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Abstract The Gram-negative bacterium Klebsiella pneumoniae is an important human pathogen. Its treatment has been complicated by the emergence of multi-drug resistant strains. The human complement system is an important part of our innate immune response that can directly kill Gram-negative bacteria by assembling membrane attack complex (MAC) pores into the bacterial outer membrane. To resist this attack, Gram-negative bacteria can modify their lipopolysaccharide (LPS). Especially the decoration of the LPS outer core with the O-antigen polysaccharide has been linked to increased bacterial survival in serum, but not studied in detail. In this study, we characterized various clinical Klebsiella pneumoniae isolates and show that expression of the LPS O1-antigen correlates with resistance to complement-mediated killing. Mechanistic data reveal that the O1-antigen does not inhibit C3b deposition and C5 conversion. In contrast, we see more efficient formation of C5a, and deposition of C6 and C9 when an O-antigen is present. Further downstream analyses revealed that the O1-antigen prevents correct insertion and polymerization of the final MAC component C9 into the bacterial membrane. Altogether, we show that the LPS O1-antigen is a key determining factor for complement resistance by K. pneumoniae and provide insights into the molecular basis of O1-mediated MAC evasion.

Abstract Biocrusts represent associations of lichens, green algae, cyanobacteria, fungi and other microorganisms, colonizing soils in varying proportions of principally arid biomes. The so-called grit crust represents a recently discovered type of biocrust situated in the Coastal Range of the Atacama Desert (Chile) made of microorganisms growing on and in granitoid pebbles, resulting in a checkerboard pattern visible to the naked eye on the landscape scale. This specific microbiome fulfills a broad range of ecosystem services, all probably driven by fog and dew-induced photosynthetic activity of mainly micro-lichens. To understand its biodiversity and impact, we applied a polyphasic approach on the phototrophic microbiome of this biocrust, combining isolation and characterization of the lichen photobionts, multi-gene phylogeny of the photobionts and mycobionts based on a direct sequencing and microphotography approach, metabarcoding and determination of chlorophylla+b contents. Metabarcoding showed that yet undescribed lichens within the Caliciaceae dominated the biocrust together with Trebouxia as the most abundant eukaryote in all plots. Together with high mean chlorophylla+b contents exceeding 410 mg m−2, this distinguished the symbiotic algae Trebouxia as the main driver of the grit crust ecosystem. The trebouxioid photobionts could be assigned to the I (T. impressa/gelatinosa) and A (T. arboricola) clades and represented several lineages containing five potential species candidates, which were identified based on the unique phylogenetic position, morphological features, and developmental cycles of the corresponding isolates. These results designate the grit crust as the only known coherent soil layer with significant landscape covering impact of at least 440 km2, predominantly ruled by a single symbiotic algal genus.

Abstract Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC–MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC–MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.

Abstract Intrinsic breast cancer molecular subtyping (IBCMS) provides significant prognostic information for patients with breast cancer and helps determine treatment. This study compared IBCMS methods on various gene-expression platforms in PALOMA-2 and PALLET trials. PALOMA-2 tumor samples were profiled using EdgeSeq and nanostring and subtyped with AIMS, PAM50, and research-use-only (ruo)Prosigna. PALLET tumor biopsies were profiled using mRNA sequencing and subtyped with AIMS and PAM50. In PALOMA-2 (n = 222), a 54% agreement was observed between results from AIMS and gold-standard ruoProsigna, with AIMS assigning 67% basal-like to HER2-enriched. In PALLET (n = 224), a 69% agreement was observed between results from PAM50 and AIMS. Different IBCMS methods may lead to different results and could misguide treatment selection; hence, a standardized clinical PAM50 assay and computational approach should be used. Trial number: NCT01740427

Introduction Delirium, a clinical manifestation of acute encephalopathy, is associated with extended hospitalisation, long-term cognitive dysfunction, increased mortality and high healthcare costs. Despite intensive research, there is still no targeted treatment. Delirium is characterised by electroencephalography (EEG) slowing, increased relative delta power and decreased functional connectivity. Recent studies suggest that transcranial alternating current stimulation (tACS) can entrain EEG activity, strengthen connectivity and improve cognitive functioning. Hence, tACS offers a potential treatment for augmenting EEG activity and reducing the duration of delirium. This study aims to evaluate the feasibility and assess the efficacy of tACS in reducing relative delta power.Methods and analysis A randomised, double-blind, sham-controlled trial will be conducted across three medical centres in the Netherlands. The study comprises two phases: a pilot phase (n=30) and a main study phase (n=129). Participants are patients aged 50 years and older who are diagnosed with delirium using the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, Text Revision criteria (DSM-5-TR), that persists despite treatment of underlying causes. During the pilot phase, participants will be randomised (1:1) to receive either standardised (10 Hz) tACS or sham tACS. In the main study phase, participants will be randomised to standardised tACS, sham tACS or personalised tACS, in which tACS settings are tailored to the participant. All participants will undergo daily 30 min of (sham) stimulation for up to 14 days or until delirium resolution or hospital discharge. Sixty-four-channel resting-state EEG will be recorded pre- and post the first tACS session, and following the final tACS session. Daily delirium assessments will be acquired using the Intensive Care Delirium Screening Checklist and Delirium Observation Screening Scale. The pilot phase will assess the percentage of completed tACS sessions and increased care requirements post-tACS. The primary outcome variable is change in relative delta EEG power. Secondary outcomes include (1) delirium duration and severity, (2) quantitative EEG measurements, (3) length of hospital stay, (4) cognitive functioning at 3 months post-tACS and (5) tACS treatment burden. Study recruitment started in April 2024 and is ongoing.Ethics and dissemination The study has been approved by the Medical Ethics Committee of the Utrecht University Medical Center and the Institutional Review Boards of all participating centres. Trial results will be disseminated via peer-reviewed publications and conference presentations.Trial registration number NCT06285721.

