Combinatorial treatment rescues tumour-microenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas.

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR-MYD88-TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition of multiple aberrant signalling pathways induced higher antitumour efficacy in lymphoid organoids and implanted ABC-DLBCL patient tumours in vivo. Our studies define the complex crosstalk between malignant ABC-DLBCL cells and Ly-TME, and provide rational combinatorial therapies that rescue Ly-TME-mediated attenuation of treatment response to MALT1 inhibitors.

Synergistic interactions of essential oil components with antibiotics against multidrug-resistant Corynebacterium striatum.

AIM: In this study, it was aimed to examine the antibacterial activity of the essential oil components (EOCs), carvacrol (CAR), cinnamaldehyde (CIN), thymol (TH), alpha pinene (α-PN), eucalyptol (EU), limonene (LIM), and the antibiotics, linezolid (LZD), vancomycin (VAN), gentamicin (GEN), ciprofloxacin (CIP), clindamycin (CLN), and penicillin (PEN) against 50 multidrug resistant Corynebacterium striatum strains, and the synergistic interactions of CAR and CIN with the antibiotics against 10 randomly selected Coryne. striatum strains to explore synergistic interactions to determine if their combined use could enhance antibiotic activity and potentially reduce resistance. METHODS AND RESULTS: The activity of the EOCs and the antibiotics against Coryne. striatum strains isolated from clinical specimens, was examined by broth microdilution method. The synergistic interactions of the EOCs with the antibiotics against 10 randomly selected Coryne. striatum strains were determined by checkerboard method. EOCs, CIN, and CAR and antibiotics, LZD, VAN, GEN, CIP, and CLN were detected to have antibacterial activity against Coryne. striatum strains alone and either synergistic interactions were observed in combinations of the antibiotics with EOCs. CONCLUSIONS: All Coryne. striatum strains were determined to be susceptible to VAN and LZD and resistant to GEN, PEN, CIP, and CLN. Synergistic interactions were observed in all combinations of antibiotics tested with CAR and CIN.

Novel Lesional Transcriptional Signature Separates Atherosclerosis With and Without Diabetes in Yorkshire Swine and Humans.

[Figure: see text].

Dense transcript profiling in single cells by image correlation decoding.

Sequential barcoded fluorescent in situ hybridization (seqFISH) allows large numbers of molecular species to be accurately detected in single cells, but multiplexing is limited by the density of barcoded objects. We present correlation FISH (corrFISH), a method to resolve dense temporal barcodes in sequential hybridization experiments. Using corrFISH, we quantified highly expressed ribosomal protein genes in single cultured cells and mouse thymus sections, revealing cell-type-specific gene expression.

Role of local coronary blood flow patterns and shear stress on the development of microvascular and epicardial endothelial dysfunction and coronary plaque.

PURPOSE OF REVIEW: The natural history of coronary atherosclerosis is complex and atherosclerotic plaques exhibit large morphologic and functional variability within the same individual as well as over time. The purpose of this article is to review the role of blood flow patterns and shear stress on the development of microvascular and epicardial endothelial dysfunction and atherosclerosis progression. RECENT FINDINGS: Recent breakthroughs in cardiovascular imaging have facilitated in-vivo characterization of the anatomic and functional characteristics of atherosclerotic plaques and have highlighted the role of endothelial shear stress and epicardial and microvascular endothelial dysfunction in the natural history of coronary atherosclerosis. SUMMARY: There is an important need to identify individual lesions which may progress to vulnerable plaque in order to provide early therapeutic management. Evaluation of endothelial shear stress, local blood flow patterns, epicardial and microvascular endothelial dysfunction, as well as their complex associations might indicate those patients who have microvascular endothelial dysfunction and increased risk for upstream epicardial endothelial dysfunction and plaque progression. Such high-risk patients could potentially be targeted for more intensive therapeutic strategies to prevent the progression of both microvascular and epicardial atherosclerotic manifestations.

