Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

Obesity is associated with chronic low-grade systemic inflammation of adipose tissue and is often linked to intestinal epithelial barrier (IEB) dysfunction. The present study aimed to evaluate the effects of in vitro gastrointestinal digests of bovine milk containing A1B or A2 {beta}-casein variants on leaky IEB and adipocyte inflammation. Digests of A1B (DA1B) and A2 (DA2) milk were administered to an in vitro Caco-2/HT-29 intestinal cell co-culture mimicking a leaky gut. Intestinal absorbed fractions derived from A1B (MA1B) and A2 (MA2) were administered to hMADS adipocytes. DA1B and DA2 did not modify intestinal permeability, either in the absence or the presence of inflammation. DA1B reduced Claudin-1 mRNA, as well as zonula occludens-1 mRNA and protein expression. Both DA1B and DA2 increased interleukin-8 expression, but only DA1B increased tumor necrosis factor-. In human adipocytes, MA1B, and to a lesser extent MA2, increased the expression of pro-inflammatory markers monocyte chemoattractant protein-1 and interleukin-6, while reducing adiponectin levels. DA2 preserved in vitro leaky IEB integrity and exhibited a lower inflammatory potential in both leaky gut and adipocytes compared to DA1B. This study is the first to establish a link among A2 milk, leaky gut syndrome, and obesity.

Autophagy is a fundamental intracellular degradation pathway with vital physiological functions. Although it is well known that autophagy is activated during starvation, the extent of basal autophagy remains unclear owing to challenges in measuring autophagic flux in vivo. In this study, we developed autophagy reporter (GFP-LC3-RFP) mice and quantified basal autophagic flux across tissues by comparing normal and autophagy-deficient conditions. Comparative analyses revealed uniformly low basal autophagic flux during embryogenesis, but significant tissue-specific variation in adult mice. In contrast to previous assumptions that basal autophagy in the brain is low, the brain, along with the liver and kidney, exhibited higher basal autophagic flux than the heart, skeletal muscle, and intestine. These data serve as foundational information on basal autophagic flux in mammals and provide a plausible explanation for the severe neurological phenotypes linked to autophagy gene mutations in mice and humans.

Administration of ciliary neurotrophic factor (CNTF) reduces food intake and body weight in both humans and experimental animals, where it also ameliorates hyperglycemia, hyperinsulinemia, and dyslipidemia. To exert its anti-obesogenic and anti-diabetogenic effects, CNTF targets brain feeding centers as well as multiple peripheral organs inducing the phosphorylation of the transcription factor signal transducer and activator of transcription 3 (p-STAT3). However, data showing which peripheral cytotypes are specifically targeted by exogenous CNTF in vivo in metabolically relevant organs are currently lacking. Here, we first evaluated the gene expression levels of the subunits of the tripartite CNTF receptor (Cntfr) complex, i.e., the Cntfr, the leukemia inhibitory factor receptor {beta} (Lifr{beta}) and the glycoprotein 130 (gp130), by quantitative real-time PCR in metabolically relevant organs of adult male mice: gastrointestinal (GI) tract, pancreas, liver, visceral and subcutaneous white (WAT) and interscapular brown adipose tissue (iBAT), skeletal muscle and the sciatic nerve. We then quantified p-STAT3 by Western blotting in these organs after intraperitoneal administration of CNTF (0.3 mg/kg) or saline. Finally, we mapped CNTF-responsive cells by immunohistochemistry, followed by morphometric quantification and confocal microscopy in both CNTF- and saline-treated mice. Lifr{beta} and gp130 were ubiquitously detected across all the investigated organs; the Cntfr showed the highest expression levels in the skeletal muscle, sciatic nerve, and iBAT, whereas it was found to be expressed to a lesser extent in the other sites. Administration of CNTF led to a significant increase of p-STAT3/STAT3 protein ratio in all organs examined, except the duodenum, and induced a distinctive pattern of cell nuclear p-STAT3 immunoreactivity. Notably, along the analyzed GI tract CNTF induced nuclear STAT3 phosphorylation in neurons of the submucosal and myenteric plexuses of the enteric nervous system and in contractile cells of the muscularis externa, where the response peaked in the mesenteric gut and colon. In the pancreas, CNTF triggered a higher activation within the endocrine component compared to the exocrine parenchyma. In the liver, CNTF induced STAT3 phosphorylation not only in parenchymal cells but also in sinusoids and resident macrophages. The cytokine activated p-STAT3 in subcutaneous and visceral white adipocytes, but also in brown adipocytes, with a prominent response observed in the beige subcutaneous adipocytes; adipose resident macrophages and endothelial cells of numerous blood vessels were also CNTF-responsive. Lastly, in skeletal muscle, a major site for glucose/lipid utilization, CNTF induced widespread nuclear p-STAT3 immunoreactivity in muscle fibers and in connective and Schwann cells of the peripheral nerves, including the sciatic nerve, supplying the gastrocnemius. In conclusion, our data indicate that CNTF acts across diverse cytotypes within metabolically relevant organs and tissues, likely fostering its peripheral metabolic effects through this cellular heterogeneity.

