A Multi-Stage Dyadic Qualitative Analysis to Disentangle How Dietary Behaviors of Asian American Young Adults are Influenced by Family.

The dietary behaviors of Asian American (AA) young adults, who face a growing non-communicable disease burden, are impacted by complex socio-ecological forces. Family plays a crucial role in the lifestyle behaviors of AA young adults; however, little is known on the methods, contributors, and impact of familial dietary influence. This study aims to deconstruct the mechanisms of AA young adult familial dietary influence through a multi-perspective qualitative assessment. A five-phase method of dyadic analysis adapted from past research was employed to extract nuanced insights from dyadic interviews with AA young adults and family members, and ground findings in behavioral theory (the Social Cognitive Theory, SCT). 37 interviews were conducted: 18 young adults, comprising 10 different AA ethnic subgroups, and 19 family members (10 parents, 9 siblings). Participants described dietary influences that were both active (facilitating, shaping, and restricting) and passive (e.g., sharing foods or environment, mirroring food behaviors). Influences connected strongly with multiple SCT constructs (e.g., behavioral capacity, reinforcements for active influences, and expectations, observational learning for passive influences). Familial influence contributed to changes in the total amount, variety, and healthfulness of foods consumed. Intra-family dynamics were crucial; family members often leveraged each other's persuasiveness or food skills to collaboratively influence diet. AA family-based interventions should consider incorporating both passive and active forms of dietary influence within a family unit, involve multiple family members, and allow for individualization to the unique dynamics and dietary behaviors within each family unit.

Magnetic Silica-Coated Fluorescent Microspheres (MagSiGlow) for Simultaneous Detection of Tumor-Associated Proteins.

Multiplexed bead assays for solution-phase biosensing often encounter cross-over reactions during signal amplification steps, leading to unwanted false positive and high background signals. Current solutions involve complex custom-designed and costly equipment, limiting their application in simple laboratory setup. In this study, we introduce a straightforward protocol to adapt a multiplexed single-bead assay to standard fluorescence imaging plates, enabling the simultaneous analysis of thousands of reactions per plate. This approach focuses on the design and synthesis of bright fluorescent and magnetic microspheres (MagSiGlow) with multiple fluorescent wavelengths serving as unique detection markers. The imaging-based, single-bead assay, combined with a scripted algorithm, allows the detection, segmentation, and co-localization on average of 7500 microspheres per field of view across five imaging channels in less than one second. We demonstrate the effectiveness of this method with remarkable sensitivity at low protein detection limits (100 pg/mL). This technique showed over 85 % reduction in signal cross-over to the solution-based method after the concurrent detection of tumor-associated protein biomarkers. This approach holds the promise of substantially enhancing high throughput biosensing for multiple targets, seamlessly integrating with rapid image analysis algorithms.

Isolated Ocular Mpox without Skin Lesions, United States.

We report a case of a 53-year-old HIV-negative patient in San Francisco, California, USA, with no classic mpox prodromal symptoms or skin lesions who experienced fulminant, vision-threatening scleritis, keratitis, and uveitis. Deep sequence analysis identified monkeypox virus RNA in the aqueous humor. We confirmed the virus on the cornea and sclera by PCR.

Expanding the pipeline for multipurpose prevention technologies: compounds with potential activity to prevent or treat HIV and other STIs.

BACKGROUND: Continued high incidence of HIV and other STIs, paired with rising antibiotic resistance to a number of existing treatments, warrants the development of new pharmaceutical approaches for STI prevention. Multipurpose prevention technologies (MPTs) offer an innovative approach for expanding HIV/STI prevention. The majority of MPT product candidates currently in development include HIV prevention, while only half include compounds active against non-HIV STIs. METHODS: This narrative review focuses on compounds in preclinical development (in vitro and in vivo) through phase 3 clinical trials with activity against one or more of the following infections: HIV, HSV-1, HSV-2, Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, and Trichomonas vaginalis. Bacterial vaginosis is included due to its association with increased risk of STIs. The focus is on compounds with novel mechanisms of action and prophylactic and/or therapeutic potential. Articles published in PubMed between 2011 and 2021, NIH RePorter and conference abstracts and proceedings between 2020 and 2021 were searched. Excluded from the review are compounds that are already being used in MPT product candidates. MAIN RESULTS: There is a growing pipeline of compounds targeting viral STIs, many of which have successfully transitioned from preclinical to clinical stages of development. However, the product development pipeline remains limited for compounds that target bacterial STIs. CONCLUSIONS: The paucity of new pharmaceutical approaches for STI prevention, particularly non-HIV STIs, remains a public health gap. Future funding priorities should include STI prevention research. Despite limited attention to STI prevention in the development of MPTs, many research institutions worldwide are working on discoveries of new compounds, exploring new indications for existing drugs or on innovative drug delivery mechanisms. Our findings can be used to connect researchers across the globe to advance the development of compounds that have potential as active pharmaceutical ingredients in future MPTs.