Heterogeneity of Coronary Plaque Morphology and Natural History: Current Understanding and Clinical Significance.

PURPOSE OF REVIEW: Despite the important progress in identifying high-risk atherosclerotic plaques, many key elements are elusive. Advanced imaging modalities provide valuable information about the anatomic and functional plaque characteristics and underscore the presence of multiple plaque morphologies. However, how the heterogeneity of atherosclerotic plaque can alter our current understanding of coronary artery disease is not fully understood. RECENT FINDINGS: Along the length of an individual plaque, the morphology patterns display marked heterogeneity. Contrary to previous beliefs, plaque morphology is also highly dynamic over time, with the vast majority of high-risk plaques becoming quiescent and mild plaques becoming severely obstructive in a short period of time. Endothelial shear stress, a local hemodynamic factor known for its critical effects in plaque initiation and progression, also displays longitudinal heterogeneity contributing to the arterial wall response in all time points. Risk stratification of plaques based on the morphological characteristics at one region of the plaque, usually the minimal lumen diameter, and at one point in time may be misleading. The evaluation of both morphological and hemodynamic characteristics along the length of a plaque will improve the risk assessment of individual plaques.

Imaging without lenses: achievements and remaining challenges of wide-field on-chip microscopy.

We discuss unique features of lens-free computational imaging tools and report some of their emerging results for wide-field on-chip microscopy, such as the achievement of a numerical aperture (NA) of ∼0.8-0.9 across a field of view (FOV) of more than 20 mm(2) or an NA of ∼0.1 across a FOV of ∼18 cm(2), which corresponds to an image with more than 1.5 gigapixels. We also discuss the current challenges that these computational on-chip microscopes face, shedding light on their future directions and applications.

Thin-capped atheromata with reduced collagen content in pigs develop in coronary arterial regions exposed to persistently low endothelial shear stress.

OBJECTIVE: The mechanisms promoting the focal formation of rupture-prone coronary plaques in vivo remain incompletely understood. This study tested the hypothesis that coronary regions exposed to low endothelial shear stress (ESS) favor subsequent development of collagen-poor, thin-capped plaques. APPROACH AND RESULTS: Coronary angiography and 3-vessel intravascular ultrasound were serially performed at 5 consecutive time points in vivo in 5 diabetic, hypercholesterolemic pigs. ESS was calculated along the course of each artery with computational fluid dynamics at all 5 time points. At follow-up, 184 arterial segments with previously identified in vivo ESS underwent histopathologic analysis. Compared with other plaque types, eccentric thin-capped atheromata developed more in segments that experienced lower ESS during their evolution. Compared with lesions with higher preceding ESS, segments persistently exposed to low ESS (<1.2 Pa) exhibited reduced intimal smooth muscle cell content; marked intimal smooth muscle cell phenotypic modulation; attenuated procollagen-I gene expression; increased gene and protein expression of the interstitial collagenases matrix-metalloproteinase-1, -8, -13, and -14; increased collagenolytic activity; reduced collagen content; and marked thinning of the fibrous cap. CONCLUSIONS: Eccentric thin-capped atheromata, lesions particularly prone to rupture, form more frequently in coronary regions exposed to low ESS throughout their evolution. By promoting an imbalance of attenuated synthesis and augmented collagen breakdown, low ESS favors the focal evolution of early lesions toward plaques with reduced collagen content and thin fibrous caps-2 critical determinants of coronary plaque vulnerability.

Oxidation of cyanobenzocycloheptatrienes: Synthesis, photooxygenation reaction and carbonic anhydrase isoenzymes inhibition properties of some new benzotropone derivatives.

