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Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth.

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it’s C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms. Mitochondria are like tiny bean-shaped “power stations” that provide our cells with the vast majority of the energy that they need. These structures, however, are not self-sufficient and instead rely on proteins and chemicals that are imported from elsewhere in the cell. Two layers of membrane enclose the mitochondria, and transporting proteins across the inner and outer membranes requires large molecular machines embedded within the membranes. One such complex, the TIM22 complex, organizes tunnel-like carrier proteins that in turn ferry chemicals across the inner membrane to fuel metabolism. The TIM22 complex is vitally important as it allows mitochondria to adapt their metabolism – that is, how and when they generate energy – to match the cell’s needs during development. Yet, while the TIM22 complex has been studied extensively in yeast, less is known about how it works in human cells. Now, Kang et al. have identified a new piece of the human equivalent of the TIM22 machinery, a protein called Tim29, which helps to assemble the TIM22 complex in human cells. Experiments reveal that Tim29 also creates a link between human TIM22 and the TOM complex, a complex that serves as the general entry point through the outer mitochondrial membrane. Sequence analysis revealed that Tim29 is found in other animals, such as chimpanzees and cows, but not in yeast. This suggests that the mitochondrial machinery has changed during evolution. Kang et al. plan to further investigate how human carrier proteins reach the mitochondria, and exactly how Tim29 helps human TIM22 to cooperate with TOM. Overall, the discovery of Tim29 highlights the importance of looking at mitochondrial machinery across different species in the hope of revealing new components and mechanisms. A future challenge will be to determine how relevant these machines are in human development and diseases.

In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.

Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1 ∆ or chs3 ∆ or the vps23 ∆ chs3 ∆ double knockout strain were unable to rescue the yeast cells as compared to vps23 ∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling. Kening Zhao et al. show that yeast extracellular vesicles are depleted of ESCRT proteins but enriched with Fks1 and Chs3. The toxic effect of antifungal agents can be diminished by exposure to the Fks1- and Chs3- rich extracellular vesicles, suggesting a role for yeast extracellular vesicles in cell wall remodelling.

Histone deacetylase 3 ( Hdac3 ) regulates the expression of lipid metabolism genes in multiple tissues, however its role in regulating lipid metabolism in the intestinal epithelium is unknown. Here we demonstrate that intestine-specific deletion of Hdac3 ( Hdac3 IKO ) protects mice from diet induced obesity. Intestinal epithelial cells (IECs) from Hdac3 IKO mice display co-ordinate induction of genes and proteins involved in mitochondrial and peroxisomal β-oxidation, have an increased rate of fatty acid oxidation, and undergo marked remodelling of their lipidome, particularly a reduction in long chain triglycerides. Many HDAC3-regulated fatty oxidation genes are transcriptional targets of the PPAR family of nuclear receptors, Hdac3 deletion enhances their induction by PPAR-agonists, and pharmacological HDAC3 inhibition induces their expression in enterocytes. These findings establish a central role for HDAC3 in co-ordinating PPAR-regulated lipid oxidation in the intestinal epithelium, and identify intestinal HDAC3 as a potential therapeutic target for preventing obesity and related diseases. Histone deacetylase 3 (HDAC3) is a regulator of lipid homeostasis in several tissues, however, its role in intestinal lipid metabolism was not yet known. Here the authors study intestine specific HDAC3 knock out mice and report that these animals have increased fatty acid oxidation and undergo remodeling of the intestinal epithelial cell lipidome.

Neuroendocrine prostate cancer (NEPC) tumours are classified by pathology into several distinct subtypes. Gene expression profiling has revealed transcriptional heterogeneity across NEPC, but this is rarely considered in the context of variation between pathologies. Diagnosis typically relies on immunohistochemical markers (CHGA, SYP, NCAM1) and genomic alterations in RB1, PTEN and TP53. We hypothesized that NEPC pathologies have unique transcriptional features. Single‐cell RNA sequencing of 18 632 tumour cells from nine patient‐derived xenograft models representing five pathologies (small‐cell and large‐cell neuroendocrine carcinomas, focal neuroendocrine differentiation (Focal NED), low‐grade neuroendocrine and amphicrine) demonstrated pathway‐specific enrichment. Focal NED and amphicrine tumours exhibited cellular subpopulations enriched for KRAS, IL2‐STAT5 and TNF signalling pathways, absent in small‐ and large‐cell carcinomas, which were instead enriched for Myc and E2F pathways. Furthermore, focal NED cells exhibited minimal clonal divergence from adjacent adenocarcinoma cells, while small cell carcinoma cells were clonally distinct. These data underscore significant transcriptional variation among NEPC pathologies, highlighting focal NED's unique biological context and its clinical implications. Single‐cell transcriptomics of prostate cancer patient‐derived xenografts reveals distinct features of neuroendocrine (NE) subtypes. Tumours with focal NE differentiation (NED) share transcriptional programmes with adenocarcinoma, differing from large and small cell neuroendocrine prostate cancer (NEPC). Our work defines the molecular landscape of NEPC, revealing layers of transcriptional heterogeneity that provide a basis to develop new therapeutic opportunities for this subtype of prostate cancer.

