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Abstract Superantigens (sAgs) are bacterial virulence factors that induce a state of immune hyperactivation by forming a bridge between certain subsets of T cell receptor (TCR) β chains on T lymphocytes, and class II major histocompatibility complex (MHC-II) molecules; this cross-linking leads to indiscriminate T cell activation, cytokine storm and toxic shock. Here we show that sAg exposure drives the preferential expansion of naive and central memory T cell subsets, but not effector or resident memory T cells, which instead, hyper release pro-inflammatory cytokines. A targeted therapeutic approach to minimise cytokine release by effector memory T cells attenuated sAg-induced cytokine release. Irrespective of antigen experience, sAg activation does not render mature T cells permanently dysfunctional, and full restoration of effector function is observed following a transient and reversible anergy. Moreover, we show that in the face of sAg induced immune hyperactivation, an intact bacterium-specific CD4+ T cell response can be mounted.

Abstract Dynamin-like GTPase proteins, including myxoma (Mx) and guanylate-binding proteins (GBPs), are among the many interferon stimulated genes induced following viral infections. While studies report that human (h)GBPs inhibit different viruses in vitro, few have convincingly demonstrated that mouse (m)GBPs mediate antiviral activity, although mGBP-deficient mice have been used extensively to define their importance in immunity to diverse intracellular bacteria and protozoa. Herein, we demonstrate that individual (overexpression) or collective (knockout (KO) mice) mGBPs of the chromosome 3 cluster (mGBPchr3) do not inhibit replication of five viruses from different virus families in vitro, nor do we observe differences in virus titres recovered from wild type versus mGBPchr3 KO mice after infection with three of these viruses (influenza A virus, herpes simplex virus type 1 or lymphocytic choriomeningitis virus). These data indicate that mGBPchr3 do not appear to be a major component of cell-intrinsic antiviral immunity against the diverse viruses tested in our studies.

Purpose Pruritus is an unpleasant sensation that creates the urge to scratch. In many chronic conditions, relentless pruritus and scratching perpetuates a vicious itch-scratch cycle. Uncontrolled itch can detrimentally affect quality of life and may lead to sleep disturbance, impaired concentration, financial burden, and psychological suffering. Recent strides have been made to develop guidelines and investigate new therapies to treat some of the most common severely pruritic conditions, however, a large group of diseases remains underrecognized and undertreated. The purpose of this article is to provide a comprehensive review of the challenges hindering the treatment of pruritus.Methods An online search was performed using PubMed, Web of Science, Google Scholar, and ClinicalTrials.gov from 1994 to 2024. Included studies were summarized and assessed for quality and relevance in treating pruritus.Results Several barriers to treating pruritus emerged, including variable presentation, objective measurement of itch, and identifying therapeutic targets. Itch associated with autoimmune conditions, connective tissue diseases, genodermatoses, cutaneous T-cell lymphoma, and pruritus of unknown origin were among the etiologies with the greatest unmet needs.Conclusion Treating pruritus poses many challenges and there are many itchy conditions that have no yet been addressed. There is an urgent need for large-scale controlled studies to investigate potential targets for these conditions and novel therapies.

Abstract Myxovirus resistance (Mx) proteins are products of interferon stimulated genes (ISGs) and Mx proteins of different species have been reported to mediate antiviral activity against a number of viruses, including influenza A viruses (IAV). Ferrets are widely considered to represent the ‘gold standard’ small animal model for studying pathogenesis and immunity to human IAV infections, however little is known regarding the antiviral activity of ferret Mx proteins. Herein, we report induction of ferret (f)Mx1/2 in a ferret lung cell line and in airway tissues from IAV-infected ferrets, noting that fMx1 was induced to higher levels that fMx2 both in vitro and in vivo. Overexpression confirmed cytoplasmic expression of fMx1 as well as its ability to inhibit infection and replication of IAV, noting that this antiviral effect of fMx1was modest when compared to cells overexpressing either human MxA or mouse Mx1. Together, these studies provide the first insights regarding the role of fMx1 in cell innate antiviral immunity to influenza viruses. Understanding similarities and differences in the antiviral activities of human and ferret ISGs provides critical context for evaluating results when studying human IAV infections in the ferret model.