The oxidation of some cyanocycloheptatrienes with CrO3 and pyridine was investigated and a few new nitrile functionalised benzotropone derivatives were obtained. Photooxygenation reaction of these products was also studied. The structures of the formed products were determined on the basis of NMR spectroscopy and the formation mechanism of unusual products was discussed. Human carbonic anhydrase isoenzymes I, and II (hCA I and hCA II) inhibition properties of nitrile functionalized new benzotropone derivatives were also studied. Both CA isozymes were inhibited in the low micromolar range by these nitrile functionalized benzotropone analogues. The newly synthesized benzotropone derivatives showed inhibition constants in the sub-micromolar range (2.51-4.06µM). The best hCA I inhibition was observed in 5H-benzocycloheptene-7-carbonitrile (Ki: 2.88±0.86µM). On the other hand, 5-oxo-5H-benzocycloheptatriene-7-carbonitrile showed the powerful inhibitory effect against hCA II (Ki: 2.51±0.34µM).

Spatial Morphoproteomic Features Predict Uniqueness of Immune Microarchitectures and Responses in Lymphoid Follicles.

Multiplex imaging technologies allow the characterization of single cells in their cellular environments. Understanding the organization of single cells within their microenvironment and quantifying disease-status related biomarkers is essential for multiplex datasets. Here we proposed SNOWFLAKE, a graph neural network framework pipeline for the prediction of disease-status from combined multiplex cell expression and morphology in human B-cell follicles. We applied SNOWFLAKE to a multiplex dataset related to COVID-19 infection in humans and showed better predictive power of the SNOWFLAKE pipeline compared to other machine learning and deep learning methods. Moreover, we combined morphological features inside graph edge features to utilize attribution methods for extracting disease-relevant motifs from single-cell spatial graphs. The underlying subgraphs were further analyzed and associated with disease status across the dataset. We showed that SNOWFLAKE successfully extracted significant low dimensional embedding from subgraphs with a clear separation between disease status and helped characterize unique cellular interactions in the subgraphs. SNOWFLAKE is a generalizable pipeline for the analysis of multiplex imaging data modality by extracting disease-relevant subgraphs guided by graph-level prediction.

Comprehensive biomechanical and anatomical atherosclerotic plaque metrics predict major adverse cardiovascular events: A new tool for clinical decision making.

BACKGROUND AND AIMS: Anatomical imaging alone of coronary atherosclerotic plaques is insufficient to identify risk of future adverse events and guide management of non-culprit lesions. Low endothelial shear stress (ESS) and high plaque structural stress (PSS) are associated with events, but individually their predictive value is insufficient for risk prediction. We determined whether combining multiple complementary, biomechanical and anatomical plaque characteristics improves outcome prediction sufficiently to inform clinical decision-making. METHODS: We examined baseline ESS, ESS gradient (ESSG), PSS, and PSS heterogeneity index (HI), and plaque burden in 22 lesions that developed subsequent events and 64 control lesions that remained quiescent from the PROSPECT study. RESULTS: 86 fibroatheromas were analysed from 67 patients. Lesions with events showed higher PSS HI (0.32 vs. 0.24, p<0.001), lower local ESS (0.56Pa vs. 0.91Pa, p = 0.007), and higher ESSG (3.82 Pa/mm vs. 1.96 Pa/mm, p = 0.007), while high PSS HI (hazard ratio [HR] 3.9, p = 0.006), high ESSG (HR 3.4, p = 0.007) and plaque burden>70 % (HR 2.6, p = 0.02) were independent outcome predictors in multivariate analysis. Combining low ESS, high ESSG, and high PSS HI gave both high positive predictive value (80 %), which increased further combined with plaque burden>70 %, and negative predictive value (81.6 %). Low ESS, high ESSG, and high PSS HI co-localised spatially within 1 mm in lesions with events, and importantly, this cluster was distant from the minimum lumen area site. CONCLUSIONS: Combining complementary biomechanical and anatomical metrics significantly improves risk-stratification of individual coronary lesions. If confirmed from larger prospective studies, our results may inform targeted revascularisation vs. conservative management strategies.

Multi-Niche Human Bone Marrow On-A-Chip for Studying the Interactions of Adoptive CAR-T Cell Therapies with Multiple Myeloma.