Extracellular Vesicle Analysis Allows for Identification of Invasive IPMN.

BACKGROUND AND AIMS: Advances in cross-sectional imaging have resulted in increased detection of intraductal papillary mucinous neoplasms (IPMNs), and their management remains controversial. At present, there is no reliable noninvasive method to distinguish between indolent and high risk IPMNs. We performed extracellular vesicle (EV) analysis to identify markers of malignancy in an attempt to better stratify these lesions. METHODS: Using a novel ultrasensitive digital extracellular vesicle screening technique (DEST), we measured putative biomarkers of malignancy (MUC1, MUC2, MUC4, MUC5AC, MUC6, Das-1, STMN1, TSP1, TSP2, EGFR, EpCAM, GPC1, WNT-2, EphA2, S100A4, PSCA, MUC13, ZEB1, PLEC1, HOOK1, PTPN6, and FBN1) in EV from patient-derived cell lines and then on circulating EV obtained from peripheral blood drawn from patients with IPMNs. We enrolled a total of 133 patients in two separate cohorts: a clinical discovery cohort (n = 86) and a validation cohort (n = 47). RESULTS: From 16 validated EV proteins in plasma samples collected from the discovery cohort, only MUC5AC showed significantly higher levels in high-grade lesions. Of the 11 patients with invasive IPMN (inv/HG), 9 had high MUC5AC expression in plasma EV of the 11 patients with high-grade dysplasia alone, only 1 had high MUC5AC expression (sensitivity of 82%, specificity of 100%). These findings were corroborated in a separate validation cohort. The addition of MUC5AC as a biomarker to imaging and high-riskstigmata allowed detection of all cases requiring surgery, whereas imaging and high-risk stigmata alone would have missed 5 of 14 cases (36%). CONCLUSIONS: MUC5AC in circulating EV can predict the presence of invasive carcinoma within IPMN. This approach has the potential to improve the management and follow-up of patients with IPMN including avoiding unnecessary surgery.

The Impact of Human Lipoaspirate and Adipose Tissue-Derived Stem Cells Contact Culture on Breast Cancer Cells: Implications in Breast Reconstruction.

BACKGROUND: Autologous fat transfer in the form of lipoaspirates for the reconstruction of the breast after breast cancer surgery is a commonly used procedure in plastic surgery. However, concerns regarding the oncologic risk of nutrient-rich fat tissue are widely debated. Previous studies have primarily focused on studying the interaction between adipose-derived stem cells (ASCs) and breast cancer cells. METHODS: In this study, we performed a comprehensive analysis of the paracrine- and contact-based interactions between lipoaspirates, ASCs and breast cancer cell lines. An inverted flask culture method was used to study the contact-based interaction between lipoaspirates and breast cancer cells, while GFP-expressing breast cancer cell lines were generated to study the cell-cell contact interaction with ASCs. Three different human breast cancer cell lines, MCF-7, MDA-MB-231 and BT-474, were studied. We analyzed the impact of these interactions on the proliferation, cell cycle and epithelial-to-mesenchymal (EMT) transition of the breast cancer cells. RESULTS: Our results revealed that both lipoaspirates and ASCs do not increase the proliferation rate of the breast cancer cells either through paracrine- or contact-dependent interactions. We observed that lipoaspirates selectively inhibit the proliferation of MCF-7 cells in contact co-culture, driven by the retinoblastoma (Rb) protein activity mediating cell cycle arrest. Additionally, ASCs inhibited MDA-MB-231 breast cancer cell proliferation in cell-cell contact-dependent interactions. Quantitative real-time PCR revealed no significant increase in the EMT-related genes in breast cancer cells upon co-culture with ASCs. CONCLUSION: In conclusion, this study provides evidence of the non-oncogenic character of lipoaspirates and supports the safety of clinical fat grafting in breast reconstruction after oncological surgical procedures. In vivo studies in appropriate animal models and long-term post-operative clinical data from patients are essential to reach the final safety recommendations.

Quantitating drug-target engagement in single cells in vitro and in vivo.

Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.

Spatiotemporal pharmacodynamics of meropenem- and tobramycin-treated Pseudomonas aeruginosa biofilms.

