J Thromb Haemost. 2023 Dec 20:S1538-7836(23)00912-1. doi: 10.1016/j.jtha.2023.12.014. Online ahead of print.

ABSTRACT

BACKGROUND: Blood clots are living tissues that release inflammatory mediators including IL-8/CXCL8 and MCP-1/CCL2. A deeper understanding of blood clots is needed to develop new therapies for pro-thrombotic disease states and regenerative medicine.

OBJECTIVES: Identify a common transcriptional shift in cultured blood clot leukocytes.

METHODS: Differential gene expression of whole blood and cultured clots (4h 37°C) was assessed by RNA sequencing (RNAseq), RT-PCR, proteomics, and histology (23 diverse healthy human donors). Cultured clot serum bioactivity was tested in endothelial barrier functional assays.

RESULTS: All cultured clots developed a polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) signature, including upregulation of OLR1 (mRNA encoding lectin-like oxidized low density lipoprotein receptor 1, Lox-1), IL-8/CXCL8, CXCL2, CCL2, IL10, IL1A, SPP1, TREM1 and DUSP4/MKP. Lipopolysaccharide enhanced PMN-MDSC gene expression and specifically induced a Type II interferon response with IL-6 production. Lox-1 was specifically expressed by cultured clot CD15+ neutrophils. Cultured clot neutrophils, but not activated platelets, shed copious amounts of soluble Lox-1 (sLox-1) with a donor-dependent amplitude. sLox-1 shedding was enhanced by phorbol ester and suppressed by heparin and by beta-glycerol phosphate, a phosphatase inhibitor. Cultured clot serum significantly enhanced endothelial cell monolayer barrier function, consistent with a pro-resolving bioactivity.

CONCLUSIONS: This study suggests that PMN-MDSC activation is part of the innate immune response to coagulation which may have a protective role in inflammation. The cultured blood clot is an innovative thrombus model that can be used to study both sterile and nonsterile inflammatory states and could be used as a personalized medicine tool for drug screening.

PMID:38135253 | DOI:10.1016/j.jtha.2023.12.014

Matrix Biol. 2023 Dec 20:S0945-053X(23)00129-4. doi: 10.1016/j.matbio.2023.12.006. Online ahead of print.

ABSTRACT

Tissue transglutaminase 2 (TG2) plays a vital role in stabilizing extracellular matrix (ECM) proteins through enzymatic crosslinking during tissue growth, repair, and inflammation. TG2 also binds non-covalently to fibronectin (FN), an essential component of the ECM, facilitating cell adhesion, migration, proliferation, and survival. However, the interaction between TG2 and fibrillar FN remains poorly understood, as most studies have focused on soluble or surface-adsorbed FN or FN fragments, which differ in their conformations from insoluble FN fibers. Using a well-established in vitro FN fiber stretch assay, we discovered that the binding of a crosslinking enzyme to ECM fibers is mechano-regulated. TG2 binding to FN is tuned by the mechanical tension of FN fibers, whereby TG2 predominantly co-localizes to low-tension FN fibers, while fiber stretching reduces their affinity for TG2. This mechano-regulated binding relies on the proximity between the N-terminal β-sandwich and C-terminal β-barrels of TG2. Crosslinking mass spectrometry (XL-MS) revealed a novel TG2-FN synergy site within TG2's C-terminal β-barrels that interacts with FN regions located outside of the canonical gelatin binding domain, specifically FNI2 and FNIII14-15. Combining XL-MS distance restraints with molecular docking revealed the mechano-regulated binding mechanism between TG2 and modules FNI7-9 by which mechanical forces regulate TG2-FN interactions. This highlights a previously unrecognized role of TG2 as a tension sensor for FN fibers. This novel interaction mechanism has significant implications in physiology and mechanobiology, including how forces regulate cell adhesion, spreading, migration, phenotype modulation, depending on the tensional state of ECM fibers. Data are available via ProteomeXchange with identifier PXD043976.

PMID:38135164 | DOI:10.1016/j.matbio.2023.12.006

J Dairy Sci. 2023 Dec 20:S0022-0302(23)02009-X. doi: 10.3168/jds.2023-23963. Online ahead of print.