Tumor development and progression reshape the physical properties of the surrounding tumor microenvironment (TME), including its biomechanical traits. This is driven by a prominent cell type in the TME, cancer‐associated fibroblasts (CAFs), which increases tissue stiffness via extracellular matrix deposition and remodeling. Currently, it is unclear whether there are also physical changes to CAFs at the cellular level and, if so, how they relate to patient outcome. Here, it is shown that CAFs have distinct morphological and biomechanical features from normal fibroblasts. Matched, patient‐derived CAFs and non‐malignant prostate fibroblasts (NPFs) from 35 patients with primary prostate cancer are examined. Morphologically, CAFs have more aligned stress fibers and larger and more elongated nuclei, based on quantitative image analysis of confocal microscopy images. In addition, single‐cell mechanical measurements using real‐time deformability cytometry showed that CAFs are larger and stiffer than NPFs. These changes are consistent across patients and validated with atomic force microscopy. A combined morphomechanical score encompassing these features is significantly associated with patient outcome. In transcriptomic analyses, the score is correlated with microtubule dynamics and a myofibroblast phenotype. Importantly, it is also demonstrated that morphomechanical features of prostate fibroblasts are modified by approved treatments for prostate cancer, such as docetaxel, and other small molecular inhibitors, particularly those targeting FGFR. In summary, changes in cellular morphomechanical properties are a consistent feature of CAFs and are associated with patient outcome. Moreover, cellular morphomechanical properties can be therapeutically targeted, potentially providing a new strategy for manipulating the TME to control cancer progression. Cancer‐associated fibroblasts (CAFs) in prostate tumors exhibit distinct morphomechanical traits vs normal fibroblasts, including greater stiffness and volume, more elongated stress fibres, and larger and more elongated nuclei. These features, quantified through imaging and real‐time deformability cytometry, correlate with patient outcomes and can be modified with therapeutics, providing a strategy to target the tumor microenvironment during cancer progression.

Intellectual Disability (ID) disorders, defined by an IQ below 70, are genetically and phenotypically highly heterogeneous. Identification of common molecular pathways underlying these disorders is crucial for understanding the molecular basis of cognition and for the development of therapeutic intervention strategies. To systematically establish their functional connectivity, we used transgenic RNAi to target 270 ID gene orthologs in the Drosophila eye. Assessment of neuronal function in behavioral and electrophysiological assays and multiparametric morphological analysis identified phenotypes associated with knockdown of 180 ID gene orthologs. Most of these genotype-phenotype associations were novel. For example, we uncovered 16 genes that are required for basal neurotransmission and have not previously been implicated in this process in any system or organism. ID gene orthologs with morphological eye phenotypes, in contrast to genes without phenotypes, are relatively highly expressed in the human nervous system and are enriched for neuronal functions, suggesting that eye phenotyping can distinguish different classes of ID genes. Indeed, grouping genes by Drosophila phenotype uncovered 26 connected functional modules. Novel links between ID genes successfully predicted that MYCN, PIGV and UPF3B regulate synapse development. Drosophila phenotype groups show, in addition to ID, significant phenotypic similarity also in humans, indicating that functional modules are conserved. The combined data indicate that ID disorders, despite their extreme genetic diversity, are caused by disruption of a limited number of highly connected functional modules.

Under both normal and pathological conditions, cells secrete variety of proteins through classical and non-classical secretory pathways into the extracellular space. Majority of these proteins represent pathophysiology of the cell from which it is secreted. Recently, though more than 92% of the protein coding genes has been mapped by human proteome map project, but number of those proteins that constitutes secretome of the cell still remains elusive. Secreted proteins or the secretome can be accessible in bodily fluids and hence are considered as potential biomarkers to discriminate between healthy and diseased individuals. In order to facilitate the biomarker discovery and to further aid clinicians and scientists working in these arenas, we have compiled and catalogued secreted proteins from the human proteome using integrated bioinformatics approach. In this study, nearly 14% of the human proteome is likely to be secreted through classical and non-classical secretory pathways. Out of which, ~38% of these secreted proteins were found in extracellular vesicles including exosomes and shedding microvesicles. Among these secreted proteins, 94% were detected in human bodily fluids including blood, plasma, serum, saliva, semen, tear and urine. We anticipate that this high confidence list of secreted proteins could serve as a compendium of candidate biomarkers. In addition, the catalogue may provide functional insights in understanding the molecular mechanisms involved in various physiological and pathophysiological conditions of the cell.