CD4 T Cell-Derived IL-21 Is Critical for Sustaining Plasmodium Infection-Induced Germinal Center Responses and Promoting the Selection of Memory B Cells with Recall Potential.

Development of Plasmodium-specific humoral immunity is critically dependent on CD4 Th cell responses and germinal center (GC) reactions during blood-stage Plasmodium infection. IL-21, a cytokine primarily produced by CD4 T cells, is an essential regulator of affinity maturation, isotype class-switching, B cell differentiation, and maintenance of GC reactions in response to many infection and immunization models. In models of experimental malaria, mice deficient in IL-21 or its receptor IL-21R fail to develop memory B cell populations and are not protected against secondary infection. However, whether sustained IL-21 signaling in ongoing GCs is required for maintaining GC magnitude, organization, and output is unclear. In this study, we report that CD4+ Th cells maintain IL-21 expression after resolution of primary Plasmodium yoelii infection. We generated an inducible knockout mouse model that enabled cell type-specific and timed deletion of IL-21 in peripheral, mature CD4 T cells. We found that persistence of IL-21 signaling in active GCs had no impact on the magnitude of GC reactions or their capacity to produce memory B cell populations. However, the memory B cells generated in the absence of IL-21 exhibited reduced recall function upon challenge. Our data support that IL-21 prevents premature cellular dissolution within the GC and promotes stringency of selective pressures during B cell fate determination required to produce high-quality Plasmodium-specific memory B cells. These data are additionally consistent with a temporal requirement for IL-21 in fine-tuning humoral immune memory responses during experimental malaria.

The Influence of Rural Healthcare Systems and Communities on Surgery and Recovery: A Qualitative Study.

INTRODUCTION: Successful recovery after surgery is complex and highly individual. Rural patients encounter greater barriers to successful surgical recovery than urban patients due to varying healthcare and community factors. Although studies have previously examined the recovery process, rural patients' experiences with recovery have not been well-studied. The rural socioecological context can provide insights into potential barriers or facilitators to rural patient recovery after surgery. METHODS: We conducted semi-structured qualitative interviews with a purposeful sample of 30 adult general surgery patients from rural areas in the Mountain West region of the United States. We used the socioecological framework to analyze their responses. Interviews focused on rural participants' experiences accessing healthcare and the impact of family and community support during postoperative recovery. Interviews were transcribed verbatim and coded using content and thematic analysis. RESULTS: All participants commented on the quality of their rural healthcare systems and its influence on postoperative care. Some enjoyed the trust developed through long-standing relationships with providers in their communities. However, participants described community providers' lack of money, equipment, and/or knowledge as barriers to care. Following surgery, participants recognized that there are advantages and disadvantages to receiving family and community support. Some participants worried about being stigmatized or judged by their community. CONCLUSIONS: Future interventions aimed at improving access to and recovery from surgery for rural patients should take into account the unique perspectives of rural patients. Addressing the socioecological factors surrounding rural surgery patients, such as healthcare, family, and community resources, will be key to improving postoperative recovery.

Computationally directed manipulation of cross-linked covalent organic frameworks for membrane applications.

Two-dimensional covalent organic frameworks (2D-COFs) exhibit characteristics ideal for membrane applications, such as high stability, tunability and porosity along with well-ordered nanopores. However, one of the many challenges with fabricating these materials into membranes is that membrane wetting can result in layer swelling. This allows molecules that would be excluded based on pore size to flow around the layers of the COF, resulting in reduced separation. Cross-linking between these layers inhibits swelling to improve the selectivity of these membranes. In this work, computational models were generated for a quinoxaline-based COF cross-linked with oxalyl chloride (OC) and hexafluoroglutaryl chloride (HFG). Enthalpy of formation and cohesive energy calculations from these models show that formation of these COFs is thermodynamically favorable and the resulting materials are stable. The cross-linked COF with HFG was synthesized and characterized with Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis with differential scanning calorimetry (TGA-DSC), and water contact angles. Additionally, these frameworks were fabricated into membranes for permeance testing. The experimental data supports the presence of cross-linking and demonstrates that varying the amount of HFG used in the reaction does not change the amount of cross-linking present. Computational models indicate that varying the cross-linking concentration has a negligible effect on stability and less cross-linking still results in stable materials. This work sheds light on the nature of the cross-linking in these 2D-COFs and their application in membrane technologies.