Cholesterol elimination in mammals depends largely on the biliary secretion of cholesterol and its conversion to bile acids, followed by their fecal loss. Human studies suggest an association between blood vitamin D levels and blood cholesterol; however, the mechanistic impact of sustained elevation of 1,25(OH)2D3 (active vitamin D) on cholesterol flux remains unclear. Here, we used two complementary mouse models--a genetic model with chronically elevated plasma 1,25(OH)2D3 (-klotho KO mice) and a pharmacological model of repeated 1,25(OH)2D3 administration in wild-type mice--to define the mechanism by which 1.25(OH)2D3 regulates the hepatic-intestinal programs controlling cholesterol elimination. -klotho KO mice showed increased fecal excretion of both cholesterol and total bile acids. Hepatically, Sr-b1, Abcg5/Abcg8, Abca1, Cyp7a1, and Mrp2 transcriptions were increased, whereas Cyp27a1 and Bsep was unchanged. Duodenal Npc1l1 was reduced, and ileal Asbt showed a decreasing trend. In the administration model, fecal bile acid levels increased by day 3, consistent with the induction of hepatic Mrp2 expression from day 3. Bsep exhibited a biphasic change, enhanced at early phase and downregulated to basal levels later and Asbt was unchanged. Increased fecal cholesterol emerged later (day 15), accompanied by late-phase induction of Abcg5/Abcg8 and suppression of Npc1l1. Together, we propose that sustained elevation of 1.25(OH)2D3 is associated with coordinated hepatic and intestinal transcriptional remodeling that promotes cholesterol disposal, with an early increase in fecal bile acid loss preceding the enhanced fecal cholesterol excretion.

Liver sinusoidal endothelial cells (LSECs) separate the blood from the hepatic parenchyma and thus are at the frontline as scavengers of blood-borne waste, pathogens and metabolic stimuli. LSECs are also critical for sensing systemic iron availability by controlling the synthesis of bone morphogenetic protein (BMP) 6, which is essential for hepcidin expression in hepatocytes. Hepcidin maintains systemic iron homeostasis by inhibiting dietary iron uptake and iron release from iron recycling macrophages. Hepcidin is also an acute-phase protein and its activation by inflammation requires active BMP signaling. It is incompletely understood how signals derived from inflammation, cellular damage and iron are integrated by the liver to assure adequate hepcidin expression. Here, we show that Bmp6 expression is activated in primary LSEC cultures upon their exposure to danger-associated molecular patterns (DAMPs), such as heme and myoglobin, pathogen-associated molecular pattern (PAMPs), such as lipopolysaccharide (LPS) and Fibroblast-Stimulating Lipopeptide-1 (FSL1), or oxidative stress inducers (H2O2). Interestingly, all regulatory cues converge at the MAPK signaling pathway, although the specific signaling branches involved are stimulus-specific. Of note, Bmp6 upregulation in LSECs in response to all signals tested is strongly enhanced by the hepatocyte secretome. As hepatocytes critically depend on active BMP/SMAD signaling to control hepcidin activation, our results reveal that multiple sources of signaling input activating Bmp6 in LSECs and hepcidin in hepatocytes serve to determine BMP/SMAD signaling strength. Furthermore, our findings identify hypoferremia (low plasma iron levels), the result of high hepcidin levels due to elevated Bmp6, as a convergent response in conditions of inflammation, oxidative stress and cellular damage. HighlightsO_LIDAMPs (heme and myoglobin), PAMPs (LPS) and oxidative stress activate Bmp6 mRNA expression via the MAPK signaling pathway C_LIO_LIThe TLR/MAPK/BMP6 regulatory axis integrates inflammatory and iron signals C_LIO_LIOur work uncovers a novel connection between innate immune sensing, oxidative stress and hepatic iron homeostasis C_LI

