Systems-Level Modeling of Cancer-Fibroblast Interaction

Cancer cells interact with surrounding stromal fibroblasts during tumorigenesis, but the complex molecular rules that govern these interactions remain poorly understood thus hindering the development of therapeutic strategies to target cancer stroma. We have taken a mathematical approach to begin defining these rules by performing the first large-scale quantitative analysis of fibroblast effects on cancer cell proliferation across more than four hundred heterotypic cell line pairings. Systems-level modeling of this complex dataset using singular value decomposition revealed that normal tissue fibroblasts variably express at least two functionally distinct activities, one which reflects transcriptional programs associated with activated mesenchymal cells, that act either coordinately or at cross-purposes to modulate cancer cell proliferation. These findings suggest that quantitative approaches may prove useful for identifying organizational principles that govern complex heterotypic cell-cell interactions in cancer and other contexts

Improved in Vivo Whole-Animal Detection Limits of Green Fluorescent Protein–Expressing Tumor Lines by Spectral Fluorescence Imaging

Green fluorescent protein (GFP) has been used for cell tracking and imaging gene expression in superficial or surgically exposed structures. However, in vivo murine imaging is often limited by several factors, including scatter and attenuation with depth and overlapping autofluorescence. The autofluorescence signals have spectral profiles that are markedly different from the GFP emission spectral profile. The use of spectral imaging allows separation and quantitation of these contributions to the total fluorescence signal seen in vivo by weighting known pure component profiles. Separation of relative GFP and autofluorescence signals is not readily possible using epifluorescent continuous-wave single excitation and emission bandpass imaging (EFI). To evaluate detection thresholds using these two methods, nude mice were subcutaneously injected with a series of GFP-expressing cells. For EFI, optimized excitation and emission bandpass filters were used. Owing to the ability to separate autofluorescence contributions from the emission signal using spectral imaging compared with the mixed contributions of GFP and autofluorescence in the emission signal recorded by the EFI system, we achieved a 300-fold improvement in the cellular detection limit. The detection limit was 3 × 103 cells for spectral imaging versus 1 × 106 cells for EFI. Despite contributions to image stacks from autofluorescence, a 100-fold dynamic range of cell number in the same image was readily visualized. Finally, spectral imaging was able to separate signal interference of red fluorescent protein from GFP images and vice versa. These findings demonstrate the utility of the approach in detecting low levels of multiple fluorescent markers for whole-animal in vivo applications

Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut

Given mounting evidence of the importance of gut-microbiota/immune-cell interactions in immune homeostasis and responsiveness, surprisingly little is known about leukocyte movements to, and especially from, the gut. We address this topic in a minimally perturbant manner using Kaede transgenic mice, which universally express a photoconvertible fluorescent reporter. Transcutaneous exposure of the cervical lymph nodes to violet light permitted punctual tagging of immune cells specifically therein, and subsequent monitoring of their immigration to the intestine; endoscopic flashing of the descending colon allowed specific labeling of intestinal leukocytes and tracking of their emigration. Our data reveal an unexpectedly broad movement of leukocyte subsets to and from the gut at steady state, encompassing all lymphoid and myeloid populations examined. Nonetheless, different subsets showed different trafficking proclivities (e.g., regulatory T cells were more restrained than conventional T cells in their exodus from the cervical lymph nodes). The novel endoscopic approach enabled us to evidence gut-derived Th17 cells in the spleens of K/BxN mice at the onset of their genetically determined arthritis, thereby furnishing a critical mechanistic link between the intestinal microbiota, namely segmented filamentous bacteria, and an extraintestinal autoinflammatory disease

Real-Time Multichannel Imaging Framework for Endoscopy, Catheters, and Fixed Geometry Intraoperative Systems

To address the need for a clinically applicable intravital optical imaging system, we developed a new hardware and software framework. We demonstrate its utility by applying it to an endoscope-based white light and fluorescent imaging system. The capabilities include acquisition and visualization algorithms that perform registration, segmentation, and histogram-based autoexposure of two imaging channels (full-spectrum white light and near-infrared fluorescence), all in real time. Data are processed and saved as 12-bit files, matching the standards of clinical imaging. Dynamic range is further improved by the evaluation of flux as a quantitative parameter. The above features are demonstrated in a series of in vitro experiments, and the in vivo application is shown with the visualization of fluorescent-labeled vasculature of a mouse peritoneum. The approach may be applied to diverse systems, including handheld devices, fixed geometry intraoperative devices, catheter-based imaging, and multimodal systems

