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The tumour suppressor PTEN is a negative regulator of the PI3K/AKT signalling pathway. Liver-specific deletion of Pten in mice results in the hyper-activation PI3K/AKT signalling accompanied by enhanced genome duplication (polyploidization), marked lipid accumulation (steatosis) and formation of hepatocellular carcinomas. However, it is unknown whether polyploidization in this model has an impact on the development of steatosis and the progression towards liver cancer. Here, we used a liver-specific conditional knockout approach to delete Pten in combination with deletion of E2f7/8, known key inducers of polyploidization. As expected, Pten deletion caused severe steatosis and liver tumours accompanied by enhanced polyploidization. Additional deletion of E2f7/8 inhibited polyploidization, alleviated Pten-induced steatosis without affecting lipid species composition and accelerated liver tumour progression. Global transcriptomic analysis showed that inhibition of polyploidization in Pten-deficient livers resulted in reduced expression of genes involved in energy metabolism, including PPAR-gamma signalling. However, we find no evidence that deregulated genes in Pten-deficient livers are direct transcriptional targets of E2F7/8, supporting that reduction in steatosis and progression towards liver cancer are likely consequences of inhibiting polyploidization. Lastly, flow cytometry and image analysis on isolated primary wildtype mouse hepatocytes provided further support that polyploid cells can accumulate more lipid droplets than diploid hepatocytes. Collectively, we show that polyploidization promotes steatosis and function as an important barrier against liver tumour progression in Pten-deficient livers.

Cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and PR-1 (CAP) superfamily that is characterized by the presence of a conserved CAP domain. Two conserved histidines in the CAP domain are proposed to function as a Zn2+-binding site with unknown function. Human CRISP1 is, however, one of the few family members that lack one of these characteristic histidine residues. The Zn2+-dependent oligomerization properties of human CRISP1 were investigated using a maltose-binding protein (MBP)-tagging approach in combination with low expression levels in XL-1 Blue bacteria. Moderate yields of soluble recombinant MBP-tagged human CRISP1 (MBP-CRISP1) and the MBP-tagged CAP domain of CRISP1 (MBP-CRISP1ΔC) were obtained. Zn2+ specifically induced oligomerization of both MBP-CRISP1 and MBP-CRISP1ΔC in vitro. The conserved His142 in the CAP domain was essential for this Zn2+ dependent oligomerization process, confirming a role of the CAP metal-binding site in the interaction with Zn2+. Furthermore, MBP-CRISP1 and MBP-CRISP1ΔC oligomers dissociated into monomers upon Zn2+ removal by EDTA. Condensation of proteins is characteristic for maturing sperm in the epididymis and this process was previously found to be Zn2+-dependent. The Zn2+-induced oligomerization of human recombinant CRISP1 may shed novel insights into the formation of functional protein complexes involved in mammalian fertilization.

Resistance to platinum-based chemotherapy represents a major clinical challenge for many tumors, including epithelial ovarian cancer. Patients often experience several response-relapse events, until tumors become resistant and life expectancy drops to 12-15 months. Despite improved knowledge of the molecular determinants of platinum resistance, the lack of clinical applicability limits exploitation of many potential targets, leaving patients with limited options. Serine biosynthesis has been linked to cancer growth and poor prognosis in various cancer types, however its role in platinum-resistant ovarian cancer is not known. Here, we show that a subgroup of resistant tumors decreases phosphoglycerate dehydrogenase (PHGDH) expression at relapse after platinum-based chemotherapy. Mechanistically, we observe that this phenomenon is accompanied by a specific oxidized nicotinamide adenine dinucleotide (NAD+) regenerating phenotype, which helps tumor cells in sustaining Poly (ADP-ribose) polymerase (PARP) activity under platinum treatment. Our findings reveal metabolic vulnerabilities with clinical implications for a subset of platinum resistant ovarian cancers. ispartof: NATURE COMMUNICATIONS vol:13 issue:1 ispartof: location:England status: published

Trained innate immunity can be induced in human macrophages by microbial ligands, but it is unknown if exposure to endogenous alarmins such as cathelicidins can have similar effects. Previously, we demonstrated sustained protection against infection by the chicken cathelicidin-2 analog DCATH-2. Thus, we assessed the capacity of cathelicidins to induce trained immunity. PMA-differentiated THP-1 (dTHP1) cells were trained with cathelicidin analogs for 24 hours and restimulated after a 3-day rest period. DCATH-2 training of dTHP-1 cells amplified their proinflammatory cytokine response when restimulated with TLR2/4 agonists. Trained cells displayed a biased cellular metabolism towards mTOR-dependent aerobic glycolysis and long-chain fatty acid accumulation and augmented microbicidal activity. DCATH-2-induced trained immunity was inhibited by histone acetylase inhibitors, suggesting epigenetic regulation, and depended on caveolae/lipid raft-mediated uptake, MAPK p38 and purinergic signaling. To our knowledge, this is the first report of trained immunity by host defense peptides.

The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)–refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9–based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI–refractory multiple myeloma. Significance: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.

CD4+ conventional T cells (Tconvs) mediate adaptive immune responses, whereas regulatory T cells (Tregs) suppress those responses to safeguard the body from autoimmunity and inflammatory diseases. The opposing activities of Tconvs and Tregs depend on the stage of the immune response and their environment, with an orchestrating role for cytokine- and costimulatory receptors. Nutrient availability also impacts T-cell functionality via metabolic and biosynthetic processes that are largely unexplored. Many data argue that costimulation by Tumor Necrosis Factor Receptor 2 (TNFR2) favors support of Treg over Tconv responses and therefore TNFR2 is a key clinical target. Here, we review the pertinent literature on this topic and highlight the newly identified role of TNFR2 as a metabolic regulator for thymus-derived (t)Tregs. We present novel transcriptomic and metabolomic data that show the differential impact of TNFR2 on Tconv and tTreg gene expression and reveal distinct metabolic impact on both cell types.

