Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

OBJECTIVE: In the present study, we introduce human lacrimal gland imaging using an ultrasound biomicroscopy (UBM) with a soft cover and show their findings.
METHODS: The representative UBM findings of palpebral lobes in seven subjects (four with non-Sjögren dry eye syndrome, one with Sjögren syndrome, and two healthy subjects) were described in this study. To prolapse the palpebral lobe, the examiner pulled the temporal part of the upper eyelid in the superotemporal direction and directed the subject to look in the inferonasal direction. We scanned the palpebral lobes longitudinally and transversely using UBM. We used an Aviso UBM with a 50 MHz linear probe and ClearScan.
RESULTS: In UBM of two healthy subjects, the echogenicity of the lacrimal gland was lower than that of the sclera and homogeneous. But the parenchyma of a patient with Sjögren dry eye syndrome was quite inhomogeneous compared to the healthy subjects. In two patients with dry eye syndrome, we were able to observe some lobules in the parenchyma. We could find excretory ducts running parallel at the surface of the longitudinal section in some subjects. In the longitudinal UBM scan of a subject, we observed a tubular structure at a depth of 1,500 μm that was considered a blood vessel. It ran from the superonasal to the inferotemporal direction. In a subject, we observed a large cyst beneath the conjunctiva.
CONCLUSIONS: Lacrimal gland imaging using UBM has both advantages of optical coherence tomography and sonography, and could be useful for evaluating dry eye syndrome.

Multiple abiotic stresses such as drought, salinity, heat, and cold stress prevailing in natural habitats affect plant growth and development. Different species modify their structural and functional traits to combat these abiotic stresses while growing in stressful environments. Cenchrus species, i.e., Cenchrus pennisetiformis, C. setiger, and C. prieurii are widely distributed grasses found growing all over the world. Samples from natural populations were collected from different ecological regions in the Punjab and Khyber Pakhtoonkhwa that were exposed to aridity, salinity, and cold, while one site was designated as normal control. In the present study, structural and functional modifications of three Cenchrus species under abiotic stresses were evaluated. It was expected that each Cenchrus species may evolve different strategies to cope with multiple abiotic stresses. All Cenchrus species responded differently whether growing in normal environment or stressful conditions. The most remarkable feature for survival in C. pennisetiformis under cold stress was increased inflorescence and increased stem and root lignification. C. prieurii showed better tolerance to saline and cold environments. C. setiger showed better development of leaf sheath anatomical traits. The structural and functional modifications in Cenchrus species such as development of mechanical tissues provided structural support, while dermal and parenchymatous tissues increased water storage capacity and minimized water loss. An increase in the concentration of organic osmolytes and ionic content aids turgor pressure maintenance and ionic content crucial for plant growth and development. It was concluded that structural and functional alterations in all Cenchrus species were very specific and critical for survival under different environmental stresses. The ecological fitness of these species relied on maintenance of growth and biomass production, and the development of mechanical, vascular, dermal and parenchyma tissues under stressful environmental conditions. Moreover, accumulation of beneficial ions (K+ and Ca2+) and organic osmolytes were critical in turgor maintenance, hence survival of Cenchrus spp.

UNASSIGNED: The automatic segmentation of brain parenchyma and cerebrospinal fluid-filled spaces such as the ventricular system is the first step for quantitative and qualitative analysis of brain CT data. For clinical practice and especially for diagnostics, it is crucial that such a method is robust to anatomical variability and pathological changes such as (hemorrhagic or neoplastic) lesions and chronic defects. This study investigates the increase in overall robustness of a deep learning algorithm that is gained by adding hemorrhage training data to an otherwise normal training cohort.
UNASSIGNED: A 2D U-Net is trained on subjects with normal appearing brain anatomy. In a second experiment the training data includes additional subjects with brain hemorrhage on image data of the RSNA Brain CT Hemorrhage Challenge with custom reference segmentations. The resulting networks are evaluated on normal and hemorrhage test casesseparately, and on an independent test set of patients with brain tumors of the publicly available GLIS-RT dataset.
UNASSIGNED: Adding data with hemorrhage to the training set significantly improves the segmentation performance over an algorithm trained exclusively on normally appearing data, not only in the hemorrhage test set but also in the tumor test set. The performance on normally appearing data is stable. Overall, the improved algorithm achieves median Dice scores of 0.98 (parenchyma), 0.91 (left ventricle), 0.90 (right ventricle), 0.81 (third ventricle), and 0.80 (fourth ventricle) on the hemorrhage test set. On the tumor test set, the median Dice scores are 0.96 (parenchyma), 0.90 (left ventricle), 0.90 (right ventricle), 0.75 (third ventricle), and 0.73 (fourth ventricle).
UNASSIGNED: Training on an extended data set that includes pathologies is crucial and significantly increases the overall robustness of a segmentation algorithm for brain parenchyma and ventricular system in CT data, also for anomalies completely unseen during training. Extension of the training set to include other diseases may further improve the generalizability of the algorithm.