Abstract SABRE (Sodium iodide with Active Background REjection) is a direct detection dark matter experiment based on arrays of radio-pure NaI(Tl) crystals. The experiment aims at achieving an ultra-low background rate and its primary goal is to confirm or refute the results from the DAMA/LIBRA experiment. The SABRE Proof-of-Principle phase was carried out in 2020–2021 at the Gran Sasso National Laboratory (LNGS), in Italy. The next phase consists of two full-scale experiments: SABRE South at the Stawell Underground Physics Laboratory, in Australia, and SABRE North at LNGS. This paper focuses on SABRE South and presents a detailed simulation of the detector, which is used to characterise the background for dark matter searches including DAMA/LIBRA-like modulation. We estimate an overall background of 0.72 cpd/kg/ $$\hbox {keV}_{\hbox {{ee}}}$$ keV ee in the energy range 1–6 $$\hbox {keV}_{\hbox {{ee}}}$$ keV ee primarily due to radioactive contamination in the crystals. Given this level of background and considering that the SABRE South has a target mass of 50 kg, we expect to exclude (confirm) DAMA/LIBRA modulation at $$4~(5)\sigma $$ 4 ( 5 ) σ within 2.5 years of data taking.

Abstract Many chronic disease symptomatologies involve desynchronized sleep-wake cycles, indicative of disrupted biorhythms. This can be interrogated using body temperature rhythms, which have circadian as well as sleep-wake behavior/environmental evoked components. Here, we investigated the association of wrist temperature amplitudes with a future onset of disease in the UK Biobank one year after actigraphy. Among 425 disease conditions (range n = 200-6728) compared to controls (range n = 62,107-91,134), a total of 73 (17%) disease phenotypes were significantly associated with decreased amplitudes of wrist temperature (Benjamini-Hochberg FDR q

Abstract A 47‐year‐old man with a background history of gastroesophageal reflux disease (GERD) and seasonal asthma underwent a gastroscopy for further investigation. Endoscopy revealed numerous polypoid lesions diffusely distributed in the lower third of the esophagus, with histology revealing squamous papilloma with occasional intraepithelial lymphocytes. The diagnosis was esophageal squamous papillomatosis (ESP), which is a rare condition characterized by exophytic and circumferential projections with friable mucosa diffusely spread through the esophagus with unclear etiology and malignancy risk.

Highlights The (011) facet has excellent charge transport properties achieving closer alignment of energy levels. By exploiting the (011) facet a device (0.06 cm2) and a module (29 cm2) achieved power conversion efficiencies of 25.24% and 21.12%, respectively.

Abstract Background Lipids may influence cellular penetrance by viral pathogens and the immune response that they evoke. We deeply phenotyped the lipidomic response to SARs‐CoV‐2 and compared that with infection with other pathogens in patients admitted with acute respiratory distress syndrome to an intensive care unit (ICU). Methods Mass spectrometry was used to characterise lipids and relate them to proteins, peripheral cell immunotypes and disease severity. Results Circulating phospholipases (sPLA2, cPLA2 (PLA2G4A) and PLA2G2D) were elevated on admission in all ICU groups. Cyclooxygenase, lipoxygenase and epoxygenase products of arachidonic acid (AA) were elevated in all ICU groups compared with controls. sPLA2 predicted severity in COVID‐19 and correlated with TxA2, LTE4 and the isoprostane, iPF2α‐III, while PLA2G2D correlated with LTE4. The elevation in PGD2, like PGI2 and 12‐HETE, exhibited relative specificity for COVID‐19 and correlated with sPLA2 and the interleukin‐13 receptor to drive lymphopenia, a marker of disease severity. Pro‐inflammatory eicosanoids remained correlated with severity in COVID‐19 28 days after admission. Amongst non‐COVID ICU patients, elevations in 5‐ and 15‐HETE and 9‐ and 13‐HODE reflected viral rather than bacterial disease. Linoleic acid (LA) binds directly to SARS‐CoV‐2 and both LA and its di‐HOME products reflected disease severity in COVID‐19. In healthy marines, these lipids rose with seroconversion. Eicosanoids linked variably to the peripheral cellular immune response. PGE2, TxA2 and LTE4 correlated with T cell activation, as did PGD2 with non‐B non‐T cell activation. In COVID‐19, LPS stimulated peripheral blood mononuclear cell PGF2α correlated with memory T cells, dendritic and NK cells while LA and DiHOMEs correlated with exhausted T cells. Three high abundance lipids – ChoE 18:3, LPC‐O‐16:0 and PC‐O‐30:0 – were altered specifically in COVID. LPC‐O‐16:0 was strongly correlated with T helper follicular cell activation and all three negatively correlated with multi‐omic inflammatory pathways and disease severity. Conclusions A broad based lipidomic storm is a predictor of poor prognosis in ARDS. Alterations in sPLA2, PGD2 and 12‐HETE and the high abundance lipids, ChoE 18:3, LPC‐O‐16:0 and PC‐O‐30:0 exhibit relative specificity for COVID‐19 amongst such patients and correlate with the inflammatory response to link to disease severity.

