Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of major facilitator superfamily transporters. With a conformation-selective nanobody, we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. The available outward-facing sugar-bound structures showed that the N- and C-terminal residues of the inner barrier contribute to the sugar selectivity. The inward-open conformation shows that the sugar selectivity pocket is also broken when the inner barrier is broken. Isothermal titration calorimetry measurements revealed that this inward-facing conformation trapped by this nanobody exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is supported by molecular dynamics simulations. Furthermore, the hydron/deuterium exchange mass spectrometry analyses allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

Phenotyping of Severe Asthma in the Era of Broad-Acting Anti-Asthma Biologics.

Severe asthma is associated with significant morbidity and mortality despite the maximal use of inhaled corticosteroids and additional controller medications, and has a high economic burden. Biologic therapies are recommended for the management of severe, uncontrolled asthma to help to prevent exacerbations and to improve symptoms and health-related quality of life. The effective management of severe asthma requires consideration of clinical heterogeneity that is driven by varying clinical and inflammatory phenotypes, which are reflective of distinct underlying disease mechanisms. Phenotyping patients using a combination of clinical characteristics such as the age of onset or comorbidities and biomarker profiles, including blood eosinophil counts and levels of fractional exhaled nitric oxide and serum total immunoglobulin E, is important for the differential diagnosis of asthma. In addition, phenotyping is beneficial for risk assessment, selection of treatment, and monitoring of the treatment response in patients with asthma. This review describes the clinical and inflammatory phenotypes of asthma, provides an overview of biomarkers routinely used in clinical practice and those that have recently been explored for phenotyping, and aims to assess the value of phenotyping in severe asthma management in the current era of biologics.

Clinical Epidemiology and Risk Factors for Critical Outcomes Among Vaccinated and Unvaccinated Adults Hospitalized With COVID-19-VISION Network, 10 States, June 2021-March 2023.

BACKGROUND: The epidemiology of coronavirus disease 2019 (COVID-19) continues to develop with emerging variants, expanding population-level immunity, and advances in clinical care. We describe changes in the clinical epidemiology of COVID-19 hospitalizations and risk factors for critical outcomes over time. METHODS: We included adults aged ≥18 years from 10 states hospitalized with COVID-19 June 2021-March 2023. We evaluated changes in demographics, clinical characteristics, and critical outcomes (intensive care unit admission and/or death) and evaluated critical outcomes risk factors (risk ratios [RRs]), stratified by COVID-19 vaccination status. RESULTS: A total of 60 488 COVID-19-associated hospitalizations were included in the analysis. Among those hospitalized, median age increased from 60 to 75 years, proportion vaccinated increased from 18.2% to 70.1%, and critical outcomes declined from 24.8% to 19.4% (all P < .001) between the Delta (June-December, 2021) and post-BA.4/BA.5 (September 2022-March 2023) periods. Hospitalization events with critical outcomes had a higher proportion of ≥4 categories of medical condition categories assessed (32.8%) compared to all hospitalizations (23.0%). Critical outcome risk factors were similar for unvaccinated and vaccinated populations; presence of ≥4 medical condition categories was most strongly associated with risk of critical outcomes regardless of vaccine status (unvaccinated: adjusted RR, 2.27 [95% confidence interval {CI}, 2.14-2.41]; vaccinated: adjusted RR, 1.73 [95% CI, 1.56-1.92]) across periods. CONCLUSIONS: The proportion of adults hospitalized with COVID-19 who experienced critical outcomes decreased with time, and median patient age increased with time. Multimorbidity was most strongly associated with critical outcomes.

Analysis of comorbidities and multimorbidity in adult patients in the International Severe Asthma Registry.