Abstract Background Cancer predisposition syndromes (CPS) impact about 10% of patients with pediatric cancer. Genetic testing (CPS‐GT) has multiple benefits, but few studies have described parent and child knowledge and attitudes regarding CPS‐GT decision‐making. This study examined parent and patient CPS‐GT decision‐making knowledge and attitudes. Procedure English‐ or Spanish‐speaking parents of children with pediatric cancer and patients with pediatric cancer ages 15–18 within 12 months of diagnosis or relapse were eligible to participate. Seventy‐five parents and 19 parent‐patient dyads (N = 94 parents, 77.7% female, 43.6% Latino/a/Hispanic; 19 patients, 31.6% female) completed surveys measuring CPS‐GT‐related beliefs. Independent samples t‐tests compared parent responses across sociodemographic characteristics and parent‐patient responses within dyads. Results Spanish‐speaking parents were significantly more likely than English‐speaking parents to believe that CPS‐GT not being helpful (p

IntroductionSome cyanobacteria can use far-red light (FRL) to drive oxygenic photosynthesis, a phenomenon known as Far-Red Light Photoacclimation (FaRLiP). It can expand photosynthetically active radiation beyond the visible light (VL) range. Therefore, it holds promise for biotechnological applications and may prove useful for the future human exploration of outer space. Typically, FaRLiP relies on a cluster of ~20 genes, encoding paralogs of the standard photosynthetic machinery. One of them, a highly divergent D1 gene known as chlF (or psbA4), is the synthase responsible for the formation of the FRL-absorbing chlorophyll f (Chl f) that is essential for FaRLiP. The minimum gene set required for this phenotype is unclear. The desert cyanobacterium Chroococcidiopsis sp. CCMEE 010 is unusual in being capable of FaRLiP with a reduced gene cluster (15 genes), and it lacks most of the genes encoding FR-Photosystem I.MethodsHere we investigated whether the reduced gene cluster of Chroococcidiopsis sp. CCMEE 010 is transcriptionally regulated by FRL and characterized the spectral changes that occur during the FaRLiP response of Chroococcidiopsis sp. CCMEE 010. In addition, the heterologous expression of the Chl f synthase from CCMEE 010 was attempted in three closely related desert strains of Chroococcidiopsis.ResultsAll 15 genes of the FaRLiP cluster were preferentially expressed under FRL, accompanied by a progressive red-shift of the photosynthetic absorption spectrum. The Chl f synthase from CCMEE 010 was successfully expressed in two desert strains of Chroococcidiopsis and transformants could be selected in both VL and FRL.DiscussionIn Chroococcidiopsis sp. CCME 010, all the far-red genes of the unusually reduced FaRLiP cluster, are transcriptionally regulated by FRL and two closely related desert strains heterologously expressing the chlF010 gene could grow in FRL. Since the transformation hosts had been reported to survive outer space conditions, such an achievement lays the foundation toward novel cyanobacteria-based technologies to support human space exploration.

There are many potential approaches to marine carbon dioxide removal (mCDR), of which ocean iron fertilization (OIF) has the longest history of study. However, OIF studies to date were not primarily designed to quantify the durability of carbon (C) storage, nor how wise OIF might be as an mCDR approach. To quantify C sequestration, we introduce a metric called the “centennial tonne,” defined as 1,000 kg of C isolated from atmospheric contact for on average at least 100 years. We present the activities needed to assess OIF from a scientific and technological perspective, and additionally, how it might be responsibly studied and potentially deployed. The five activities include: field studies in the Northeast Pacific; improved modeling for field studies, data assimilation and predictions at larger scales; improvements in monitoring, reporting and verification (MRV) for C, and also MRV for tracking ecological and environmental impacts; and developing new iron sources and their delivery, to increase efficiencies and reduce costs. The fifth activity is to understand whether public and community support exists for OIF, and what governance structures might support further research and possible deployment of OIF. This article is written by a multidisciplinary experts group called Exploring Ocean Iron Solutions (ExOIS) that is organized around a responsible code of conduct. Of the mCDR approaches, OIF has the potential to be low cost, scalable, and rapidly deployable. Reducing CO2 emissions must lead the way, but there is also an urgency to decide under what conditions and whether OIF might be deployed or not.

Abstract Alpha-synuclein (αS)-rich Lewy bodies and neurites in the cerebral cortex correlate with the presence of dementia in Parkinson disease (PD) and Dementia with Lewy bodies (DLB), but whether αS influences synaptic vesicle dynamics in human cortical neurons is unknown. Using a new iPSC-based assay platform for measuring synaptic vesicle cycling, we found that in human cortical glutamatergic neurons, increased αS from either transgenic expression or triplication of the endogenous locus in patient-derived neurons reduced synaptic vesicle cycling under both stimulated and spontaneous conditions. Thus, using a robust, easily adopted assay platform, we show for the first time αS-induced synaptic dysfunction in human cortical neurons, a key cellular substrate for PD dementia and DLB.

Preventing the introduction of Listeria monocytogenes, subsequent biofilm formation, and persistence in food processing environments is important for reducing the risk of cross-contamination of ready-to-eat foods. This study determined the effect of Ɛ-poly-lysine (EPL), hydrogen peroxide (HP), and lauric arginate (LAE) on L. monocytogenes biofilm formation and the inactivation of mature biofilms. For inhibition studies, biofilms of L. monocytogenes Scott A (serotype 4b) and 2014L-6025 (serotype 1/2b) were developed separately at 37 °C for 48 h in the presence of sub-inhibitory concentrations (SIC) of either EPL (10 ppm), HP (2 ppm), or LAE (1.5 ppm) on polystyrene plates and stainless-steel rounds. Inactivation was determined by exposing mature biofilms on each surface to each antimicrobial at their minimum bactericidal concentration (MBC), 10xMBC, or 100xMBC for 24 h at 37 °C. The presence of these antimicrobials at SIC did not inhibit biofilm formation on either surface and their effect on mature biofilms varied by strain and surface. Application of EPL at 1xMBC (100 ppm) for 24 h resulted in greater reductions in counts of both strains on polystyrene than HP (40 ppm) and LAE (5 ppm) under the same conditions at 1xMBC (P ≤ 0.0243). Exposure of mature biofilms to LAE at 10xMBC (50 ppm) for 1 h was more effective in reducing counts on polystyrene than HP at 10xMBC (400 ppm) for the same duration (P ≤ 0.0136), and both HP and LAE applied at 100xMBC (4,000 and 500 ppm, respectively) for 24 h more effectively inactivated mature biofilms of L. monocytogenes Scott A on polystyrene compared to EPL (10,000 ppm) (P ≤ 0.0307). Application of LAE at 10xMBC for 24 h was more effective at inactivating strain Scott A on stainless steel compared to 10xMBC of EPL (1,000 ppm) or HP (P ≤ 0.0430). Future studies are needed to determine the efficacy of these and other antimicrobials on additional strains and serotypes of L. monocytogenes at temperatures relevant to food production and storage.