COVID-19 vaccination single cell datasets

The datasets presented here comprise the sequencing data featured in the research paper titled: "Multimodal single-cell datasets characterize antigen-specific CD8+ T cells across SARS-CoV-2 vaccination and infection": https://www.nature.com/articles/s41590-023-01608-9 Peripheral Blood Mononuclear Cell (PBMC) samples utilized for both CITE-seq and ASAP-seq were systematically collected at four distinct time intervals: Pre-vaccination (Day 0) Post-primary vaccination (Day 2 and Day 10. Seven days post-boost vaccination (Day 28). The count matrix folder contains count matrices for each experimental type, specifically CITE-seq, ASAP-seq, and ECCITE-seq. In addition, we have included the fully integrated, processed Seurat objects for downstream analysis. Details of the content within the count matrix folder are as follows: The RNA, ATAC, and TCR modality outputs were generated using the 10x Cellranger pipeline. HTO and ADT modalities were mapped with Alevin. Outlined below are the three processed single-cell datasets: PBMC_vaccine_CITE.rds: 3' RNA and surface proteins (173 TotalSeq-A antibodies) PBMC_vaccine_ASAP.rds: Chromatin accessibility and surface proteins (173 TotalSeq-A antibodies) PBMC_vaccine_ECCITE_TCR.rds: 5' RNA, surface proteins (137 TotalSeq-C antibodies), TCR and dextramer loaded with peptides of SARS-CoV-2 spike protein. antigen_module_genes.rds: This file contains the vaccine-induced gene sets. antigen_module_peaks.rds: This file contains the DE peaks specific for vaccine-induced cells. To map the scRNA-seq query dataset onto our CITE-seq reference: library(Seurat) PBMC_CITE <- readRDS("/zenedo/PBMC_vaccine_CITE.rds") query_scRNA <- readRDS("/home/xx/your_own_data.rds") anchors <- FindTransferAnchors( reference = PBMC_CITE, query = query_scRNA, normalization.method = "SCT", k.anchor = 5, reference.reduction = "spca", dims = 1:50) query_scRNA <- MapQuery( anchorset = anchors, query = query_scRNA, reference = PBMC_CITE, refdata = list( l1 = "celltypel1", l2 = "celltypel2", l3 = "celltypel3"), reference.reduction = "spca", reduction.model = "wnn.umap") To use the scATAC-seq data, please run the commands below to update the path of the fragment file for the object. Vaccine_ASAP <- readRDS("PBMC_vaccine_ASAP.rds") # remove fragment file information Fragments(Vaccine_ASAP) <- NULL # Update the path of the fragment file Fragments(Vaccine_ASAP) <- CreateFragmentObject(path = "download/PBMC_vaccine_ASAP_fragments.tsv.gz", cells = Cells(Vaccine_ASAP)

Door-to-balloon time and the determining factors in a tertiary cardiac center in Nepal

Background: Door-to-balloon (DTB) time of 90 min during primary angioplasty is considered as the benchmark duration. Shorter DTB time is preferable, and longer duration can have poor clinical outcomes. Methods: A cross-sectional observational study of three months in Shahid Gangalal National Heart Center was conducted in which all patients undergoing primary angioplasty were included. The DTB time was calculated, and the different determining factors were studied. Results: Seventy-nine patients undergoing primary percutaneous intervention were studied. The median DTB time was 79 minutes (Interquartile range [IQR] 59–115 min). Forty-six (58.2%) patients had a DTB time of less than 90 min. DTB time varied significantly with direct visit vs transfer (p = 0.029) and office time visit (9 am–5 pm) vs off time (5 pm–9 am) (p = 0.012). DTB time did not differ between any infarct-related vessels (p = 0.471), number of vessels involved (p = 0.638), and the added procedures (defibrillation, thrombosuction, and temporary pacemaker insertion) (p = 0.682) during angioplasty. No significant differences were recorded according to age (p = 0.330), gender (p = 0.254), hypertension (p = 0.073), diabetes (p = 0.487), heart failure (p = 0.316), and baseline left ventricular ejection fraction (LVEF) (p = 0.819). Conclusion: The median DTB time in primary angioplasty was less than 90 minutes. The significant determining factors were timing of hospital visit (office vs off time) and type of visit (direct vs transfer). There can be improvement in factors determining DTB time to lower it further. Keywords: Direct vs. transfer, Door-to-balloon time, Office time vs. off time, Radial vs. femora

Table_1_An evaluation of energy and carbon budgets in diverse cropping systems for sustainable diversification of rainfed uplands in India's eastern hill and plateau region.docx

With increasing cost and use of energy in agriculture, the traditional practice of mono-cropping of rice in upland is neither sustainable nor eco-friendly. It is necessary to identify crop diversification options with high energy efficiency, productivity, and low global warming potential (GWP). In this experiment, an inclusive system analysis was accomplished for 3 years (2016–2019) of five mono-cropping production (MCP) systems namely rice (R), finger millet (FM), black gram (BG), horse gram (HG), pigeon pea (PP), and four intercropped systems viz. R+BG, R+HG, FM+ BG, and FM + HG. The key objective was to evaluate the flow of energy, carbon balance, and GWP of these varied production systems. Puddled rice was recorded as an energy-exhaustive crop (27,803 MJ ha−1), while horse gram was noted to have the lowest energy use (26,537 MJ ha−1). The total energy output from pigeon pea (130,312 MJ ha−1) and diversified intercropped systems (142,135 MJ ha−1) was 65.3% and 80.3% higher than mono-cultured systems, respectively. Rice and rice-based intercropping production systems showed higher carbon footprints (1,264–1,392 kg CO2 eq. ha−1). Results showed that R+BG and R+HG were the most energy-efficient production systems, having higher energy ratio (5.8 and 6.0), higher carbon efficiency (7.41 and 8.24), and carbon sustainability index (6.41 and 7.24) as against 3.30, 3.61, and 2.61 observed under sole cropping production systems. On average, rice and rice-based production systems had 7.4 times higher GWP than other production systems. In productivity terms, pigeon pea and FM+HG had higher rice equivalent yields of 8.81 and 5.79 t ha−1 and benefit-cost ratios of 2.29 and 1.87, respectively. Thus, the present study suggests that pigeon pea and finger millet-based intercropping systems were the most appropriate crop diversification options for the rainfed upland agro-ecosystem of the eastern region of India.</p