Multiple myeloma (MM), a cancer of bone marrow plasma cells, is the second-most common hematological malignancy. However, despite immunotherapies like chimeric antigen receptor (CAR)-T cells, relapse is nearly universal. The bone marrow (BM) microenvironment influences how MM cells survive, proliferate, and resist treatment. Yet, it is unclear which BM niches give rise to MM pathophysiology. Here, we present a 3D microvascularized culture system, which models the endosteal and perivascular bone marrow niches, allowing us to study MM-stroma interactions in the BM niche and model responses to therapeutic CAR-T cells. We demonstrated the prolonged survival of cell line-based and patient-derived multiple myeloma cells within our in vitro system and successfully flowed in donor-matched CAR-T cells. We then measured T cell survival, differentiation, and cytotoxicity against MM cells using a variety of analysis techniques. Our MM-on-a-chip system could elucidate the role of the BM microenvironment in MM survival and therapeutic evasion and inform the rational design of next-generation therapeutics. TEASER: A multiple myeloma model can study why the disease is still challenging to treat despite options that work well in other cancers.

Prediction of progression of coronary artery disease and clinical outcomes using vascular profiling of endothelial shear stress and arterial plaque characteristics: the PREDICTION Study.

BACKGROUND: Atherosclerotic plaques progress in a highly individual manner. The purposes of the Prediction of Progression of Coronary Artery Disease and Clinical Outcome Using Vascular Profiling of Shear Stress and Wall Morphology (PREDICTION) Study were to determine the role of local hemodynamic and vascular characteristics in coronary plaque progression and to relate plaque changes to clinical events. METHODS AND RESULTS: Vascular profiling, using coronary angiography and intravascular ultrasound, was used to reconstruct each artery and calculate endothelial shear stress and plaque/remodeling characteristics in vivo. Three-vessel vascular profiling (2.7 arteries per patient) was performed at baseline in 506 patients with an acute coronary syndrome treated with a percutaneous coronary intervention and in a subset of 374 (74%) consecutive patients 6 to 10 months later to assess plaque natural history. Each reconstructed artery was divided into sequential 3-mm segments for serial analysis. One-year clinical follow-up was completed in 99.2%. Symptomatic clinical events were infrequent: only 1 (0.2%) cardiac death; 4 (0.8%) patients with new acute coronary syndrome in nonstented segments; and 15 (3.0%) patients hospitalized for stable angina. Increase in plaque area (primary end point) was predicted by baseline large plaque burden; decrease in lumen area (secondary end point) was independently predicted by baseline large plaque burden and low endothelial shear stress. Large plaque size and low endothelial shear stress independently predicted the exploratory end points of increased plaque burden and worsening of clinically relevant luminal obstructions treated with a percutaneous coronary intervention at follow-up. The combination of independent baseline predictors had a 41% positive and 92% negative predictive value to predict progression of an obstruction treated with a percutaneous coronary intervention. CONCLUSIONS: Large plaque burden and low local endothelial shear stress provide independent and additive prediction to identify plaques that develop progressive enlargement and lumen narrowing. CLINICAL TRIAL REGISTRATION: URL: http:www.//clinicaltrials.gov. Unique Identifier: NCT01316159.

Analysis of direct expansion heat recovery ventilation devices by orthogonal optimization method.