BACKGROUND: Investigation for the presence of asthma comorbidities is recommended by the Global Initiative for Asthma because their presence can complicate asthma management. OBJECTIVE: To understand the prevalence and pattern of comorbidities and multimorbidity in adults with severe asthma and their association with asthma-related outcomes. METHODS: This was a cross-sectional study using data from the International Severe Asthma Registry from 22 countries. A total of 30 comorbidities were identified and categorized a priori as any of the following: (1) potentially type 2-related comorbidities, (2) potentially oral corticosteroid (OCS)-related comorbidities, or (3) comorbidities mimicking or aggravating asthma. The association between comorbidities and asthma-related outcomes was investigated using multivariable models adjusted for country, age at enrollment, and sex (ie male or female). RESULTS: Of the 11,821 patients, 69%, 67%, and 55% had at least 1 potentially type 2-related, potentially OCS-related, or mimicking or aggravating comorbidities, respectively; 57% had 3 or more comorbidities, and 33% had comorbidities in all 3 categories. Patients with allergic rhinitis, nasal polyposis, and chronic rhinosinusitis experienced 1.12 (P = .003), 1.16 (P < .001), and 1.29 times (P < .001) more exacerbations per year, respectively, than those without. Patients with nasal polyposis and chronic rhinosinusitis were 40% and 46% more likely (P < .001), respectively, to have received long-term (LT) OCS. All assessed potential OCS-related comorbidities (except obesity) were associated with a greater likelihood of LTOCS use (odds ratios [ORs]: 1.23-2.77) and, except for dyslipidemia, with a greater likelihood of uncontrolled asthma (ORs: 1.29-1.68). All mimicking or aggravating comorbidities assessed were associated with more exacerbations (1.24-1.68 times more), all (except bronchiectasis) with increased likelihood of uncontrolled asthma (ORs: 1.57-1.81), and all (except chronic obstructive pulmonary disease) with increased likelihood of LTOCS use (ORs: 1.37-1.57). A greater number of comorbidities was associated with worse outcomes. CONCLUSION: In a global study, comorbidity or multimorbidity is reported in most adults with severe asthma and is associated with poorer asthma-related outcomes. CLINICAL TRIAL REGISTRATION: The International Severe Asthma Registry database has ethical approval from the Anonymous Data Ethics Protocols and Transparency (ADEPT) committee (ADEPT0218) and is registered with the European Union Electronic Register of Post-Authorization Studies (European Network Centres for Pharmacoepidemiology and Pharmacovigilance [ENCEPP]/DSPP/23720). The study was designed, implemented, and reported in compliance with the European Network Centres for Pharmacoepidemiology and Pharmacovigilance (ENCEPP) Code of Conduct (EMA 2014; EUPAS44024) and with all applicable local and international laws and regulations, and registered with ENCEPP (https://www.encepp.eu/encepp/viewResource.htm?id=48848). Governance was provided by ADEPT (registration number: ADEPT1121).

Association Between T2-related Comorbidities and Effectiveness of Biologics in Severe Asthma.

Rationale: Previous studies investigating the impact of comorbidities on the effectiveness of biologic agents have been relatively small and of short duration and have not compared classes of biologic agents. Objectives: To determine the association between type 2-related comorbidities and biologic agent effectiveness in adults with severe asthma (SA). Methods: This cohort study used International Severe Asthma Registry data from 21 countries (2017-2022) to quantify changes in four outcomes before and after biologic therapy-annual asthma exacerbation rate, FEV1% predicted, asthma control, and long-term oral corticosteroid daily dose-in patients with or without allergic rhinitis, chronic rhinosinusitis (CRS) with or without nasal polyps (NPs), NPs, or eczema/atopic dermatitis. Measurements and Main Results: Of 1,765 patients, 1,257, 421, and 87 initiated anti-IL-5/5 receptor, anti-IgE, and anti-IL-4/13 therapies, respectively. In general, pre- versus post-biologic therapy improvements were noted in all four asthma outcomes assessed, irrespective of comorbidity status. However, patients with comorbid CRS with or without NPs experienced 23% fewer exacerbations per year (95% CI, 10-35%; P < 0.001) and had 59% higher odds of better post-biologic therapy asthma control (95% CI, 26-102%; P < 0.001) than those without CRS with or without NPs. Similar estimates were noted for those with comorbid NPs: 22% fewer exacerbations and 56% higher odds of better post-biologic therapy control. Patients with SA and CRS with or without NPs had an additional FEV1% predicted improvement of 3.2% (95% CI, 1.0-5.3; P = 0.004), a trend that was also noted in those with comorbid NPs. The presence of allergic rhinitis or atopic dermatitis was not associated with post-biologic therapy effect for any outcome assessed. Conclusions: These findings highlight the importance of systematic comorbidity evaluation. The presence of CRS with or without NPs or NPs alone may be considered a predictor of the effectiveness of biologic agents in patients with SA.