Wood performs several functions to ensure tree survival and carbon allocation to a finite stem volume leads to trade-offs among cell types. It is not known to what extent these trade-offs modify functional trade-offs and if they are consistent across climates and evolutionary lineages. Twelve wood traits were measured in stems and coarse roots across 60 adult angiosperm tree species from temperate, Mediterranean and tropical climates. Regardless of climate, clear trade-offs occurred among cellular fractions, but did not translate into specific functional trade-offs. Wood density was negatively related to hydraulic conductivity (Kth ) in stems and roots, but was not linked to nonstructural carbohydrates (NSC), implying a functional trade-off between mechanical integrity and transport but not with storage. NSC storage capacity was positively associated with Kth in stems and negatively in roots, reflecting a potential role for NSC in the maintenance of hydraulic integrity in stems but not in roots. Results of phylogenetic analyses suggest that evolutionary histories cannot explain covariations among traits. Trade-offs occur among cellular fractions, without necessarily modifying trade-offs in function. However, functional trade-offs are driven by coordinated changes among xylem cell types depending on the dominant role of each cell type in stems and roots.

Extracellular matrix (ECM) remodeling has been associated with chronic lung diseases. However, information about specific age-associated differences in lung ECM is currently limited. In this study, we aimed to identify and localize age-associated ECM differences in human lungs using comprehensive transcriptomic, proteomic, and immunohistochemical analyses. Our previously identified age-associated gene expression signature of the lung was re-analyzed limiting it to an aging signature based on 270 control patients (37-80 years) and focused on the Matrisome core geneset using geneset enrichment analysis. To validate the age-associated transcriptomic differences on protein level, we compared the age-associated ECM genes (false discovery rate, FDR < 0.05) with a profile of age-associated proteins identified from a lung tissue proteomics dataset from nine control patients (49-76 years) (FDR < 0.05). Extensive immunohistochemical analysis was used to localize and semi-quantify the age-associated ECM differences in lung tissues from 62 control patients (18-82 years). Comparative analysis of transcriptomic and proteomic data identified seven ECM proteins with higher expression with age at both gene and protein levels: COL1A1, COL6A1, COL6A2, COL14A1, FBLN2, LTBP4, and LUM. With immunohistochemistry, we demonstrated higher protein levels with age for COL6A2 in whole tissue, parenchyma, airway wall, and blood vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in lung parenchyma. Our study revealed that higher age is associated with lung ECM remodeling, with specific differences occurring in defined regions within the lung. These differences may affect lung structure and physiology with aging and as such may increase susceptibility to developing chronic lung diseases.NEW & NOTEWORTHY We identified seven age-associated extracellular matrix (ECM) proteins, i.e., COL1A1, COL6A1, COL6A2 COL14A1, FBLN2, LTBP4, and LUM with higher transcript and protein levels in human lung tissue with age. Extensive immunohistochemical analysis revealed significant age-associated differences for COL6A2 in whole tissue, parenchyma, airway wall, and vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in parenchyma. Our findings lay a new foundation for the investigation of ECM differences in age-associated chronic lung diseases.

There is an urgent need to stimulate agricultural output in many tropical and subtropical countries of the world to combat hunger and malnutrition. The starchy crop cassava (Manihot esculenta), growing even under sub-optimal conditions, is a key staple food in these regions, providing millions of people with food. Cassava biotechnology is an important technique benefiting agricultural progress, but successful implementation of many biotechnological concepts depends on the availability of the right spatiotemporal expression tools. Yet, well-characterized cassava promoters are scarce in the public domain. In this study, we investigate the promoter activity and tissue specificity of 24 different promoter elements in stably transformed cassava plants. We show that many of the investigated promoters, especially from other species, have surprisingly low activity and/or tissue specificity, but feature several promoter sequences that can drive tissue-specific expression in either autotrophic-, transport- or storage tissues. We especially highlight pAtCAB1, pMePsbR, and pSlRBCS2 as strong and specific source promoters, pAtSUC2, pMeSWEET1-like, and pMeSUS1 as valuable tools for phloem and phloem parenchyma expression, and pStB33, pMeGPT, pStGBSS1, as well as pStPatatin Class I, as strong and specific promoters for heterotrophic storage tissues. We hope that the provided information and sequences prove valuable to the cassava community by contributing to the successful implementation of biotechnological concepts aimed at the improvement of cassava nutritional value and productivity.