OBJECTIVES: The selection and dose of antibiotic therapy for biofilm-related infections are based on traditional pharmacokinetic studies using planktonic bacteria. The objective of this study was to characterize the time course and spatial activity of human exposure levels of meropenem and tobramycin against Pseudomonas aeruginosa biofilms grown in an in vitro flow-chamber model. METHODS: Pharmacokinetic profiles of meropenem and tobramycin used in human therapy were administered to GFP-labelled P. aeruginosa PAO1 grown in flow chambers for 24 or 72 h. Images were acquired using confocal laser scanning microscopy throughout antibiotic treatment. Bacterial biomass was measured using COMSTAT and pharmacokinetic/pharmacodynamic models were fitted using NONMEM7. RESULTS: Meropenem treatment resulted in more rapid and sustained killing of both the 24 and 72 h PAO1 biofilm compared with tobramycin. Biofilm regrowth after antibiotic treatment occurred fastest with tobramycin. Meropenem preferentially killed subpopulations within the mushroom cap of the biofilms, regardless of biofilm maturity. The spatial killing by tobramycin varied with biofilm maturity. A tobramycin-treated 24 h biofilm resulted in live and dead cells detaching from the biofilm, while treatment of a 72 h biofilm preferentially killed subpopulations on the periphery of the mushroom stalk. Regrowth occurred primarily on the mushroom caps. Combination meropenem and tobramycin therapy resulted in rapid and efficient killing of biofilm cells, with a spatial pattern similar to meropenem alone. CONCLUSIONS: Simulated human concentrations of meropenem and tobramycin in young and mature PAO1 biofilms exhibited differences in temporal and spatial patterns of killing and antibiotic tolerance development.

Use of isavuconazole in a patient with voriconazole-induced QTc prolongation.

A 22-year-old woman with cystic fibrosis developed QTc interval prolongation following lung transplantation in the setting of voriconazole therapy. After the discontinuation of voriconazole and initiation of isavuconazole, her QTc interval normalized. This case highlights the unique property of QTc interval shortening by isavuconazole among the triazole antifungals.

Impact of a Guideline for the Management of Antimicrobial/Warfarin Interactions in the Inpatient Setting and Across Transition of Care.

BACKGROUND: Drug-drug interactions (DDIs) with warfarin and antimicrobial agents are a common cause of international normalized ratio (INR) instability, which can affect the risk for bleeding and thrombotic events. OBJECTIVE: The purpose of this study was to assess the impact of a comprehensive guideline for the management of warfarin-antimicrobial DDIs across transitions of care. The guideline emphasizes improving identification of significant antimicrobial-warfarin DDIs during hospitalization, empirical warfarin dose modification based on DDI and baseline INR, patient education, documentation of the DDI, communication with outpatient providers regarding the DDI and anticipated antimicrobial stop date, and warfarin dose adjustment on discontinuation of antimicrobial. METHODS: This retrospective, single-center, quasiexperimental, pre-post study compared end points 3 months before and after guideline implementation. The primary outcome was time within therapeutic range (TTR). RESULTS: The study included 78 preguideline and 31 postguideline patients; baseline characteristics were similar between groups. Implementation of the guideline resulted in greater in-hospital TTR (72% vs 50%, P = 0.04) and improved TTR across transition of care (70% vs 46%, P = 0.01). Documentation of DDI in the pharmacy anticoagulation discharge summary significantly improved in the postguideline group (40% vs 14%, P = 0.02) and numerically improved within the daily pharmacy progress notes (94% vs 82%, P = 0.13). The implementation of the guideline was associated with a nonsignificant, numerical reduction in bleeding events compared with the preguideline group (0 vs 4 events, P = 0.11). CONCLUSION: This single-center approach to optimize the comprehensive management of significant antimicrobial-warfarin DDIs resulted in improved communication with outpatient providers and improved INR TTR.

New in vitro model to study the effect of human simulated antibiotic concentrations on bacterial biofilms.

A new in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms utilizing flow cell technology and confocal laser scanning microscopy is described. The device has the ability to simulate the changing antibiotic concentrations in humans associated with intravenous dosing on bacterial biofilms grown under continuous culture conditions. The free drug concentrations of a single 2-g meropenem intravenous bolus dose and first-order elimination utilizing a half-life of 0.895 h (elimination rate constant, 0.776 h(-1)) were simulated. The antibacterial activity of meropenem against biofilms of Pseudomonas aeruginosa PAO1 and three clinical strains isolated from patients with cystic fibrosis was investigated. Additionally, the effect of meropenem on PAO1 biofilms cultured for 24 h versus that on biofilms cultured for 72 h was examined. Using confocal laser scanning microscopy, rapid biofilm killing was observed in the first hour of the dosing interval for all biofilms. However, for PAO1 biofilms cultured for 72 h, only bacterial subpopulations at the periphery of the biofilm were affected, with subpopulations at the substratum remaining viable, even at the conclusion of the dosing interval. The described model is a novel method to investigate antimicrobial killing of bacterial biofilms using human simulated concentrations.