Contains fulltext : 286723.pdf (Publisher’s version ) (Open Access) Animal models continue to be used to investigate cartilage repair strategies. Adequate anaesthesia and pain management are essential in order to guarantee acceptable animal welfare as well as reproducible experimental results. This systematic review evaluates reporting of anaesthesia and pain management in surgical large animal models (horse, pig, dog, goat and sheep) of (osteo)chondral repair. Manuscripts published between 2015 and 2020 were included after a comprehensive search strategy. Data were evaluated using descriptive statistics and qualitative review. Out of 223 eligible studies, 220 studies contained incomplete information on anaesthetic and pain management. Pre-, intra- and post-operative analgesia were not mentioned in 68%, 94%, and 64% of manuscripts respectively. A total of 176 studies reported that animals underwent general anaesthesia during surgery. Surprisingly, 30% of these articles did not provide any detail on anaesthetic management, while 37% reported using inhalant, hypnotic or sedative drugs only, without mention of analgesics. Pain monitoring was not reported in 87% of manuscripts. The vast majority of preclinical large animal studies on cartilage repair did not meet veterinary clinical standards for anaesthesia and analgesia, and failed to report according to the ARRIVE international guidelines. In light of serious welfare, ethical and translational validity concerns, improvement is urgently needed.

Endocrine disrupting chemicals (EDCs) can interfere with normal hormonal action and regulation. Exposure of women to EDCs has been associated with adverse reproductive health outcomes. The assays currently used to identify EDCs that elicit female reproductive toxicity lack screening tests that address effects on the maturation of oocytes, a process that enables them to be fertilized and develop into embryos. Here, a screening method employing the bovine model of in vitro oocyte maturation and embryo production is described. Endpoints explored address important events in oocyte maturation and developmental competence acquisition. To test the method, the effects of the known human EDC diethylstilbestrol (DES; an estrogen receptor agonist) were evaluated in a range of concentrations (10–9 M, 10–7 M, 10–5 M). Bovine oocytes were exposed to DES during in vitro maturation (IVM) or embryos were exposed during in vitro embryo culture (IVC). The endpoints evaluated included nuclear maturation, mitochondrial redistribution, cumulus cell expansion, apoptosis, and steroidogenesis. DES-exposed oocytes were fertilized to record embryo cleavage and blastocyst rates to uncover effects on developmental competence. Similarly, the development of embryos exposed to DES during IVC was monitored to assess the impact on early embryo development. Exposure to 10–9 M or 10–7 M DES did not affect the endpoints addressing oocyte maturation or embryo development. However, there were considerable detrimental effects observed in oocytes exposed to 10–5 M DES. Specifically, compared to vehicle-treated oocytes, there was a statistically significant reduction in nuclear maturation (3% vs 84%), cumulus expansion (2.8-fold vs 3.6-fold) and blastocyst rate (3% vs 32%). Additionally, progesterone and pregnenolone concentrations measured in IVM culture media were increased. The screening method described here shows that bovine oocytes were sensitive to the action of this particular chemical (i.e., DES), albeit at high concentrations. In principle, this method provides a valuable tool to assess the oocyte maturation process and early embryo development that can be used for reproductive toxicity screening and possibly EDC identification. Further studies should include EDCs with different mechanisms of action and additional endpoints to further demonstrate the applicability of the bovine oocyte model for chemical risk assessment purposes and EDC identification.

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy1,2. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ3–5. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone6,7, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes—which we term ‘W-bumps’—showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape. Tryptophan depletion in melanoma cells after prolonged treatment with interferon-γ (IFNγ) results in ribosomal frameshifting and the production of aberrant peptides that can be presented to T cells and induce an immune response.

In a previous study, we reported that porcine sperm cysteine-rich secretory protein 2 (CRISP2) is localized in the post-acrosomal sheath-perinuclear theca (PT) as reduction-sensitive oligomers. In the current study, the decondensation and removal of CRISP2 was investigated during in vitro sperm capacitation, after both the induction of the acrosome reaction and in vitro fertilization. Confocal immunofluorescent imaging revealed that additional CRISP2 fluorescence appeared on the apical ridge and on the equatorial segment (EqS) of the sperm head following capacitation, likely due to cholesterol removal. After an ionophore A23187-induced acrosome reaction, CRISP2 immunofluorescence disappeared from the apical ridge and the EqS area partly not only owing to the removal of the acrosomal shroud vesicles, but to its presence in a subdomain of EqS. The fate of sperm head CRISP2 was further examined post-fertilization. In vitro matured porcine oocytes were co-incubated with boar sperm cells for 6–8 h and the zygotes were processed for CRISP2 immunofluorescent staining. Notably, decondensation of CRISP2, and thus of the sperm PT, occurred while the sperm nucleus was still fully condensed. CRISP2 was no longer detectable in fertilized oocytes in which sperm nuclear decondensation and paternal pronucleus formation were apparent. This rapid dispersal of CRISP2 in the PT is likely regulated by redox reactions for which its cysteine-rich domain is sensitive. Reduction of disulfide bridges within CRISP2 oligomers may be instrumental for PT dispersal and elimination.

Extracellular vesicles (EVs) and their cargo microRNAs (miRNAs) are important regulators of embryo development to the blastocyst stage and beyond. Before implantation can take place, hatching of blastocysts from their zona pellucida is required. However, underlying mechanisms by which blastocyst formation and hatching are initiated remain largely unknown. Here, we provide evidence that embryonic EVs containing bta-miR-378a-3p play a crucial role in blastocyst hatching, using a bovine model. A customized procedure was used to isolate EV-miRNAs from culture droplets conditioned by individual bovine embryos that either developed to the blastocyst stage or did not (nonblastocyst). RNA sequencing identified 69 differentially expressed miRNAs between EVs derived from blastocyst conditioned medium (CM) and nonblastocyst CM. Among the miRNAs up-regulated in blastocyst CM, we selected bta-miR-378a-3p for further validation by functionality testing on developing in vitro embryos by means of mimics and inhibitors. Supplementing the embryo culture medium with miR-378a-3p mimic significandy improved blastocyst quality, with higher cell numbers and reduced apoptosis, and improved hatching, while the opposite was found after supplementation with miR-378a-3p inhibitor (P < 0.01). Transcriptomic analysis of embryos treated with miR-378 mimic/inhibitor showed differential expression (P < 0.01) of genes associated with embryo development and implantation, including RAPT GAP, ARFGEF2, SLC7A6, CENPA, SP1, LDLR, PYCRJ, MYD88, TPPJ, and NCOA3. In conclusion, miR-378a-3p is up-regulated in EVs secreted by embryos that develop to the blastocyst stage, and this EV-derived miR-378a-3p increases blastocyst quality and regulates embryo hatching, which is essential for embryo implantation.

