Wildland firefighter work history and clinical indicators of cardiovascular health.

OBJECTIVE: To determine the association between the occupational history as a wildland firefighter (WFF) and clinical indicators of cardiovascular health. METHODS: Among 2,862 WFFs we evaluated associations between the number of total days assigned on fire and high-risk categories of three clinically measured cardiovascular indicators. RESULTS: Almost one-third (32%) of WFFs had one or more clinical measures that would place them in high-risk categories for BMI, blood pressure, and total cholesterol. WFF work history was associated with some of these measures: odds ratio (and 95% confidence interval) for highest versus lowest tertile of days on fire were 1.4 (1.2, 1.8) and 1.2 (1.0, 1.5) for high-risk categories of BMI and cholesterol, respectively. CONCLUSION: More frequent screening and targeted health promotion programs for WFFs are warranted to increase awareness of cardiovascular risk and prevention strategies.

Novel cell states arise in embryonic cells devoid of key reprogramming factors.

The capacity for embryonic cells to differentiate relies on a large-scale reprogramming of the oocyte and sperm nucleus into a transient totipotent state. In zebrafish, this reprogramming step is achieved by the pioneer factors Nanog, Pou5f3, and Sox19b (NPS). Yet, it remains unclear whether cells lacking this reprogramming step are directed towards wild type states or towards novel developmental canals in the Waddington landscape of embryonic development. Here we investigate the developmental fate of embryonic cells mutant for NPS by analyzing their single-cell gene expression profiles. We find that cells lacking the first developmental reprogramming steps can acquire distinct cell states. These states are manifested by gene expression modules that result from a failure of nuclear reprogramming, the persistence of the maternal program, and the activation of somatic compensatory programs. As a result, most mutant cells follow new developmental canals and acquire new mixed cell states in development. In contrast, a group of mutant cells acquire primordial germ cell-like states, suggesting that NPS-dependent reprogramming is dispensable for these cell states. Together, these results demonstrate that developmental reprogramming after fertilization is required to differentiate most canonical developmental programs, and loss of the transient totipotent state canalizes embryonic cells into new developmental states in vivo.

The efficacy of 1-allyloxy-4-propoxybenzene (3c{3,6}) against Varroa destructor mites in honey bee colonies from Maryland, USA.

Varroa destructor Oud (Acari: Varroidae) is a harmful ectoparasite of Apis mellifera L. honey bees causing widespread colony losses in Europe and North America. To control populations of these mites, beekeepers have an arsenal of different treatments, including both chemical and nonchemical options. However, nonchemical treatments can be labor intensive, and Varroa has gained resistance to some conventional pesticides, and the use of other chemical treatments is restricted temporally (e.g., cannot be applied during periods of honey production). Thus, beekeepers require additional treatment options for controlling mite populations. The compound 1-allyloxy-4-propoxybenzene (3c{3,6}) is a diether previously shown to be a strong feeding deterrent against Lepidopteran larvae and a repellent against mosquitoes and showed promise as a novel acaricide from laboratory and early field trials. Here we test the effect of the compound, applied at 8 g/brood box on wooden release devices, on honey bees and Varroa in field honey bee colonies located in Maryland, USA, and using a thymol-based commercial product as a positive control. 3c{3,6} had minimal effect on honey bee colonies, but more tests are needed to determine whether it affected egg production by queens. Against Varroa3c{3,6} had an estimated efficacy of 78.5%, while the positive control thymol product showed an efficacy of 91.3%. 3c{3,6} is still in the development stage, and the dose or application method needs to be revisited.

Metaphyseal Fixation Using Cones and Sleeves for Severe Proximal Tibial Bone Loss.