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli, the GSH-specific {gamma}-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na/H antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB, are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA, chaB, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA, but not chaB or chaC, contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli.

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is characterized and initiated by the excessive accumulation of triacylglycerols (TG) and cholesteryl esters (CE) in the liver. Hepatic TG and CE synthesis, lipolysis and transport are tightly regulated by nutritional status, and disruption of this homeostasis contributes to MASLD pathogenesis. We have found that an endoplasmic reticulum-localized arylacetamide deacetylase (AADAC) catalyzes hepatic TG/CE turnover, and suppresses SREBP- and LXR-regulated lipogenesis and fatty acid esterification. Consequently, AADAC deficiency in mice leads to increased hepatic lipid synthesis, exacerbated steatosis, and impaired whole-body metabolism during Western-type diet feeding. These findings implicate AADAC as an important regulator of hepatic neutral lipid metabolism, linking endoplasmic reticulum cholesteryl ester hydrolysis as a modulator of lipid synthesis, and suggest its potential role in limiting MASLD pathogenesis under conditions of chronic overnutrition.

A proteomic analysis of Ixodes scapularis nymph saliva identified 252 proteins, including six tubular lipid-binding proteins (TULIPs). Comparing nymphs fed on mice that were uninfected or infected with Borrelia burgdorferi, twelve salivary proteins showed significant differences in the amounts detected, including XP_040079658.2, which we refer to as TULIP2. Considering the known immunity-related functions of some TULIPs, we expressed and purified TULIP2 from Escherichia coli and analyzed its interaction with B. burgdorferi lipids. The purification of TULIP2 from E. coli presented many obstacles, due to insolubility, which is consistent with previous reports from studies of other TULIP family members. The binding results showed specificity for B. burgdorferi lipids, with evidence for cholesteryl {beta}-galactoside as a major binding target. Molecular modeling of TULIP2 did not show any strong lipid binding sites. We used molecular dynamics simulation of TULIP2 to explore its conformational landscape by thermal unfolding. The earliest unfolding intermediate opened a hydrophobic pocket to which cholesteryl {beta}-galactoside was predicted to bind strongly. We propose that a specific lipid bilayer interaction with TULIP2 triggers the opening of the ligand-binding site.

Genetically identical cells grown in the same environment show variation in gene expression known as expression noise. Expression noise can be heritable and impact fitness, making it subject to natural selection. Increasing expression noise for the Saccharomyces cerevisiae TDH3 gene was shown to be beneficial in glucose-based media when mean TDH3 expression was far from the fitness optimum but deleterious when it was close to this optimum. Here, we show that growth on different carbon sources alters the effects of new mutations on TDH3 expression noise and examine the fitness effects of changing expression noise. In galactose-based media, we observed the same relationship between expression noise and fitness seen in glucose-based media, but in glycerol- and ethanol-based media, we observed the opposite relationship or no significant relationship, respectively. Using simulations of single-cell organisms, we found that these differences were most likely explained by environment-specific relationships between gene expression and fitness. We also found that, far from the optimum, the fitness effects of noise were greatest when expression was highly heritable between mother and daughter cells. The empirical observations and simulations reported in this study show how environments influence both the production of expression noise and its impacts on fitness.