In vitro production (IVP) embryos have a reduced quality and poor cryotolerance in comparison to in vivo embryos. This study investigated whether free fatty acid (FFA) conditions, fatty acid free (FAF)- synthetic oviduct fluid (SOF) without or with 25 μM of saturated stearic (C18:0) or unsaturated oleic (C18:1) acid during the first 5 IVP days, relate to quality and cryosurvival of day 8 blastocysts. Apart from the blastocyst scores, both 1) number and size of lipid droplets of fresh blastocysts and 2) total number and apoptotic and necrotic cells, before and after freezing-thawing, were scored by confocal microscopy. Blastocyst rates were significantly lower in the FAF SOF condition in comparison to other groups. Interestingly, blastocysts originating from the C18:1 group, with a significantly higher lipid content, and blastocysts from the FAF SOF group demonstrated a high cryosurvival rate (70.1 and 67.4%, respectively) comparable with in vivo blastocysts (68%), in contrast to the poor cryosurvival of C18:0 exposed embryos (17.6%). In all freeze-thawed embryos the average amount of apoptotic and necrotic cells increased albeit that the C18:0 condition rates were higher (43.2%) when compared to C18:1 (26.0%) and FAF SOF conditions (26.5%). The current data show that FFA administered during early embryonic development significantly affect the cryotolerance of blastocysts.

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

The importance of fatty acid (FA) metabolism in cancer is well-established, yet the mechanisms underlying metabolic reprogramming remain elusive. Here, we identify tetraspanin CD37, a prognostic marker for aggressive B-cell lymphoma, as essential membrane-localized inhibitor of FA metabolism. Deletion of CD37 on lymphoma cells results in increased FA oxidation shown by functional assays and metabolomics. Furthermore, CD37-negative lymphomas selectively deplete palmitate from serum in mouse studies. Mechanistically, CD37 inhibits the FA transporter FATP1 through molecular interaction. Consequently, deletion of CD37 induces uptake and processing of exogenous palmitate into energy and essential building blocks for proliferation, and inhibition of FATP1 reverses this phenotype. Large lipid deposits and intracellular lipid droplets are observed in CD37-negative lymphoma tissues of patients. Moreover, inhibition of carnitine palmitoyl transferase 1 A significantly compromises viability and proliferation of CD37-deficient lymphomas. Collectively, our results identify CD37 as a direct gatekeeper of the FA metabolic switch in aggressive B-cell lymphoma.Tetraspanin CD37 deficiency has been reported as a prognostic marker for aggressive B-cell lymphoma. Here, the authors show that CD37 interacts with the fatty acid transporter 1 to inhibit palmitate uptake and its deficiency leads to increased fatty acid metabolism which promotes tumorigenesis in B-cell lymphoma.

The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus, yet little is known regarding the overall proteomic composition of the PT. Here, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa following the implementation of a long-established subcellular fractionation protocol designed to increase the detection of low abundance proteins. A total of 1,802 proteins were identified, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire annotated proteome of the Sus scrofa species so far. In the PT structure itself, we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data revealed that the PT is replete in proteins critical for sperm-egg fusion and egg activation, including: Izumo family members 1–4 (IZUMO1-4) and phosphoinositide specific phospholipase ζ (PLCZ1). Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. This is particularly intriguing as it is currently held that the ER structure is lost during testicular sperm maturation. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. It remains to be explored whether proteasome subunits within the PT actively assist in the protein degradation of paternal cell structures post-fertilization and how aberrations in PT protein content may delay embryonic development.

During the development of drug resistance, multiple myeloma (MM) cells undergo changes to their metabolism. However, how these metabolic changes can be exploited to improve treatment efficacy is not known. Here we demonstrate that targeting coenzyme Q10 (CoQ) biosynthesis through the mevalonate pathway works in synergy with the proteasome inhibitor bortezomib (BTZ) in MM. We show that gene expression signatures relating to the mitochondrial tricarboxylic acid (TCA) cycle and electron transport chain (ETC) predispose to clinical BTZ resistance and poor prognosis in MM patients. Mechanistically, BTZ-resistant cells show increased activity of glutamine-driven TCA cycle and oxidative phosphorylation, together with an increased vulnerability towards ETC inhibition. Moreover, BTZ resistance is accompanied by high levels of the mitochondrial electron carrier CoQ, while the mevalonate pathway inhibitor simvastatin increases cell death and decreases CoQ levels, specifically in BTZ-resistant cells. Both in vitro and in vivo, simvastatin enhances the effect of bortezomib treatment. Our study links CoQ synthesis to drug resistance in MM and provides a novel avenue for improving BTZ responses through statin-induced inhibition of mitochondrial metabolism.

Metabolic and toxic liver disorders, such as fatty liver disease (steatosis) and drug-induced liver injury, are highly prevalent and potentially life-threatening. To allow for the study of these disorders from the early stages onward, without using experimental animals, we collected porcine livers in a slaughterhouse and perfused these livers normothermically. With our simplified protocol, the perfused slaughterhouse livers remained viable and functional over five hours of perfusion, as shown by hemodynamics, bile production, indocyanine green clearance, ammonia metabolism, gene expression and histology. As a proof-of-concept to study liver disorders, we show that an infusion of free fatty acids and acetaminophen results in early biochemical signs of liver damage, including reduced functionality. In conclusion, the present platform offers an accessible system to perform research in a functional, relevant large animal model while avoiding using experimental animals. With further improvements to the model, prolonged exposure could make this model a versatile tool for studying liver diseases and potential treatments.

The idea that amyloid fibrils and other types of protein aggregates are toxic for cells has been challenged by the discovery of a variety of functional aggregates. However, an identification of crucial differences between pathological and functional aggregation remains to be explored. Functional protein aggregation is often reversible by nature in order to respond properly to changing physiological conditions of the cell. In addition, increasing evidence indicates that fast fibril growth is a feature of functional amyloids, providing protection against the long-term existence of potentially toxic oligomeric intermediates. It is becoming clear that functional protein aggregation is a complexly organized process that can be mediated by a multitude of biomolecular factors. In this overview, we discuss the roles of diverse biomolecules, such as lipids/membranes, glycosaminoglycans, nucleic acids and metal ions, in regulating functional protein aggregation. Our studies on the protein GAPR-1 revealed that several of these factors influence the amyloidogenic properties of this protein. These observations suggest that GAPR-1, as well as the cysteine-rich secretory proteins, antigen 5 and pathogenesis-related proteins group 1 (CAP) superfamily of proteins that it belongs to, require the assembly into an amyloid state to exert several of their functions. A better understanding of functional aggregate formation may also help in the prevention and treatment of amyloid-related diseases.