Synthesis, characterization and absolute configurations of methyl ladderanoates.

Methyl esters of [5]-ladderanoic acid and [3]-ladderanoic acid were prepared by esterification of the acids isolated from biomass at a wastewater treatment plant. Optical rotations at six different wavelengths (633, 589, 546, 436, 405 and 365 nm) and vibrational circular dichroism (VCD) spectra in the 1800-900 cm-1 region were measured in CDCl3 solvent and compared with quantum chemical (QC) predictions using B3LYP functional and 6-311++G(2d,2p) basis set with polarizing continuum model representing the solvent. QC predictions gave negative optical rotations at all six wavelengths for (R)-methyl [5]-ladderanoate and positive optical rotations for (R)-methyl [3]-ladderanoate, the same signs as previously reported for the corresponding acids. The crystal structure of (-)-methyl [5]-ladderanoate independently confirmed (R) configuration. The QC-predicted VCD spectra using Boltzmann population weighted spectra of individual conformers did not provide satisfactory quantitative agreement with the experimental VCD spectra. An improved quantitative agreement for VCD spectra could be obtained when conformer populations were optimized to maximize the similarity between experimental and predicted VCD spectra, but more improvements in VCD predictions are needed.

Ultrasound for the diagnosis of necrotizing fasciitis: A systematic review of the literature.

BACKGROUND: Necrotizing fasciitis (NF) is a deadly disorder that can be challenging to diagnose on history and examination alone. Point-of-care ultrasound (POCUS) is widely available and has been increasingly used for diagnosing skin and soft tissue infections. We performed a systematic review to determine the accuracy of POCUS for diagnosing NF with subgroup analyses of the accuracy of specific POCUS examination components. METHODS: PubMed, Scopus, CINAHL, LILACS, the Cochrane databases, Google Scholar, and bibliographies of selected articles were assessed for all retrospective, prospective, and randomized control trials evaluating the accuracy of POCUS for diagnosing NF. Data were dual extracted into a predefined worksheet and quality analysis was performed with the QUADAS-2 tool. Data were summarized and an overall summary was completed. RESULTS: We identified three papers (n = 221 patients; 33% NF) that met our inclusion criteria. The overall sensitivity ranged from 85.4%-100% while the specificity ranged from 44.7% to 98.2%. Fluid accumulation along the fascial plane was the most sensitive (85.4%; 95% CI 72.2% - 93.9%), while subcutaneous emphysema was the most specific (100%; 95% CI 92.5% - 100%). CONCLUSIONS: POCUS has good sensitivity and specificity for the diagnosis of NF. POCUS should be considered as an adjunct to the initial clinical decision making for the diagnosis of NF.

An analysis of the impact of annual cancer genetic testing guideline updates on a past patient population.

Germline genetic testing for cancer predisposition genes has become an essential component of cancer treatment and risk reduction. The National Comprehensive Cancer Network (NCCN) releases annual genetic testing guidelines that identify characteristics of patients that could be affected by a hereditary cancer syndrome. These guidelines have broadened over time and the implications for past patients of cancer genetics clinics are not well understood. This study is a retrospective chart review aimed at determining the percentage and characteristics of past patients that meet updated NCCN guidelines (Breast, Ovarian, and Pancreas [BOP] v1.2022 and Colorectal [CRC] v1.2021), patients that attended a follow-up appointment, and patients who went on to receive genetic testing. Clinical data and characteristics were compared between the study population as a whole and the cohort of patients that met updated NCCN guidelines BOP v1.2022 and CRC v1.2021. The study population consisted of 280 patients with 76 (27.1%) patients meeting updated NCCN guidelines BOP v1.2022 and CRC v1.2021. The year of initial cancer genetic counseling appointment was statistically significant (p = 0.023) with patients more likely to meet NCCN guidelines BOP v1.2022 and CRC v1.2021 with earlier initial cancer genetic counseling appointments. In the cohort that met updated NCCN guidelines BOP v1.2022 and CRC v1.2021, the most common reason was a change in the NCCN guidelines (BOP or CRC) (54/76, 71.1%) with triple-negative breast cancer diagnosed at any age being the most impactful guideline change (19/54, 35.2%). Twenty-one patients attended a follow-up appointment (7.5%) and of those that received genetic testing (17/21, 81%) most received negative results (13/17, 61.9%), with one pathogenic, low penetrance result (1/17, 5.9%, CHEK2 p.I157T). Provider-initiated follow-up was attributed to most follow-up appointments (16/21, 76.2%) implying patients do not tend to follow-up on their own. Education to non-genetics providers as well as targeted implementation of follow-up protocols possibly managed by genetic counseling assistants and utilizing electronic medical record (EMR) patient messaging could lead to improved patient follow-up.