Interleukin-6 (IL-6), produced by skeletal muscle and extramuscular tissues, regulates skeletal muscle function through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. However, the interaction between intrinsic (locally produced) IL-6 and extrinsic (circulating) IL-6 in skeletal muscle remains unclear. We investigated whether and how intrinsic expression of IL-6 in cultured primary human myoblasts influences their response to extrinsic stimulation with recombinant human IL-6 (rhIL-6). Using gene silencing, we found that suppression of intrinsic IL-6 enhanced rhIL-6-induced phosphorylation of STAT1 and STAT3. Silencing STAT3 also increased rhIL-6-induced STAT1 phosphorylation, but silencing STAT1 had no effect on STAT3 phosphorylation. Pretreatment of myoblasts with neutralising anti-IL-6 antibodies increased phosphorylation of STAT1 and STAT3 induced by 50 ng/mL rhIL-6, whereas pretreatment with 5 ng/mL rhIL-6 reduced this response. Despite increased JAK/STAT signalling, IL-6 silencing decreased glucose and oleic acid uptake and oxidation under both basal and rhIL-6-stimulated conditions. Collectively, our results imply that intrinsic IL-6 restrains activation of the JAK/STAT pathway by extrinsic IL-6, but acts synergistically with it to promote myoblast energy metabolism.

Mitochondrial homeostasis, governed by the balance between biogenesis and mitophagy, is essential for steroidogenesis in adrenocortical cells. While the requirement of active mitochondria for steroid synthesis is well-established, the hormonal regulation of genes governing mitochondrial function remains poorly understood. This study investigated whether angiotensin II (Ang II) and the cAMP/PKA pathway modulate the expression of key regulatory factors involved in mitochondrial biogenesis and redox status in the human adrenocortical H295R cell line. Using real-time qPCR and Western blot, we show that Ang II and 8Br-cAMP --a permeant analogue of cAMP-- modulate NRF-1, Nrf2, UCP2, and ANT1 impacting on mitochondrial biogenesis, antioxidant defense, and respiratory activity. These molecular changes correlated with increased mitochondrial membrane polarization, as confirmed by MitoTracker red staining. Interestingly, Ang II stimulation promoted a time-dependent increase in TFAM levels, a key transcription factor in mitochondria, which correlates with the increase in mitochondrial DNA (mtDNA) content. The rate of oxygen consumption (OCR) and mitochondrial parameters were determined, with results showing that Ang II led to a significant increase in basal and maximum respiration, ATP production, and proton leak. These findings suggest that hormone stimulation favors mitochondrial activity, thereby enhancing the bioenergetic capacity of adrenocortical cells. Furthermore, treatment with the uncoupler CCCP triggered a retrograde signaling response, upregulating nuclear-encoded mitochondrial genes to counteract mitochondrial membrane depolarization. Our findings demonstrate for the first time that hormonal signals directly modulate the mitochondrial genetic program in H295R human adrenocortical cells, optimizing the bioenergetic platform required for efficient steroidogenic function.

Circadian rhythms regulate diverse physiological processes, including metabolism, and their disruption has been implicated in metabolic disorders such as obesity. However, the tissue-specific effects of obesity on peripheral circadian clocks remain incompletely understood. Here, we investigated the impact of high-fat diet (HFD)-induced obesity on circadian gene expression in skeletal muscle, liver, and white adipose tissue (WAT). Mice were fed either a regular diet (RD) or HFD for 6 weeks, followed by tissue collection at 4-hour intervals over a 24-hour period. Under RD conditions, key circadian regulators and their downstream targets exhibited robust 24-hour oscillations across all tissues. In contrast, HFD feeding induced distinct, tissue-specific alterations. In the liver, Per2, Dbp, and Rev-erb showed phase-advanced expression patterns, whereas in WAT, rhythmic expression was markedly attenuated. Notably, skeletal muscle largely preserved circadian gene expression patterns, indicating relative resistance to HFD-induced circadian disruption. In addition, HFD feeding altered metabolic gene expression in adipose tissue, characterized by reduced Pgc1 expression and increased Leptin expression. Together, these findings demonstrate that HFD-induced obesity differentially disrupts peripheral circadian clocks in a tissue-specific manner and highlight skeletal muscle as a relatively resilient tissue. These results provide insight into how circadian dysregulation contributes to metabolic abnormalities in obesity.