Highlights • Sub-lethal PMAP-36 treatment of bacteria increases outer membrane vesicle release. • Lipidomic analysis revealed the OMV lipidome upon PMAP-36 or heat treatment. • Supplementation with PMAP-36 attenuated undesirable OMV-induced immune responses., Host defense peptides (HDPs), such as cathelicidins, are small, cationic, amphipathic peptides and represent an important part of the innate immune system. Most cathelicidins, including the porcine PMAP-36, are membrane active and disrupt the bacterial membrane. For example, a chicken cathelicidin, CATH-2, has been previously shown to disrupt both Escherichia coli membranes and to release, at sub-lethal concentrations, outer membrane vesicles (OMVs). Since OMVs are considered promising vaccine candidates, we sought to investigate the effect of sub-bactericidal concentrations of PMAP-36 on both OMV release by a porcine strain of Bordetella bronchiseptica and on the modulation of immune responses to OMVs. PMAP-36 treatment of bacteria resulted in a slight increase in OMV release. The characteristics of PMAP-36-induced OMVs were compared with those of spontaneously released OMVs and OMVs induced by heat treatment. The stability of both PMAP-36- and heat-induced OMVs was decreased compared to spontaneous OMVs, as shown by dynamic light scattering. Furthermore, treatment of bacteria with PMAP-36 or heat resulted in an increase in negatively charged phospholipids in the resulting OMVs. A large increase in lysophospholipid content was observed in heat-induced OMVs, which was at least partially due to the activity of the outer-membrane phospholipase A (OMPLA). Although PMAP-36 was detected in OMVs isolated from PMAP-36-treated bacteria, the immune response of porcine bone-marrow-derived macrophages to these OMVs was similar as those against spontaneous or heat-induced OMVs. Therefore, the effect of PMAP-36 addition after OMV isolation was investigated. This did decrease cytokine expression of OMV-stimulated macrophages. These results indicate that PMAP-36 is a promising molecule to attenuate undesirable immune responses, for instance in vaccines., Graphical abstract Image, graphical abstract

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

Significance Spatial organization of mitochondria is vital for cellular function. In many specialized cell types, mitochondria are immobilized at specific subcellular loci through interactions with the cytoskeleton. One of the most striking mitochondrial configurations occurs in mammalian sperm, where mitochondria wrap around the flagellum. Malformation of the mitochondrial sheath causes infertility, but the molecular structures underlying this intricate arrangement are unknown. Here, we analyzed the mitochondrial sheath in sperm from three mammalian species. We find that although mitochondrial dimensions and cristae architecture vary across species, molecular assemblies mediating intermitochondria and mitochondria–cytoskeleton interactions are conserved. These findings yield important insight into sperm physiology and evolution and are relevant for other polarized cell types, such as muscles, neurons, photoreceptors, and hair cells., Mitochondria–cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria–cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

Molenaar et al. show that seipin does not play a role in the biogenesis of lipid droplets composed mostly or only of the neutral lipid retinyl ester, even in cells that produce triacylglycerols., Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid-liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn2+ metal ions enhances inclusion formation. Furthermore, Zn2+ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.

Multiple myeloma (MM) is a hematological malignancy that exhibits aberrantly high levels of proteasome activity. While treatment with the proteasome inhibitor bortezomib substantially increases overall survival of MM patients, acquired drug resistance remains the main challenge for MM treatment. Using a combination treatment of docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) and bortezomib, it was demonstrated previously that pretreatment with DHA/EPA significantly increased bortezomib chemosensitivity in MM cells. In the current study, both transcriptome and metabolome analysis were performed to comprehensively evaluate the underlying mechanism. It was demonstrated that pretreating MM cells with DHA/EPA before bortezomib potently decreased the cellular glutathione (GSH) level and altered the expression of the related metabolites and key enzymes in GSH metabolism, whereas simultaneous treatment only showed minor effects on these factors, thereby suggesting the critical role of GSH degradation in overcoming bortezomib resistance in MM cells. Moreover, RNA-seq results revealed that the nuclear factor erythroid 2-related factor 2 (NRF2)-activating transcription factor 3/4 (ATF3/4)-ChaC glutathione specific gamma-glutamylcyclotransferase 1 (CHAC1) signaling pathway may be implicated as the central player in the GSH degradation. Pathways of necroptosis, ferroptosis, p53, NRF2, ATF4, WNT, MAPK, NF-κB, EGFR, and ERK may be connected to the tumor suppressive effect caused by pretreatment of DHA/EPA prior to bortezomib. Collectively, this work implicates GSH degradation as a potential therapeutic target in MM and provides novel mechanistic insights into its significant role in combating bortezomib resistance.

Host defense peptides (HDPs) are part of the innate immune system and constitute a first line of defense against invading pathogens. They possess antimicrobial activity against a broad spectrum of pathogens. However, pathogens have been known to adapt to hostile environments. Therefore, the bacterial response to treatment with HDPs was investigated. Previous observations suggested that sublethal concentrations of HDPs increase the release of outer membrane vesicles (OMVs) in Escherichia coli. First, the effects of sublethal treatment with HDPs CATH-2, PMAP-36, and LL-37 on OMV release of several Gram-negative bacteria were analyzed. Treatment with PMAP-36 and CATH-2 induced release of OMVs, but treatment with LL-37 did not. The OMVs were further characterized with respect to morphological properties. The HDP-induced OMVs often had disc-like shapes. The beneficial effect of bacterial OMV release was studied by determining the susceptibility of E. coli toward HDPs in the presence of OMVs. The minimal bactericidal concentration was increased in the presence of OMVs. It is concluded that OMV release is a means of bacteria to dispose of HDP-affected membrane. Furthermore, OMVs act as a decoy for HDPs and thereby protect the bacterium. IMPORTANCE Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2050. Antimicrobial peptides, also known as host defense peptides (HDPs), and HDP-derived antimicrobials have potent antimicrobial activity and high potential as alternatives to antibiotics due to low resistance development. Some resistance mechanisms have developed in bacteria, and complete understanding of bacterial defense against HDPs will aid their use in the clinic. This study provides insight into outer membrane vesicles (OMVs) as potential defense mechanisms against HDPs, which will allow anticipation of unforeseen resistance to HDPs in clinical use and possibly prevention of bacterial resistance by the means of OMVs.