BACKGROUND: Tibial bone defects are commonly encountered in revision total knee arthroplasty (rTKA) and can be managed with metaphyseal cones or sleeves. Few studies have directly compared tibial cones and sleeves in rTKA, and none have limited this comparison to the most severe tibial defects. The purpose of this study was to evaluate and compare the outcomes of metaphyseal cones and sleeves for tibial reconstruction in rTKA regarding implant fixation and clinical outcomes. METHODS: A retrospective review was conducted on patients undergoing rTKA in which metaphyseal cones or sleeves were utilized for addressing metaphyseal bone loss (34 cones and 18 sleeves). Tibial bone loss was classified according to the Anderson Orthopaedic Research Institute bone defect classification, with types 2B and 3 being included. Patient-reported outcomes and postoperative complications were collected, and a radiographic evaluation of osseointegration or loosening was performed. RESULTS: There were 52 knees included (34 cones, 18 sleeves), with a median follow-up of 41.0 months. All-cause implant survival was 100% at 2 years and 96% (95% confidence interval: 76 to 99%) at 4 years, with 98% of tibial components demonstrating osseointegration at the final follow-up. During follow-up, there were a total 11 revisions, of which 1 sleeve was revised secondary to implant loosening. Tibial sleeves had a higher risk of revision compared to tibial cones (P < .01), and sleeves fixed with a hybrid technique were more likely to need revision than cones fixed by the same method (P = .01). CONCLUSIONS: Porous metaphyseal tibial cones and tibial metaphyseal sleeves both performed well at a 41-month median follow-up with no difference in aseptic survivorship between the 2 constructs. Both demonstrate high rates of osseointegration, low rates of aseptic failure, and significant improvement in Knee Society Scores in patients with severe tibial defects in rTKA.

Droplet-based proteomics reveals CD36 as a marker for progenitors in mammary basal epithelium.

Deep proteomic profiling of rare cell populations has been constrained by sample input requirements. Here, we present DROPPS (droplet-based one-pot preparation for proteomic samples), an accessible low-input platform that generates high-fidelity proteomic profiles of 100-2,500 cells. By applying DROPPS within the mammary epithelium, we elucidated the connection between mitochondrial activity and clonogenicity, identifying CD36 as a marker of progenitor capacity in the basal cell compartment. We anticipate that DROPPS will accelerate biology-driven proteomic research for a multitude of rare cell populations.

DNA Released by Adeno-Associated Virus Strongly Alters Capsid Aggregation Kinetics in a Physiological Solution.

While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.

Granulocytic myeloid-derived suppressor cell activity during biofilm infection is regulated by a glycolysis/HIF1a axis.

Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI). A primary contributor to infection chronicity is an expansion of granulocytic myeloid-derived suppressor cells (G-MDSCs), which are critical for orchestrating the antiinflammatory biofilm milieu. Single-cell sequencing and bioinformatic metabolic algorithms were used to explore the link between G-MDSC metabolism and S. aureus PJI outcome. Glycolysis and the hypoxia response through HIF1a were significantly enriched in G-MDSCs. Interfering with both pathways in vivo, using a 2-deoxyglucose nanopreparation and granulocyte-targeted Hif1a conditional KO mice, respectively, attenuated G-MDSC-mediated immunosuppression and reduced bacterial burden in a mouse model of S. aureus PJI. In addition, single-cell RNA-Seq (scRNA-Seq) analysis of granulocytes from PJI patients also showed an enrichment in glycolysis and hypoxia-response genes. These findings support the importance of a glycolysis/HIF1a axis in promoting G-MDSC antiinflammatory activity and biofilm persistence during PJI.

Novel Technique for the Identification of Hip Implants Using Artificial Intelligence.

BACKGROUND: The anticipated growth of total hip arthroplasty will result in an increased need for revision total hip arthroplasty. Preoperative planning, including identifying current implants, is critical for successful revision surgery. Artificial intelligence (AI) is promising for aiding clinical decision-making, including hip implant identification. However, previous studies have limitations such as small datasets, dissimilar stem designs, limited scalability, and the need for AI expertise. To address these limitations, we developed a novel technique to generate large datasets, tested radiographically similar stems, and demonstrated scalability utilizing a no-code machine learning solution. METHODS: We trained, validated, and tested an automated machine learning-implemented convolutional neural network to classify 9 radiographically similar femoral implants with a metaphyseal-fitting wedge taper design. Our novel technique uses computed tomography-derived projections of a 3-dimensional scanned implant model superimposed within a computed tomography pelvis volume. We employed computer-aided design modeling and MATLAB to process and manipulate the images. This generated 27,020 images for training (22,957) and validation (4,063) sets. We obtained 786 test images from various sources. The performance of the model was evaluated by calculating sensitivity, specificity, and accuracy. RESULTS: Our machine learning model discriminated the 9 implant models with a mean accuracy of 97.4%, sensitivity of 88.4%, and specificity of 98.5%. CONCLUSIONS: Our novel hip implant detection technique accurately identified 9 radiographically similar implants. The method generates large datasets, is scalable, and can include historic or obscure implants. The no-code machine learning model demonstrates the feasibility of obtaining meaningful results without AI expertise, encouraging further research in this area.