ABSTRACT

Reproductive performance is a key determinant of cow longevity in a pasture-based, seasonal dairy system. Unfortunately, direct fertility phenotypes such as inter-calving interval or pregnancy rate tend to have low heritabilities and occur relatively late in an animal's life. In contrast, age at puberty (AGEP) is a moderately heritable, early-in-life trait, that may be estimated using an animal's age at first measured elevation in blood plasma progesterone (AGEP4) concentrations. Understanding the genetic architecture of AGEP4 in addition to genetic relationships between AGEP4 and fertility traits in lactating cows is important, as is its relationship with body size in the growing animal. Thus, the objectives of this research were 3-fold. First, to estimate the genetic and phenotypic (co)variances between AGEP4 and subsequent fertility during first and second lactations. Second, to quantify the associations between AGEP4 and height, length, and body weight (BW) measured when animals were around 11 mo old (SD = 0.5). Third, to identify genomic regions that are likely to be associated with variation in AGEP4. We measured AGEP4, height, length, and BW in around 5,000 Holstein-Friesian or Holstein-Friesian x Jersey crossbred yearling heifers, across 54 pasture-based herds managed in seasonal calving farm systems. We also obtained calving rate (CR42: success or failure to calve within the first 42 d of the seasonal calving period), breeding rate (PB21: success or failure to be presented for breeding within the first 21 d of the seasonal breeding period) and pregnancy rate (PR42: success or failure to become pregnant within the first 42 d of the seasonal breeding period) phenotypes from their first and second lactations. The animals were genotyped using the Weatherby's Versa 50K SNP array (Illumina, USA). The estimated heritabilities of AGEP4, height, length, and BW were 0.34 (0.30, 0.37), 0.28 (0.25, 0.31), 0.21 (0.18, 0.23), and 0.33 (0.30, 0.36), respectively. In contrast, the heritabilities of CR42, PB21 and PR42 were all < 0.05 in both first and second lactations. The genetic correlations between AGEP4 and these fertility traits were generally moderate ranging from 0.11 to 0.60, whereas genetic correlations between AGEP4 and yearling body conformation traits ranged from 0.02 to 0.28. Our genome wide association study (GWAS) highlighted a genomic window on chromosome 5 that was strongly associated with variation in AGEP4. We also identified 4 regions, located on chromosomes 14, 6, 1 and 11 (in order of decreasing importance), that exhibited suggestive associations with AGEP4. Our results show that AGEP4 is a reasonable predictor of estimated breeding values (EBVs) for fertility traits in lactating cows. While the GWAS provided insights into genetic mechanisms underpinning AGEP4, further work is required to test genomic predictions of fertility that use this information.

PMID:38135043 | DOI:10.3168/jds.2023-23963

Cell Metab. 2023 Dec 14:S1550-4131(23)00445-X. doi: 10.1016/j.cmet.2023.11.013. Online ahead of print.

ABSTRACT

Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy in vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.

PMID:38134929 | DOI:10.1016/j.cmet.2023.11.013

Comput Biol Med. 2023 Dec 13;169:107810. doi: 10.1016/j.compbiomed.2023.107810. Online ahead of print.

ABSTRACT

Non-silent single nucleotide genetic variants, like nonsense changes and insertion-deletion variants, that affect protein function and length substantially are prevalent and are frequently misclassified. The low sensitivity and specificity of existing variant effect predictors for nonsense and indel variations restrict their use in clinical applications. We propose the Pathogenic Mutation Prediction (PMPred) method to predict the pathogenicity of single nucleotide variations, which impair protein function by prematurely terminating a protein's elongation during its synthesis. The prediction starts by monitoring functional effects (Gene Ontology annotation changes) of the change in sequence, using an existing ensemble machine learning model (UniGOPred). This, in turn, reveals the mutations that significantly deviate functionally from the wild-type sequence. We have identified novel harmful mutations in patient data and present them as motivating case studies. We also show that our method has increased sensitivity and specificity compared to state-of-the-art, especially in single nucleotide variations that produce large functional changes in the final protein. As further validation, we have done a comparative docking study on such a variation that is misclassified by existing methods and, using the altered binding affinities, show how PMPred can correctly predict the pathogenicity when other tools miss it. PMPred is freely accessible as a web service at https://pmpred.kansil.org/, and the related code is available at https://github.com/kansil/PMPred.