Characterizing History of Health Sciences Organizations at Academic Health Sciences Centers.

Questions of how to sustain interest in the history of medicine and broader health sciences (HOM/HS) in a changing institutional environment, and how to collaborate with stakeholders to offer activities to do so, are on the radar for many academic health sciences centers and their libraries. This essay is an initial exploratory study of non-curricular HOM/HS efforts at United States medical schools ranked in the top thirty in primary care or research. In 2019, we collected public information pertinent to any presence of an on-campus HOM/HS community and the group's structure, including funding, activities, and the library's involvement with the group. Seventeen of forty-five institutions in the sample presented information about an institutional HOM/HS group. All posted a mission statement. Their funding varied in nature; some collected fees from members, while others relied on university support. Half were student-led. Most groups hosted regular lecture series, with fifteen groups hosting at least one annually. Six groups sponsored publications or awards. These findings indicate that several institutions with active programs offer potential models and lessons for sustaining HOM/HS communities. Beyond providing a physical or digital space in which HOM/HS groups connect, libraries play an active role in fostering some of these communities.

Blocking RAGE improves wound healing in diabetic pigs.

Receptor for Advanced Glycated End-products (RAGE) is highly expressed in diabetes and impairs wound healing. We proposed that administering an antibody that blocks RAGE will hasten the healing of dorsal wounds in diabetic pigs compared with a non-immune IgG. Two purpose-bred diabetic (D) Yucatan minipigs (Sinclair, Auxvasse MO) each underwent 12 2 × 2 cm full thickness dorsal wounds: four wounds received decellularized porcine skin patches (Xylyx Bio, Bklyn NY): four anti-RAGE Ab (CR-3) infused patches, four saline infused patches and four wounds were left open. One pig received anti-RAGE Ab (CR-3) 1 mg/kg IM q 10 days and other received non-immune IgG. Wounds were measured at 2 and 4 weeks followed by euthanasia and wound harvesting. At 2 weeks few of the patches appeared to be incorporated into the wound. By 4 weeks all patches in pigs treated systemically with CR-3 were detached and the wounds almost healed. For all 24 wounds for both pigs regardless of presence of patch or type of patch, the average IgG treated pig wound size at 4 weeks was 69.2 ± 14.6% of initial size and the average CR-3 treated pig wound size was 40.9 ± 11.3% of initial size (P = 0.0002). Quantitative immunohistology showed greater staining for collagen in the CR-3 treated wounds compared with IgG treated. Staining was positive for RAGE, Mac, and IL-6 in the IgG treated wounds and negative in the CR-3 treated wounds. From these pilot experiments, we conclude that a RAGE blocking antibody given parenterally improved wound healing in a diabetic pig while patches were not effective.

Low lysophosphatidylcholine induces skeletal muscle myopathy that is aggravated by high-fat diet feeding.