Subclinical Measures of Cardiovascular Health Among Wildland Firefighters.

OBJECTIVE: The aim of the study is to compare subclinical measures of cardiovascular health among wildland firefighters (WFFs) to the US general population. METHODS: Our cross-sectional study compared body mass index, total cholesterol, and blood pressure in 11,051 WFFs aged 17 to 64 years using Department of the Interior Medical Screening Program clinical screening examinations between 2014-2018 to National Health and Nutrition Examination Survey of 2015-2016 cycle using adjusted logistic regression analyses. RESULTS: The logistic regression model shows significantly higher odds of hypertension and prehypertension in WFFs (2.84 times more with 95% CI: 2.28-3.53) than US general population. There were no consistent differences in body mass index or total cholesterol between the two population. CONCLUSIONS: Hypertension and prehypertension were more prevalent in WFFs compared with the US general population, which suggests the need for actions for protecting against cardiovascular disease among WFFs.

FGL2 promotes tumour growth and attenuates infiltration of activated immune cells in melanoma and ovarian cancer models.

The tumour microenvironment is infiltrated by immunosuppressive cells, such as regulatory T cells (Tregs), which contribute to tumour escape and impede immunotherapy outcomes. Soluble fibrinogen-like protein 2 (sFGL2), a Treg effector protein, inhibits immune cell populations, via receptors FcγRIIB and FcγRIII, leading to downregulation of CD86 in antigen presenting cells and limiting T cell activation. Increased FGL2 expression is associated with tumour progression and poor survival in several different cancers, such as glioblastoma multiforme, lung, renal, liver, colorectal, and prostate cancer. Querying scRNA-seq human cancer data shows FGL2 is produced by cells in the tumour microenvironment (TME), particularly monocytes and macrophages as well as T cells and dendritic cells (DCs), while cancer cells have minimal expression of FGL2. We studied the role of FGL2 exclusively produced by cells in the TME, by leveraging Fgl2 knockout mice. We tested two murine models of cancer in which the role of FGL2 has not been previously studied: epithelial ovarian cancer and melanoma. We show that absence of FGL2 leads to a more activated TME, including activated DCs (CD86+, CD40+) and T cells (CD25+, TIGIT+), as well as demonstrating for the first time that the absence of FGL2 leads to more activated natural killer cells (DNAM-1+, NKG2D+) in the TME. Furthermore, the absence of FGL2 leads to prolonged survival in the B16F10 melanoma model, while the absence of FGL2 synergizes with oncolytic virus to prolong survival in the ID8-p53-/-Brca2-/- ovarian cancer model. In conclusion, targeting FGL2 is a promising cancer treatment strategy alone and in combination immunotherapies.

Differential DNA damage repair and PARP inhibitor vulnerability of the mammary epithelial lineages.

It is widely assumed that all normal somatic cells can equally perform homologous recombination (HR) and non-homologous end joining in the DNA damage response (DDR). Here, we show that the DDR in normal mammary gland inherently depends on the epithelial cell lineage identity. Bioinformatics, post-irradiation DNA damage repair kinetics, and clonogenic assays demonstrated luminal lineage exhibiting a more pronounced DDR and HR repair compared to the basal lineage. Consequently, basal progenitors were far more sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis) in both mouse and human mammary epithelium. Furthermore, PARPi sensitivity of murine and human breast cancer cell lines as well as patient-derived xenografts correlated with their molecular resemblance to the mammary progenitor lineages. Thus, mammary epithelial cells are intrinsically divergent in their DNA damage repair capacity and PARPi vulnerability, potentially influencing the clinical utility of this targeted therapy.

Apollo-NADP+ reveals in vivo adaptation of NADPH/NADP+ metabolism in electrically activated pancreatic ß cells.