Summary: CD4 T cells are central effectors of anti-cancer immunity and immunotherapy, yet the regulation of CD4 tumor-specific T (TTS) cells is unclear. We demonstrate that CD4 TTS cells are quickly primed and begin to divide following tumor initiation. However, unlike CD8 TTS cells or exhaustion programming, CD4 TTS cell proliferation is rapidly frozen in place by a functional interplay of regulatory T cells and CTLA4. Together these mechanisms paralyze CD4 TTS cell differentiation, redirecting metabolic circuits, and reducing their accumulation in the tumor. The paralyzed state is actively maintained throughout cancer progression and CD4 TTS cells rapidly resume proliferation and functional differentiation when the suppressive constraints are alleviated. Overcoming their paralysis established long-term tumor control, demonstrating the importance of rapidly crippling CD4 TTS cells for tumor progression and their potential restoration as therapeutic targets.

Introduction Postburn breast deformities pose functional and aesthetic concerns for burn patients, particularly when injury occurs before puberty, as normal breast development may be hindered. Postburn breast reconstruction aims at restoration of native anatomic features, as well as re-establishment of symmetry. The objectives of this scoping review are to map the literature on scar management and breast reconstruction, highlighting strategies that are particular to postburn deformities, as well as to establish optimal timing principles. Methods A comprehensive search of the English literature across MEDLINE and EMBASE databases, including the grey literature, was conducted. Literature of all study designs were eligible, provided it discussed the treatment of postburn breast deformities. Results A total of 64 studies were included. The most common study design was case series (58%) followed by retrospective cohorts (28%). Scar contracture release with split thickness skin grafts (26%) and various techniques for nipple-areola reconstruction (22%) were the most common reconstructive procedures. Discussion Scar contracture releases predominate when there is normal breast development under a contracted skin envelope, and should be performed as soon as breast mound development is restricted. Surgical techniques widely used for postmastectomy reconstruction are required for patients with amastia or hypoplastic breasts. Conclusion Given the heterogeneity of defects, availability of donor sites, and patient preference, no standardized guideline is available. Surgeons should combine basic scar management principles with postmastectomy techniques, adapting the surgical approach to features that are particular to thermally injured patients, as well as taking into account ideal timing considerations. Lay Summary Breast deformities secondary to burn scars pose functional and aesthetic concerns for burn patients, particularly when injury occurs before puberty, as normal breast development may be hindered. Postburn breast reconstruction aims at restoration of native anatomic features, as well as re-establishment of symmetry. This literature review aimed at summarizing the available techniques to treat postburn breast deformities, as well as establishing optimal timing guidelines, given these issues may occur at any phase of breast development. When there is breast development under a scarred skin envelope, treatment entails scar contracture release and should be recommended as soon as the diagnosis is established, in order to allow the breast to further develop in an unrestricted manner. When there is absence of breast tissue, surgical techniques widely utilized for breast cancer reconstruction are warranted, and should be delayed until no further breast development is expected. Given the heterogeneity of deformities, availability of donor sites, and patience preference, no standardized guideline is available. Treatment options include several surgical techniques, in addition to non-surgical scar management, and timing considerations must take into account the patient's developmental phase and psychosocial wellness.

Optimal lung repair and regeneration are essential for recovery from viral infections, including influenza A virus (IAV). We have previously demonstrated that acute inflammation and mortality induced by IAV is under circadian control. However, it is not known whether the influence of the circadian clock persists beyond the acute outcomes. Here, we utilize the UK Biobank to demonstrate an association between poor circadian rhythms and morbidity from lower respiratory tract infections, including the need for hospitalization and mortality after discharge; this persists even after adjusting for common confounding factors. Furthermore, we use a combination of lung organoid assays, single-cell RNA sequencing, and IAV infection in different models of clock disruption to investigate the role of the circadian clock in lung repair and regeneration. We show that lung organoids have a functional circadian clock and the disruption of this clock impairs regenerative capacity. Finally, we find that the circadian clock acts through distinct pathways in mediating lung regeneration — in tracheal cells via the Wnt/β-catenin pathway and through IL-1β in alveolar epithelial cells. We speculate that adding a circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes.