Defects in Coenzyme Q (CoQ) metabolism have been associated with primary mitochondrial disorders, neurodegenerative diseases and metabolic conditions. The consequences of CoQ deficiency have not been fully addressed, and effective treatment remains challenging. Here, we use mice with primary CoQ deficiency (Coq9R239X), and we demonstrate that CoQ deficiency profoundly alters the Q-junction, leading to extensive changes in the mitochondrial proteome and metabolism in the kidneys and, to a lesser extent, in the brain. CoQ deficiency also induces reactive gliosis, which mediates a neuroinflammatory response, both of which lead to an encephalopathic phenotype. Importantly, treatment with either vanillic acid (VA) or β-resorcylic acid (β-RA), two analogs of the natural precursor for CoQ biosynthesis, partially restores CoQ metabolism, particularly in the kidneys, and induces profound normalization of the mitochondrial proteome and metabolism, ultimately leading to reductions in gliosis, neuroinflammation and spongiosis and, consequently, reversing the phenotype. Together, these results provide key mechanistic insights into defects in CoQ metabolism and identify potential disease biomarkers. Furthermore, our findings clearly indicate that the use of analogs of the CoQ biosynthetic precursor is a promising alternative therapy for primary CoQ deficiency and has potential for use in the treatment of more common neurodegenerative and metabolic diseases that are associated with secondary CoQ deficiency., MCIN/AEI, Spain, European Commission RTI2018093503-B-100, Muscular Dystrophy Association MDA-602322, Junta de Andalucia P20_00134 PEER-00832020, EPIC-XS - Horizon 2020 programme of the European Union 823839, "Plan Propio de Investigacion" from the University of Granada Junta de Andalucia

Lysophospholipids (LPLs) are crucial for regulating epithelial integrity and homeostasis in eukaryotes, however the effects of LPLs produced by bacteria on host cells is largely unknown. The membrane of the human bacterial pathogen Campylobacter jejuni is rich in LPLs. Although C. jejuni possesses several virulence factors, it lacks traditional virulence factors like type III secretion systems, present in most enteropathogens. Here, we provide evidence that membrane lipids lysophosphatidylethanolamines (lysoPEs) of C. jejuni are able to lyse erythrocytes and are toxic for HeLa and Caco-2 cells. Lactate dehydrogenase (LDH) release assays and confocal microscopy revealed that lysoPE permeabilizes the cells. LysoPE toxicity was partially rescued by oxidative stress inhibitors, indicating that intracellular reactive oxygen species may contribute to the cell damage. Our results show that especially the short-chain lysoPEs (C:14) which is abundantly present in the C. jejuni membrane may be considered as a novel virulence factor.

The risk of coastal storm flooding is deteriorating under global warming, especially for the heavily urbanized deltaic cities, like Shanghai. The Nature-Based Flood Defense (NBFD), as an eco-friendly design alternative for hard infrastructure against coastal flooding, is gaining attention. Nevertheless, the vulnerability of saltmarsh due to the biological instability, resulting in the uncertainties on coastal protection, is considered the bottleneck challenge that hinders the broad application of the NBFD concept. We argue that except for direct wave attenuations by the above-ground vegetation during storms, the gradual sediment trapping and consolidating during the non-storm period is a more crucial function of coastal saltmarsh, which mitigates storm waves by forming a broader and higher intertidal morphology. This benefit is an important value of saltmarsh-based coastal protection but is largely neglected in many NBFD studies. Taking Chongming Dongtan Shoal (CDS) as a case study, we demonstrated that over 2/3th wave attenuation during storms is contributed by the saltmarsh morphology, and less than 1/3th is from the saltmarsh vegetation. The relative contribution of the saltmarsh morphology on wave mitigation is even enhanced under the increasing storm grades from 100 yrs. to 5000 yrs. return levels. To promote this idea for broader application, the cost-benefit analysis of three artificial NBFD solutions (e.g., submerged breakwater, timber piles, and sand nourishment) are compared. We identified an optimal measure of the submerged breakwater for CDS, which minimizes the ecological impact and maximizes the cost-benefit. Moreover, the wave-free zone behind the breakwater increases the chance of vegetation establishment, helps suspended sediment trapping, hence fostering a beneficent cycle for saltmarsh restoration. In summary, ignoring the contribution of saltmarsh morphology on wave attenuation largely underestimated the benefits of vegetation-based coastal protection, which should be greatly emphasized to provide a solid basis for developing NBFD.

Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and in their energy metabolism strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid β-oxidation is essential for egg production by adult femaleSchistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism ofS. mansoni. Incubations with [14C]-labelled fatty acids demonstrated that adults, eggs and miracidia ofS. mansonidid not oxidize fatty acids, as no14CO2production could be detected. We then re-examined theS. mansonigenome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding β-oxidation enzymes, which demonstrates thatS. mansonicannot use this pathway in any of its lifecycle stages. The same was true forS. japonicum.Absence of β-oxidation, however, does not imply that fatty acids from the host are not metabolized by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate them in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid β-oxidation does not occur in schistosomes. Our analyses ofS. mansonifurther revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicates that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium.

SummaryTo become fertilization-competent, mammalian sperm must undergo a complex series of biochemical and morphological changes in the female reproductive tract. These changes, collectively called capacitation, culminate in the exocytosis of the acrosome, a large vesicle overlying the nucleus. Acrosomal exocytosis is not an all-or-nothing event, but rather a regulated process in which vesicle cargo disperses gradually. However, the structural mechanisms underlying this controlled release remain undefined. In addition, unlike other exocytotic events, fusing membranes are shed as vesicles; the cell thus loses the entire anterior two-thirds of its plasma membrane and yet remains intact while the remaining non-vesiculated plasma membrane becomes fusogenic. Precisely how cell integrity is maintained through-out this drastic vesiculation process is unclear, as is how it ultimately leads to the acquisition of fusion competence. Here, we use cryo-electron tomography to visualize these processes in unfixed, unstained, fully-hydrated sperm. We show that crystalline structures within the acrosome disassemble during capacitation and acrosomal exocytosis, representing a plausible mechanism for gradual dispersal of the acrosomal matrix. We find that the architecture of the sperm head supports an atypical membrane fission-fusion pathway that maintains cell integrity. Finally, we detail how the acrosome reaction transforms both the micron-scale topography and the nano-scale protein landscape of the sperm surface, thus priming the sperm for fertilization.SignificanceMammalian sperm must undergo a complex series of biochemical and morphological changes in the female reproductive tract in order to become fertilization-competent. These changes culminate in acrosomal exocytosis, during which multiple membrane fusions destabilize the acrosomal vesicle and liberate its contents, which include proteins implicated in penetrating and binding to the egg vestments. Here, we use cryo-electron tomography to visualize acrosomal exocytosis intermediates in unfixed, unstained sperm. Our results suggest structural bases for how gradual dispersal of acrosome contents is regulated, as well as for how the cell remains intact after losing much of its plasma membrane, We also show that acrosomal exocytosis transforms both the micron-scale topography and the nano-scale molecular landscape of the sperm surface, thus priming it for interaction and fusion with the egg. These findings yield important insights into sperm physiology and contribute to our understanding of the fundamental yet enigmatic process of mammalian fertilization.

