Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.

The application of an external magnetic field has been shown to improve the Cd phytoremediation efficiency of F. arundinacea by leaf harvesting. However, the influencing mechanisms of the promoting effect have not yet been revealed. This study evaluated variations in the Cd subcellular allocation and fractions in various F. arundinacea leaves, with or without magnetized water irrigation. Over 50 % of the metal were sequestered within the cell wall in all tissues under all treatments, indicating that cell wall binding was a critical detoxification pathway for Cd. After magnetized water treatment, the metal stored in the cytoplasm of roots raised from 33.1 % to 45.3 %, and the quantity of soluble Cd in plant roots enhanced from 53.4 % to 59.0 %. The findings suggested that magnetized water mobilized Cd in the roots, and thus drove it into the leaves. In addition, the proportion of Cd in the organelles, and the concentration of ethanol-extracted Cd in emerging leaves, decreased by 13.0 % and 47.1 %, respectively, after magnetized water treatment. These results explained why an external field improved the phytoextraction effect of the plant through leaf harvesting.

In this study, wild barley (Hordeum brevisubulatum) infected (E+) and uninfected (E-) by Epichloë bromicola were used for hydroponic experiments during the seedling stage. Various attributes, such as the effect of fungal endophyte on the growth and development of wild barley, the absorption of cadmium (Cd) and mineral elements (Ca, Mg, Fe, Mn, Cu, Zn), subcellular distribution, and chemical forms were investigated under CdCl2 stress. The results showed that the fungal endophy significantly reduced the Ca content and percentage of plant roots under Cd stress. The Fe and Mn content of roots, the mineral element content of soluble fractions, and the stems in the pectin acid or protein-chelated state increased significantly in response to fungal endophy. Epichloë endophyte helped Cd2+ to enter into plants; and reduced the positive correlation of Ca-Fe and Ca-Mn in roots. In addition, it also decreased the correlation of soluble components Cd-Cu, Cd-Ca, Cd-Mg in roots, and the negative correlation between pectin acid or protein-chelated Cd in stems and mineral elements, to increase the absorbance of host for mineral elements. In conclusion, fungal endophy regulated the concentration and distribution of mineral elements, while storing more Cd2+ to resist the damage caused by Cd stress. The study could provide a ground for revealing the Cd tolerance mechanism of endophytic fungal symbionts.
The present study is the first to study the effect of fungal endophy on essential mineral elements of plants under heavy metal stress, filling a gap in the existing research. The study could be helpful to reveal the mechanism of endophytic fungi to improve the host\'s tolerance to heavy metals and provide a foundation for the grass-endophyte symbionts to improve heavy metal-contaminated soils as ecological grasses.

Multimodal mass spectrometry (MMS) incorporates an imaging modality with probe-based mass spectrometry (MS) to enable precise, targeted data acquisition and provide additional biological and chemical data not available by MS alone. Two categories of MMS are covered; in the first, an imaging modality guides the MS probe to target individual cells and to reduce acquisition time by automatically defining regions of interest. In the second category, imaging and MS data are coupled in the data analysis pipeline to increase the effective spatial resolution using a higher resolution imaging method, correct for tissue deformation, and incorporate fine morphological features in an MS imaging dataset. Recent methodological and computational developments are covered along with their application to single-cell and imaging analyses.

BACKGROUND: Poly C Binding Protein 1 (PCBP1) belongs to the heterogeneous nuclear ribonucleoprotein family. It is a multifunctional protein that participates in several functional circuits and plays a variety of roles in cellular processes. Although PCBP1 has been identified in several mammals, its function in porcine was unclear.
RESULTS: In this study, we cloned the gene of porcine PCBP1 and analyzed its evolutionary relationships among different species. We found porcine PCBP1 protein sequence was similar to that of other animals. The subcellular localization of PCBP1 in porcine kidney cells 15 (PK-15) cells was analyzed by immunofluorescence assay (IFA) and revealed that PCBP1 was mainly localized to the nucleus. Reverse transcription-quantitative PCR (RT-qPCR) was used to compare PCBP1 mRNA levels in different tissues of 30-day-old pigs. Results indicated that PCBP1 was expressed in various tissues and was most abundant in the liver. Finally, the effects of PCBP1 on cell cycle and apoptosis were investigated following its overexpression or knockdown in PK-15 cells. The findings demonstrated that PCBP1 knockdown arrested cell cycle in G0/G1 phase, and enhanced cell apoptosis.
CONCLUSIONS: Porcine PCBP1 is a highly conserved protein, plays an important role in determining cell fate, and its functions need further study.

Glycolysis is the central metabolic pathway across all kingdoms of life. Intensive research efforts have been devoted to understanding the tightly orchestrated processes of converting glucose into energy in health and disease. Our review highlights the advances in knowledge of how metabolic and gene networks are integrated through the precise spatiotemporal compartmentalization of rate-limiting enzymes. We provide an overview of technically innovative approaches that have been applied to study phosphofructokinase-1 (PFK1), which represents the fate-determining step of oxidative glucose metabolism. Specifically, we discuss fast-acting chemical biology and optogenetic tools that have delineated new links between metabolite fluxes and transcriptional reprogramming, which operate together to enact tissue-specific processes. Finally, we discuss how recent paradigm-shifting insights into the fundamental basis of glycolytic regulatory control have shed light on the mechanisms of tumorigenesis and could provide insight into new therapeutic vulnerabilities in cancer.