PMID:38134749 | DOI:10.1016/j.compbiomed.2023.107810

Bioinformatics. 2023 Dec 22:btad773. doi: 10.1093/bioinformatics/btad773. Online ahead of print.

ABSTRACT

SUMMARY: CellularPotts.jl is a software package written in Julia to simulate biological cellular processes such as division, adhesion, and signaling. Accurately modeling and predicting these simple processes is crucial because they facilitate more complex biological phenomena related to important disease states like tumor growth, wound healing, and infection. Here we take advantage of Cellular Potts Modeling (CPM) to simulate cellular interactions and combine them with differential equations to model dynamic cell signaling patterns. These models are advantageous over other approaches because they retain spatial information about each cell while remaining computationally efficient at larger scales. Users of this package define three key inputs to create valid model definitions: a 2- or 3-dimensional space, a table describing the cells to be positioned in that space, and a list of model penalties that dictate cell behaviors. Models can then be evolved over time to collect statistics, simulated repeatedly to investigate how changing a specific property impacts cellular behavior, and visualized using any of the available plotting libraries in Julia.

AVAILABILITY AND IMPLEMENTATION: The CellularPotts.jl package is released under the MIT license and is available at https://github.com/RobertGregg/CellularPotts.jl. An archived version of the code (v0.3.2) at time of submission can also be found at https://doi.org/10.5281/zenodo.10407783.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PMID:38134421 | DOI:10.1093/bioinformatics/btad773

J Am Chem Soc. 2023 Dec 22. doi: 10.1021/jacs.3c08205. Online ahead of print.

ABSTRACT

The self-assembly of DNA-based monomers into higher-order structures has significant potential for realizing various biomimetic behaviors including algorithmic assembly, ultrasensitive detection, and self-replication. For these behaviors, it is desirable to implement high energetic barriers to undesired spurious nucleation, where such barriers can be bypassed via seed-initiated assembly. Joint-neighbor capture is a mechanism enabling the construction of such barriers while allowing for algorithmic behaviors, such as bit-copying. Cycles of polymerization with division could accordingly be used for implementing exponential growth in self-replicating materials. Previously, we demonstrated crisscross polymerization, a strategy that attains robust seed-dependent self-assembly of single-stranded DNA and DNA-origami monomers via joint-neighbor capture. Here, we expand the crisscross assembly to achieve autonomous, isothermal exponential amplification of ribbons through their concurrent growth and scission via toehold-mediated strand displacement. We demonstrate how this crisscross chain reaction, or 3CR, can be used as a detection strategy through coupling to single- and double-stranded nucleic acid targets and introduce a rule-based stochastic modeling approach for simulating molecular self-assembly behaviors such as crisscross-ribbon scission.

PMID:38133996 | DOI:10.1021/jacs.3c08205

Microbiol Resour Announc. 2023 Dec 22:e0082523. doi: 10.1128/mra.00825-23. Online ahead of print.

ABSTRACT

We present the genome sequence of a polerovirus (family Solemoviridae) isolated from wild oat (Avena fatua) in Australia. The genome sequence consists of 5,631 nucleotides and shares 87% nucleotide identity with its closest relative, cereal yellow dwarf virus RPV isolate 010 (GenBank accession number EF521830).

PMID:38133456 | DOI:10.1128/mra.00825-23

Toxics. 2023 Dec 7;11(12):998. doi: 10.3390/toxics11120998.