Mammalian sperm carry a variety of highly condensed insoluble protein structures such as the perinuclear theca, the fibrous sheath and the outer dense fibers, which are essential to sperm function. We studied the role of cysteine rich secretory protein 2 (CRISP2); a known inducer of non-pathological protein amyloids, in pig sperm with a variety of techniques. CRISP2, which is synthesized during spermatogenesis, was localized by confocal immunofluorescent imaging in the tail and in the post-acrosomal region of the sperm head. High-resolution localization by immunogold labeling electron microscopy of ultrathin cryosections revealed that CRISP2 was present in the perinuclear theca and neck region of the sperm head, as well as in the outer dense fibers and the fibrous sheath of the sperm tail. Interestingly, we found that under native, non-reducing conditions CRISP2 formed oligomers both in the tail and the head but with different molecular weights and different biochemical properties. The tail oligomers were insensitive to reducing conditions but nearly complete dissociated into monomers under 8 M urea treatment, while the head 250 kDa CRISP2 positive oligomer completely dissociated into CRISP2 monomers under reducing conditions. The head specific dissociation of CRISP2 oligomer is likely a result of the reduction of various sulfhydryl groups in the cysteine rich domain of this protein. The sperm head CRISP2 shared typical solubilization characteristics with other perinuclear theca proteins as was shown with sequential detergent and salt treatments. Thus, CRISP2 is likely to participate in the formation of functional protein complexes in both the sperm tail and sperm head, but with differing oligomeric organization and biochemical properties. Future studies will be devoted to the understand the role of CRISP2 in sperm protein complexes formation and how this contributes to the fertilization processes. Summary sentence Biochemical properties of CRISP2 oligomers in boar spermatozoa.

The membrane phospholipid composition is not a stable bacterial characteristic but can change in response to altered environmental conditions. Here we provide the dataset of the phospholipidome and transcriptome of the microaerophilic human pathogen Campylobacter jejuni under different environmental conditions. These data have been used in Cao (2020), The unique phospholipidome of the enteric pathogen C. jejuni: Lysolipids are required for motility at low oxygen availability. Here the abundance of each phospholipid is shown during the growth of C. jejuni for 0-108 h under low and high oxygen conditions (0.3 vs 10% O2). The phospholipid data were obtained by applying high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS). The transcriptomic data obtained by RNA-seq show the differential expressed genes between logarithmic and stationary grown bacteria. In addition, our data might serve as a reference information for further in-depth investigation to understand the relation between specific phospholipids and the activity of membrane associated proteins.

Following activation, conventional T (T-conv) cells undergo an mTOR-driven glycolytic switch. Regulatory T (T-reg) cells reportedly repress the mTOR pathway and avoid glycolysis. However, here we demonstrate that human thymus-derived T-reg (tT(reg)) cells can become glycolytic in response to tumour necrosis factor receptor 2 (TNFR2) costimulation. This costimulus increases proliferation and induces a glycolytic switch in CD3-activated tT(reg) cells, but not in T-conv cells. Glycolysis in CD3-TNFR2-activated tT(reg) cells is driven by PI3-kinase-mTOR signalling and supports tT(reg) cell identity and suppressive function. In contrast to glycolytic T-conv cells, glycolytic tT(reg) cells do not show net lactate secretion and shuttle glucose-derived carbon into the tricarboxylic acid cycle. Ex vivo characterization of blood-derived TNFR2(hi)CD4(+)CD25(hi)CD127(lo) effector T cells, which were FOXP3(+)IKZF2(+), revealed an increase in glucose consumption and intracellular lactate levels, thus identifying them as glycolytic tT(reg) cells. Our study links TNFR2 costimulation in human tT(reg) cells to metabolic remodelling, providing an additional avenue for drug targeting.

In response to changes in their environment bacteria need to change both their protein and phospholipid repertoire to match environmental requirements, but the dynamics of bacterial phospholipid composition under different growth conditions is still largely unknown. In the present study, we investigated the phospholipidome of the bacterial pathogen Campylobacter jejuni. Transcription profiling on logarithmic and stationary phase grown cells of the microaerophilic human pathogen C. jejuni using RNA-seq revealed differential expression of putative phospholipid biosynthesis genes. By applying high-performance liquid chromatography tandem–mass spectrometry, we identified 203 phospholipid species representing the first determination of the phospholipidome of this pathogen. We identified nine different phospholipid classes carrying between one and three acyl chains. Phospholipidome analysis on bacteria of different ages (0–5 days) showed rapid changes in the ratio of phospholipids containing ethanolamine, or glycerol as phospholipid head group and in the number of cyclopropane bond containing fatty acids. Oxygen concentration influenced the percentage of lysophospholipids, and cyclo-propane bonds containing acyl chains. We show that large amounts of the phospholipids are lysophospholipids (30–45%), which mutant studies reveal are needed for normal C. jejuni motility at low oxygen conditions. C. jejuni possesses an unusual phospholipidome that is highly dynamic in response to environmental changes.

Vitamin A (retinol) is important for normal growth, vision and reproduction. It has a role in the immune response and the development of metabolic syndrome. Most of the retinol present in the body is stored as retinyl esters within lipid droplets in hepatic stellate cells (HSCs). In case of liver damage, HSCs release large amounts of stored retinol, which is partially converted to retinoic acid (RA). This surge of RA can mediate the immune response and enhance the regeneration of the liver. If the damage persists activated HSCs change into myofibroblast-like cells producing extracellular matrix, which increases the chance of tumorigenesis to occur. RA has been shown to decrease proliferation and metastasis of hepatocellular carcinoma. The levels of RA and RA signaling are influenced by the possibility to esterify retinol towards retinyl esters. This suggests a complex regulation between different retinoids, with an important regulatory role for HSCs.