Obesity alters skeletal muscle lipidome and promotes myopathy, but it is unknown whether aberrant muscle lipidome contributes to the reduction in skeletal muscle contractile force-generating capacity. Comprehensive lipidomic analyses of mouse skeletal muscle revealed a very strong positive correlation between the abundance of lysophosphatidylcholine (lyso-PC), a class of lipids that is known to be downregulated with obesity, with maximal tetanic force production. The level of lyso-PC is regulated primarily by lyso-PC acyltransferase 3 (LPCAT3), which acylates lyso-PC to form phosphatidylcholine. Tamoxifen-inducible skeletal muscle-specific overexpression of LPCAT3 (LPCAT3-MKI) was sufficient to reduce muscle lyso-PC content in both standard chow diet- and high-fat diet (HFD)-fed conditions. Strikingly, the assessment of skeletal muscle force-generating capacity ex vivo revealed that muscles from LPCAT3-MKI mice were weaker regardless of diet. Defects in force production were more apparent in HFD-fed condition, where tetanic force production was 40% lower in muscles from LPCAT3-MKI compared to that of control mice. These observations were partly explained by reductions in the cross-sectional area in type IIa and IIx fibers, and signs of muscle edema in the absence of fibrosis. Future studies will pursue the mechanism by which LPCAT3 may alter protein turnover to promote myopathy.

Chiral Molecular Structure Determination for a Desired Compound Just from Its Molecular Formula and Vibrational Optical Activity Spectra.

A novel proof-of-concept model for chiral molecular structure determination using just the molecular formula and vibrational optical activity (VOA) spectra is presented. To verify this concept, the molecular formula of a desired compound is used to generate all possible chiral structural isomers and their VOA spectra are predicted. The similarity analyses of predicted VOA spectra were then carried out in two different ways: (a) similarity between VOA spectrum of one structural isomer with those of the rest, referred to as cross-correlations; (b) similarity between VOA spectra of all chiral structural isomers with the experimental VOA spectra of the desired compound. Three different molecular formulae, C4H8O, C3H5ClO, and C6H10O, and their chiral structural isomers (6, 9, and 75 respectively), were investigated. In each case, the correct chiral molecular structure of the desired compound was identified without ambiguity. Cross-correlation analysis revealed the uniqueness of VOA spectra in deducing the chiral molecular structure solely from its molecular formula. Different chiral structural isomers associated with the molecular formula CH3NO2 were also found to have no significant cross-correlations between their VOA spectra, opening a pathway to detect and identify the elusive chiral N-hydroxyoxaziridine from its VOA spectra.

Absolute configuration of seco-eudesmanolide inuloxin D from experimental and predicted chiroptical studies of its 4-O-acetyl derivative.

Sesquitepenoids inuloxins A-D, belonging to different subgroups, were isolated from Dittrichia viscosa and showed potential biocontrol of some parasitic plants as Pelipanche, Orobanche, and Cuscuta species. The absolute configurations of the first three inuloxins A-C were previously determined by using experimental and computational chiroptical spectroscopic methods. The absolute configuration of inuloxin D remains to be established. The bioactive inuloxin E, closely related to inuloxin D, was recently isolated from the same plant organic extract. The same relative configuration of inuloxin D was assigned to inuloxin E by comparison of their NMR spectroscopic data. The absolute configurations of inuloxin D and inuloxin E are suggested in this work by analysis of the experimental and predicted chiroptical properties of the 4-O-acetyl derivative of inuloxin D.

Effect of a split-flow physician in triage model on abdominal CT ordering rate and yield.

OBJECTIVE: The objective of this study was to compare the rate and clinical yield of computed tomography (CT) imaging between patients presenting with abdominal pain initially seen by a physician in triage (PIT) versus those seen only by physicians working in the main emergency department (ED). METHODS: A retrospective study was conducted of all self-arrivals >18 years old presenting to a single ED with abdominal pain. Nine-hundred patients were randomly selected from both the PIT and traditional patient flow groups and rates and yields of CT imaging were compared, both alone and in a model controlling for potential confounders. Predetermined criteria for CT significance included need for admission, consult, or targeted medications. RESULTS: The overall rate of CT imaging (unadjusted) did not differ between the PIT and traditional groups, 48.7% (95% CI 45.4-51.9) vs. 45.1% (95% CI 41.8-48.4), respectively (p = .13). The CT yield for patients seen in in the PIT group was also similar to that of the traditional group: 49.1% (95% CI 44.4-53.8) vs. 50.5% (95% CI 45.6-55.4) (p = .68). In the logistic regression model, when controlling for age, gender, ESI-acuity, race and insurance payor, PIT vs. traditional was not a predictor of CT ordering (OR 1.14, 95% CI 0.94-1.38). CONCLUSIONS: For patients with abdominal pain, we found no significant differences in rates of CT ordering or CT yield for patients seen in a PIT vs. traditional models, suggesting the increased efficiencies offered by PIT models do not come at the cost of increased or decreased imaging utilization.