Intramuscular lipids are stored as subsarcolemmal or intramyofibrillar droplets with potential diverse roles in energy metabolism. We examined intramuscular lipid utilization through transmission electron microscopy during repeated high-intensity intermittent exercise, an aspect that is hitherto unexplored. Seventeen moderately to well-trained males underwent three periods (EX1-EX3) of 10 × 45-s high-intensity cycling [∼100%-120% Wattmax (Wmax)] combined with maximal repeated sprints (∼250%-300% Wmax). M. vastus lateralis biopsies were obtained at baseline, after EX1, and EX3. During the complete exercise session, no net decline in either subsarcolemmal or intermyofibrillar lipid volume density occurred. However, a temporal relationship emerged for subsarcolemmal lipids with an ∼11% increase in droplet size after EX1 (P = 0.024), which reverted to baseline levels after EX3 accompanied by an ∼30% reduction in the numerical density of subsarcolemmal lipid droplets compared with both baseline (P = 0.019) and after EX1 (P = 0.018). Baseline distinctions were demonstrated with an approximately twofold higher intermyofibrillar lipid volume in type 1 versus type 2 fibers (P = 0.008), mediated solely by a higher number rather than the size of lipid droplets (P < 0.001). No fiber-type-specific differences were observed in subsarcolemmal lipid volume although type 2 fibers exhibited ∼17% larger droplets (P = 0.034) but a lower numerical density (main effect; P = 0.010) including 3% less droplets at baseline. Collectively, these findings suggest that intramuscular lipids do not serve as an important substrate during high-intensity intermittent exercise; however, the repeated exercise pattern mediated a temporal remodeling of the subsarcolemmal lipid pool. Furthermore, fiber-type- and compartment-specific differences were found at baseline underscoring the heterogeneity in lipid droplet deposition.NEW & NOTEWORTHY Undertaking a severe repeated high-intensity intermittent exercise protocol led to no net decline in neither subsarcolemmal nor intermyofibrillar lipid content in the thigh muscle of young moderately to well-trained participants. However, a temporal remodeling of the subsarcolemmal pool of lipid droplets did occur indicative of potential transient lipid accumulation. Moreover, baseline fiber-type distinctions in subcellular lipid droplet deposition were present underscoring the diversity in lipid droplet storage among fiber types and subcellular regions.

The integration of robust single-pot, solid-phase-enhanced sample preparation with powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely used to define the extracellular vesicle (EV) proteome landscape and underlying biology. However, EV proteome studies are often limited by sample availability, requiring upscaling cell cultures or larger volumes of biofluids to generate sufficient materials. Here, we have refined data independent acquisition (DIA)-based MS analysis of EV proteome by optimizing both protein enzymatic digestion and chromatography gradient length (ranging from 15 to 44 min). Our short 15 min gradient length can reproducibly quantify 1168 (from as little as 500 pg of EV peptides) to 3882 proteins groups (from 50 ng peptides), including robust quantification of 22 core EV marker proteins. Compared to data-dependent acquisition, DIA achieved significantly greater EV proteome coverage and quantification of low abundant protein species. Moreover, we have achieved optimal magnetic bead-based sample preparation tailored to low quantities of EVs (0.5 to 1 µg protein) to obtain sufficient peptides for MS quantification of 1908-2340 protein groups. We demonstrate the power and robustness of our pipeline in obtaining sufficient EV proteomes granularity of different cell sources to ascertain known EV biology. This underscores the capacity of our optimised workflow to capture precise and comprehensive proteome of EVs, especially from ultra-low sample quantities (sub-nanogram), an important challenge in the field where obtaining in-depth proteome information is essential.

Multi-component drugs (MCDs) can induce various cellular changes covering multiple levels, from molecular and subcellular structure to cell morphology. A \"non-invasive\" method for comprehensively detecting the dynamic changes of cellular fine structure and chemical components on the subcellular level is highly desirable for MCD studies. In this study, the subcellular dynamic processes of gastric cancer BGC823 cells after treatment with a multi-component drug, Compound Kushen Injection (CKI), were investigated using a homemade, high-resolution, confocal Raman spectroscopy (RS) device combined with bright-field imaging. The Raman spectra of the nucleus, cytoplasm and intracellular vesicles (0.4-1 μm) were collected simultaneously for each cell treated with CKI at different times and doses. The RS measurements showed that CKI decreased the DNA signatures, which the drug is known to inhibit. Meanwhile, the CKI-induced subcellular dynamic changes in the appearance of numerous intracellular vesicles and the deconstruction of cytoplasm components were observed and discussed. The results demonstrated that high-resolution subcellular micro-Raman spectroscopy has potential for detecting fine cellular dynamic variation induced by drugs and the screening of MCDs in cancer therapy.

Climate change jeopardizes soybean production by declining seed yield and quality. In this review, the morphophysiological alterations of soybean in response to abiotic stress are summarized, followed by illustrations of cellular metabolisms and regulatory mechanisms to organellar stress based on subcellular proteomics. This highlights the communications associated with reactive oxygen species scavenging, molecular chaperones, and phytohormone signals among subcellular compartments. Given the complexity of climate change and the limitations of plants in coping with multiple abiotic stresses, a generic response to environmental constraints is proposed between calcium and abscisic acid signals in subcellular organelles. This review summarizes the findings of subcellular proteomics in stressed soybean and discusses the future prospects of subcellular proteomics for promoting the improvement of climate-tolerant crops.