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Introduction: Bovine mastitis caused by Escherichia coli compromises animal health and inflicts substantial product losses in dairy farming. It may manifest as subclinical through severe acute disease and can be transient or persistent in nature. Little is known about bacterial factors that impact clinical outcomes or allow some strains to outcompete others in the mammary gland (MG) environment. Mastitis-associated E. coli (MAEC) may have distinctive characteristics which may contribute to the varied nature of the disease. Given their high levels of intraspecies genetic variability, virulence factors of commonly used MAEC model strains may not be relevant to all members of this group. Methods: In this study, we sequenced the genomes of 96 MAEC strains isolated from cattle with clinical mastitis (CM). We utilized clinical severity data to perform genome-wide association studies to identify accessory genes associated with strains isolated from mild or severe CM, or with high or low competitive fitness during in vivo competition assays. Genes associated with mastitis pathogens or commensal strains isolated from bovine sources were also identified. Results: A type-2 secretion system (T2SS) and a chitinase (ChiA) exported by this system were strongly associated with pathogenic isolates compared with commensal strains. Deletion of chiA from MAEC isolates decreased their adherence to cultured bovine mammary epithelial cells. Discussion: The increased fitness associated with strains possessing this gene may be due to better attachment in the MG. Overall, these results provide a much richer understanding of MAEC and suggest bacterial processes that may underlie the clinical diversity associated with mastitis and their adaptation to this unique environment.

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Multiple infiltrative disorders can affect the small bowel, often resulting in diffuse small bowel wall thickening. These infiltrative disorders can manifest owing to various factors such as an influx of immunologic or neoplastic cells or the accumulation of substances within one or more layers of the intestinal wall. Although there can be considerable overlap in the appearances of infiltrative diseases on cross-sectional images, a comprehensive understanding of more specific ancillary imaging features and clinicopathologic correlation can substantially narrow the differential diagnosis. The radiologist can be instrumental in synthesizing the clinical and imaging information and guiding subsequent workup. The authors present a comprehensive review of the infiltrative disorders that commonly involve the small bowel. These disorders are organized on the basis of their pathophysiologic features, with multiple illustrative case examples to enhance understanding of these entities. CT and MRI are currently the most commonly used imaging modalities for evaluating small bowel disorders, and this review is focused on these two modalities. Detailed information regarding the pathologic features, clinical presentation, and imaging findings of these infiltrative disorders is provided to aid radiologists in recognizing and differentiating these conditions. ©RSNA, 2024.

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Because of the decreasing supply of new antibiotics, recent outbreaks of infectious diseases, and the emergence of antibiotic-resistant microorganisms, it is imperative to develop new effective strategies for deactivating a broad spectrum of microorganisms and viruses. We have implemented electrically polarized nanoscale metallic (ENM) coatings that deactivate a wide range of microorganisms including Gram-negative and Gram-positive bacteria with greater than 6-log reduction in less than 10 minutes of treatment. The electrically polarized devices were also effective in deactivating lentivirus and Candida albicans. The key to the high deactivation effectiveness of ENM devices is electrochemical production of micromolar cuprous ions, which mediated reduction of oxygen to hydrogen peroxide. Formation of highly damaging species, hydroxyl radicals and hypochlorous acid, from hydrogen peroxide contributed to antimicrobial properties of the ENM devices. The electric polarization of nanoscale coatings represents an unconventional tool for deactivating a broad spectrum of microorganisms through in situ production of reactive oxygenated and chlorinated species.

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Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis.

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Background: West Nile virus (WNV), Everglades virus (EVEV), and five species of Orthobunyavirus were isolated from mosquitoes collected in the Everglades in 2016-2017. Prior studies of blood meals of mosquitoes in southern Florida have related findings to acquisition and transmission of EVEV, St. Louis encephalitis virus, and WNV, but not the Orthobunyavirus viruses associated with the subgenus Melanoconion of the genus Culex. Materials and Methods: In the present study, blood-fed mosquitoes were collected in the Everglades in 2016, 2017, 2021, and 2022, and from an industrial site in Naples, FL in 2017. Blood meals were identified to host species by PCR assays using mitochondrial cytochrome b gene. Results: Blood meals were identified from Anopheles crucians complex and 11 mosquito species captured in the Florida Everglades and from 3 species collected from an industrial site. The largest numbers of blood-fed specimens were from Culex nigripalpus, Culex erraticus, Culex cedecei, and Aedes taeniorhynchus. Cx. erraticus fed on mammals, birds, and reptiles, particularly American alligator. This mosquito species could transmit WNV to American alligator in the wild. Cx. nigripalpus acquired blood meals primarily from birds and mammals and frequently fed on medium-sized mammals and white-tailed deer. Water and wading birds were the primary avian hosts for Cx. nigripalpus and Cx. erraticus in the Everglades. Wading birds are susceptible to WNV and could serve as reservoir hosts. Cx. cedecei fed on five species of rodents, particularly black and hispid cotton rats. EVEV and three different species of Orthobunyavirus have been isolated from the hispid cotton rat and Cx. cedecei in the Everglades. Cx. cedecei is likely acquiring and transmitting these viruses among hispid cotton rats and other rodents. The marsh rabbit was a frequent host for An. crucians complex. An. crucians complex, and other species could acquire Tensaw virus from rabbits. Conclusions: Our study contributes to a better understanding of the host and viral associations of mosquito species in southwestern Florida.