Comparative Transcriptomic Response of Primary and Immortalized Macrophages to Murine Norovirus Infection.

Murine norovirus (NoV) is genetically similar to human NoV and offers both an efficient in vitro cell culture system and an animal model by which to investigate the molecular basis of replication. In this study, we present a detailed global view of host alterations to cellular pathways that occur during the progression of a NoV infection. This was accomplished for both Mus musculus BALB/c-derived RAW264.7 (RAW) cells, an immortalized cell line widely used in in vitro replication studies, and primary bone marrow-derived macrophages (BMDM), representing a permissive in vivo target cell in the host. Murine NoV replicated in both cell types, although detected genome copies were approximately one log lower in BMDM compared with RAW cells. RAW and BMDM cells shared an IRF3/7-based IFN response that occurred early in infection. In RAW cells, transcriptional upregulation and INF-ß expression were not coupled in that a significant delay in the detection of secreted INF-ß was observed. In contrast, primary BMDM showed an early upregulation of transcripts and immediate release of INF-ß that might account for lower virus yield. Differences in the transcriptional pathway responses included a marked decrease in expression of key genes in the cell cycle and lipid pathways in RAW cells compared with that of BMDM. Our comparative analysis indicates the existence of varying host responses to virus infection in populations of permissive cells. Awareness of these differences at the gene level will be important in the application of a given permissive culture system to the study of NoV immunity, pathogenesis, and drug development.

Natural Product-Derived Chiral Pyrrolidine-2,5-diones, Their Molecular Structures and Conversion to Pharmacologically Important Skeletons.

The versatility of the natural products (2S,3S)- and (2S,3R)-3-hydroxy-5-oxotetrahydrofuran-2,3-dicarboxylic acids (1 and 2), isolated in large amounts from tropical plant sources, has been demonstrated by the construction of 3-substituted and 3,4-disubstituted chiral pyrrolidine-2,5-diones. The absolute configurations of chiral pyrrolidine-2,5-diones have been ascertained using chiroptical spectroscopic methods and/or single-crystal XRD data. A combination of different reaction strategies delivering a diverse matrix of fused heterocyclic ring systems is presented. The pyrrolo[2,1-a]isoquinoline alkaloid (+)-crispine A possesses a wide range of pharmacological activities including antidepressant, antiplatelet, antileukemic, and anticancer activities. The analogues of indolizino[8,7-b]indole alkaloids (+)- and (-)-harmicine show strong antileishmanial, antinociceptive, PDE5-inhibitory, antimalarial, and antiviral activities. The bicyclic furo[2,3-b]pyrrolo skeleton is present in many natural products. Thus, the uniqueness of relatively cheap, naturally occurring chiral 2-hydroxycitric acid lactones as chirons has been demonstrated by the construction of some important molecular skeletons that are otherwise difficult to synthesize.

Static and Evolving Norovirus Genotypes: Implications for Epidemiology and Immunity.

Noroviruses are major pathogens associated with acute gastroenteritis worldwide. Their RNA genomes are diverse, with two major genogroups (GI and GII) comprised of at least 28 genotypes associated with human disease. To elucidate mechanisms underlying norovirus diversity and evolution, we used a large-scale genomics approach to analyze human norovirus sequences. Comparison of over 2000 nearly full-length ORF2 sequences representing most of the known GI and GII genotypes infecting humans showed a limited number (≤5) of distinct intra-genotypic variants within each genotype, with the exception of GII.4. The non-GII.4 genotypes were comprised of one or more intra-genotypic variants, with each variant containing strains that differed by only a few residues over several decades (remaining "static") and that have co-circulated with no clear epidemiologic pattern. In contrast, the GII.4 genotype presented the largest number of variants (>10) that have evolved over time with a clear pattern of periodic variant replacement. To expand our understanding of these two patterns of diversification ("static" versus "evolving"), we analyzed using NGS the nearly full-length norovirus genome in healthy individuals infected with GII.4, GII.6 or GII.17 viruses in different outbreak settings. The GII.4 viruses accumulated mutations rapidly within and between hosts, while the GII.6 and GII.17 viruses remained relatively stable, consistent with their diversification patterns. Further analysis of genetic relationships and natural history patterns identified groupings of certain genotypes into larger related clusters designated here as "immunotypes". We propose that "immunotypes" and their evolutionary patterns influence the prevalence of a particular norovirus genotype in the human population.