International audience; Currently available test methods are not well-suited for the identification of chemicals that disturb hormonal processes involved in female reproductive development and function. This renders women's reproductive health at increasing risk globally, which, coupled with increasing incidence rates of reproductive disorders, is of great concern. A woman's reproductive health is largely established during embryonic and fetal development and subsequently matures during puberty. The endocrine system influences development, maturation, and function of the female reproductive system, thereby making appropriate hormone levels imperative for correct functioning of reproductive processes. It is concerning that the effects of human-made chemicals on the endocrine system and female reproductive health are poorly addressed in regulatory chemical safety assessment, partly because adequate test methods are lacking. Our EU-funded project FREIA aims to address this need by increasing understanding of how endocrine disrupting chemicals (EDCs) can impact female reproductive health. We will use this information to provide better test methods that enable fit-for-purpose chemical regulation and then share our knowledge, promote a sustainable society, and improve the reproductive health of women globally.

The dependency of cancer cells on glutamine may be exploited therapeutically as a new strategy for treating cancers that lack druggable driver genes. Here we found that human liver cancer was dependent on extracellular glutamine. However, targeting glutamine addiction using the glutaminase inhibitor CB-839 as monotherapy had a very limited anticancer effect, even against the most glutamine addicted human liver cancer cells. Using a chemical library, we identified V-9302, a novel inhibitor of glutamine transporter ASCT2, as sensitizing glutamine dependent (GD) cells to CB-839 treatment. Mechanically, a combination of CB-839 and V-9302 depleted glutathione and induced reactive oxygen species (ROS), resulting in apoptosis of GD cells. Moreover, this combination also showed tumor inhibition in HCC xenograft mouse models in vivo. Our findings indicate that dual inhibition of glutamine metabolism by targeting both glutaminase and glutamine transporter ASCT2 represents a potential novel treatment strategy for glutamine addicted liver cancers.

Members of the CAP superfamily (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-Related 1 proteins) are characterized by the presence of a structurally conserved CAP domain. The common structure-function relationship of this domain is still poorly understood. In this study, we unravel specific molecular mechanisms modulating the quaternary structure of the mammalian CAP protein GAPR-1 (Golgi-Associated plant Pathogenesis-Related protein 1). Copper ions are shown to induce a distinct amyloid-like aggregation pathway of GAPR-1 in the presence of heparin. This involves an immediate shift from native multimers to monomers which are prone to form amyloid-like fibrils. The Cu2+-induced aggregation pathway is independent of a conserved metal-binding site and involves the formation of disulfide bonds during the nucleation process. The elongation process occurs independently of the presence of Cu2+ ions, and amyloid-like aggregation can proceed under oxidative conditions. In contrast, the Zn2+-dependent aggregation pathway was found to be independent of cysteines and was reversible upon removal of Zn2+ ions. Together, our results provide insight into the regulation of the quaternary structure of GAPR-1 by metal ions and redox homeostasis with potential implications for regulatory mechanisms of other CAP proteins.

Diagnosis of cystic echinococcosis (CE) is at present mainly based on imaging techniques. Serology has a complementary role, partly due to the small number of standardized and commercially available assays. Therefore we examined the clinical performance of the SERION ELISA classic Echinococcus IgG test. Using 10 U/ml as a cut-off point, and serum samples from 50 CE patients and 105 healthy controls, the sensitivity and specificity were 98.0% and 96.2%, respectively. If patients with other infectious diseases were used as negative controls, the specificity decreased to 76.9%, which causes poor positive predictive values. However, if results between 10 and 15 U/ml are classified as indecisive, the specificity of positive results (≥15 U/ml) increased to 92.5% without greatly affecting the sensitivity (92.0%). Using this approach in combination with imaging studies, the SERION ELISA classic Echinococcosis IgG test can be a useful aid in the diagnosis of CE.

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) cleaves von Willebrand Factor (VWF) multimers to control their thrombogenicity. The fibrinolytic enzyme plasmin can cleave VWF in a similar manner. However, plasmin can also cleave ADAMTS13, which ultimately inactivates it. This leaves the overall role of plasmin in primary haemostasis uncertain.We investigated the combined molecular effects of plasmin on VWF and ADAMTS13. We first identified that plasmin destroys FRETS-VWF73 substrate by cleaving the ADAMTS13 binding region in a buffered system. We next investigated how plasmin affects both VWF and ADAMTS13 under static conditions in plasma by western blotting. We found that globular VWF is largely protected from plasmin cleavage. However, ADAMTS13 is rapidly cleaved under these conditions, suggesting inactivation. Surprisingly, we observed that plasmin enhances ADAMTS13 activity in a modified two-stage FRETS-VWF73 assay that protects FRETS-VWF73 substrate from degradation. In direct binding studies under the same conditions, we found that plasmin generates multiple C-terminally truncated forms of ADAMTS13 with VWF-binding capacity. In an effort to seek evidence for this mechanism in vivo, we analysed plasma from patients with systemic amyloidosis, which is hallmarked by a hyperfibrinolytic state. We found that their plasma contained increased levels of C-terminally truncated forms of ADAMTS13, which correlated with their hyperfibrinolytic state.We propose that truncation of ADAMTS13 by plasmin abolishes intramolecular self-association, which improves interaction with unfolded VWF.

Bacteria have evolved different mechanisms to catabolize carbon sources from nutrient mixtures. They first consume their preferred carbon source, before others are used. Regulatory mechanisms adapt the metabolism accordingly to maximize growth and to outcompete other organisms. The human pathogen Campylobacter jejuni is an asaccharolytic Gram-negative bacterium that catabolizes amino acids and organic acids for growth. It prefers serine and aspartate as carbon sources, however it lacks all regulators known to be involved in regulating carbon source utilization in other organisms. In which manner C. jejuni adapts its metabolism towards the presence or absence of preferred carbon sources is unknown. In this study, we show with transcriptomic analysis and enzyme assays how C. jejuni adapts its metabolism in response to its preferred carbon sources. In the presence of serine as well as lactate and pyruvate C. jejuni inhibits the utilization of other carbon sources, by repressing the expression of a number of central metabolic enzymes. The regulatory proteins RacR, Cj1000 and CsrA play a role in the regulation of these metabolic enzymes. This metabolism dependent transcriptional repression correlates with an accumulation of intracellular succinate. Hence, we propose a demand-based catabolite repression mechanism in C. jejuni, depended on intracellular succinate levels. This article is protected by copyright. All rights reserved.