Optimized Near-IR Fluorescent Agents for in Vivo Imaging of Btk Expression.

Bruton's tyrosine kinase (Btk) is intricately involved in anti-apoptotic signaling pathways in cancer and in regulating innate immune response. A number of Btk inhibitors are in development for use in treating B-cell malignancies and certain immunologic diseases. To develop robust companion imaging diagnostics for in vivo use, we set out to explore the effects of red wavelength fluorochrome modifications of two highly potent irreversible Btk inhibitors, Ibrutinib and AVL-292. Surprisingly, we found that subtle chemical differences in the fluorochrome had considerable effects on target localization. Based on iterative designs, we developed a single optimized version with superb in vivo imaging characteristics enabling single cell Btk imaging in vivo. This agent (Ibrutinib-SiR-COOH) is expected to be a valuable chemical tool in deciphering Btk biology in cancer and host cells in vivo.

Imaging cellular distribution of Bcl inhibitors using small molecule drug conjugates.

Overexpression of anti-apoptotic proteins such as Bcl-2 is a cellular mechanism to evade apoptosis; consequently, Bcl-2 inhibitors are being developed as anticancer agents. In this work, we have synthesized a fluorescent version of ABT-199 in an effort to visualize a drug surrogate by high resolution imaging. We show that this fluorescent conjugate has comparable Bcl-2 binding efficacy and cell line potency to the parent compound and can be used as an imaging agent in several cancer cell types. We anticipate that this agent will be a valuable tool for studying the single-cell distribution and pharmacokinetics of ABT-199 as well the broader group of BH3-mimetics.

Metformin and adipose-derived stem cell combination therapy alleviates radiation-induced skin fibrosis in mice.

BACKGROUND: Radiation therapy often leads to late radiation-induced skin fibrosis (RISF), causing movement impairment and discomfort. We conducted a comprehensive study to assess the effectiveness of metformin and adipose-derived stem cells (ASCs), whether autologous or allogeneic, individually or in combination therapy, in mitigating RISF. METHODS: Using a female C57BL/6J mouse model subjected to hind limb irradiation as a representative RISF model, we evaluated metformin, ASCs, or their combination in two contexts: prophylactic (started on day 1 post-irradiation) and therapeutic (initiated on day 14 post-irradiation, coinciding with fibrosis symptoms). We measured limb movement, examined skin histology, and analyzed gene expression to assess treatment efficacy. RESULTS: Prophylactic metformin and ASCs, whether autologous or allogeneic, effectively prevented late fibrosis, with metformin showing promising results. However, combination therapy did not provide additional benefits when used prophylactically. Autologous ASCs, alone or with metformin, proved most effective against late-stage RISF. Prophylactic intervention outperformed late therapy for mitigating radiation skin damage. Co-culture studies revealed that ASCs and metformin downregulated inflammation and fibrotic gene expression in both mouse and human fibroblasts. CONCLUSIONS: Our study suggests metformin's potential as a prophylactic measure to prevent RISF, and the combination of ASCs and metformin holds promise for late-stage RISF treatment. These findings have clinical implications for improving the quality of life for those affected by radiation-induced skin fibrosis.

Colocalization of Cancer-Associated Biomarkers on Single Extracellular Vesicles for Early Detection of Cancer.

Detection of cancer early, when it is most treatable, remains a significant challenge because of the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. Detection of their presence is made more difficult by a lack of abundant tumor-specific indicators (ie, protein biomarkers, circulating tumor DNA) that would enable detection using a noninvasive diagnostic assay. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. We demonstrate the technical feasibility of this approach in human cancer cell line-derived EVs, where we show strong correlations between assay signal and cell line gene/protein expression for the ovarian cancer-associated biomarkers bone marrow stromal antigen-2, folate receptor-α, and mucin-1. Furthermore, we demonstrate that detecting distinct colocalized biomarkers on the surface of EVs significantly improves discrimination performance relative to single biomarker measurements. Using this approach, we observe promising discrimination of high-grade serous ovarian cancer versus benign ovarian masses and healthy women in a proof-of-concept clinical study.