Targeted overexpression of catalase to mitochondria does not prevent cardioskeletal myopathy in Barth syndrome.

Barth Syndrome (BTHS) is an X-linked recessive disorder characterized by cardiomyopathy and muscle weakness. The underlying cause of BTHS is a mutation in the tafazzin (TAZ) gene, a key enzyme of cardiolipin biosynthesis. The lack of CL arising from loss of TAZ function results in destabilization of the electron transport system, promoting oxidative stress that is thought to contribute to development of cardioskeletal myopathy. Indeed, in vitro studies demonstrate that mitochondria-targeted antioxidants improve contractile capacity in TAZ-deficient cardiomyocytes. The purpose of the present study was to determine if resolving mitochondrial oxidative stress would be sufficient to prevent cardiomyopathy and skeletal myopathy in vivo using a mouse model of BTHS. To this end we crossed mice that overexpress catalase in the mitochondria (MCAT mice) with TAZ-deficient mice (TAZKD) to produce TAZKD mice that selectively overexpress catalase in the mitochondria (TAZKD+MCAT mice). TAZKD+MCAT mice exhibited decreased mitochondrial H2O2 emission and lipid peroxidation compared to TAZKD littermates, indicating decreased oxidative stress. Despite the improvements in oxidative stress, TAZKD+MCAT mice developed cardiomyopathy and mild muscle weakness similar to TAZKD littermates. These findings indicate that resolving oxidative stress is not sufficient to suppress cardioskeletal myopathy associated with BTHS.

Absolute Configurations of Naturally Occurring [5]- and [3]-Ladderanoic Acids: Isolation, Chiroptical Spectroscopy, and Crystallography.

We have isolated mixtures of [5]- and [3]-ladderanoic acids 1a and 2a from the biomass of an anammox bioreactor and have separated the acids and their phenacyl esters for the first time by HPLC. The absolute configurations of the naturally occurring acids and their phenacyl esters are assigned as R at the site of side-chain attachment by comparison of experimental specific rotations with corresponding values predicted using quantum chemical (QC) methods. The absolute configurations for 1a and 2a were independently verified by comparison of experimental Raman optical activity spectra with corresponding spectra predicted using QC methods. The configurational assignments of 1a and 2a and of the phenacyl ester of 1a were also confirmed by X-ray crystallography.

(R)-Metacycloprodigiosin-HCl: Chiroptical properties and structure.

(R)-Metacycloprodigiosin can exist in three different tautomeric forms, each with hydrogens at C9' and C12 in syn or anti orientation. With the addition of HCl, this structural diversity reduces to syn-(R)-metacycloprodigiosin-HCl (1a) and anti-(R)-metacycloprodigiosin-HCl (1b), each with multiple conformers. Energetics and chiroptical properties, namely, electronic circular dichroism (ECD) and specific optical rotation (SOR), of (R)-metacycloprodigiosin-HCl have been investigated at B3LYP/6-311++G(2d,2p) level. The experimental ECD spectra of (R)-metacycloprodigiosin-HCl have also been measured. Calculations indicated that the lowest energy conformer of 1b is approximately 2.7 kcal/mol lower in energy than that of 1a, and the energy barrier for anti to syn conversion is approximately 13 kcal/mol. The population weighted calculated SORs of 1a and 1b are, respectively, positive and negative. The respective calculated ECD spectra of these pseudoenantiomers show an almost mirror image relationship between them. The experimental SOR and ECD compare well with those predicted for 1b. Thus, 1b is expected to be predominant, a situation confirmed also by nuclear Overhauser effect (NOE) data, with a similar conclusion reached for prodigiosin R1.