Several genetically encoded sensors have been developed to study live cell NADPH/NADP+ dynamics, but their use has been predominantly in vitro. Here, we developed an in vivo assay using the Apollo-NADP+ sensor and microfluidic devices to measure endogenous NADPH/NADP+ dynamics in the pancreatic ß cells of live zebrafish embryos. Flux through the pentose phosphate pathway, the main source of NADPH in many cell types, has been reported to be low in ß cells. Thus, it is unclear how these cells compensate to meet NADPH demands. Using our assay, we show that pyruvate cycling is the main source of NADP+ reduction in ß cells, with contributions from folate cycling after acute electrical activation. INS1E ß cells also showed a stress-induced increase in folate cycling and further suggested that this cycling requires both increased glycolytic intermediates and cytosolic NAD+. Overall, we show in vivo application of the Apollo-NADP+ sensor and reveal that ß cells are capable of adapting NADPH/NADP+ redox during stress.

Role of Domain-Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt.

Monoclonal antibodies (mAbs) make up a major class of biotherapeutics with a wide range of clinical applications. Their physical stability can be affected by various environmental factors. For instance, an acidic pH can be encountered during different stages of the mAb manufacturing process, including purification and storage. Therefore, understanding the behavior of flexible mAb molecules in acidic solution environments will benefit the development of stable mAb products. This study used small-angle X-ray scattering (SAXS) and complementary biophysical characterization techniques to investigate the conformational flexibility and protein-protein interactions (PPI) of a model mAb molecule under near-neutral and acidic conditions. The study also characterized the interactions between Fab and Fc fragments under the same buffer conditions to identify domain-domain interactions. The results suggest that solution pH significantly influences mAb flexibility and thus could help mAbs remain physically stable by maximizing local electrostatic repulsions when mAbs become crowded in solution. Under acidic buffer conditions, both Fab and Fc contribute to the repulsive PPI observed among the full mAb at a low ionic strength. However, as ionic strength increases, hydrophobic interactions lead to the self-association of Fc fragments and, subsequently, could affect the aggregation state of the mAb.

Chromatin expansion microscopy reveals nanoscale organization of transcription and chromatin.

Nanoscale chromatin organization regulates gene expression. Although chromatin is notably reprogrammed during zygotic genome activation (ZGA), the organization of chromatin regulatory factors during this universal process remains unclear. In this work, we developed chromatin expansion microscopy (ChromExM) to visualize chromatin, transcription, and transcription factors in vivo. ChromExM of embryos during ZGA revealed how the pioneer factor Nanog interacts with nucleosomes and RNA polymerase II (Pol II), providing direct visualization of transcriptional elongation as string-like nanostructures. Blocking elongation led to more Pol II particles clustered around Nanog, with Pol II stalled at promoters and Nanog-bound enhancers. This led to a new model termed "kiss and kick", in which enhancer-promoter contacts are transient and released by transcriptional elongation. Our results demonstrate that ChromExM is broadly applicable to study nanoscale nuclear organization.

Liposome-tethered supported lipid bilayer platform for capture and release of heterogeneous populations of circulating tumor cells.

Because of scarcity, vulnerability, and heterogeneity in the population of circulating tumor cells (CTCs), the CTC isolation system relying on immunoaffinity interaction exhibits inconsistent efficiencies for all types of cancers and even CTCs with different phenotypes in individuals. Moreover, releasing viable CTCs from an isolation system is of importance for molecular analysis and drug screening in precision medicine, which remains a challenge for current systems. In this work, a new CTC isolation microfluidic platform was developed and contains a coating of the antibody-conjugated liposome-tethered-supported lipid bilayer in a developed chaotic-mixing microfluidic system, referred to as the "LIPO-SLB" platform. The biocompatible, soft, laterally fluidic, and antifouling properties of the LIPO-SLB platform offer high CTC capture efficiency, viability, and selectivity. We successfully demonstrated the capability of the LIPO-SLB platform to recapitulate different cancer cell lines with different antigen expression levels. In addition, the captured CTCs in the LIPO-SLB platform can be detached by air foam to destabilize the physically assembled bilayer structures due to a large water/air interfacial area and strong surface tension. More importantly, the LIPO-SLB platform was constructed and used for the verification of clinical samples from 161 patients with different primary cancer types. The mean values of both single CTCs and CTC clusters correlated well with the cancer stages. Moreover, a considerable number of CTCs were isolated from patients' blood samples in the early/localized stages. The clinical validation demonstrated the enormous potential of the universal LIPO-SLB platform as a tool for prognostic and predictive purposes in precision medicine.