Freshwater algae represent an underexplored source of naturally occurring bioactive metabolites with potential applications in pharmaceutical and biomedical research. This study investigated the phytochemical composition, antioxidant capacity, and preliminary cytotoxic potential of ethanolic and n-hexane extracts of freshwater algal species collected at Jilani Park, Lahore, Pakistan. Algal species were identified morphologically by Dr. Ghazal Yasmeen (Institute of Botany, Punjab University, Lahore). Extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) and qualitative phytochemical screening. Antioxidant activity was evaluated using DPPH radical scavenging, hydrogen peroxide scavenging, and reducing power assays. Cytotoxic potential was assessed using MTT and cell adhesion assays on HeLa and SF767 cell lines as preliminary indicators of bioactivity. GC-MS analysis identified 25 compounds, including sterols, fatty acid esters, terpenoids, phenolic compounds, and volatile metabolites. Phytochemical screening confirmed the presence of flavonoids, phenolics, tannins, and terpenoids in the extracts. Among the tested extracts, the n-hexane fraction demonstrated comparatively higher antioxidant activity across multiple assays. Ethanolic extracts showed moderate reductions in HeLa cell viability, whereas limited effects were observed in SF767 cells. These findings suggest that freshwater algae are promising natural reservoirs of antioxidant metabolites with potential relevance for future isolation and characterization of bioactive compounds for biomedical applications. Further purification and mechanistic studies are required to identify specific active constituents.

BACKGROUNGBisphenol A (BPA) has been linked to hypertension and disturbances in lipid metabolism; however, limited evidence is available regarding its association with hypertensive intracerebral hemorrhage (ICH). METHODSA multicenter, retrospective case-control study was conducted involving 129 participants, including individuals from an ICH group and healthy controls. Standard assays were employed to assess serum thyroid function, lipid profiles, serum fatty acid-binding [x]protein 4 (FABP4), oxidative stress markers, gap junction proteins, Wnt/{beta}-catenin signaling pathway activity, and expression changes of S100A8-mediated inflammatory cytokines involved in gut-brain interactions. Correlation analyses using Pearson and Spearman methods revealed that both BPA exposure and low T3 levels were significantly associated with elevated diastolic blood pressure, altered lipid metabolism, gut microbiota composition, and microglial activation. RESULTSGender-based disparities in lipid metabolism were identified. Changes in {beta}3-adrenergic receptor and neuromodulin-1 expression appear to influence fat regulation and attenuate oxidative stress responses. Subsequently, increased expression of gap junction proteins and activation of the Wnt/{beta}-catenin signaling pathway contribute to metabolic reprogramming and alterations in biochemical kinetics. Gut microbiota analysis demonstrated that, compared to controls, the ICH group exhibited significant dysbiosis and reduced alpha diversity. Further correlation analyses indicated that BPA levels were positively associated with FABP4 and oxidative stress markers, while S100A8 showed a strong dependence on microglial expression. CONCLUSIONThe interplay between lipid metabolism dysfunction and pro-inflammatory cytokines enhances vascular vulnerability. Collectively, BPA exposure, oxidative stress, and microglia-mediated neuroinflammation are significantly associated with an elevated risk of hypertensive ICH. China Clinical Trial Registry registration noticeFrom: China Clinical Trials Registry <chictr@vip.qq.com>+To:guopingwang60a<guopingwang60a@163.com> yunyanshuangfei <yunyanshuangfei@126.com> FUNDINGThis work was supported by the Natural Science Foundation of Shanxi Province (grant no. 201701D121177) Key informationGender-specific differences were observed in lipid metabolism and oxidative stress parameters; BPA exposure was shown to induce lipid metabolic disturbances, promote excessive production of oxidative stress byproducts, and consequently elevate oxidative stress responses; BPA was associated with stress-induced alterations in thyroid hormone function, further exacerbating dysregulation of lipid metabolism and oxidative stress; Fatty acid binding protein 4 (FABP4), a key adipokine implicated in metabolic disorders and adipose tissue inflammation, exhibited a significant positive correlation with serum BPA levels, whereas low levels of triiodothyronine (T3) were negatively correlated with FABP4. These findings suggest that serum FABP4 may serve as a biochemical marker for chronic low-grade adipose tissue inflammation and metabolic dysfunction; Gap junction proteins and the Wnt/{beta}-catenin signaling pathway may contribute to microglial activation and mediate neuroinflammatory responses, nerve injury, and secondary pathological processes in obesity-related cerebral hemorrhage.