This research examined the histological micro-structure of tendril vasculature in cucurbitaceous taxa. In this research, the tendril anatomy of 17 taxa of Cucurbitaceae categorized into seven genera, including Cucumis (five species), Cucurbita and Luffa (three species each), Citrullus and Momordica (two species each) while Lagenaria and Praecitrullus (one species each), collected from different areas of the Thal desert were examined via microscopic imaging to explore its taxonomic significance. Tendril transverse sections were cut with a Shandon Microtome to prepare slides. The distinctive characteristics of taxonomic value (qualitative and quantitative) include tendril and vascular bundle shape, variation in the number of vascular bundles, tendril diameter length, layers of sclerenchyma, and shape of collenchyma and epidermal cells. Tendril shapes observed are irregular, slightly oval-shaped, slightly C shaped, angular (4-angled, 6-angled, or polygonal), and star shaped. Quantitative measurements were taken to analyze the data statistically using SPSS software. Cucurbita pepo had a maximum tendril diameter length of 656.1 µm and a minimum in Momordica balsamina of 123.05 µm. The highest number of vascular bundles (12) were noticed in Luffa acutangula var.amara. Angular type was prominent in collenchyma, and irregular shape was dominant in sclerenchyma cells. A maximum of seven to nine sclerenchyma layers were present in Lagenaria siceraria and a minimum of two or three layers in Cucumis melo subsp. agrestis, Cucumis melo var. flexuosus, and Cucumis melo var.cantalupensis. Epidermis cells also show great variations with a rectangular shape being dominant. Statistical UPGMA dendrogram clustering of tendril vasculature traits shows that histological sections studied with microscopic techniques can be used to identify species and will play a vital role in future taxonomic and phylogenic linkages.

Brain slice culture (BSC) is a well-known three-dimensional model of the brain. In this study, we use organotypic slices for studying neuro-lymphatic physiology, to directly test the longstanding assumption that the brain is not a hospitable milieu for typical lymphatic vessels. An additional objective is to model fluid egress through brain perivascular space systems and to visualize potential cellular interactions among cells in the leptomeninges including alterations of cellular geometry and number of processes. Immortalized lymphatic rat cell lines were used to seed organotypic brain slices. The brain slice model was characterized by monitoring morphologies, growth rates, degree of apoptosis, and transport properties of brain slices with or without a lymphatic component. The model was then challenged with fibroblast co-cultures, as a control cell that is not normally found in the brain. Immortalized lymphatic cells penetrated the brain slices within 2-4 days. Typical cell morphology is spindly with bipolar and tripolar forms well represented. Significantly more indigo carmine marker passed through lymphatic seeded BSCs compared to arachnoid BSCs. Significantly more indigo carmine passed through brain slices co-cultured with fibroblast compared to lymphatic and arachnoid BSCs alone. We have developed an organotypic model in which lymphatic cells are able to interact with parenchymal cells in the cerebrum. Their presence appears to alter the small molecule transport ability of whole-brain slices. Lymphatic cells decreased dye transport in BSCs, possibly by altering the perivascular space. Given their direct contact with the CSF, they may affect convectional and diffusional processes. Our model shows that a decrease in lymphatic cell growth may reduce the brain slice\'s transport capabilities.

The advent of high-throughput sequencing and population genomics has enabled researchers to investigate selection pressure at hypervariable genomic loci encoding pathogen-associated molecular pattern (PAMP) molecules like lipopolysaccharide (LPS). Xanthomonas is a model and a major group of phytopathogenic bacteria that infect hosts in tissue-specific manner. Our in-depth population-based genomic investigation revealed the emergence of major lineages in two Xanthomonas pathogens that infect xylem of rice and sugarcane is associated with the acquisition and later large-scale replacement by distinct type of LPS cassettes. In the population of the rice xylem pathogen, Xanthomonas oryzae pv. oryzae (Xoo) and sugarcane pathogens Xanthomonas sacchari (Xsac) and Xanthomonas vasicola (Xvv), the BXO8 type of LPS cassette is replaced by a BXO1 type of cassette in Xoo and by Xvv type LPS cassette in Xsac and Xvv. These findings suggest a wave of parallel evolution at an LPS locus mediated by horizontal gene transfer (HGT) events during its adaptation and emergence. Aside from xylem pathogens, two closely related lineages of Xoo that infect parenchyma of rice and Leersia hexandra grass have acquired an LPS cassette from Xanthomonas pathogens that infect parenchyma of citrus, walnut, and strawberries, indicating yet another instance of parallel evolution mediated by HGT at an LPS locus. Our targeted and megapopulation-based genome dynamic studies revealed the acquisition and dominance of specific types of LPS cassettes in adaptation and success of a major group of phytopathogenic bacteria. IMPORTANCE Lipopolysaccharide (LPS) is a major microbe associated molecular pattern and hence a major immunomodulator. As a major and outer member component, it is expected that LPS is a frontline defense mechanism to deal with different host responses. Limited studies have indicated that LPS loci are also highly variable at strain and species level in plant-pathogenic bacteria, suggesting strong selection pressure from plants and associated niches. The advent of high-throughput genomics has led to the availability of a large set of genomic resources at taxonomic and population levels. This provides an exciting and important opportunity to carryout megascale targeted and population-based comparative genomic/association studies at important loci like those encoding LPS biosynthesis to understand their role in the evolution of the host, tissue specificity, and also predominant lineages. Such studies will also fill major gap in understanding host and tissue specificity in pathogenic bacteria. Our pioneering study uses the Xanthomonas group of phytopathogens that are known for their characteristic host and tissue specificity. The present deep phylogenomics of diverse Xanthomonas species and its members revealed lineage association and dominance of distinct types of LPS in accordance with their origin, host, tissue specificity, and evolutionary success.