Background:. Cytomegalovirus (CMV) infection causes significant morbidity and mortality in lung transplant recipients. Current guidelines use pretransplant donor and recipient CMV serostatus to predict the risk of subsequent CMV replication and length of antiviral prophylaxis. Immunological monitoring may better inform the risk of CMV infection in patients, thereby allowing for improved tailoring of antiviral prophylaxis. In this study, we compared 2 commercially available assays, the QuantiFERON-CMV (QFN-CMV) and T-Track-CMV (enzyme-linked immunosorbent spot assay), to predict the risk of CMV disease in lung transplant recipients. Methods:. We performed CMV immunity assays on 32 lung transplant recipients at risk of CMV disease as defined by serostatus (CMV-seropositive recipients, n = 26; or CMV-seronegative lung transplant recipient receiving a CMV-seropositive donor organ, n = 6). QFN-CMV and T-Track were performed on peripheral blood mononuclear cells, and episodes of CMV replication in both serum and bronchoalveolar lavage were found to be correlated to the CMV immune assays. The predictive ability of the assays was determined using Kaplan–Meier curves. Results:. There was a degree of concordance between tests, with 44% of recipients positive for both tests and 28% negative for both tests; however, test results were discordant in 28% of cases. A negative result in either the QFN-CMV (P < 0.01) or T-Track (P < 0.05) assays was obtained in a significantly higher number of recipients who experienced CMV replication in the blood. Using these assays together gave higher predictability of CMV replication, with only 1 recipient experiencing CMV replication in the blood who obtained a positive test result for both assays. Neither assay was able to predict recipients who experienced CMV replication in the lung allograft. Conclusions:. Our study demonstrates that CMV immunity assays can predict viremia; however, the lack of association with allograft infection suggests that CMV-specific T-cell immunity in the circulation is not associated with the control of CMV replication within the transplanted lung allograft.

Salt passivation of perovskite often results in formation of 2D perovskite layers, which impaired charge transport behaviour. Here, the authors study the energy barrier of 2D perovskite formation upon passivation by different iodide salt, and provide insight how to manipulate this to maximise device performance.

High-precision bioengineering and synthetic biology require fine-tuning gene expression at both transcriptional and posttranscriptional levels. Gene transcription is tightly regulated by promoters and terminators. Promoters determine the timing, tissues and cells, and levels of the expression of genes. Terminators mediate transcription termination of genes and affect mRNA levels posttranscriptionally, e.g., the 3′-end processing, stability, translation efficiency, and nuclear to cytoplasmic export of mRNAs. The promoter and terminator combination affects gene expression. In the present article, we review the function and features of plant core promoters, proximal and distal promoters, and terminators, and their effects on and benchmarking strategies for regulating gene expression.

Abstract Background The accurate interpretation of RNA-Seq data presents a moving target as scientists continue to introduce new experimental techniques and analysis algorithms. Simulated datasets are an invaluable tool to accurately assess the performance of RNA-Seq analysis methods. However, existing RNA-Seq simulators focus on modeling the technical biases and artifacts of sequencing, rather than on simulating the original RNA samples. A first step in simulating RNA-Seq is to simulate RNA. Results To fill this need, we developed the Configurable And Modular Program Allowing RNA Expression Emulation (CAMPAREE), a simulator using empirical data to simulate diploid RNA samples at the level of individual molecules. We demonstrated CAMPAREE’s use for generating idealized coverage plots from real data, and for adding the ability to generate allele-specific data to existing RNA-Seq simulators that do not natively support this feature. Conclusions Separating input sample modeling from library preparation/sequencing offers added flexibility for both users and developers to mix-and-match different sample and sequencing simulators to suit their specific needs. Furthermore, the ability to maintain sample and sequencing simulators independently provides greater agility to incorporate new biological findings about transcriptomics and new developments in sequencing technologies. Additionally, by simulating at the level of individual molecules, CAMPAREE has the potential to model molecules transcribed from the same genes as a heterogeneous population of transcripts with different states of degradation and processing (splicing, editing, etc.). CAMPAREE was developed in Python, is open source, and freely available at https://github.com/itmat/CAMPAREE .

Although circulating tumour DNA (ctDNA) can predict immune checkpoint blockade (ICB) responses, its association with tumour biomarkers remains unknown. Here, the authors use ctDNA to inform exome and transcriptome profiling of >100 patients with 30 cancer types on a single clinical ICB trial and identify tumour microenvironment features associated with response.

Summary: This is a cytometry by time-of-flight (CyTOF) staining protocol for hematopoietic-derived cells, that leverages live-cell barcoding using receptor-type tyrosine-protein phosphatase C (CD45) antibodies conjugated to metal isotopes in combination with DNA-based palladium barcoding to multiplex up to 40 samples. In this protocol, DNA-based barcoding is performed before surface and intracellular immunostaining, which reduces the batch effects that result from day-to-day variations in staining and instrument sensitivity. This protocol also reduces antibody consumption and eliminates the need for repeated instrument adjustment. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The immunology of Indigenous populations is generally understudied outside the context of diseases that are prevalent in these communities. Here the authors identify prevalence of influenza CD8+ T cell epitopes in an Indigenous Australian population expressing the susceptibility allomorph HLA A*24:02 and validate immunodominance of some of these epitopes in mice.