ABSTRACT

Tobacco smoke contains between 7000 and 10,000 constituents, and only an evanescently low number of which have been identified, let alone been evaluated for their toxicity. Recently, the Food and Drug Administration has published a list of 93 chemical tobacco constituents that are harmful or potentially harmful to a number of cellular processes. However, their effect on developing skeletal cells is unknown. In this study, we used ToxPI, a computational tool, to prioritize constituents on this list for screening in osteogenically differentiating human embryonic stem cells and fibroblasts. In selected endpoint assays, we evaluated the potential of these chemicals to inhibit osteogenic differentiation success as well as their cytotoxicity. Six of these chemicals, which were ascribed an embryotoxic potential in our screen, as well as nicotine, which was not found to be osteotoxic in vitro, were then evaluated in combinatorial exposures, either in pairs of two or three. No one single chemical could be pinpointed as the culprit of reduced calcification in response to tobacco exposure. Combining chemicals at their half-maximal inhibitory concentration of differentiation often elicited expected decreases in calcification over the individual exposures; however, cytotoxicity was improved in many of the dual combinations. A reverse response was also noted, in which calcification output improved in combinatorial exposures. Results from ternary combinations reflected those from double combinations. Thus, the results from this study suggest that it may be difficult to isolate single chemicals as the primary drivers of skeletal embryotoxicity and that the full combination of chemicals in tobacco smoke may produce the hypomineralization phenotype that we have so far observed in vitro in human embryonic stem cells as well as in vivo in zebrafish.

PMID:38133399 | DOI:10.3390/toxics11120998

Pathogens. 2023 Nov 28;12(12):1395. doi: 10.3390/pathogens12121395.

ABSTRACT

BACKGROUND: Human and wild rodent infection rates with Leishmania (Viannia) braziliensis are needed to differentiate transmission pathways in anthropogenically altered habitats.

METHODS: Human participants in northeast Brazil were tested by the leishmanin skin test (LST) and inspected for lesions/scars characteristic of American clinical leishmaniasis (ACL). Molecular (PCR/qPCR) test records of free-ranging rodents were available from a concurrent capture-mark-recapture study. Force of Infection (λ) and recovery (ρ) rates were estimated from cross-sectional and longitudinal datasets.

RESULTS: Cumulative prevalences of human LST+ves and ACL scar+ves were 0.343-0.563 (n = 503 participants) and 0.122-0.475 (n = 503), respectively. Active ACL lesions were not detected. Annual rates of LST conversions were λ = 0.03-0.15 and ρ = 0.02-0.07. The probability of infection was independent of sex and associated with increasing age in addition to the period of exposure. Rodents (n = 596 individuals of 6 species) showed high rates of exclusively asymptomatic infection (λ = 0.222/month) and potential infectiousness to the sand fly vector. Spatially concurrent rodent and household human infection prevalences were correlated.

CONCLUSIONS: Human exposure to L. (V.) braziliensis continues to be high despite the substantial drop in reported ACL cases in recent years. Spill-over transmission risk to humans from rodents in peridomestic habitats is likely supported by a rodent infection/transmission corridor linking houses, plantations, and the Atlantic Forest.

PMID:38133280 | DOI:10.3390/pathogens12121395

Metabolites. 2023 Dec 18;13(12):1203. doi: 10.3390/metabo13121203.

ABSTRACT

Huntington's disease (HD) is a progressive, fatal neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms. The precise mechanisms of HD progression are poorly understood; however, it is known that there is an expansion of the trinucleotide cytosine-adenine-guanine (CAG) repeat in the Huntingtin gene. Important new strategies are of paramount importance to identify early biomarkers with predictive value for intervening in disease progression at a stage when cellular dysfunction has not progressed irreversibly. Metabolomics is the study of global metabolite profiles in a system (cell, tissue, or organism) under certain conditions and is becoming an essential tool for the systemic characterization of metabolites to provide a snapshot of the functional and pathophysiological states of an organism and support disease diagnosis and biomarker discovery. This review briefly highlights the historical progress of metabolomic methodologies, followed by a more detailed review of the use of metabolomics in HD research to enable a greater understanding of the pathogenesis, its early prediction, and finally the main technical platforms in the field of metabolomics.

PMID:38132886 | DOI:10.3390/metabo13121203

Insects. 2023 Dec 5;14(12):924. doi: 10.3390/insects14120924.