Recent studies on the use of plant-derived and other bioactive compounds and antimicrobials in food have challenged the idea that exposure to antimicrobials at sublethal or subinhibitory concentrations (SICs) increases the virulence potential of bacterial pathogens including Listeria monocytogenes. The objective of this study was to determine the effect of exposure to SICs of Ɛ-polylysine (EPL), hydrogen peroxide (HP), and lauric arginate (LAE) on L. monocytogenes virulence. For all assays, L. monocytogenes strains Scott A and 2014L-6025 were grown to mid-log phase in the presence of SICs of EPL, HP, or LAE. Motility was determined by spot inoculating cultures on soft brain heart infusion agar (0.3% agar). Cultures grown in SICs of antimicrobials were also inoculated onto Caco-2 cells (10:1 MOI) to determine the effects on subsequent adhesion and invasion. Last, the relative expression of key virulence genes (prfA, plcB, hlyA, actA, inlA, inlB, sigB, and virR) following growth in SICs was determined by RT-qPCR. Results indicate that L. monocytogenes growth in the presence of SICs of EPL, HP, or LAE did not affect the motility, adhesion, or invasion capacity of either strain. Changes in gene expression were observed for both L. monocytogenes strains. More specifically, SICs of EPL and LAE reduced hlyA expression in Scott A, whereas SICs of EPL and HP increased the expression of virR. The upregulation of sigB and actA in the presence of EPL and LAE, respectively, was observed in strain 2014L-6025. These findings indicate that exposure to SICs of these antimicrobials has varying effects on L. monocytogenes that differ by strain. Although no phenotypic effects were observed in terms of motility, adhesion, and invasion, the observed changes in virulence gene expression warrant further investigation.

Microalgae, known for their land-sparing cultivation and remarkable photosynthetic efficiency, play a crucial role in advancing sustainable development goals. This study explored the use of cost-effective inorganic carbon (the ionic form of CO2) and organic carbon sources (lactose, a primary sugar in whey wastewater) to support the growth of Chlorella sorokiniana UTEX 1230, and improved protein content through nitrogen supply optimization. NaHCO3 proved to be more effective than Na2CO3, resulting in a 3.4-fold increase in biomass concentration, with an initial 5 mM HCO3‾ concentration compared to the basal medium. Further addition of 0.75 % lactose led to a 4.05-fold increase in biomass concentration, with the maximum specific growth rate at 1.28/d, maximum biomass concentration at 695.88 mg/L, and maximum biomass productivity at 250.91 mg/L/d. Nitrogen supplementation greatly increased protein concentration from 16.41 % (250 mg/L NaNO3) to 52.95 % (1000 mg/L NaNO3). These results support the potential for utilizing whey wastewater bioremediation to produce high-value protein via microalgal cultivation.

AbstractThe measurement of the disparate impact of a particular de facto discriminatory policy on a minority or otherwise legally protected group has been of importance since passage of the Civil Rights Act of 1964. When the data available for the measurement of disparate impact, as embodied in the so-called “disparity ratio,” come from samples, a statistical approach naturally suggests itself. This article reviews both the law and statistics literature with regard to statistical inference applicable to the disparity ratio and related measures of disparate impact. From that review, three primary approaches are evaluated, the difference in so-called “rejection” rates for the protected and non-protected groups, their ratio (the disparity ratio), and the natural logarithm of the disparity ratio. For various reasons, the direct ratio estimator is recommended for use in all but small samples, where the log-ratio approach is to be preferred. The main points are illustrated with two fair housing examples, one being the possible discriminatory effect by race owing to a landlord’s refusal to accept Section 8 housing vouchers in lieu of cash rent, and the other being the effects of occupancy restrictions on families with children. Various methodological issues that arise in the application of these three estimation approaches are addressed in the context of the more complex sample designs that underlie the data utilized.

Lysosomal function is impaired in Niemann-Pick disease type C1 (NPC1), a rare and inherited neurodegenerative disorder, resulting in late endosomal/lysosomal accumulation of unesterified cholesterol. The precise pathogenic mechanism of NPC1 remains incompletely understood. In this study, we employed metabolomics to uncover secondary accumulated substances in NPC1. Our findings unveiled a substantial elevation in the levels of three alkyl-lysophosphatidylcholine [alkyl-LPC, also known as lyso-platelet activating factor (PAF)] species in NPC1 compared to controls across various tissues, including brain tissue from individuals with NPC1, liver, spleen, cerebrum, cerebellum, and brain stem from NPC1 mice, as well as in both brain and liver tissue from NPC1 cats. The three elevated alkyl-LPC species were as follows: LPC O-16:0, LPC O-18:1, and LPC O-18:0. However, the levels of PAF 16:0, PAF 18:1, and PAF 18:0 were not altered in NPC1. In the NPC1 feline model, the brain and liver alkyl-LPC levels were reduced following 2-hydroxypropyl-β-cyclodextrin (HPβCD) treatment, suggesting that alkyl-LPCs are secondary storage metabolites in NPC1 disease. Unexpectedly, cerebrospinal fluid (CSF) levels of LPC O-16:0 and LPC O-18:1 were decreased in individuals with NPC1 compared to age-appropriate comparison samples, and their levels were increased in 80% of participants 2 years after intrathecal HPβCD treatment. The fold increases in CSF LPC O-16:0 and LPC O-18:1 levels were more pronounced in responders compared to nonresponders. This study identified alkyl-LPC species as secondary storage metabolites in NPC1 and indicates that LPC O-16:0 and LPC O-18:1, in particular, could serve as potential biomarkers for tracking treatment response in NPC1 patients.

IntroductionMaternal intervillous monocytes (MIMs) and fetal Hofbauer cells (HBCs) are myeloid-derived immune cells at the maternal-fetal interface. Maternal reproductive history is associated with differential risk of pregnancy complications. The molecular phenotypes and roles of these distinct monocyte/macrophage populations and the influence of gravidity on these phenotypes has not been systematically investigated.MethodsHere, we used RNA sequencing to study the transcriptional profiles of MIMs and HBCs in normal term pregnancies.ResultsOur analyses revealed distinct transcriptomes of MIMs and HBCs. Genes involved in differentiation and cell organization pathways were more highly expressed in MIMs vs. HBCs. In contrast, HBCs had higher expression of genes involved in inflammatory responses and cell surface receptor signaling. Maternal gravidity influenced monocyte programming, as expression of pro-inflammatory molecules was significantly higher in MIMs from multigravidae compared to primigravidae. In HBCs, multigravidae displayed enrichment of gene pathways involved in cell-cell signaling and differentiation.DiscussionOur results demonstrated that MIMs and HBCs have highly divergent transcriptional signatures, reflecting their distinct origins, locations, functions, and roles in inflammatory responses. Furthermore, maternal gravidity influences the gene signatures of MIMs and HBCs, potentially modulating the interplay between tolerance and trained immunity. The phenomenon of reproductive immune memory may play a novel role in the differential susceptibility of primigravidae to pregnancy complications.

