is primarily expressed. Therefore, targeting CD44 in (pre)adipocytes may provide therapeutic potential to treat obesity-associated metabolic complications.

Early, rapidly progressive vasculopathy in a transplanted heart: A possible complication of COVID-19.

The epidemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has had a significant global impact, especially on immunosuppressed populations such as heart transplant recipients. While SARS-CoV-2 initially infects the respiratory system, cardiovascular complications induced by coronavirus disease 2019 (COVID-19) include cardiac arrest, myocardial infarction, heart failure, myocarditis, arrhythmia, acute myocyte injury, thrombotic events, and cardiogenic shock. Here, we present a case of a 45-year-old African American male who tested positive for COVID-19 infection six months after receiving a heart transplant. The patient was asymptomatic initially, but two weeks later he developed dyspnea, early satiety, and abdominal bloating. The patient was admitted to the hospital for acute renal failure and subsequently diagnosed with moderate acute T cell-mediated allograft rejection (Grade 2R) by endomyocardial biopsy. Three months after testing positive for COVID-19, the patient suffered a sudden cardiac death. At autopsy, the epicardium was diffusely edematous and showed vascular congestion. The coronary arteries showed a striking concentric narrowing of lumens and diffusely thickened arterial walls of all major extramural arteries deemed consistent with a rapidly progressive form of cardiac allograft vasculopathy (CAV). SARS-CoV-2 nucleocapsid protein was localized by immunohistochemistry (IHC) in endothelial cells of venules and capillaries within the epicardium. Our localization of SARS-CoV-2 in coronary vessel endothelial cells by IHC suggests that endothelial cell infection, endotheliitis, and immune-related inflammation may be a primary mechanism of vascular injury. The present case represents an early onset rapidly progressive form of CAV. This case may be the first case of post-transplant arteriopathy occurring in such a short time that includes corresponding autopsy, surgical pathology, and IHC data.

Evidence of reassortment of avian influenza A (H2) viruses in Brazilian shorebirds.

Influenza A viruses of the H2 subtype represent a zoonotic and pandemic threat to humans due to a lack of widespread specific immunity. Although A(H2) viruses that circulate in wild bird reservoirs are distinct from the 1957 pandemic A(H2N2) viruses, there is concern that they could impact animal and public health. There is limited information on AIVs in Latin America, and next to nothing about H2 subtypes in Brazil. In the present study, we report the occurrence and genomic sequences of two influenza A viruses isolated from wild-caught white-rumped sandpipers (Calidris fuscicollis). One virus, identified as A(H2N1), was isolated from a bird captured in Restinga de Jurubatiba National Park (PNRJ, Rio de Janeiro), while the other, identified as A(H2N2), was isolated from a bird captured in Lagoa do Peixe National Park (PNLP, Rio Grande do Sul). DNA sequencing and phylogenetic analysis of the obtained sequences revealed that each virus belonged to distinct subtypes. Furthermore, the phylogenetic analysis indicated that the genomic sequence of the A(H2N1) virus isolated from PNRJ was most closely related to other A(H2N1) viruses isolated from North American birds. On the other hand, the A(H2N2) virus genome recovered from the PNLP-captured bird exhibited a more diverse origin, with some sequences closely related to viruses from Iceland and North America, and others showing similarity to virus sequences recovered from birds in South America. Viral genes of diverse origins were identified in one of the viruses, indicating local reassortment. This suggests that the extreme South of Brazil may serve as an environment conducive to reassortment between avian influenza virus lineages from North and South America, potentially contributing to an increase in overall viral diversity.

Thyroid hormone analogues: Promising therapeutic avenues to improve the neurodevelopmental outcomes of intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a pregnancy complication impairing fetal growth and development. The compromised development is often attributed to disruptions of oxygen and nutrient supply from the placenta, resulting in a number of unfavourable physiological outcomes with impaired brain and organ growth. IUGR is associated with compromised development of both grey and white matter, predisposing the infant to adverse neurodevelopmental outcomes, including long-lasting cognitive and motor difficulties. Cerebral thyroid hormone (TH) signalling, which plays a crucial role in regulating white and grey matter development, is dysregulated in IUGR, potentially contributing to the neurodevelopmental delays associated with this condition. Notably, one of the major TH transporters, monocarboxylate transporter-8 (MCT8), is deficient in the fetal IUGR brain. Currently, no effective treatment to prevent or reverse IUGR exists. Management strategies involve close antenatal monitoring, management of maternal risk factors if present and early delivery if IUGR is found to be severe or worsening in utero. The overall goal is to determine the most appropriate time for delivery, balancing the risks of preterm birth with further fetal compromise due to IUGR. Drug candidates have shown either adverse effects or little to no benefits in this vulnerable population, urging further preclinical and clinical investigation to establish effective therapies. In this review, we discuss the major neuropathology of IUGR driven by uteroplacental insufficiency and the concomitant long-term neurobehavioural impairments in individuals born IUGR. Importantly, we review the existing clinical and preclinical literature on cerebral TH signalling deficits, particularly the impaired expression of MCT8 and their correlation with IUGR. Lastly, we discuss the current evidence on MCT8-independent TH analogues which mimic the brain actions of THs by being metabolised in a similar manner as promising, albeit underappreciated approaches to promote grey and white matter development and improve the neurobehavioural outcomes following IUGR.

Intrauterine inflammation and postnatal intravenous dopamine alter the neurovascular unit in preterm newborn lambs.