ABSTRACT

The global loss of biodiversity is an urgent concern requiring the implementation of effective monitoring. Flying insects, such as pollinators, are vital for ecosystems, and establishing their population dynamics has become essential in conservation biology. Traditional monitoring methods are labour-intensive and show time constraints. In this work, we explore the use of bioacoustic sensors for monitoring flying insects. Data collected at four Italian farms using traditional monitoring methods, such as hand netting and pan traps, and bioacoustic sensors were compared. The results showed a positive correlation between the average number of buzzes per hour and insect abundance measured by traditional methods, primarily by pan traps. Intraday and long-term analysis performed on buzzes revealed temperature-related patterns of insect activity. Passive acoustic monitoring proved to be effective in estimating flying insect abundance, while further development of the algorithm is required to correctly identify insect taxa. Overall, innovative technologies, such as bioacoustic sensors, do not replace the expertise and data quality provided by professionals, but they offer unprecedented opportunities to ease insect monitoring to support conservation biodiversity efforts.

PMID:38132598 | DOI:10.3390/insects14120924

Biology (Basel). 2023 Dec 12;12(12):1516. doi: 10.3390/biology12121516.

ABSTRACT

The innate immune system is the first line of defense in multicellular organisms. Danio rerio is widely considered a promising model for IIS-related research, with the most amount of scRNAseq data available among Teleostei. We summarized the scRNAseq and spatial transcriptomics experiments related to the IIS for zebrafish and other Teleostei from the GEO NCBI and the Single-Cell Expression Atlas. We found a considerable number of scRNAseq experiments at different stages of zebrafish development in organs such as the kidney, liver, stomach, heart, and brain. These datasets could be further used to conduct large-scale meta-analyses and to compare the IIS of zebrafish with the mammalian one. However, only a small number of scRNAseq datasets are available for other fish (turbot, salmon, cavefish, and dark sleeper). Since fish biology is very diverse, it would be a major mistake to use zebrafish alone in fish immunology studies. In particular, there is a special need for new scRNAseq experiments involving nonmodel Teleostei, e.g., long-lived species, cancer-resistant fish, and various fish ecotypes.

PMID:38132342 | DOI:10.3390/biology12121516

Biology (Basel). 2023 Dec 11;12(12):1509. doi: 10.3390/biology12121509.

ABSTRACT

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and/or defective insulin production in the human body. Although the antidiabetic action of corn silk (CS) is well-established, the understanding of the mechanism of action (MoA) behind this potential is lacking. Hence, this study aimed to elucidate the MoA in different samples (raw and three extracts: aqueous, hydro-ethanolic, and ethanolic) as a therapeutic agent for the management of T2DM using metabolomic profiling and computational techniques. Ultra-performance liquid chromatography-mass spectrometry (UP-LCMS), in silico techniques, and density functional theory were used for compound identification and to predict the MoA. A total of 110 out of the 128 identified secondary metabolites passed the Lipinski's rule of five. The Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analysis revealed the cAMP pathway as the hub signaling pathway, in which ADORA1, HCAR2, and GABBR1 were identified as the key target genes implicated in the pathway. Since gallicynoic acid (-48.74 kcal/mol), dodecanedioc acid (-34.53 kcal/mol), and tetradecanedioc acid (-36.80 kcal/mol) interacted well with ADORA1, HCAR2, and GABBR1, respectively, and are thermodynamically stable in their formed compatible complexes, according to the post-molecular dynamics simulation results, they are suggested as potential drug candidates for T2DM therapy via the maintenance of normal glucose homeostasis and pancreatic β-cell function.

PMID:38132335 | DOI:10.3390/biology12121509

Biology (Basel). 2023 Dec 8;12(12):1505. doi: 10.3390/biology12121505.