Abstract Mutations in the α-Synuclein (αS) gene promote αS monomer aggregation that causes neurodegeneration in familial Parkinson’s disease (fPD). However, most mouse models expressing single-mutant αS transgenes develop neuronal aggregates very slowly, and few have dopaminergic cell loss, both key characteristics of PD. To accelerate neurotoxic aggregation, we previously generated fPD αS E46K mutant mice with rationally designed triple mutations based on the α-helical repeat motif structure of αS (fPD E46K→3 K). The 3 K variant increased αS membrane association and decreased the physiological tetramer:monomer ratio, causing lipid- and vesicle-rich inclusions and robust tremor-predominant, L-DOPA responsive PD-like phenotypes. Here, we applied an analogous approach to the G51D fPD mutation and its rational amplification (G51D → 3D) to generate mutant mice. In contrast to 3 K mice, G51D and 3D mice accumulate monomers almost exclusively in the cytosol while also showing decreased αS tetramer:monomer ratios. Both 1D and 3D mutant mice gradually accumulate insoluble, higher-molecular weight αS oligomers. Round αS neuronal deposits at 12 mos immunolabel for ubiquitin and pSer129 αS, with limited proteinase K resistance. Both 1D and 3D mice undergo loss of striatal TH+ fibers and midbrain dopaminergic neurons by 12 mos and a bradykinesia responsive to L-DOPA. The 3D αS mice have decreased tetramer:monomer equilibria and recapitulate major features of PD. These fPD G51D and 3D mutant mice should be useful models to study neuronal αS-toxicity associated with bradykinetic motor phenotypes.

Abstract Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.

In this invited editorial, two long-tenured editors for the Journal of Patient-Centered Research and Reviews recount the publication’s many impactful contributions to the medical literature over its first 10 years.

Chylopericardium is a rare entity in veterinary medicine. In this report we document the development of chylopericardium in two dogs undergoing chronic hemodialysis. An 11-year-old female spayed Labrador retriever (Case 1) presented with acute coughing and lethargy 2 months following initial dialysis catheter placement and initiation of dialysis therapy for severe azotemia. Echocardiography demonstrated severe pericardial effusion and cardiac tamponade. Pericardial fluid analysis was consistent with chylous effusion. The dog underwent a subtotal pericardiectomy with thoracic duct ligation, and a PleuralPort™ was placed. The patient continued to receive outpatient hemodialysis therapy after pericardiectomy for several months until she died acutely at home. A 4-year-old male neutered Doberman (Case 2) was being treated for 2 months with outpatient hemodialysis for management of chronic kidney disease. On presentation for the 17th hemodialysis treatment, the patient had increased respiratory rate. Echocardiography demonstrated pleural and pericardial effusions, and fluid analysis in both cavities was consistent with chylous effusion. Use of tissue plasminogen activator (TPA), clot removal and replacement of the catheter was attempted; however pleural and pericardial effusion continued. The patient was euthanized after 25 hemodialysis sessions as owners elected not to pursue more procedures. In both cases, the cause of the chylopericardium was suspected to be secondary to catheter-associated thrombosis and/or stenosis based on multiple imaging modalities. Despite use of rivaroxaban and clopidogrel concurrently in each case, the chylous effusion persisted. This case report describes clinical details of a rare complication of long-term indwelling dialysis catheters in two dogs.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45–65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Feline infectious peritonitis (FIP) is a devastating and often fatal disease caused by feline coronavirus (FCoV). Currently, there is no widely used vaccine for FIP, and many attempts using a variety of platforms have been largely unsuccessful due to the disease’s highly complicated pathogenesis. One such complication is antibody-dependent enhancement (ADE) seen in FIP, which occurs when sub-neutralizing antibody responses to viral surface proteins paradoxically enhance disease. A novel vaccine strategy is presented here that can overcome the risk of ADE by instead using a lipid nanoparticle-encapsulated mRNA encoding the transcript for the internal structural nucleocapsid (N) FCoV protein. Both wild type and, by introduction of silent mutations, GC content-optimized mRNA vaccines targeting N were developed. mRNA durability in vitro was characterized by quantitative reverse-transcriptase PCR and protein expression by immunofluorescence assay for one week after transfection of cultured feline cells. Both mRNA durability and protein production in vitro were improved with the GC-optimized construct as compared to wild type. Immune responses were assayed by looking at N-specific humoral (by ELISA) and stimulated cytotoxic T cell (by flow cytometry) responses in a proof-of-concept mouse vaccination study. These data together demonstrate that an LNP–mRNA FIP vaccine targeting FCoV N is stable in vitro, capable of eliciting an immune response in mice, and provides justification for beginning safety and efficacy trials in cats.

Abstract Due to multi-drug resistance, physicians increasingly use the last-resort antibiotic colistin to treat infections with the Gram-negative bacterium Klebsiella pneumoniae. Unfortunately, K. pneumoniae can also develop colistin resistance. Interestingly, colistin resistance has dual effects on bacterial clearance by the immune system. While it increases resistance to antimicrobial peptides, colistin resistance has been reported to sensitize certain bacteria for killing by human serum. Here we investigate the mechanisms underlying this increased serum sensitivity, focusing on human complement which kills Gram-negatives via membrane attack complex (MAC) pores. Using in vitro evolved colistin resistant strains and a fluorescent MAC-mediated permeabilization assay, we showed that two of the three tested colistin resistant strains, Kp209_CSTR and Kp257_CSTR, were sensitized to MAC. Transcriptomic and mechanistic analyses focusing on Kp209_CSTR revealed that a mutation in the phoQ gene locked PhoQ in an active state, making Kp209_CSTR colistin resistant and MAC sensitive. Detailed immunological assays showed that complement activation on Kp209_CSTR in human serum required specific IgM antibodies that bound Kp209_CSTR but did not recognize the wild-type strain. Together, our results show that developing colistin resistance affected recognition of Kp209_CSTR and its killing by the immune system.

Abstract Somatic activating MAP2K1 mutations in endothelial cells (ECs) cause extracranial arteriovenous malformation (AVM). We previously reported the generation of a mouse line allowing inducible expression of constitutively active MAP2K1 (p.K57N) from the Rosa locus (R26 GT-Map2k1-GFP/+) and showed, using Tg-Cdh5CreER, that EC expression of mutant MAP2K1 is sufficient for the development of vascular malformations in the brain, ear, and intestines. To gain further insight into the mechanism by which mutant MAP2K1 drives AVM development, we induced MAP2K1 (p.K57N) expression in ECs of postnatal-day-1 pups (P1) and investigated the changes in gene expression in P9 brain ECs by RNA-seq. We found that over-expression of MAP2K1 altered the transcript abundance of > 1600 genes. Several genes had > 20-fold changes between MAP2K1 expressing and wild-type ECs; the highest were Col15a1 (39-fold) and Itgb3 (24-fold). Increased expression of COL15A1 in R26 GT-Map2k1-GFP/+; Tg-Cdh5CreER +/− brain ECs was validated by immunostaining. Ontology showed that differentially expressed genes were involved in processes important for vasculogenesis (e.g., cell migration, adhesion, extracellular matrix organization, tube formation, angiogenesis). Understanding how these genes and pathways contribute to AVM formation will help identify targets for therapeutic intervention.