Bioorthogonal approach to identify unsuspected drug targets in live cells.

A proteomics method to pull down secondary drug targets from live cells is described. The drug of interest is modified with trans-cyclooctene (TCO) and incubated with live cells. Upon cell lysis, the modified drug bound to the protein is pulled down using magnetic beads decorated with a cleavable tetrazine-modified linker. Samples are then run on an SDS-PAGE gel and isolated bands are submitted for mass spectrometry analysis to identify drug targets.

Extracellular vesicle analysis of plasma allows differential diagnosis of atypical pancreatic serous cystadenoma.

Increased use of cross-sectional imaging has resulted in frequent detection of incidental cystic pancreatic lesions. Serous cystadenomas (SCAs) are benign cysts that do not require surgical intervention unless symptomatic. Unfortunately, up to half of SCAs do not have typical imaging findings ("atypical SCAs"), overlap with potentially malignant precursor lesions, and thus pose a diagnostic challenge. We tested whether the analysis of circulating extracellular vesicle (EV) biomarkers using a digital EV screening technology (DEST) could enhance the discrimination of cystic pancreatic lesions and avoid unnecessary surgical intervention in these atypical SCAs. Analysis of 25 different protein biomarkers in plasma EV from 68 patients identified a putative biomarker signature of Das-1, Vimentin, Chromogranin A, and CAIX with high discriminatory power (AUC of 0.99). Analysis of plasma EV for multiplexed markers may thus be helpful in clinical decision-making.

Multiplexed analysis of EV reveals specific biomarker composition with diagnostic impact.

Exosomes and extracellular vesicles (EV) are increasingly being explored as circulating biomarkers, but their heterogenous composition will likely mandate the development of multiplexed EV technologies. Iteratively multiplexed analyses of near single EVs have been challenging to implement beyond a few colors during spectral sensing. Here we developed a multiplexed analysis of EV technique (MASEV) to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers. Contrary to the common belief, we show that: several markers proposed to be ubiquitous are less prevalent than believed; multiple biomarkers concur in single vesicles but only in small fractions; affinity purification can lead to loss of rare EV subtypes; and deep profiling allows detailed analysis of EV, potentially improving the diagnostic content. These findings establish the potential of MASEV for uncovering fundamental EV biology and heterogeneity and increasing diagnostic specificity.

Human adipose ECM alleviates radiation-induced skin fibrosis via endothelial cell-mediated M2 macrophage polarization.

Radiation therapy can lead to late radiation-induced skin fibrosis (RISF), causing movement restriction, pain, and organ dysfunction. This study evaluated adipose-derived extracellular matrix (Ad-ECM) as a mitigator of RISF. Female C57BL/6J mice that were irradiated developed fibrosis, which was mitigated by a single local Ad-ECM injection, improving limb movement and reducing epithelium thickness and collagen deposition. Ad-ECM treatment resulted in decreased expression of pro-inflammatory and fibrotic genes, and upregulation of anti-inflammatory cytokines, promoting M2 macrophage polarization. Co-culture of irradiated human fibroblasts with Ad-ECM down-modulated fibrotic gene expression and enhanced bone marrow cell migration. Ad-ECM treatment also increased interleukin (IL)-4, IL-5, and IL-15 expression in endothelial cells, stimulating M2 macrophage polarization and alleviating RISF. Prophylactic use of Ad-ECM showed effectiveness in mitigation. This study suggests Ad-ECM's potential in treating chronic-stage fibrosis.

Inferring Viral Transmission Pathways from Within-Host Variation.

Genome sequencing can offer critical insight into pathogen spread in viral outbreaks, but existing transmission inference methods use simplistic evolutionary models and only incorporate a portion of available genetic data. Here, we develop a robust evolutionary model for transmission reconstruction that tracks the genetic composition of within-host viral populations over time and the lineages transmitted between hosts. We confirm that our model reliably describes within-host variant frequencies in a dataset of 134,682 SARS-CoV-2 deep-sequenced genomes from Massachusetts, USA. We then demonstrate that our reconstruction approach infers transmissions more accurately than two leading methods on synthetic data, as well as in a controlled outbreak of bovine respiratory syncytial virus and an epidemiologically-investigated SARS-CoV-2 outbreak in South Africa. Finally, we apply our transmission reconstruction tool to 5,692 outbreaks among the 134,682 Massachusetts genomes. Our methods and results demonstrate the utility of within-host variation for transmission inference of SARS-CoV-2 and other pathogens, and provide an adaptable mathematical framework for tracking within-host evolution.