Abstract Background Full-length isoform quantification from RNA-Seq is a key goal in transcriptomics analyses and has been an area of active development since the beginning. The fundamental difficulty stems from the fact that RNA transcripts are long, while RNA-Seq reads are short. Results Here we use simulated benchmarking data that reflects many properties of real data, including polymorphisms, intron signal and non-uniform coverage, allowing for systematic comparative analyses of isoform quantification accuracy and its impact on differential expression analysis. Genome, transcriptome and pseudo alignment-based methods are included; and a simple approach is included as a baseline control. Conclusions Salmon, kallisto, RSEM, and Cufflinks exhibit the highest accuracy on idealized data, while on more realistic data they do not perform dramatically better than the simple approach. We determine the structural parameters with the greatest impact on quantification accuracy to be length and sequence compression complexity and not so much the number of isoforms. The effect of incomplete annotation on performance is also investigated. Overall, the tested methods show sufficient divergence from the truth to suggest that full-length isoform quantification and isoform level DE should still be employed selectively.

Host cell restriction factors are intracellular proteins that can inhibit virus replication. Characterisation of novel host cell restriction factors can provide potential targets for host-directed therapies. In this study, we aimed to assess a member of the Tripartite-motif family protein (TRIM) family, TRIM16, as a putative host cell restriction factor. To this end, we utilized constitutive or doxycycline-inducible systems to overexpress TRIM16 in HEK293T epithelial cells and then tested for its ability to inhibit growth by a range of RNA and DNA viruses. In HEK293T cells, overexpression of TRIM16 resulted in potent inhibition of multiple viruses, however, when TRIM16 was overexpressed in other epithelial cell lines (A549, Hela, or Hep2), virus inhibition was not observed. When investigating the antiviral activity of endogenous TRIM16, we report that siRNA-mediated knockdown of TRIM16 in A549 cells also modulated the mRNA expression of other TRIM proteins, complicating the interpretation of results using this method. Therefore, we used CRISPR/Cas9 editing to knockout TRIM16 in A549 cells and demonstrate that endogenous TRIM16 did not mediate antiviral activity against the viruses tested. Thus, while initial overexpression in HEK293T cells suggested that TRIM16 was a host cell restriction factor, alternative approaches did not validate these findings. These studies highlight the importance of multiple complementary experimental approaches, including overexpression analysis in multiple cell lines and investigation of the endogenous protein, when defining host cell restriction factors with novel antiviral activity.

KIR2DL2 and KIR2DL3 are two inhibitory members of the killer-cell immunoglobulin-like receptors (KIR) family that share a common HLA-I preference in binding HLA from the C1 group. However, it is still unclear to what extent binding and function is equivalent between KIR2DL2 and 2DL3. Here, the authors present the crystal structures of KIR2DL2 and 2DL3 in complex with HLA-C*07:02 and observe differences in HLA-C recognition between KIR2DL2 and 2DL3, which correlates with differences in HLA-C binding preference as they show with mutagenesis and binding studies.

Background Combining immunotherapy and antiangiogenic agents is a promising treatment strategy in endometrial cancer. To date, no biomarkers for response have been identified and data on post-immunotherapy progression are lacking. We explored the combination of a checkpoint inhibitor (nivolumab) and an antiangiogenic agent (cabozantinib) in immunotherapy-naïve endometrial cancer and in patients whose disease progressed on previous immunotherapy with baseline biopsy for immune profiling.Patients and methods In this phase II trial (ClinicalTrials.gov NCT03367741, registered December 11, 2017), women with recurrent endometrial cancer were randomized 2:1 to nivolumab with cabozantinib (Arm A) or nivolumab alone (Arm B). The primary endpoint was Response Evaluation Criteria in Solid Tumors-defined progression-free survival (PFS). Patients with carcinosarcoma or prior immune checkpoint inhibitor received combination treatment (Arm C). Baseline biopsy and serial peripheral blood mononuclear cell (PBMC) samples were analyzed and associations between patient outcome and immune data from cytometry by time of flight (CyTOF) and PBMCs were explored.Results Median PFS was 5.3 (90% CI 3.5 to 9.2) months in Arm A (n=36) and 1.9 (90% CI 1.6 to 3.4) months in Arm B (n=18) (HR=0.59, 90% CI 0.35 to 0.98; log-rank p=0.09, meeting the prespecified statistical significance criteria). The most common treatment-related adverse events in Arm A were diarrhea (50%) and elevated liver enzymes (aspartate aminotransferase 47%, alanine aminotransferase 42%). In-depth baseline CyTOF analysis across treatment arms (n=40) identified 35 immune-cell subsets. Among immunotherapy-pretreated patients in Arm C, non-progressors had significantly higher proportions of activated tissue-resident (CD103+CD69+) ɣδ T cells than progressors (adjusted p=0.009).Conclusions Adding cabozantinib to nivolumab significantly improved outcomes in heavily pretreated endometrial cancer. A subgroup of immunotherapy-pretreated patients identified by baseline immune profile and potentially benefiting from combination with antiangiogenics requires further investigation.

Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity.

New adverse reactions to the COVID-19 vaccines are being identified as vaccination rates increase worldwide. Recently, there have been two reports of Moderna (mRNA-1273) vaccine induced relapse of chronic spontaneous urticaria (CSU) that was previously well controlled. Herein, we report a case of AstraZeneca/Oxford (ChAdOx1) vaccine triggered CSU in a patient with no history of CSU with achieved remission.

MARCH1 and MARCH8 are closely related E3 ubiquitin ligases that ubiquitinate an overlapping spectrum of host proteins and restrict replication of certain viruses. While the antiviral activity of MARCH8 has been intensively studied, less is known regarding virus inhibition by MARCH1. Isoforms 1 and 2 of MARCH1 are very similar in overall structure but show major differences in their N-terminal cytoplasmic domain (N-CT). Herein, we used a doxycycline-inducible overexpression system to demonstrate that MARCH1.1 reduces titres of influenza A virus (IAV) released from infected cells whereas MARCH1.2 does not. The deletion of the entire N-CT of MARCH1.2 restored its ability to restrict IAV infectivity and sequential deletions mapped the restoration of IAV inhibition to delete the 16 N-terminal residues within the N-CT of MARCH1.2. While only MARCH1.1 mediated anti-IAV activity, qPCR demonstrated the preferential expression of MARCH1.2 over MARCH1.1 mRNA in unstimulated human peripheral blood mononuclear cells and also in monocyte-derived macrophages. Together, these studies describe the differential ability of MARCH1 isoforms to restrict IAV infectivity for the first time. Moreover, as published immunological, virological and biochemical studies examining the ability of MARCH1 to target particular ligands generally use only one of the two isoforms, these findings have broader implications for our understanding of how MARCH1 isoforms might differ in their ability to modulate particular host and/or viral proteins.

The antiviral activity of MARCH8 has been associated with the downregulation of envelope glycoproteins from a range of different viruses, resulting in reduced incorporation into nascent virions. Here, we show that MARCH8 restricts IAV at a late stage in virus replication, but this was not associated with reduced expression of IAV envelope glycoproteins on the surfaces of infected cells, pointing to a distinct mechanism of antiviral activity.

Background: Beginning March 2020, the COVID-19 pandemic has disrupted different aspects of life. The impact on children's rate of weight gain has not been analysed. Methods: In this retrospective cohort study, we used United States (US) Electronic Health Record (EHR) data from Optum® to calculate the age- and sex- adjusted change in BMI (∆BMIadj) in individual 6-to-17-year-old children between two well child checks (WCCs). The mean of individual ∆BMIadj during 2017–2020 was calculated by month. For September-December WCCs, the mean of individual ∆BMIadj (overall and by subgroup) was reported for 2020 and 2017–2019, and the impact of 2020 vs 2017–2019 was tested by multivariable linear regression. Findings: The mean [95% Confidence Interval - CI] ∆BMIadj in September-December of 2020 was 0·62 [0·59,0·64] kg/m2, compared to 0·31 [0·29, 0·32] kg/m2 in previous years. The increase was most prominent in children with pre-existing obesity (1·16 [1·07,1·24] kg/m2 in 2020 versus 0·56 [0·52,0·61] kg/m2 in previous years), Hispanic children (0·93 [0·84,1·02] kg/m2 in 2020 versus 0·41 [0·36,0·46] kg/m2 in previous years), and children who lack commercial insurance (0·88 [0·81,0·95] kg/m2 in 2020 compared to 0·43 [0·39,0·47] kg/m2 in previous years). ∆BMIadj accelerated most in ages 8–12 and least in ages 15–17. Interpretation: Children's rate of unhealthy weight gain increased notably during the COVID-19 pandemic across demographic groups, and most prominently in children already vulnerable to unhealthy weight gain. This data can inform policy decisions critical to child development and health as the pandemic continues to unfold. Funding: Amgen, Inc.