Heat recovery ventilation devices include rectangular plate cross-flow, hexagonal plate combined counter and cross-flow, rotary wheel sensible, sorption rotor hybrid sensible and latent heat exchanger. Currently, existing studies have produced no clear findings on which climatic conditions latent heat recovery would be optimal, and therefore sought to determine in which climatic conditions it would be suitable to use devices that perform latent heat recovery. This study analysed the performance of different heat recovery devices in different climatic conditions in a ventilation project of a sample hotel building. In the case study, while there is a useful heat recovery between 44.01 and 58.68 kW at low outdoor temperatures in devices with only sensible heat transfer, this value increases up to 158.42 kW, as the outdoor temperature rises. In the heat recovery device providing latent heat transfer, the amount of useful heat recovery varies between 51.34 and 352.16 kW at low outdoor temperatures, depending on the outdoor relative humidity, while this amount increases to 411.26 from 773.25 kW at high outdoor temperatures. Outdoor temperature and humidity levels required for latent heat recovery was also determined by orthogonal optimization method. By using the orthogonal optimization, the study found that under conditions of high temperature that exceeds 35 °C in outdoor ambient temperature, and high humidity that exceeds 60% relative humidity, usage of latent heat recovery devices caused significant differences in total heat recovery ratio. Analysis also concludes that these devices can be used under these conditions.

Subcellular spatially resolved gene neighborhood networks in single cells.

Image-based spatial omics methods such as fluorescence in situ hybridization (FISH) generate molecular profiles of single cells at single-molecule resolution. Current spatial transcriptomics methods focus on the distribution of single genes. However, the spatial proximity of RNA transcripts can play an important role in cellular function. We demonstrate a spatially resolved gene neighborhood network (spaGNN) pipeline for the analysis of subcellular gene proximity relationships. In spaGNN, machine-learning-based clustering of subcellular spatial transcriptomics data yields subcellular density classes of multiplexed transcript features. The nearest-neighbor analysis produces heterogeneous gene proximity maps in distinct subcellular regions. We illustrate the cell-type-distinguishing capability of spaGNN using multiplexed error-robust FISH data of fibroblast and U2-OS cells and sequential FISH data of mesenchymal stem cells (MSCs), revealing tissue-source-specific MSC transcriptomics and spatial distribution characteristics. Overall, the spaGNN approach expands the spatial features that can be used for cell-type classification tasks.

Single-Cell and Spatial Analysis of Emergent Organoid Platforms.

Organoids have emerged as a promising advancement of the two-dimensional (2D) culture systems to improve studies in organogenesis, drug discovery, precision medicine, and regenerative medicine applications. Organoids can self-organize as three-dimensional (3D) tissues derived from stem cells and patient tissues to resemble organs. This chapter presents growth strategies, molecular screening methods, and emerging issues of the organoid platforms. Single-cell and spatial analysis resolve organoid heterogeneity to obtain information about the structural and molecular cellular states. Culture media diversity and varying lab-to-lab practices have resulted in organoid-to-organoid variability in morphology and cell compositions. An essential resource is an organoid atlas that can catalog protocols and standardize data analysis for different organoid types. Molecular profiling of individual cells in organoids and data organization of the organoid landscape will impact biomedical applications from basic science to translational use.

The Role of Shear Stress in Coronary Artery Disease.

Coronary artery disease is the leading cause of morbidity and mortality worldwide, especially in developed countries, with an increasing incidence in developing countries. Despite the advances in cardiology, there are yet many unanswered questions about the natural history of coronary atherosclerosis. However, it has not been fully explained why some coronary artery plaques remain quiescent over time, whereas others evolve to a high-risk, "vulnerable" plaque with a predisposition to destabilize and induce a cardiac event. Furthermore, approximately half of the patients with acute coronary syndromes demonstrate no prior symptoms of ischemia or angiographically evident disease. Recent findings have indicated that apart from cardiovascular risk factors, genetics, and other unknown factors, local hemodynamic forces, such as endothelial shear stress, blood flow patterns, and endothelial dysfunction of the epicardial and microvascular coronary arteries, are associated with the progression of coronary plaque and the development of cardiovascular complications with complex interactions. In this review article, we summarize the mechanisms that affect coronary artery plaque progression, indicating the importance of endothelial shear stress, endothelial dysfunction of epicardial and microvascular vessels, inflammation, and their complex associations, underlying in parallel the clinical perspectives of these findings.

Single-cell spatial metabolomics with cell-type specific protein profiling for tissue systems biology.