Polyomavirus-associated Disseminated T-cell Lymphoma in a Colony of Zebra Finches (Taeniopygia guttata).

Four zebra finches in a closed research colony presented with variable clinical signs, including masses, skin lesions, shivering, and/or ruffled feathers. These birds were not responsive to treatment efforts; 3 died and one was euthanized. All 4 were submitted for necropsy to determine the cause of the clinical signs. Gross necropsy and histopathologic findings from all birds resulted in a diagnosis of round cell neoplasia in multiple organs, including the skin, liver, kidney, and reproductive tract, with intranuclear inclusion bodies in the neoplastic cells. In all 4 cases, immunohistochemical staining showed strong immunoreactivity for CD3 in 70% to 80% of the neoplastic round cells, with a relatively small subset that were immunopositive for Pax5. These findings supported a diagnosis of T-cell lymphoma. Frozen liver tissue from one case was submitted for next-generation sequencing (NGS), which revealed viral RNA with 100% sequence homology to canary polyomavirus strain 34639 that had originally been identified in a European goldfinch. Formalin-fixed paraffin-embedded scrolls from another case were also submitted for NGS, which revealed viral RNA with 97.2% sequence homology to canary polyomavirus strain 37273 that had originally been identified in a canary. To localize the virus in situ, RNAscope hybridization was performed using a probe designed to target the VP1 gene of the sequenced virus in frozen liver tissue. In all 4 cases, disseminated and robust hybridization signals were detected in neoplastic cells. These findings indicate that polyomaviruses have the potential to be oncogenic in zebra finches.

Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter.

While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na+-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined a low-sugar affinity inward-facing Na+-bound cryoEM structure. Collectively with the available outward-facing sugar-bound structures, both the outer and inner barriers were localized. The N- and C-terminal residues of the inner barrier contribute to the sugar selectivity pocket. When the inner barrier is broken as shown in the inward-open conformation, the sugar selectivity pocket is also broken. The binding assays by isothermal titration calorimetry revealed that this inward-facing conformation trapped by the conformation-selective Nb exhibited a greatly decreased sugar-binding affinity, suggesting the mechanisms for the substrate intracellular release and accumulation. While the inner/outer barrier shift directly regulates the sugar-binding affinity, it has little or no effect on the cation binding, which is also supported by molecular dynamics simulations. Furthermore, the use of this Nb in combination with the hydron/deuterium exchange mass spectrometry allowed us to identify dynamic regions; some regions are involved in the functionally important inner barrier-specific salt-bridge network, which indicates their critical roles in the barrier switching mechanisms for transport. These complementary results provided structural and dynamic insights into the mobile barrier mechanism for cation-coupled symport.

Cystic retroperitoneal dedifferentiated liposarcoma: A case report.

Liposarcoma is the most common primary retroperitoneal sarcoma in adults. We report the case of an 86-year-old male who presented to the emergency department with frequent falls and unexplained weight loss that was found to have a cystic retroperitoneal dedifferentiated liposarcoma. Initial computed tomography revealed a large heterogeneous complex cystic hypoenhancing lesion in the left retroperitoneum. Subsequent magnetic resonance imaging demonstrates a multilocular cystic mass with microscopic lipid content, diffusion restriction, and enhancing nodular soft-tissue components. Histologic examination of the tissue sample following biopsy is consistent with cystic retroperitoneal dedifferentiated liposarcoma. Further management was not pursued due to the patient's advanced age and frailty.