Abstract Background Immune checkpoint inhibitors (ICIs) demonstrate unprecedented efficacy in multiple malignancies; however, the mechanisms of sensitivity and resistance are poorly understood and predictive biomarkers are scarce. INSPIRE is a phase 2 basket study to evaluate the genomic and immune landscapes of peripheral blood and tumors following pembrolizumab treatment. Methods Patients with incurable, locally advanced or metastatic solid tumors that have progressed on standard therapy, or for whom no standard therapy exists or standard therapy was not deemed appropriate, received 200 mg pembrolizumab intravenously every three weeks. Blood and tissue samples were collected at baseline, during treatment, and at progression. One core biopsy was used for immunohistochemistry and the remaining cores were pooled and divided for genomic and immune analyses. Univariable analysis of clinical, genomic, and immunophenotyping parameters was conducted to evaluate associations with treatment response in this exploratory analysis. Results Eighty patients were enrolled from March 21, 2016 to June 1, 2017, and 129 tumor and 382 blood samples were collected. Immune biomarkers were significantly different between the blood and tissue. T cell PD-1 was blocked (≥98%) in the blood of all patients by the third week of treatment. In the tumor, 5/11 (45%) and 11/14 (79%) patients had T cell surface PD-1 occupance at weeks six and nine, respectively. The proportion of genome copy number alterations and abundance of intratumoral 4-1BB+ PD-1+ CD8 T cells at baseline (P

Adverse early-life exposures have a lasting negative impact on health. Neonatal hyperoxia that is a risk factor for bronchopulmonary dysplasia confers susceptibility to influenza A virus (IAV) infection later in life. Given our previous findings that the circadian clock protects against IAV, we asked if the long-term impact of neonatal hyperoxia vis-à-vis IAV infection includes circadian disruption. Here, we show that neonatal hyperoxia abolishes the clock-mediated time of day protection from IAV in mice, independent of viral burden through host tolerance pathways. We discovered that the lung intrinsic clock (and not the central or immune clocks) mediated this dysregulation. Loss of circadian protein, Bmal1, in alveolar type 2 (AT2) cells recapitulates the increased mortality, loss of temporal gating, and other key features of hyperoxia-exposed animals. Our data suggest a novel role for the circadian clock in AT2 cells in mediating long-term effects of early-life exposures to the lungs.

2′3′-cGAMP is known as a nonclassical second messenger and small immune modulator that possesses potent antitumor and antiviral activities via inducing the stimulator of IFN genes–mediated (STING-mediated) signaling pathway. However, its function in regulating type 2 immune responses remains unknown. Therefore, we sought to determine a role of STING activation by 2′3′-cGAMP in type 2 inflammatory reactions in multiple mouse models of eosinophilic asthma. We discovered that 2′3′-cGAMP administration strongly attenuated type 2 lung immunopathology and airway hyperreactivity induced by IL-33 and a fungal allergen, Aspergillus flavus. Mechanistically, upon the respiratory delivery, 2′3′-cGAMP was mainly internalized by alveolar macrophages, in which it activated the STING/IFN regulatory factor 3/type I IFN signaling axis to induce the production of inhibitory factors containing IFN-α, which blocked the IL-33–mediated activation of group 2 innate lymphoid (ILC2) cells in vivo. We further demonstrated that 2′3′-cGAMP directly suppressed the proliferation and function of both human and mouse ILC2 cells in vitro. Taken together, our findings suggest that STING activation by 2′3′-cGAMP in alveolar macrophages and ILC2 cells can negatively regulate type 2 immune responses, implying that the respiratory delivery of 2′3′-cGAMP might be further developed as an alternative strategy for treating type 2 immunopathologic diseases such as eosinophilic asthma.

RIG-I is an innate sensor of RNA virus infection and its activation induces interferon-stimulated genes (ISGs). In vitro studies using human cells have demonstrated the ability of synthetic RIG-I agonists (3pRNA) to inhibit IAV replication. However, in mouse models of IAV the effectiveness of 3pRNA reported to date differs markedly between studies. Myxoma resistance (Mx)1 is an ISG protein which mediates potent anti-IAV activity, however most inbred mouse strains do not express a functional Mx1. Herein, we utilised C57BL/6 mice that do (B6.A2G-Mx1) and do not (B6-WT) express functional Mx1 to assess the ability of prophylactic 3pRNA treatment to induce ISGs and to protect against subsequent IAV infection. In vitro, 3pRNA treatment of primary lung cells from B6-WT and B6.A2G-Mx1 mice resulted in ISG induction however inhibition of IAV infection was more potent in cells from B6.A2G-Mx1 mice. In vivo, a single intravenous injection of 3pRNA resulted in ISG induction in lungs of both B6-WT and B6.A2G-Mx1 mice, however potent and long-lasting protection against subsequent IAV challenge was only observed in B6.A2G-Mx1 mice. Thus, despite broad ISG induction, expression of a functional Mx1 is critical for potent and long-lasting RIG-I agonist-mediated protection in the mouse model of IAV infection.