Metabolic reprogramming in cancer and immune cells occurs to support their increasing energy needs in biological tissues. Here we propose Single Cell Spatially resolved Metabolic (scSpaMet) framework for joint protein-metabolite profiling of single immune and cancer cells in male human tissues by incorporating untargeted spatial metabolomics and targeted multiplexed protein imaging in a single pipeline. We utilized the scSpaMet to profile cell types and spatial metabolomic maps of 19507, 31156, and 8215 single cells in human lung cancer, tonsil, and endometrium tissues, respectively. The scSpaMet analysis revealed cell type-dependent metabolite profiles and local metabolite competition of neighboring single cells in human tissues. Deep learning-based joint embedding revealed unique metabolite states within cell types. Trajectory inference showed metabolic patterns along cell differentiation paths. Here we show scSpaMet's ability to quantify and visualize the cell-type specific and spatially resolved metabolic-protein mapping as an emerging tool for systems-level understanding of tissue biology.

Microbial Changes occurring during oronasal fistula wound healing.

The oral microbiome is a complex community that matures with dental development while oral health is also a recognized risk factor for systemic disease. Despite the oral cavity having a substantial microbial burden, healing of superficial oral wounds occurs quickly and with little scarring. By contrast, creation of an oro-nasal fistula (ONF), often occurring after surgery to correct a cleft palate, is a significant wound healing challenge that is further complicated by a connection of the oral and nasal microbiome. In this study, we characterized the changes in the oral microbiome of mice following a freshly inflicted wound in the oral palate that results in an open and unhealed ONF. Creation of an ONF in mice significantly lowered oral microbiome alpha diversity, with concurrent blooms of Enterococcus faecalis, Staphylococcus lentus, and Staphylococcus xylosus in the oral cavity. Treatment of mice with oral antibiotics one week prior to ONF infliction resulted in a reduction in the alpha diversity, prevented E. faecalis and S. lentus, and S. xylosus blooms, but did not impact ONF healing. Strikingly, delivery of the beneficial microbe Lactococcus lactis subsp. cremoris (LLC) to the wound bed of the freshly inflicted ONF via a PEG-MAL hydrogel vehicle resulted in rapid healing of the ONF. Healing of the ONF was associated with the maintenance of relatively high microbiome alpha diversity, and limited the abundance of E. faecalis and S. lentus, and S. xylosus in the oral cavity. These data demonstrate that a freshly inflicted ONF in the murine palate is associated with a dysbiotic oral microbiome state that may prevent ONF healing, and a bloom of opportunistic pathogens. The data also demonstrate that delivery of a specific beneficial microbe, LLC, to the ONF can boost wound healing, can restore and/or preserve oral microbiome diversity, and inhibit blooms of opportunistic pathogens.

Spatial subcellular organelle networks in single cells.

Organelles play important roles in human health and disease, such as maintaining homeostasis, regulating growth and aging, and generating energy. Organelle diversity in cells not only exists between cell types but also between individual cells. Therefore, studying the distribution of organelles at the single-cell level is important to understand cellular function. Mesenchymal stem cells are multipotent cells that have been explored as a therapeutic method for treating a variety of diseases. Studying how organelles are structured in these cells can answer questions about their characteristics and potential. Herein, rapid multiplexed immunofluorescence (RapMIF) was performed to understand the spatial organization of 10 organelle proteins and the interactions between them in the bone marrow (BM) and umbilical cord (UC) mesenchymal stem cells (MSCs). Spatial correlations, colocalization, clustering, statistical tests, texture, and morphological analyses were conducted at the single cell level, shedding light onto the interrelations between the organelles and comparisons of the two MSC subtypes. Such analytics toolsets indicated that UC MSCs exhibited higher organelle expression and spatially spread distribution of mitochondria accompanied by several other organelles compared to BM MSCs. This data-driven single-cell approach provided by rapid subcellular proteomic imaging enables personalized stem cell therapeutics.