Spotted fever group rickettsiae are tick-borne obligate intracellular bacteria that infect microvascular endothelial cells. Humans and mammalian infection results in endothelial cell barrier dysfunction and increased vascular permeability. We previously demonstrated that treatment of Rickettsia parkeri-infected cells with the calcium channel blocker benidipine significantly delayed vascular barrier permeability. Thus, we hypothesized that benidipine, known to be safe and effective for other clinical processes, could reduce rickettsia-induced vascular permeability in vivo in an animal model of spotted fever rickettsiosis. Based on liver, lung and brain vascular FITC-dextran extravasation studies, benidipine did not reliably impact vascular permeability. However, it precipitated a deleterious effect on responses to control sublethal R. parkeri infection. Animals treated with benidipine alone had no clinical signs or changes in histopathology and splenic immune cell distributions. Benidipine-treated infected animals had marked increases in tissue and blood bacterial loads, more extensive inflammatory histopathologic injury, and changes in splenic architecture and immune cell distributions potentially reflecting diminished Ca2+ signaling, reduced innate immune cell activation, and loss of rickettsial propagation control. Impaired T cell activation by R. parkeri antigen in the presence of benidipine was confirmed in vitro with the use of NKT cell hybridomas. The unexpected findings stand in stark contrast to recent discussions of the benefits of calcium channel blockers for viral infections and chronic infectious or inflammatory diseases. A role for calcium channel blockers in exacerbation of human rickettsiosis and acute inflammatory infections should be evaluated by a retrospective review of patient's outcomes and medications.

Auditory brainstem neurons in the lateral superior olive (LSO) receive excitatory input from the ipsilateral cochlear nucleus (CN) and inhibitory transmission from the contralateral CN via the medial nucleus of the trapezoid body (MNTB). This circuit enables sound localization using interaural level differences. Early studies have observed an additional inhibitory input originating from the ipsilateral side. However, many of its details, such as its origin, remained elusive. Employing electrical and optical stimulation of afferents in acute mouse brainstem slices and anatomical tracing, we here describe a glycinergic projection to LSO principal neurons that originates from the ipsilateral CN. This inhibitory synaptic input likely mediates inhibitory sidebands of LSO neurons in response to acoustic stimulation.

Abstract Background Studies suggest that group-based well child care—a shared medical appointment where families come together as a group to receive pediatric primary care—increases patient-reported satisfaction and adherence to recommended care. Evidence supporting the use of group well child care for mothers with opioid use disorder, however, is lacking. The overall objective of the Child Healthcare at MATER Pediatric Study (CHAMPS) trial is to evaluate a group model of well child care for mothers with opioid use disorder and their children. Methods CHAMPS is a single-site 2-arm cluster randomized controlled trial. A total of 108 mother–child dyads will be enrolled into the study. Twenty-six clusters of approximately 4 mother-infant dyads each will be randomized 1:1 to one of two study arms (intervention or control). Clustering will be based on child’s month of birth. In the intervention arm, group well child care will be provided on-site at a maternal substance use disorder treatment program. Mother–child dyads in the control arm will receive individual well child care from one nearby pediatric primary care clinic. Dyads in both study arms will be followed prospectively for 18 months, and data will be compared between the two study arms. Primary outcomes include well child care quality and utilization, child health knowledge, and parenting quality. Discussion The CHAMPS trial will provide evidence to determine if a group well child care offered on-site at an opioid treatment program for pregnant and parenting women is beneficial over individual well child care for families impacted by maternal opioid use disorder. Trial registration ClinicalTrials.gov identifier: NCT05488379. Registered on Aug. 04, 2022.

Anita Lynam,1 Charlotte Curtis,1 Brooklyn Stanley,2,3 Heath Heatley,3 Chloe Worthington,2,3 Emma-Jane Roberts,2,3 Christopher Price,2,3 Victoria Carter,2,3 John Dennis,1 Andrew McGovern,1 David Price2,3 1Momentum Data, Pendragon House, St. Albans, Hertfordshire, UK; 2Optimum Patient Care, Cambridge, UK; 3Observational and Pragmatic Research Institute, SingaporeCorrespondence: Andrew McGovern, Momentum Data, Pendragon House, St. Albans, Hertfordshire, UK, Email andy.mcgovern@momentumdata.co.ukIntroduction: Electronic medical records (EMRs) maintained in primary care in the UK and collected and stored in EMR databases offer a world-leading resource for observational clinical research. We aimed to profile one such database: the Optimum Patient Care Research Database (OPCRD).Methods and Participants: The OPCRD, incepted in 2010, is a growing primary care EMR database collecting data from 992 general practices within the UK. It covers over 16.6 million patients across all four countries within the UK, and is broadly representative of the UK population in terms of age, sex, ethnicity and socio-economic status. Patients have a mean duration of 11.7 years’ follow-up (SD 17.50), with a majority having key summary data from birth to last data entry. Data for the OPCRD are collected incrementally monthly and extracted from all of the major clinical software systems used within the UK and across all four coding systems (Read version 2, Read CTV3, SNOMED DM+D and SNOMED CT codes). Via quality-improvement programmes provided to GP surgeries, the OPCRD also includes patient-reported outcomes from a range of disease-specific validated questionnaires, with over 66,000 patient responses on asthma, COPD, and COVID-19. Further, bespoke data collection is possible by working with GPs to collect new research via patient-reported questionnaires.Findings to Date: The OPCRD has contributed to over 96 peer-reviewed research publications since its inception encompassing a broad range of medical conditions, including COVID-19.Conclusion: The OPCRD represents a unique resource with great potential to support epidemiological research, from retrospective observational studies through to embedded cluster-randomised trials. Advantages of the OPCRD over other EMR databases are its large size, UK-wide geographical coverage, the availability of up-to-date patient data from all major GP software systems, and the unique collection of patient-reported information on respiratory health.Keywords: primary care, electronic health records, medical records, datasets, demography, health outcomes

A wide-field imaging system uses birefringence to efficiently permit quantitative fluorophore concentration mapping, with clear relevance for neurosurgery.

It is increasingly recognized that very small proteins (μ-proteins) are ubiquitously found in all species of the three domains of life, and that they fulfill important functions. The halophilic archaeon Haloferax volcanii contains 282 μ-proteins of less than 70 amino acids. Notably, 43 of these contain two C(P)XCG motifs, suggesting their potential to complex a zinc ion. To explore the significance of these proteins, 16 genes encoding C(P)XCG proteins had been deleted, and the majority of mutants exhibited phenotypic differences to the wild-type. One such protein, HVO_2753, was thoroughly characterized in a previous study. In the present study an in-depth analysis of a second protein, HVO_0758, was performed. To achieve this goal, the HVO_0758 protein was produced heterologously in Escherichia coli and homologously in H. volcanii. The purified protein was characterized using various biochemical approaches and NMR spectroscopy. The findings demonstrated that HVO_0758 is indeed a bona fide zinc finger protein, and that all four cysteine residues are essential for folding. The NMR solution structure was solved, revealing that HVO_0758 is comprised of an N-terminal alpha helix containing several positively charged residues and a globular core with the zinc finger domain. The transcriptomes of the HVO_0758 deletion mutant and, for comparison, the HVO_2753 deletion mutant were analyzed with RNA-Seq and compared against that of the wild-type. In both mutants many motility and chemotaxis genes were down-regulated, in agreement to the phenotype of the deletion mutants, which had a swarming deficit. The two H. volcanii zinc-finger μ-proteins HVO_0758 and HVO_2753 showed many differences. Taken together, two zinc finger μ-proteins of H. volcanii have been characterized intensively, which emerged as pivotal contributors to swarming behavior and biofilm formation.