BACKGROUND: Intrauterine inflammation is considered a major cause of brain injury in preterm infants, leading to long-term neurodevelopmental deficits. A potential contributor to this brain injury is dysregulation of neurovascular coupling. We have shown that intrauterine inflammation induced by intra-amniotic lipopolysaccharide (LPS) in preterm lambs, and postnatal dopamine administration, disrupts neurovascular coupling and the functional cerebral haemodynamic responses, potentially leading to impaired brain development. In this study, we aimed to characterise the structural changes of the neurovascular unit following intrauterine LPS exposure and postnatal dopamine administration in the brain of preterm lambs using cellular and molecular analyses. METHODS: At 119-120 days of gestation (term = 147 days), LPS was administered into the amniotic sac in pregnant ewes. At 126-7 days of gestation, the LPS-exposed lambs were delivered, ventilated and given either a continuous intravenous infusion of dopamine at 10 µg/kg/min or isovolumetric vehicle solution for 90 min (LPS, n = 6; LPSDA, n = 6). Control preterm lambs not exposed to LPS were also administered vehicle or dopamine (CTL, n = 9; CTLDA, n = 7). Post-mortem brain tissue was collected 3-4 h after birth for immunohistochemistry and RT-qPCR analysis of components of the neurovascular unit. RESULTS: LPS exposure increased vascular leakage in the presence of increased vascular density and remodelling with increased astrocyte "end feet" vessel coverage, together with downregulated mRNA levels of the tight junction proteins Claudin-1 and Occludin. Dopamine administration decreased vessel density and size, decreased endothelial glucose transporter, reduced neuronal dendritic coverage, increased cell proliferation within vessel walls, and increased pericyte vascular coverage particularly within the cortical and deep grey matter. Dopamine also downregulated VEGFA and Occludin tight junction mRNA, and upregulated dopamine receptor DRD1 and oxidative protein (NOX1, SOD3) mRNA levels. Dopamine administration following LPS exposure did not exacerbate any effects induced by LPS. CONCLUSION: LPS exposure and dopamine administration independently alters the neurovascular unit in the preterm brain. Alterations to the neurovascular unit may predispose the developing brain to further injury.

Drp1 controls complex II assembly and skeletal muscle metabolism by Sdhaf2 action on mitochondria.

The dynamin-related guanosine triphosphatase, Drp1 (encoded by Dnm1l), plays a central role in mitochondrial fission and is requisite for numerous cellular processes; however, its role in muscle metabolism remains unclear. Here, we show that, among human tissues, the highest number of gene correlations with DNM1L is in skeletal muscle. Knockdown of Drp1 (Drp1-KD) promoted mitochondrial hyperfusion in the muscle of male mice. Reduced fatty acid oxidation and impaired insulin action along with increased muscle succinate was observed in Drp1-KD muscle. Muscle Drp1-KD reduced complex II assembly and activity as a consequence of diminished mitochondrial translocation of succinate dehydrogenase assembly factor 2 (Sdhaf2). Restoration of Sdhaf2 normalized complex II activity, lipid oxidation, and insulin action in Drp1-KD myocytes. Drp1 is critical in maintaining mitochondrial complex II assembly, lipid oxidation, and insulin sensitivity, suggesting a mechanistic link between mitochondrial morphology and skeletal muscle metabolism, which is clinically relevant in combatting metabolic-related diseases.

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation.

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

Highly pathogenic avian influenza A(H5N1) virus in a common bottlenose dolphin (Tursiops truncatus) in Florida.

Since late 2021, highly pathogenic avian influenza (HPAI) viruses of A/goose/Guangdong/1/1996 (H5N1) lineage have caused widespread mortality in wild birds and poultry in the United States. Concomitant with the spread of HPAI viruses in birds are increasing numbers of mammalian infections, including wild and captive mesocarnivores and carnivores with central nervous system involvement. Here we report HPAI, A(H5N1) of clade 2.3.4.4b, in a common bottlenose dolphin (Tursiops truncatus) from Florida, United States. Pathological findings include neuronal necrosis and inflammation of the brain and meninges, and quantitative real time RT-PCR reveal the brain carried the highest viral load. Virus isolated from the brain contains a S246N neuraminidase substitution which leads to reduced inhibition by neuraminidase inhibitor oseltamivir. The increased prevalence of A(H5N1) viruses in atypical avian hosts and its cross-species transmission into mammalian species highlights the public health importance of continued disease surveillance and biosecurity protocols.

Results of an interlaboratory study on the working curve in vat photopolymerization.

The working curve informs resin properties and print parameters for stereolithography, digital light processing, and other photopolymer additive manufacturing (PAM) technologies. First demonstrated in 1992, the working curve measurement of cure depth vs radiant exposure of light is now a foundational measurement in the field of PAM. Despite its widespread use in industry and academia, there is no formal method or procedure for performing the working curve measurement, raising questions about the utility of reported working curve parameters. Here, an interlaboratory study (ILS) is described in which 24 individual laboratories performed a working curve measurement on an aliquot from a single batch of PAM resin. The ILS reveals that there is enormous scatter in the working curve data and the key fit parameters derived from it. The measured depth of light penetration Dp varied by as much as 7x between participants, while the critical radiant exposure for gelation Ec varied by as much as 70x. This significant scatter is attributed to a lack of common procedure, variation in light engines, epistemic uncertainties from the Jacobs equation, and the use of measurement tools with insufficient precision. The ILS findings highlight an urgent need for procedural standardization and better hardware characterization in this rapidly growing field.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.