The mitochondrial fatty acid {beta}-oxidation (mFAO) is an important source of energy when carbohydrate stores are depleted. It is also involved in many diseases, including inherited fatty-acid oxidation deficiencies (mFAODs). Patients with the same genetic variant often present with clinically heterogeneous phenotypes, but the mechanisms contributing to this heterogeneity are poorly understood. To investigate the underlying pathophysiology of different mFAODs, we constructed a computational model of mFAO in human liver, based on experimentally determined enzyme kinetics. A recognised, but seldom addressed challenge in metabolic modelling is the substantial uncertainty about kinetic parameter values. Whereas experimental values of some mFAO parameters are quite reproducible, others vary by up to four orders of magnitude between different reports. To address this, we generated an ensemble of kinetic models, each with the same reaction stoichiometry and rate equations, but different kinetic parameters, sampled from distributions of literature-derived values. We also comprehensively report these values and the arguments based on which they were evaluated. The resulting models were validated against available flux data, yielding a final ensemble of 51 valid models. These models recapitulate recent findings about the accumulation of medium-chain acyl-CoAs and the concomitant depletion of free CoA (CoASH) in medium-chain acyl-CoA dehydrogenase deficiency. We applied the ensemble to a set of known mFAODs, separating them into long-chain (LC-) and short-/medium-chain (S/MC-)mFAODs. The residual activity at which clinical symptoms are known to occur corresponded well with the residual activity in the model at which pathway flux was significantly decreased in LC-mFAODs. Residual activity in S/MC-mFAODs correlated less strongly with pathway flux, but these deficiencies did show a combined flux- and CoASH-reduction effect. This comparison is of importance to researchers and clinicians, as it identifies possible ways in which insights about one mFAOD may be applied to another based on shared biochemical properties. Author SummaryWhen building computer models of metabolic pathways, it is typical to take the "best" experimental data and use that as input into the model. However, especially when working with human cells, ethical and practical constraints often mean that even the "best" experimental data is still subject to substantial uncertainty. We explicitly modelled the uncertainty about the inner workings of fat burning (fatty acid oxidation). The resulting model is known as an "ensemble". The ensemble predicts ranges instead of single outcomes, allowing us to assess the confidence level of our predictions. We assess a set of inherited diseases - enzyme deficiencies - simulating them at different levels of severity with the ensemble. We find that the model does a good job of predicting the severity of the deficiencies at which symptoms will occur. It also allows us to identify a key difference between two subgroups within this group of deficiencies: long-chain and medium-/short-chain, depending on the size of the fats being metabolised. The long-chain variant is predicted to correlate most straightforwardly with the severity of the deficiencies, due to its effect on energy generation. Medium-/short-chain deficiencies, in contrast, have more complex consequences.