The Mn3Ga material is a promising candidate for memory and computing devices owing to its rich crystalline structures of tunable ferrimagnetic and collinear and non-collinear antiferromagnetic phases. In particular, Mn3Ga with non-collinear antiferromagnetic order exhibits giant anomalous and topological Hall conductivities and is a potential material platform for hosting spin-related quantum phenomena. In this study, we demonstrate Mn3Ga films grown on thermally oxidized Si substrates, with and without the Ta buffer, under different deposition temperatures (Ts). With increasing Ts, the dominant crystalline structure across all Mn3Ga films evolves from a cubic to hybrid tetragonal and hexagonal texture, wherein the crystalline orientation of spins endows the films with in-plane magnetic anisotropy. For Ta/Mn3Ga and Mn3Ga films grown under high Ts, the inhomogeneity in surface energy of the buffer layer results in a non-uniform granular film in the former. Notably, the Mn3Ga films of hexagonal texture exhibit topological Hall signatures. The density functional theory calculations on the hexagonal Mn3Ga phase corroborated with the experimental magnetic, structural, and transport properties. These findings establish an important platform for tailoring Mn3Ga films toward multifunctional applications.

ABSTRACT Anabaena sp. PCC 7120 grows by forming filaments of communicating cells and is considered a paradigm of bacterial multicellularity. Molecular exchanges between contiguous cells in the filament take place through multiprotein channels that traverse the septal peptidoglycan through nanopores connecting their cytoplasms. Besides, the septal-junction complexes contribute to strengthen the filament. In search for proteins with coiled-coil domains that could provide for cytoskeletal functions in Anabaena, we identified SepT (All2460). SepT is characteristic of the phylogenetic clade of filamentous cyanobacteria with the ability to undergo cell differentiation. SepT-GFP fusions indicate that the protein is located at the cell periphery and, conspicuously, in the intercellular septa. During cell division, the protein is found at midcell resembling the position of the divisome. The bacterial adenylate cyclase two-hybrid analysis shows SepT interactions with itself and putative elongasome (MreB, RodA), divisome (FtsW, SepF, ZipN), and septal-junction (SepJ)-related proteins. Thus, SepT appears to rely on the divisome for localization at mature intercellular septa to form part of intercellular protein complexes. Two independently obtained mutants lacking SepT showed alterations in cell size and impaired septal and peripheral peptidoglycan incorporation during cell growth and division. Notably, both mutants showed conspicuous alterations in the array of nanopores present in the intercellular peptidoglycan disks, including aberrant nanopore morphology, number, and distribution. SepT appears, therefore, to be involved in the control of peptidoglycan growth and the formation of cell-cell communication structures that are at the basis of the multicellular character of this group of cyanobacteria. IMPORTANCE Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism.

Psychotropic medications are typically prescribed in a trial-and-error fashion, and some providers are beginning to utilize pharmacogenetic testing (PGx) as a supplemental prescribing tool in treatment decision making. PGx testing shows potential in enhancing provider insights into personalized prescribing for patients by examining genetic information related to drug metabolism. Literature points to providers’ lack of knowledge in PGx interpretation as a main barrier, including psychiatric mental health nurse practitioners (PMHNPs). The aim of this study was to measure a difference, if any, in the knowledge and perceptions of PGx after implementation using a pre-post design. This study implemented an educational intervention on graduate nursing students (n = 15). Data were collected by using a pre- and post-interventional questionnaire. Results demonstrated a significant difference in findings related to students’ knowledge (p < 0.001), students’ skills related to pharmacogenetics, (p < 0.001), as well as students’ perceived ability to implement pharmacogenetics into their practice, (p = 0.028). The authors propose that the knowledge gained from the study demonstrates the importance of introducing PGx education into the PMHNP curricula and to prepare future PMHNPs to confidently utilize PGx in their clinical practice.

Background: Ophthalmic segment aneurysms that divide the optic nerve are exceedingly rare. We present a unique case of asymptomatic optic nerve splitting secondary into internal carotid artery ophthalmic segment aneurysm that was successfully treated with flow diversion.Case Description: A 58-year-old female presented to an outside hospital with an intermittent “rubbing sound” in her right ear. Imaging was remarkable for a left anterior skull base meningioma and a left internal carotid artery ophthalmic segment aneurysm. Humphrey visual field testing confirmed lack of scotoma or visual deficit. Intraoperatively, during craniotomy for resection of the meningioma, the aneurysm dome was noted to be splitting the optic nerve with a thin layer covering the dome.Given that the patient was asymptomatic and had intact visual function, it was decided that flow diversion would be safer option than clipping to avoid any optic nerve manipulation. On follow up, the patient remained free of visual impairment with an occluded aneurysm. Conclusion: This case presentation of a unique, asymptomatic ophthalmic segment aneurysm with splitting of the optic nerve, demonstrates successful treatment with flow diversion. It is notable both for the asymptomatic presentation and for highlighting the successful treatment of this kind of aneurysm with flow diversion.

Abstract In Parkinson’s disease and other synucleinopathies, the elevation of α-synuclein phosphorylated at Serine129 (pS129) is a widely cited marker of pathology. However, the physiological role for pS129 has remained undefined. Here we use multiple approaches to show for the first time that pS129 functions as a physiological regulator of neuronal activity. Neuronal activity triggers a sustained increase of pS129 in cultured neurons (200% within 4 h). In accord, brain pS129 is elevated in environmentally enriched mice exhibiting enhanced long-term potentiation. Activity-dependent α-synuclein phosphorylation is S129-specific, reversible, confers no cytotoxicity, and accumulates at synapsin-containing presynaptic boutons. Mechanistically, our findings are consistent with a model in which neuronal stimulation enhances Plk2 kinase activity via a calcium/calcineurin pathway to counteract PP2A phosphatase activity for efficient phosphorylation of membrane-bound α-synuclein. Patch clamping of rat SNCA −/− neurons expressing exogenous wild-type or phospho-incompetent (S129A) α-synuclein suggests that pS129 fine-tunes the balance between excitatory and inhibitory neuronal currents. Consistently, our novel S129A knock-in (S129AKI) mice exhibit impaired hippocampal plasticity. The discovery of a key physiological function for pS129 has implications for understanding the role of α-synuclein in neurotransmission and adds nuance to the interpretation of pS129 as a synucleinopathy biomarker.