ABSTRACT

Plants possess an innate ability to generate vast amounts of sugar and produce a range of sugar-derived compounds that can be utilized for applications in industry, health, and agriculture. Nucleotide sugars lie at the unique intersection of primary and specialized metabolism, enabling the biosynthesis of numerous molecules ranging from small glycosides to complex polysaccharides. Plants are tolerant to perturbations to their balance of nucleotide sugars, allowing for the overproduction of endogenous nucleotide sugars to push flux towards a particular product without necessitating the re-engineering of upstream pathways. Pathways to produce even non-native nucleotide sugars may be introduced to synthesize entirely novel products. Heterologously expressed glycosyltransferases capable of unique sugar chemistries can further widen the synthetic repertoire of a plant, and transporters can increase the amount of nucleotide sugars available to glycosyltransferases. In this opinion piece, we examine recent successes and potential future uses of engineered nucleotide sugar biosynthetic, transport, and utilization pathways to improve the production of target compounds. Additionally, we highlight current efforts to engineer glycosyltransferases. Ultimately, the robust nature of plant sugar biochemistry renders plants a powerful chassis for the production of target glycoconjugates and glycans.

PMID:38132331 | DOI:10.3390/biology12121505

Biology (Basel). 2023 Nov 23;12(12):1461. doi: 10.3390/biology12121461.

ABSTRACT

A cell constantly receives signals and takes different fates accordingly. Given the uncertainty rendered by signal transduction noise, a cell may incorrectly perceive these signals. It may mistakenly behave as if there is a signal, although there is none, or may miss the presence of a signal that actually exists. In this paper, we consider a signaling system with two outputs, and introduce and develop methods to model and compute key cell decision-making parameters based on the two outputs and in response to the input signal. In the considered system, the tumor necrosis factor (TNF) regulates the two transcription factors, the nuclear factor κB (NFκB) and the activating transcription factor-2 (ATF-2). These two system outputs are involved in important physiological functions such as cell death and survival, viral replication, and pathological conditions, such as autoimmune diseases and different types of cancer. Using the introduced methods, we compute and show what the decision thresholds are, based on the single-cell measured concentration levels of NFκB and ATF-2. We also define and compute the decision error probabilities, i.e., false alarm and miss probabilities, based on the concentration levels of the two outputs. By considering the joint response of the two outputs of the signaling system, one can learn more about complex cellular decision-making processes, the corresponding decision error rates, and their possible involvement in the development of some pathological conditions.

PMID:38132287 | DOI:10.3390/biology12121461

Elife. 2023 Dec 22;12:e86365. doi: 10.7554/eLife.86365. Online ahead of print.

ABSTRACT

Elucidating the intricate neural mechanisms underlying brain functions requires integrative brain dynamics modeling. To facilitate this process, it is crucial to develop a general-purpose programming framework that allows users to freely define neural models across multiple scales, efficiently simulate, train, and analyze model dynamics, and conveniently incorporate new modeling approaches. In response to this need, we present BrainPy. BrainPy leverages the advanced just-in-time (JIT) compilation capabilities of JAX and XLA to provide a powerful infrastructure tailored for brain dynamics programming. It offers an integrated platform for building, simulating, training, and analyzing brain dynamics models. Models defined in BrainPy can be JIT compiled into binary instructions for various devices, including Central Processing Unit (CPU), Graphics Processing Unit (GPU), and Tensor Processing Unit (TPU), which ensures high running performance comparable to native C or CUDA. Additionally, BrainPy features an extensible architecture that allows for easy expansion of new infrastructure, utilities, and machine-learning approaches. This flexibility enables researchers to incorporate cutting-edge techniques and adapt the framework to their specific needs.

PMID:38132087 | DOI:10.7554/eLife.86365

Dev Dyn. 2023 Dec 22. doi: 10.1002/dvdy.683. Online ahead of print.

ABSTRACT

BACKGROUND: Spatial mapping on the single-cell level over the whole organism can uncover roles of molecular players involved in vertebrate development. Custom microscopes have been developed that use multiple objectives to view a sample from multiple views at the same time. Such multiview imaging approaches can improve resolution and uniformity of image quality as well as allow whole embryos to be imaged (Swoger et al., Opt Express, 2007;15(13):8029). However, multiview imaging is highly restricted to specialized equipment requiring multiple objectives or sample rotation with automated hardware.