Intracellular replication of Pseudomonas aeruginosa in epithelial cells requires suppression of the caspase-4 inflammasome.

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Stalled but not forgotten: Bacterial exotoxins inhibit translation to activate NLRP1.

In this issue of JEM, companion articles from Pinilla et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20230104) and Robinson et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20230105) demonstrate that ribotoxic stress induced by Pseudomonas aeruginosa and Corynebacterium diphtheriae EEF2-targeting exotoxins leads to NLRP1 inflammasome activation, representing a new mechanism of effector-triggered immunity.

First-in-human Study of AZD5153, A Small-molecule Inhibitor of Bromodomain Protein 4, in Patients with Relapsed/Refractory Malignant Solid Tumors and Lymphoma.

AZD5153, a reversible, bivalent inhibitor of the bromodomain and extraterminal family protein BRD4, has preclinical activity in multiple tumors. This first-in-human, phase I study investigated AZD5153 alone or with olaparib in patients with relapsed/refractory solid tumors or lymphoma. Adults with relapsed tumors intolerant of, or refractory to, prior therapies received escalating doses of oral AZD5153 once daily or twice daily continuously (21-day cycles), or AZD5153 once daily/twice daily continuously or intermittently plus olaparib 300 mg twice daily, until disease progression or unacceptable toxicity. Between June 30, 2017 and April 19, 2021, 34 patients received monotherapy and 15 received combination therapy. Dose-limiting toxicities were thrombocytopenia/platelet count decreased (n = 4/n = 2) and diarrhea (n = 1). The recommended phase II doses (RP2D) were AZD5153 30 mg once daily or 15 mg twice daily (monotherapy) and 10 mg once daily (intermittent schedule) with olaparib. With AZD5153 monotherapy, common treatment-emergent adverse events (TEAE) included fatigue (38.2%), thrombocytopenia, and diarrhea (each 32.4%); common grade ≥ 3 TEAEs were thrombocytopenia (14.7%) and anemia (8.8%). With the combination, common TEAEs included nausea (66.7%) and fatigue (53.3%); the most common grade ≥ 3 TEAE was thrombocytopenia (26.7%). AZD5153 had dose-dependent pharmacokinetics, with minimal accumulation, and demonstrated dose-dependent modulation of peripheral biomarkers, including upregulation of HEXIM1. One patient with metastatic pancreatic cancer receiving combination treatment had a partial response lasting 4.2 months. These results show AZD5153 was tolerable as monotherapy and in combination at the RP2Ds; common toxicities were fatigue, hematologic AEs, and gastrointestinal AEs. Strong evidence of peripheral target engagement was observed.

A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection.

Inflammasomes are cytosolic innate immune complexes that assemble upon detection of diverse pathogen-associated cues and play a critical role in host defense and inflammatory pathogenesis. Here, we find that the human inflammasome-forming sensor CARD8 senses HIV-1 infection via site-specific cleavage of the CARD8 N-terminus by the HIV protease (HIV-1PR). HIV-1PR cleavage of CARD8 induces pyroptotic cell death and the release of pro-inflammatory cytokines from infected cells, processes regulated by Toll-like receptor stimulation prior to viral infection. In acutely infected cells, CARD8 senses the activity of both de novo translated HIV-1PR and packaged HIV-1PR that is released from the incoming virion. Moreover, our evolutionary analyses reveal that the HIV-1PR cleavage site in human CARD8 arose after the divergence of chimpanzees and humans. Although chimpanzee CARD8 does not recognize proteases from HIV or simian immunodeficiency viruses from chimpanzees (SIVcpz), SIVcpz does cleave human CARD8, suggesting that SIVcpz was poised to activate the human CARD8 inflammasome prior to its cross-species transmission into humans. Our findings suggest a unique role for CARD8 inflammasome activation in response to lentiviral infection of humans.