Abstract Background KRISTINE is an open-label, phase III study of trastuzumab emtansine + pertuzumab (T-DM1 + P) versus docetaxel + carboplatin + trastuzumab + pertuzumab (TCH + P) in patients with HER2-positive, stage II–III breast cancer. We investigated the association of biomarkers with clinical outcomes in KRISTINE. Methods Patients were randomized to receive neoadjuvant T-DM1 + P or TCH + P and assessed for pathologic complete response (pCR; ypT0/is, ypN0). HER2 status (per central assessment), hormone receptor status, PIK3CA mutation status, HER2/HER3 mRNA levels, tumor-infiltrating lymphocyte levels, PD-L1 status, and NanoString data were analyzed. pCR rates by treatment arm were compared across biomarker subgroups. Analyses were descriptive. Results Biomarker analyses included data from all 444 patients (T-DM1 + P, n = 223; TCH + P, n = 221) enrolled in KRISTINE. Biomarker distribution was balanced across treatment arms. All subgroups with higher HER2 amplification/expression and immune marker levels showed numerically higher pCR rates in both arms. Mutated versus non-mutated PIK3CA tumors were associated with numerically lower pCR rates in the T-DM1 + P arm but not in the TCH + P arm. In a multivariate analysis, Prediction Analysis of Microarray with the 50-gene classifier (PAM50) HER2-enriched subtype, HER2 gene ratio ≥ 4, and PD-L1-positive status positively influenced the pCR rate. Biomarkers associated with lower pCR rates (e.g., low HER2 levels, positive hormone receptor status, mutated PIK3CA) were more likely to co-occur. Dynamic on-treatment biomarker changes were observed. Differences in the treatment effects for T-DM1 + P versus TCH + P were similar to those observed in the intent-to-treat population for the majority of the biomarker subgroups. Conclusions Although our biomarker analysis did not identify a subgroup of patients that benefited from neoadjuvant T-DM1 + P versus TCH + P, the data revealed that patients with higher HER2 amplification/expression and immune marker levels had improved response irrespective of treatment arm. These analyses confirm the role of HER2 tumor biology and the immune microenvironment in influencing pCR in the neoadjuvant setting and reaffirm the molecular diversity of HER2-positive breast cancer. Trial Registration: ClinicalTrials.gov NCT02131064. Registered 06 May 2014.

Abstract The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.

Objective: To assess the utilization of preventive health services and the prevalence of chronic health conditions among a cohort of women in treatment for opioid use disorder (OUD). Methods: Ninety-seven women who were receiving treatment for OUD from a single urban treatment program completed a self-administered anonymous online questionnaire that asked about demographics, health, receipt of preventive health services, and utilization of health care. Descriptive statistics were used to describe data. Results: More than one-third of respondents reported that their health was fair or poor, whereas one-quarter were very concerned with their health. Most participants (59%) reported at least one chronic health condition; nearly 1 in 5 reported two or more conditions. Less than half of respondents had received a routine medical examination in the past year. Vaccine uptake was low; 56% received the coronavirus disease 2019 vaccine and 36% received the annual influenza vaccine. Conclusions: Women in treatment for OUD could benefit from enhanced health care to address the high rates of chronic diseases and risk factors and underutilization of recommended preventive health services. Interventions and models of care that aim to enhance utilization of such services, and ultimately improve the health of this vulnerable population, may be worth exploring.

Alboglossiphonia pallida (Verrill, 1872) comb. nov. is resurrected and redescribed based on morphological and molecular data from specimens of the type locality (New Haven County, Connecticut, USA) that demonstrate it is distinct from North American Alboglossiphonia heteroclita, European Alboglossiphonia heteroclita, and Alboglossiphonia papillosa. Alboglossiphonia pallida is characterized by having dark chromatophores on the dorsal surface arranged lateral to patrilaterally and medially as a thin line or interrupted thin line along with three pairs of eye spots (with the first pair closest together), six pairs of crop ceca, and a united gonopore. Additional sampling of specimens of the genus Alboglossiphonia is needed to understand its phylogeny especially as many species have not been collected since their description.

Abstract Background Both the gut microbiota and chronic viral infections have profound effects on host immunity, but interactions between these influences have been only superficially explored. Cytomegalovirus (CMV), for example, infects approximately 80% of people globally and drives significant changes in immune cells. Similarly, certain gut-resident bacteria affect T-cell development in mice and nonhuman primates. It is unknown if changes imposed by CMV on the intestinal microbiome contribute to immunologic effects of the infection. Results We show that rhesus cytomegalovirus (RhCMV) infection is associated with specific differences in gut microbiota composition, including decreased abundance of Firmicutes, and that the extent of microbial change was associated with immunologic changes including the proliferation, differentiation, and cytokine production of CD8+ T cells. Furthermore, RhCMV infection disrupted the relationship between short-chain fatty acid producers and Treg/Th17 balance observed in seronegative animals, showing that some immunologic effects of CMV are due to disruption of previously existing host-microbe relationships. Conclusions Gut microbes have an important influence on health and disease. Diet is known to shape the microbiota, but the influence of concomitant chronic viral infections is unclear. We found that CMV influences gut microbiota composition to an extent that is correlated with immunologic changes in the host. Additionally, pre-existing correlations between immunophenotypes and gut microbes can be subverted by CMV infection. Immunologic effects of CMV infection on the host may therefore be mediated by two different mechanisms involving gut microbiota. Video Abstract

Acute myeloid leukaemia (AML) is maintained by self-renewing leukemic stem cells. Here the authors show that Heat Shock Transcription Factor 1 (HSF1) is specifically required for the maintenance of AML stem cells, while sparing steady-state and stressed haematopoiesis and that pharmacologically targeting HSF1 may have broad anti-leukemic effects.

Abstract α-Synuclein (αSyn) aggregation in Lewy bodies and neurites defines both familial and ‘sporadic’ Parkinson’s disease. We previously identified α-helically folded αSyn tetramers, in addition to the long-known unfolded monomers, in normal cells. PD-causing αSyn mutations decrease the tetramer:monomer (T:M) ratio, associated with αSyn hyperphosphorylation and cytotoxicity in neurons and a motor syndrome of tremor and gait deficits in transgenic mice that responds in part to L-DOPA. Here, we asked whether LRRK2 mutations, the most common genetic cause of cases previously considered sporadic PD, also alter tetramer homeostasis. Patient neurons carrying G2019S, the most prevalent LRRK2 mutation, or R1441C each had decreased T:M ratios and pSer129 hyperphosphorylation of their endogenous αSyn along with increased phosphorylation of Rab10, a widely reported substrate of LRRK2 kinase activity. Two LRRK2 kinase inhibitors normalized the T:M ratio and the hyperphosphorylation in the G2019S and R1441C patient neurons. An inhibitor of stearoyl-CoA desaturase, the rate-limiting enzyme for monounsaturated fatty acid synthesis, also restored the αSyn T:M ratio and reversed pSer129 hyperphosphorylation in both mutants. Coupled with the recent discovery that PD-causing mutations of glucocerebrosidase in Gaucher’s neurons also decrease T:M ratios, our findings indicate that three dominant genetic forms of PD involve life-long destabilization of αSyn physiological tetramers as a common pathogenic mechanism that can occur upstream of progressive neuronal synucleinopathy. Based on αSyn’s finely-tuned interaction with certain vesicles, we hypothesize that the fatty acid composition and fluidity of membranes regulate αSyn’s correct binding to highly curved membranes and subsequent assembly into metastable tetramers.