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Apoptosis is characterized by membrane blebbing and apoptotic body formation. Caspase cleavage of ROCK1 generates an active fragment that promotes actin-myosin-mediated contraction and membrane blebbing during apoptosis. Expression of caspase-resistant non-cleavable ROCK1 (Rock1 NC) prolonged survival of mice that rapidly develop B cell lymphomas due to Eµ-Myc transgene expression. Eµ-Myc; Rock1 NC mice had significantly fewer bone marrow cells relative to those in Eµ-Myc mice expressing wild-type ROCK1 (Rock1 WT), which was associated with altered cell cycle profiles. Circulating macrophage numbers were lower in Eµ-Myc; Rock1 NC mice, but there were higher levels of bone marrow macrophages, consistent with spontaneous cell death in Eµ-Myc; Rock1 NC mouse bone marrows being more inflammatory. Rock1 WT recipient mice transplanted with pre-neoplastic Eµ-Myc; Rock1 NC bone marrow cells survived longer than mice transplanted with Eµ-Myc; Rock1 WT cells, indicating that the survival benefit was intrinsic to the Eµ-Myc; Rock1 NC bone marrow cells. The results suggest that the apoptotic death of Eµ-Myc; Rock1 NC cells generates a proliferation-suppressive microenvironment in bone marrows that reduces cell numbers and prolongs B cell lymphoma mouse survival.

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Proteins often exist and function as part of higher-order complexes or networks. A challenge is to identify the universe of proximal and interacting partners for a given protein. We describe how the high-activity promiscuous biotin ligase called TurboID is fused to the actin-binding peptide LifeAct to label by biotinylation proteins that bind, or are in close proximity, to actin. The rapid enzyme kinetics of TurboID allows the profiles of actin-binding proteins to be compared under different conditions, such as acute disruption of filamentous actin structures with cytochalasin D.

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PURPOSE: To evaluate the trends in utilization and results of computed tomography pulmonary angiography (CTPA study) for detection of acute pulmonary embolism (PE) in the Emergency Department (ED) during different phases of COVID-19 public health emergency. METHODS: We conducted a retrospective review of CTPA studies ordered through our ED in the months of March through May during five consecutive years from 2019 to 2023, designated as pre-pandemic, early, ongoing, recovery, and post-pandemic periods respectively. Collected characteristics included patient age, patient sex, and result of the study. RESULTS: The utilization of CTPA studies for ED patients increased during the early, ongoing, and recovery periods. CTPA study utilization in the post-pandemic period was not significantly different from the pre-pandemic period (p = 0.08). No significant difference in CTPA study utilization was noted in the other periods when stratified by age group or sex, compared to the pre-pandemic period. The positivity rate of acute PE in ED patients was not significantly different in other periods compared to the pre-pandemic period. CONCLUSION: At our institution, the utilization and positivity rates of CTPA studies for the ED patients were not significantly different in the post-pandemic period compared to the pre-pandemic period. While studies spanning a larger timeframe and involving multiple institutions are needed to test the applicability of this observation to a wider patient population beyond our defined post-pandemic period, we conclude that our study provides some confidence to the ordering provider and the radiologist in embracing the end of COVID-19 public health emergency by the WHO and the United States HHS with respect to CTPA studies.

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Chimeric antigen receptor (CAR) T cells are an effective treatment for some blood cancers. However, the lack of tumor-specific surface antigens limits their wider use. We identified a set of surface antigens that are limited in their expression to cancer and the central nervous system (CNS). We developed CAR T cells against one of these antigens, LINGO1, which is widely expressed in Ewing sarcoma (ES). To prevent CNS targeting, we engineered LINGO1 CAR T cells lacking integrin α4 (A4ko), an adhesion molecule essential for migration across the blood-brain barrier. A4ko LINGO1 CAR T cells were efficiently excluded from the CNS but retained efficacy against ES. We show that altering adhesion behavior expands the set of surface antigens targetable by CAR T cells.