Background The physiology underlying "brain fog" in the absence of orthostatic stress in postural tachycardia syndrome (POTS) remains poorly understood. Methods and Results We evaluated cognitive and hemodynamic responses (cardiovascular and cerebral: heart rate, blood pressure, end‐tidal carbon dioxide, and cerebral blood flow velocity (CBFv) in the middle cerebral artery at baseline, after initial cognitive testing, and after (30‐minutes duration) prolonged cognitive stress test (PCST) whilst seated; as well as after 5‐minute standing in consecutively enrolled participants with POTS (n=22) and healthy controls (n=18). Symptom severity was quantified with orthostatic hypotensive questionnaire at baseline and end of study. Subjects in POTS and control groups were frequency age‐ and sex‐matched (29±11 versus 28±13 years; 86 versus 72% women, respectively; both P≥0.4). The CBFv decreased in both groups (condition, P=0.04) following PCST, but a greater reduction in CBFv was observed in the POTS versus control group (−7.8% versus −1.8%; interaction, P=0.038). Notably, the reduced CBFv following PCST in the POTS group was similar to that seen during orthostatic stress (60.0±14.9 versus 60.4±14.8 cm/s). Further, PCST resulted in greater slowing in psychomotor speed (6.1% versus 1.4%, interaction, P=0.027) and a greater increase in symptom scores at study completion (interaction, P0.05). Conclusions Reduced CBFv and cognitive dysfunction were evident in patients with POTS following prolonged cognitive stress even in the absence of orthostatic stress.

Dequalinium is used as an antimicrobial compound for oral health and other microbial infections. Derivatives of dequalinium, the bis-quinolinium cyclophanes UCL 1684 and UCL 1848, are high affinity SK potassium channel antagonists. Here we investigated these compounds as M3 muscarinic receptor (mACHR) antagonists. We used the R-CEPIAer endoplasmic reticulum calcium reporter to functionally assay for Gq-coupled receptor signaling, and investigated the bis-quinolinium cyclophanes as antagonists of M3 mACHR activation in transfected CHO cells. Given mACHR roles in airway smooth muscle (ASM) contractility, we also tested the ability of UCL 1684 to relax ASM. We find that these compounds antagonized M3 mACHRs with an IC50 of 0.27 μM for dequalinium chloride, 1.5 μM for UCL 1684 and 1.0 μM for UCL 1848. UCL 1684 also antagonized M1 (IC50 0.12 μM) and M5 (IC50 0.52 μM) mACHR responses. UCL 1684 was determined to be a competitive antagonist at M3 receptors as it increased the EC50 for carbachol without a reduction in the maximum response. The Ki for UCL1684 determined from competition binding experiments was 909 nM. UCL 1684 reduced carbachol-evoked ASM contractions (>90%, IC50 0.43 μM), and calcium mobilization in rodent and human lung ASM cells. We conclude that dequalinium and bis-quinolinium cyclophanes antagonized M3 mACHR activation at sub- to low micromolar concentrations, with UCL 1684 acting as an ASM relaxant. Caution should be taken when using these compounds to block SK potassium channels, as inhibition of mACHRs may be a side-effect if excessive concentrations are used.

Bubble surface area flux (Sb) is one of the main design parameter in flotation column that typically employed to describe the gas dispersion properties, and it has a strong correlation with the flotation rate constant. There is a limited information available in the literature regarding the effect of particle type, density, wettability and concentration on Sb. In this paper, computational fluid dynamics (CFD) simulations are performed to study the gas–liquid–solid three-phase flow dynamics in flotation column by employing the Eulerian–Eulerian formulation with k-ε turbulence model. The model is developed by writing Fortran subroutine and incorporating then into the commercial CFD code AVL FIRE, v.2014. This paper studies the effects of superficial gas velocities and particle type, density, wettability and concentration on Sb and bubble concentration in the flotation column. The model has been validated against published experimental data. It was found that the CFD model was able to predict, where the response variable as indicated by R-Square value of 0.98. These results suggest that the developed CFD model is reasonable to describe the flotation column reactor. From the CFD results, it is also found that Sb decreased with increasing solid concentration and hydrophobicity, but increased with increasing superficial gas velocity. For example, approximately 28% reduction in the surface area flux is observed when coal concentration is increased from 0 to 10%, by volume. While for the same solid concentration and gas flow rate, the bubble surface area flux is approximately increased by 7% in the presences of sphalerite. A possible explanation for this might be that increasing solid concentration and hydrophobicity promotes the bubble coalescence rate leading to the increase in bubble size. Also, it was found that the bubble concentration would decrease with addition of hydrophobic particle (i.e., coal). For instance, under the same operating conditions, approximately 23% reduction in the bubble concentration is predicted when the system was working with hydrophobic particles. The results presented are useful for understanding flow dynamics of three-phase system and provide a basis for further development of CFD model for flotation column. Keywords: CFD, Froth flotation, Bubble surface area flux, Solid properties, Bubble concentration