Evaluation of Three Automated Nontreponemal Rapid Plasma Reagin (RPR) Tests for the Laboratory Diagnosis of Syphilis.

Automated nontreponemal rapid plasma reagin (RPR) tests were recently introduced in the United States for syphilis testing and limited performance data are available. In collaboration with the Association of Public Health Laboratories, three public health laboratories (PHL) were chosen through a competitive selection process to evaluate the performance of three FDA-cleared automated RPR test systems: BioPlex 2200 Syphilis Total & RPR assay (Bio-Rad Laboratories), AIX 1000 (Gold Standard Diagnostics), and ASI Evolution (Arlington Scientific). Panels prepared at the CDC included: a qualitative panel comprised of 734 syphilis reactive/nonreactive sera; a quantitative panel of 50 syphilis reactive sera (RPR titer 1:64 to 1:1,024); and a reproducibility panel of 15 nonreactive and reactive sera (RPR titer 1:1 to 1:64). Panels were shipped frozen to the PHL and tested on the automated RPR systems following manufacturers' instructions. Prior test results were blinded to all laboratories. When compared to manual RPR (Arlington Scientific) performed at the CDC as a reference test, the qualitative panel results demonstrated an overall concordance of 95.9% for AIX 1000, 94.6% for ASI Evolution, and 92.6% for Bioplex RPR; quantitative panel showed within range titer of 2-fold for 94% of specimens for AIX 1000, 68% for ASI Evolution, and 64% for BioPlex RPR, and the reproducibility testing panel demonstrated point estimates ranging from 69 to 95%. Automated RPR instruments could reduce turnaround time and minimize interpretation errors. However, additional evaluations with more specimens could assist laboratories with implementing automated RPR tests and understanding their limitations.

The Challenge of Emerging Resistant Gram-Positive Pathogens in Hip and Knee Periprosthetic Joint Infections.

➤: An increase in resistant bacterial pathogens has occurred over the last 4 decades. ➤: Careful patient selection and improving or correcting risk factors for periprosthetic joint infection (PJI) before elective surgical treatment are strongly recommended. ➤: Appropriate microbiological methods, including those used to detect and grow Cutibacterium acnes, are recommended. ➤: Antimicrobial agents used in the prevention or management of infection should be selected appropriately and the duration of therapy should be carefully considered in order to mitigate the risk of developing bacterial resistance. ➤: Molecular methods including rapid polymerase chain reaction (PCR) diagnostics, 16S sequencing, and/or shotgun and/or targeted whole-genome sequencing are recommended in culture-negative cases of PJI. ➤: Expert consultation with an infectious diseases specialist (if available) is recommended to assist with the appropriate antimicrobial management and monitoring of patients with PJI.

Fatty acid transport protein inhibition sensitizes breast and ovarian cancers to oncolytic virus therapy via lipid modulation of the tumor microenvironment.

Introduction: Adipocytes in the tumour microenvironment are highly dynamic cells that have an established role in tumour progression, but their impact on anti-cancer therapy resistance is becoming increasingly difficult to overlook. Methods: We investigated the role of adipose tissue and adipocytes in response to oncolytic virus (OV) therapy in adipose-rich tumours such as breast and ovarian neoplasms. Results: We show that secreted products in adipocyte-conditioned medium significantly impairs productive virus infection and OV-driven cell death. This effect was not due to the direct neutralization of virions or inhibition of OV entry into host cells. Instead, further investigation of adipocyte secreted factors demonstrated that adipocyte-mediated OV resistance is primarily a lipid-driven phenomenon. When lipid moieties are depleted from the adipocyte-conditioned medium, cancer cells are re-sensitized to OV-mediated destruction. We further demonstrated that blocking fatty acid uptake by cancer cells, in a combinatorial strategy with virotherapy, has clinical translational potential to overcome adipocyte-mediated OV resistance. Discussion: Our findings indicate that while adipocyte secreted factors can impede OV infection, the impairment of OV treatment efficacy can be overcome by modulating lipid flux in the tumour milieu.