Reverse cholesterol transport or cholesterol efflux is part of an extensive plasma membrane remodeling process in spermatozoa that is imperative for fertilization. For ram spermatozoa, sheep serum is well known to support in vitro fertilization (IVF), but knowledge of its explicit role is limited. Though, it is postulated to elicit cholesterol efflux owing to the presence of high-density lipoproteins (HDLs) that interact with transmembrane cholesterol transporters, such as adenosinetriphosphate (ATP)-binding cassette transporter A1 (ABCA1) and scavenger receptor class B, type I (SR-BI). In this study, we report that both sheep serum and HDLs were able to elicit cholesterol efflux alone by up to 20–40% (as measured by the boron dipyrromethene (BODIPY)-cholesterol assay). Furthermore, when the antagonists glibenclamide and valspodar were used to inhibit the function of ABCA1 and SR-BI or ABCA1 alone, respectively, cholesterol efflux was only marginally reduced (8–15%). Nevertheless, it is likely that in ram spermatozoa, a specific facilitated pathway of cholesterol efflux is involved in the interaction between cholesterol acceptors and transporters. Interestingly, exposure to HDLs also induced hyperactivated motility, another critical event required for successful fertilization. Taken together, this study details the first report of the dual action of HDLs on ram spermatozoa, providing both an insight into the intricacy of events leading up to fertilization in vivo as well as demonstrating the possible application of HDL supplementation in media for IVF., First report of the dual action of high-density lipoproteins on ram spermatozoa in mediating critical fertilization-dependent processes.

BACKGROUND: Improvements in mass spectrometry (MS) technologies coupled with bioinformatics developments have allowed considerable advancement in the measurement and interpretation of lipidomics data in recent years. Since research areas employing lipidomics are rapidly increasing, there is a great need for bioinformatic tools that capture and utilize the complexity of the data. Currently, the diversity and complexity within the lipidome is often concealed by summing over or averaging individual lipids up to (sub)class-based descriptors, losing valuable information about biological function and interactions with other distinct lipids molecules, proteins and/or metabolites. AIM OF REVIEW: To address this gap in knowledge, novel bioinformatics methods are needed to improve identification, quantification, integration and interpretation of lipidomics data. The purpose of this mini-review is to summarize exemplary methods to explore the complexity of the lipidome. KEY SCIENTIFIC CONCEPTS OF REVIEW: Here we describe six approaches that capture three core focus areas for lipidomics: (1) lipidome annotation including a resolvable database identifier, (2) interpretation via pathway- and enrichment-based methods, and (3) understanding complex interactions to emphasize specific steps in the analytical process and highlight challenges in analyses associated with the complexity of lipidome data.

Malassezia spp. are lipid-dependent yeasts that have been related to skin mycobiota and dermatological and systemic diseases. Study of lipid droplets (LDs) is relevant to elucidate the unknown role of these organelles in Malassezia and to gain a broader overview of lipid metabolism in Malassezia. Here, we standardized two protocols for the analysis of LDs in M. pachydermatis and M. globosa. The first describes co-staining for confocal laser-scanning fluorescence microscopy, and the second details extraction and purification of LDs. The double stain is achieved with three different neutral lipid fluorophores, namely Nile Red, BODIPY™ 493/503, and HCS LipidTOX™ Deep Red Neutral, in combination with Calcofluor White. For LD extraction, cell wall rupture is conducted using Trichoderma harzianum enzymes and cycles of vortexing with zirconium beads. LD purification is performed in a three-step ultracentrifugation process. These standardizations will contribute to the study of the dynamics, morphology, and composition of LDs in Malassezia. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lipid droplet fluorescence staining Basic Protocol 2: Lipid droplet extraction and purification Support Protocol: Malassezia spp. culture conditions.

Lipids play Jekyll and Hyde in the liver. On the one hand, the lipid-laden status of hepatic stellate cells is a hallmark of healthy liver. On the other hand, the opposite is true for lipid-laden hepatocytes—they obstruct liver function. Neglected lipid accumulation in hepatocytes can progress into hepatic fibrosis, a condition induced by the activation of stellate cells. In their resting state, these cells store substantial quantities of fat-soluble vitamin A (retinyl esters) in large lipid droplets. During activation, these lipid organelles are gradually degraded. Hence, treatment of fatty liver disease is treading a tightrope—unsophisticated targeting of hepatic lipid accumulation might trigger problematic side effects on stellate cells. Therefore, it is of great importance to gain more insight into the highly dynamic lipid metabolism of hepatocytes and stellate cells in both quiescent and activated states. In this review, part of the special issue entitled “Cellular and Molecular Mechanisms underlying the Pathogenesis of Hepatic Fibrosis 2020”, we discuss current and highly versatile aspects of neutral lipid metabolism in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).

Summary Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research., Highlights • Feline organoids can be cultured from fresh and frozen liver and from needle biopsy • A feline-specific liver culture medium allows organoid expansion up to 32 passages • When fed fatty acids, feline liver organoids store more lipids than human organoids • Feline liver organoids can be used to model hepatic steatosis, In this study Kruitwagen and colleagues establish and characterize a feline liver organoid culture, which has adult stem cell properties and can be differentiated toward hepatocyte-like cells. They propose liver organoids as a tool to model hepatic steatosis and show that feline liver organoids accumulate more lipids than human organoids when provided with excess fatty acids.

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC???MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT???/??? HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT???/??? HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ?? 9.0% small (??? 700 nm) lipid droplets in LRAT???/??? HSCs and 25.6 ?? 1.4% large (1400???2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (??? 700 nm) lipid droplets strongly increased both in wild type and in LRAT???/??? HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.

Significance Here, we report that the kidney has an effective sensing and signaling mechanism to balance microbial metabolite levels in the human body. Using healthy volunteers and in vivo and in vitro assays, we show that the kidney responds to elevated endogenous metabolite levels and stimulates organic anion secretion by AhR and EGFR signaling, controlled by miR-223 and reactive oxygen species. This remote sensing function aids body homeostasis and has important implications for the identification of novel therapeutic targets to preserve kidney function in patients., Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

Pyrroline-5-carboxylate reductase 1 (PYCR1) is the final enzyme involved in the biosynthesis of proline and has been found to be upregulated in various forms of cancer. Due to the role of proline in maintaining the redox balance of cells and preventing apoptosis, PYCR1 is emerging as an attractive oncology target. Previous PYCR1 knockout studies led to a reduction in tumor growth. Accordingly, a small molecule inhibitor of PYCR1 could lead to new treatments for cancer, and a focused screening effort identified pargyline as a fragment-like hit. We report the design and synthesis of the first tool compounds as PYCR1 inhibitors, derived from pargyline, which were assayed to assess their ability to attenuate the production of proline. Structural activity studies have revealed the key determinants of activity, with the most potent compound (4) showing improved activity in vitro in enzyme (IC50 = 8.8 µM) and pathway relevant effects in cell-based assays.