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PURPOSE: With expansion of academic cancer center networks across geographically-dispersed sites, ensuring high-quality delivery of care across all network affiliates is essential. We report on the characteristics and efficacy of a radiation oncology peer-review quality assurance (QA) system implemented across a large-scale multinational cancer network. METHODS AND MATERIALS: Since 2014, weekly case-based peer-review QA meetings have been standard for network radiation oncologists with radiation oncology faculty at a major academic center. This radiotherapy (RT) QA program involves pre-treatment peer-review of cases by disease site, with disease-site subspecialized main campus faculty members. This virtual QA platform involves direct review of the proposed RT plan as well as supporting data, including relevant pathology and imaging studies for each patient. Network RT plans were scored as being concordant or nonconcordant based on national guidelines, institutional recommendations, and/or expert judgment when considering individual patient-specific factors for a given case. Data from January 1, 2014, through December 31, 2019, were aggregated for analysis. RESULTS: Between 2014 and 2019, across 8 network centers, a total of 16,601 RT plans underwent peer-review. The network-based peer-review case volume increased over the study period, from 958 cases in 2014 to 4,487 in 2019. A combined global nonconcordance rate of 4.5% was noted, with the highest nonconcordance rates among head-and-neck cases (11.0%). For centers that joined the network during the study period, we observed a significant decrease in the nonconcordance rate over time (3.1% average annual decrease in nonconcordance, P = 0.01); among centers that joined the network prior to the study period, nonconcordance rates remained stable over time. CONCLUSIONS: Through a standardized QA platform, network-based multinational peer-review of RT plans can be achieved. Improved concordance rates among newly added network affiliates over time are noted, suggesting a positive impact of network membership on the quality of delivered cancer care.

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BACKGROUND: Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking. METHODS: Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging. RESULTS: High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging. CONCLUSIONS: We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization.

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Staphylococcus aureus are gram-positive bacteria responsible for a wide array of diseases, ranging from skin and soft tissue infections to more chronic illnesses such as toxic shock syndrome, osteomyelitis, and endocarditis. Vancomycin is currently one of the most effective antibiotics available in treating patients infected with methicillin-resistant S. aureus (MRSA), however the emergence of vancomycin-resistant S. aureus (VRSA), and more commonly vancomycin-intermediate S. aureus (VISA), threaten the future efficacy of vancomycin. Intermediate resistance to vancomycin occurs due to mutations within the loci of Staphylococcal genes involved in cell wall formation such as rpoB, graS, and yycG. We hypothesized the VISA phenotype may also arise as a result of the natural stress occurring within S. aureus biofilms, and that this phenomenon is mediated by the RecA/SOS response. Wildtype and recA null mutant/lexAG94E strains of S. aureus biofilms were established in biofilm microtiter assays or planktonic cultures with or without the addition of sub-inhibitory concentrations of vancomycin (0.063 mg/l - 0.25 mg/L ciprofloxacin, 0.5 mg/l vancomycin). Efficiency of plating techniques were used to quantify the subpopulation of biofilm-derived S. aureus cells that developed vancomycin-intermediate resistance. The results indicated that a greater subpopulation of cells from wildtype biofilms (4.16 × 102 CFUs) emerged from intermediate-resistant concentrations of vancomycin (4 µg/ml) compared with the planktonic counterpart (1.53 × 101 CFUs). Wildtype biofilms (4.16 × 102 CFUs) also exhibited greater resistance to intermediate-resistant concentrations of vancomycin compared with strains deficient in the recA null mutant (8.15 × 101 CFUs) and lexA genes (8.00 × 101 CFUs). While the VISA phenotype would be an unintended consequence of genetic diversity and potentially gene transfer in the biofilm setting, it demonstrates that mutations occurring within biofilms allow for S. aureus to adapt to new environments, including the presence of widely used antibiotics.

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BACKGROUND: Septoplasty is used to correct nasal obstruction from nasal septum deviation. However, the long-term efficacy of septoplasty is unclear, and no literature reviews have examined long-term outcomes of septoplasty with or without turbinate modification. This systematic review aimed to evaluate the long-term efficacy of septoplasty with or without turbinate modification in improving nasal obstruction. DATA SOURCES: PubMed, EMBASE, Cochrane CENTRAL. METHODS: A systematic review of the literature was conducted using the aforementioned databases. Studies reporting outcomes 12+ months after functional septoplasty with or without turbinate surgery for nasal obstruction were included. Septorhinoplasties, concurrent sinus surgery, pediatric studies, and studies where septoplasty was performed for indications other than nasal obstruction were excluded. RESULTS: After screening, 35 studies with 4,432 patients were included. Mean weighted post-operative follow-up time was 29.1 months (range 12-120 months). All studies reported significant improvement in subjective and objective outcomes at long-term follow-up compared to baseline. When comparing short-term (<12 months) to long-term (≥12 months) outcomes, four studies noticed that subjective outcomes worsened slightly over time, but no study found a significant change in objective outcomes over time. In addition, 23 studies reported patient satisfaction and/or improvement rates, with 75.4% (2,348/3,113) of patients expressing satisfaction/improvement at an average of 27.0 months after surgery. CONCLUSIONS: Overall, septoplasty with or without turbinate modification shows significant improvement in obstructive symptoms at long-term follow-up per both objective and subjective measures. Whether outcomes may worsen slightly over time remains indeterminate based on mixed results in the literature. LEVEL OF EVIDENCE: N/A Laryngoscope, 2023.