In this work, we describe the formation of geometric phases during nonadiabatic frequency swept (FS) radio frequency (RF) pulses with sine amplitude modulation and cosine frequency modulation functions. The geometric phases during the FS pulse were analyzed using a Schrödinger equation formalism, and the unified analytical expression for the geometric phase was derived. We present the solutions for sub-geometric phase components incorporated in spinor wavefunctions for the RF Hamiltonian of spin ½ nuclei. We demonstrate that the geometric phases during sine/cosine RF pulses are opposite in signs for different initial conditions of the spinor and that geometric phases can accumulate in correspondence to different magnetization trajectories. The derived formalism could be extended for the evaluation of the geometric phases during a wide class of amplitude- and frequency-modulated pulses used in MRI and in high-resolution NMR.

IntroductionImmune checkpoint inhibitors (ICIs) only benefit a subset of cancer patients, underlining the need for predictive biomarkers for patient selection. Given the limitations of tumor tissue availability, flow cytometry of peripheral blood mononuclear cells (PBMCs) is considered a noninvasive method for immune monitoring. This study explores the use of spectrum flow cytometry, which allows a more comprehensive analysis of a greater number of markers using fewer immune cells, to identify potential blood immune biomarkers and monitor ICI treatment in non-small-cell lung cancer (NSCLC) patients.MethodsPBMCs were collected from 14 non-small-cell lung cancer (NSCLC) patients before and after ICI treatment and 4 healthy human donors. Using spectrum flow cytometry, 24 immune cell markers were simultaneously monitored using only 1 million PBMCs. The results were also compared with those from clinical flow cytometry and bulk RNA sequencing analysis. ResultsOur findings showed that the measurement of CD4+ and CD8+ T cells by spectrum flow cytometry matched well with those by clinical flow cytometry (Pearson R ranging from 0.75 to 0.95) and bulk RNA sequencing analysis (R=0.80, P=1.3 x 10-4). A lower frequency of CD4+ central memory cells before treatment was associated with a longer median progression-free survival (PFS) [Not reached (NR) vs. 5 months; hazard ratio (HR)=8.1, 95% confidence interval (CI) 1.5–42, P=0.01]. A higher frequency of CD4-CD8- double-negative (DN) T cells was associated with a longer PFS (NR vs. 4.45 months; HR=11.1, 95% CI 2.2–55.0, P=0.003). ICIs significantly changed the frequency of cytotoxic CD8+PD1+ T cells, DN T cells, CD16+CD56dim and CD16+CD56- natural killer (NK) cells, and CD14+HLDRhigh and CD11c+HLADR + monocytes. Of these immune cell subtypes, an increase in the frequency of CD16+CD56dim NK cells and CD14+HLADRhigh monocytes after treatment compared to before treatment were associated with a longer PFS (NR vs. 5 months, HR=5.4, 95% CI 1.1-25.7, P=0.03; 7.8 vs. 3.8 months, HR=5.7, 95% CI 169 1.0-31.7, P=0.04), respectively. ConclusionOur preliminary findings suggest that the use of multicolor spectrum flow cytometry helps identify potential blood immune biomarkers for ICI treatment, which warrants further validation.

Case series summary A 2-year-old female spayed Abyssinian cat was evaluated for lethargy and inappetence that first occurred approximately 4 days prior. In addition, urination had not been observed by the owner for 5 days. A Lilium species plant had been brought into the house approximately 5 days before initial evaluation, and intoxication was therefore suspected. Bloodwork revealed anemia, severe azotemia and hyperkalemia. As the cat was anuric with severe azotemia, hyperkalemia and fluid overload, intermittent hemodialysis was recommended. Attempts were made to place a hemodialysis catheter into the external jugular veins bilaterally, but after initial successful venipuncture, the instrumentation would not pass into either vein. During fluoroscopic angiography, no internal jugular veins or external jugular veins caudal to the thoracic inlet were visualized and venous drainage from the head occurred via the vertebral veins. Owing to the anomalous anatomy in the cervical region, a hemodialysis catheter could not be placed into either jugular vein. Alternative sites for the placement of an extracorporeal catheter were discussed with the owner, but humane euthanasia was elected. Relevance and novel information To the authors’ knowledge, this is the first report of a cat with bilateral external jugular vein anomalies resulting in blind endings that did not communicate with the vena cava. This was discovered during attempts to place a hemodialysis catheter for the management of anuric renal failure secondary to Lilium species intoxication. While this anatomical variation is likely uncommon, it is an important differential to consider when faced with challenging external jugular vein catheterizations in feline patients.

Hemoglobin (Hb) present in the lung epithelium is of unknown significance. However Hb being an nitric oxide (NO) scavenger can bind to NO and reduce its deleterious effects. Hence we postulated an NO scavenging role for this lung Hb. Doing transwell co-culture with bronchial epithelial cells, A549/16-HBE (apical) and human airway smooth muscle cells (HASMCs as basal), we found that Hb can protect the smooth muscle soluble guanylyl cyclase (sGC) from excess NO. Inducing the apical A549/16-HBE cells with cytokines to trigger iNOS expression and NO generation caused a time dependent increase in SNO-sGC and this was accompanied with a concomitant drop in sGC-α1β1 heterodimerization. Silencing Hbαβ in the apical cells further increased the SNO on sGC with a faster drop in the sGC heterodimer and these effects were additive along with further silencing of thioredoxin 1 (Trx1). Since heme of Hb is critical for NO scavenging we determined the Hb heme in a mouse model of allergic asthma (OVA) and found that Hb in the inflammed OVA lungs was low in heme or heme-free relative to those of naïve lungs. Further we established a direct correlation between the status of the sGC heterodimer and the Hb heme from lung samples of human asthma, iPAH, COPD and cystic fibrosis. These findings present a new mechanism of protection of lung sGC by the epithelial Hb, and suggests that this protection maybe lost in asthma or COPD where lung Hb is unable to scavenge the NO due to it being heme-deprived.