Host-specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome.

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8's ability to sense coronavirus 3CLpros and, instead, enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intraspecies variation in inflammasome-mediated viral sensing and immunopathology.

Volume reduction with surgery for ICH: when is it effective? Analysis of the CT scans from the STICH II trial.

BACKGROUND: The surgical trial of lobar intracerebral haemorrhage (STICH II) was a randomised controlled trial evaluating early surgical removal of a clot. This paper investigates volume change in both arms of the trial with respect to Extended Glasgow Outcome Scale (GOSE) groups. METHODS: Patients randomised into STICH II had an initial diagnostic CT and a second CT 5 days after randomisation. Each scan was anonymously assessed by at least two central readers. An analysis of agreement between the two readers was conducted using kappa tests and intraclass correlation. The change in volume in both the early surgery (ES) and the initial conservative treatment (ICT) arms were analysed with respect to the six-month GOSE outcome. RESULTS: Of the 597 patients randomised in the trial there were 582 pre-randomisation scans and 566 5-day scans available for analysis of agreement. There was good agreement between the assessors for the radiological inclusion criteria including volume (ICC = 0.87) and this was better than the agreement between the assessor and local investigator (ICC = 0.73). There were 526 patients with two scans available for analysis of change in volume measurement. The median percentage change in volume for the ES group was a reduction of 92.4% (IQR 75.6%, 99.0%) while for the ICT group, in which some cases crossed over to delayed surgery, it was only 5.7% (IQR 16.4% increase, 29.5% reduction). ES patients with almost complete removal (99-100%) had the best outcome with only 30% dead or lower severely disabled. For the ICT group outcome was related to the final volume: the smaller the final volume the better the outcome. CONCLUSIONS: This analysis provides evidence for central assessments of scans in exploratory analyses and further information regarding the potential advantage of early and more complete clot removal on outcome in ICH and should inform the planning of future trials.Clinical trials registration: ISRCTN22153967.

Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.

Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4-3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.

Associations between ABO non-identical platelet transfusions and patient outcomes-A multicenter retrospective analysis.

BACKGROUND: Due to platelet availability limitations, platelet units ABO mismatched to recipients are often transfused. However, since platelets express ABO antigens and are collected in plasma which may contain ABO isohemagglutinins, it remains controversial as to whether ABO non-identical platelet transfusions could potentially pose harm and/or have reduced efficacy. STUDY DESIGN AND METHODS: The large 4-year publicly available Recipient Epidemiology and Donor Evaluation Study-III (REDS-III) database was used to investigate patient outcomes associated with ABO non-identical platelet transfusions. Outcomes included mortality, sepsis, and subsequent platelet transfusion requirements. RESULTS: Following adjustment for possible confounding factors, no statistically significant association between ABO non-identical platelet transfusion and increased risk of mortality was observed in the overall cohort of 21,176 recipients. However, when analyzed by diagnostic category and recipient ABO group, associations with increased mortality for major mismatched transfusions were noted in two of eight subpopulations. Hematology/Oncology blood group A and B recipients (but not group O) showed a Hazard Ratio (HR) of 1.29 (95%CI: 1.03-1.62) and intracerebral hemorrhage group O recipients (but not groups A and B) showed a HR of 1.75 (95%CI: 1.10-2.80). Major mismatched transfusions were associated with increased odds of receiving additional platelet transfusion each post-transfusion day (through day 5) regardless of the recipient blood group. DISCUSSION: We suggest that prospective studies are needed to determine if specific patient populations would benefit from receiving ABO identical platelet units. Our findings indicate that ABO-identical platelet products minimize patient exposure to additional platelet doses.