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During the second meiotic cell division, egg cells discard one set of chromatids to the polar body to produce a large haploid gamete. Meiotic spindle rotation is a critical step to ensure proper polar body extrusion. In this issue, Bourdais et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202211029) have identified MRCKß as an essential kinase for efficient spindle rotation. MRCK activates cortical myosin II rings overlying the spindle to prevent the notoriously sticky interaction between the cell cortex and chromatin to facilitate spindle rotation. Furthermore, Bourdais et al. found that the same MRCK-myosin II pathway also operates in zygotes to promote parental genome unification.

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T cells expressing chimeric antigen receptors (CARs) have shown remarkable therapeutic activity against different types of cancer. However, the wider use of CAR T cells has been hindered by the potential for life-threatening toxicities due to on-target off-tumor killing of cells expressing low amounts of the target antigen. CD229, a signaling lymphocyte-activation molecule (SLAM) family member, has previously been identified as a target for CAR T cell-mediated treatment of multiple myeloma (MM) due to its high expression on the surfaces of MM cells. CD229 CAR T cells have shown effective clearance of MM cells in vitro and in vivo. However, healthy lymphocytes also express CD229, albeit at lower amounts than MM cells, causing their unintended targeting by CD229 CAR T cells. To increase the selectivity of CD229 CAR T cells for MM cells, we used a single amino acid substitution approach of the CAR binding domain to reduce CAR affinity. To identify CARs with increased selectivity, we screened variant binding domains using solid-phase binding assays and biolayer interferometry and determined the cytotoxic activity of variant CAR T cells against MM cells and healthy lymphocytes. We identified a CD229 CAR binding domain with micromolar affinity that, when combined with overexpression of c-Jun, confers antitumor activity comparable to parental CD229 CAR T cells but lacks the parental cells' cytotoxic activity toward healthy lymphocytes in vitro and in vivo. The results represent a promising strategy to improve the efficacy and safety of CAR T cell therapy that requires clinical validation.

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Cytotoxic T lymphocytes (CTLs) fight intracellular pathogens and cancer by identifying and destroying infected or transformed target cells1. To kill, CTLs form a specialized cytotoxic immune synapse (IS) with a target of interest and then release toxic perforin and granzymes into the interface to elicit programmed cell death2-5. The IS then dissolves, enabling CTLs to search for additional prey and professional phagocytes to clear the corpse6. While the mechanisms governing IS assembly have been studied extensively, far less is known about target cell release. Here, we applied time-lapse imaging to explore the basis for IS dissolution and found that it occurred concomitantly with the cytoskeletal contraction of apoptotic targets. Genetic and pharmacological perturbation of this contraction response indicated that it was both necessary and sufficient for CTL dissociation. We also found that mechanical amplification of apoptotic contractility promoted faster CTL detachment and serial killing. Collectively, these results establish a biophysical basis for IS dissolution and highlight the importance of mechanosensory feedback in the regulation of cell-cell interactions.

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De Neys argues against assigning exclusive capacities to automatic versus controlled processes. The dual implicit process model provides a theoretical rationale for the exclusivity of automatic threat processing, and corresponding data provide empirical evidence of such exclusivity. De Neys's dismissal of exclusivity is premature and based on a limited sampling of psychological research.

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PURPOSE OF REVIEW: Heart failure (HF) is a growing public health concern that impairs the quality of life and is associated with significant mortality. As the prevalence of heart failure increases, multidisciplinary care is essential to provide comprehensive care to individuals. RECENT FINDINGS: The challenges of implementing an effective multidisciplinary care team can be daunting. Effective multidisciplinary care begins at the initial diagnosis of heart failure. The transition of care from the inpatient to the outpatient setting is critically important. The use of home visits, case management, and multidisciplinary clinics has been shown to decrease mortality and heart failure hospitalizations, and major society guidelines endorse multidisciplinary care for heart failure patients. Expanding heart failure care beyond cardiology entails incorporating primary care, advanced practice providers, and other disciplines. Patient education and self-management are fundamental to multidisciplinary care, as is a holistic approach to effectively address comorbid conditions. Ongoing challenges include navigating social disparities within heart failure care and limiting the economic burden of the disease.

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