O_LIPlants and fungi are major sources of natural products beneficial to society, making the study of distinct species essential for discovering new drugs and bioactive compounds. The medicinal mushroom "Lingzhi" or "Reishi" (Ganoderma lingzhi) is widely used in traditional medicine and extensively studied for its bioactive triterpenoids, yet it is commonly identified as Ganoderma lucidum, the type species of the genus, which lacks a type specimen. C_LIO_LIWe sequenced a G. lucidum specimen preserved in the Kew fungarium, which agreed with the original description and was collected from wood of Corylus avellana in southern England. Using this reference specimen, we compiled genomic and ITS barcoding datasets to explore the genetic and geographic variation within this species. C_LIO_LIWe showed that G. lingzhi and G. lucidum diverged more than 12 million years ago and that all seven "G. lucidum" genomes deposited in public databases belong to other species. More than 1000 barcoding sequences showed that the widely used homology-based ITS barcoding is not working in this group, which can be mitigated by a phylogenetic placement approach. The 149 sequences assigned to G. lucidum with high confidence showed a Eurasian distribution and introductions to North and South America and Africa. C_LIO_LIOur study underscores the importance of accurate species identification and provides guidance for a group of pharmaceutical and socially significant species. To further support future studies and the wider public in differentiating between G. lingzhi and G. lucidum, we propose using "False Lingzhi" as the English name for G. lucidum. C_LI Societal Impact StatementTraditional Chinese Medicine has expanded far beyond Asia, with growing markets in North America and Europe for supplements and functional foods. Lingzhi or Reishi (Ganoderma lingzhi), a well-known medicinal mushroom, is valued for its anti-inflammatory and anticancer properties. However, it is often misidentified with species that may not provide the same health benefits. This confusion poses risks to consumer safety, product regulation, and research. Here, we establish a reference using morphological and molecular tools for the most commonly misidentified species (Ganoderma lucidum) and propose the name "False Lingzhi" to distinguish it, supporting accurate identification, safer product development, and reliable research.

BackgroundHepatic stellate cells (HSC) are Vitamin A storing non-parenchymal cells of the liver. During injury and inflammation, HSCs are the major contributors of excessive extracellular matrix (ECM) leading to Liver Fibrosis (LF). Emerging evidence suggests a fibrosis-independent role of these cells as key regulators of liver homeostasis and liver regeneration, emphasising on the dual role of HSCs in liver. HSCs are known to secrete several growth factors through which they largely execute their functions. However, the role of secretome (exosomes) from early activated or undifferentiated HSCs in a fibrotic milieu nor its composition are completely understood. MethodsLX-2 cells were cultured in low to no serum conditions and their isolated exosomes were transplanted into fibrotic severe combined immune deficient (SCID) mice livers, followed by post-transplantation analysis of the liver tissue and compared to the untreated controls. Total proteomic profiling of cell and exosomal cargo was performed using mass spectrometry and the data analysed and compared with the total HSC cell proteome. ResultsSignificant reduction in collagen in the transplanted mice livers compared to untreated fibrotic controls was observed with both the cells and exosomes transplantation. Comparative analysis revealed distinct enrichment of proteins and signaling pathways associated with extracellular matrix regulation, cellular communication, and metabolism in exosomes. Notably, these pathways are prominently represented in the exosomal fraction, suggesting a selective packaging of functional mediators. ConclusionThis study suggests the potential role of HSCs in regulating the complex liver homeostasis via exosomal network of proteins that contribute significantly to liver repair by ECM remodelling and growth factor-mediated signalling to regulate metabolism, fibrosis and liver regeneration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/721862v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@99bbf4org.highwire.dtl.DTLVardef@1029dd0org.highwire.dtl.DTLVardef@c6f578org.highwire.dtl.DTLVardef@1dba81_HPS_FORMAT_FIGEXP M_FIG C_FIG