RESULTS: Our approach uses a standard single-objective confocal microscope to perform serial multiview imaging. Multiple views are imaged sequentially by mounting the fixed sample in an agarose tetrahedron that is manually rotated in between imaging each face. Computational image fusion allows for a joint 3D image to be create from multiple tiled Z-stacks acquired from different angles. The resulting fused image has improved resolution and imaging extent.

CONCLUSION: With this technique, multiview imaging can be performed on a variety of common single-objective microscopes to allow for whole-embryo, high-resolution imaging.

PMID:38131490 | DOI:10.1002/dvdy.683

Curr Protoc. 2023 Dec;3(12):e952. doi: 10.1002/cpz1.952.

ABSTRACT

Plant sample preparation for analyses is a fundamental step in high-throughput omics strategies. Especially for plant metabolomics, quenching of hydrolytic enzymes able to affect metabolite concentrations is crucial for the accuracy of results. Given that DNA is usually less labile than metabolites, most sampling and shipment procedures able to preserve the metabolome are also suitable for preventing the degradation of plant DNA or of DNA of pathogens in the plant tissue. In this article, we describe all the steps of sample collection, shipment (including the phytosanitary issues of moving plant samples), and processing for combined genomics and metabolomics from a single sample, as well as the protocols used in our laboratories for downstream approaches for crop plants, allowing collection of multi-omic datasets in large experimental setups. The protocols have been adjusted to apply to both freeze-dried and fresh-frozen material to allow the processing of crop plant samples that will require long-distance transport. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of freeze-dried leaf disks for multiplexed PCR or DArT-Seq genotyping Basic Protocol 2: Medium-throughput preparation of pathogen-free nucleic acids for most genotyping-resequencing applications or pathogen detection Alternate Protocol: Low-throughput extraction of high-quality DNA for resequencing using commercial kits Support Protocol: DNA quality control Basic Protocol 3: Preparation of freeze-dried plant material for metabolomics Basic Protocol 4: Preparation of fresh-frozen plant material for metabolomics Basic Protocol 5: Preparation and shipment of metabolite extracts for metabolomic analyses Basic Protocol 6: Sample shipping and long-term storage.

PMID:38131272 | DOI:10.1002/cpz1.952

Int J Oncol. 2024 Feb;64(2):18. doi: 10.3892/ijo.2023.5606. Epub 2023 Dec 22.

ABSTRACT

Fatty acid‑binding protein 5 (FABP5) and androgen receptor (AR) are critical promoters of prostate cancer. In the present study, the effects of knocking out the FABP5 or AR genes on malignant characteristics of prostate cancer cells were investigated, and changes in the expression of certain key proteins in the FABP5 (or AR)‑peroxisome proliferator activated receptor‑γ (PPARγ)‑vascular endothelial growth factor (VEGF) signaling pathway were monitored. The results obtained showed that FABP5‑ or AR‑knockout (KO) led to a marked suppression of the malignant characteristics of the cells, in part, through disrupting this signaling pathway. Moreover, FABP5 and AR are able to interact with each other to regulate this pathway, with FABP5 controlling the dominant AR splicing variant 7 (ARV7), and AR, in return, regulates the expression of FABP5. Comparisons of the RNA profiles revealed the existence of numerous differentially expressed genes (DEGs) comparing between the parental and the FABP5‑ or AR‑KO cells. The six most abundant changes in DEGs were found to be attributable to the transition from androgen‑responsive to androgen‑unresponsive, castration‑resistant prostate cancer (CRPC) cells. These findings have provided novel insights into the complex molecular pathogenesis of CRPC cells, and have demonstrated that interactions between FABP5 and AR contribute to the transition of prostate cancer cells to an androgen‑independent state. Moreover, gene enrichment analysis revealed that the most highly enriched biological processes associated with the DEGs included those responsive to fatty acids, cholesterol and sterol biosynthesis, as well as to lipid and fatty acid transportation. Since these pathways regulated by FABP5 or AR may be crucial in terms of transducing signals for cancer cell progression, targeting FABP5, AR and their associated pathways, rather than AR alone, may provide a new avenue for the development of therapeutic strategies geared towards suppressing the malignant progression to CRPC cells.

PMID:38131188 | DOI:10.3892/ijo.2023.5606