Adipogenesis is the process of adipose-derived stem cells (ADSCs) responding to extracellular signals from the stem cell niche to differentiate into adipocytes (fat cells) and may be studied in vitro using a cocktail of chemicals that promote adipogenic differentiation to produce differentiated ADSCs (dADSCs). The global membrane N- and O-glycosylation changes of this process have been previously analysed and compared to native adipocytes as a benchmark for a true adipocyte profile, and revealed that bisecting GlcNAc type N-glycans are characteristic of adipogenesis. As stem cell differentiation has been widely reported to result in cellular protein changes, the same cells (ADSCs, dADSCs and mature adipocytes) were characterised for their membrane proteome here using label-free quantitative shotgun proteomics analysis. The membrane proteome displayed more differences in protein numbers between the cell types compared to the previously reported N-glycome which had shown high identical glycomes between stem cells and in vitro dADSCs, suggesting that the proteome is more dynamic during in vitro adipogenesis. Following the global shotgun proteomics analysis, a more targeted approach of carrying out proteomic analysis of de-N-glycosylated peptides of gel-separated proteins unearthed new glycoproteins not detected in the shotgun proteomic analysis. This approach identified the adipogenic marker, CD36, to be under-represented in the shotgun proteome analysis, but as the dominant (glyco)protein in the adipocyte membrane proteome that was also up-regulated at the mRNA transcript level in both the in vitro differentiated ADSCs (7.1-fold increase) and mature adipocytes (102.9-fold increase). A comparison of CD36 sequence coverage in the global shotgun analysis with the de-N-glycosylated CD36 revealed a 41% increase when N-glycans were removed prior to trypsin digestion, explaining its observed increased abundance and highlights the crucial need for de-N-glycosylation of proteins in proteomics experiments for increased identification of glycoproteins. The systems glycobiology approach by the integration of previously reported glycomics data and the proteomics and transcriptomics analyses in this work extended the investigation of membrane protein glycosylation changes in adipose-derived stem cell differentiation. The work provides a framework for future glycoproteomics-based investigations into the differentiation of stem cells into adipocytes, and will allow their related pathologies and potential therapeutic applications to be discovered. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/722121v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@189a786org.highwire.dtl.DTLVardef@5563b8org.highwire.dtl.DTLVardef@5cb5borg.highwire.dtl.DTLVardef@69e11f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fast cytoplasmic streaming enables extensive chloroplast movements in the giant cells of unicellular, siphonous macroalgae. Here, we studied chloroplast movements in two such algae: the Dasycladalean Acetabularia acetabulum and the Bryopsidales Bryopsis sp.. We hypothesised that chloroplast movements function as a protective avoidance mechanism under excess light, particularly in Bryopsis sp., which lacks capacity for fast induction of photoprotective non-photochemical quenching (NPQ) and state transitions. In addition, we also investigated whether chloroplast movements are involved in responses to wounding and herbivory. The movements were studied by light microscopy, photography and pulse modulated chlorophyll a fluorescence quenching analysis. Chemical inhibitors of actin polymerization and microtubules assembly were used to confirm that the observed effects were active responses controlled by the cytoskeleton. A. acetabulum responded to high light by reversible chloroplast aggregation, probed by macro-imaging; and chemical inhibition of chloroplast movements led to an enhancement of Photosystem II photoinhibition, as probed by the fluorescence parameter FV/FM. No chloroplast movements were observed in Bryopsis sp. in response to high light. In A. acetabulum, wounding caused either by cutting or due to feeding by the sap-sucking sea slug Elysia timida triggered aggregation of chloroplasts within minutes of incurring the damage. Interestingly, the aggregation also occurred in intact cells away from the cutting site. Furthermore, the addition of media collected from the vicinity of cut algae was sufficient to induce chloroplast aggregation in intact algae, suggesting that water-borne cues or signals triggered the aggregation response in A. acetabulum. Bryopsis sp., however, responded to cutting by only local chloroplast aggregation. The relevance of chloroplast movements in protection against both abiotic and biotic stressors in A. acetabulum, and the potential reasons behind the different defence strategies of the algae, are discussed.