Biomolecules

Over the past decades, pathway analysis has become one of the most commonly used approaches for the functional interpretation of metabolomics data. Although the approach is widely used, it is not well standardized and the impact of different methodologies on the functional outcome is not well understood. Using four publicly available datasets, we investigated two main aspects of topological pathway analysis, namely the consideration of non-human native enzymatic reactions (e.g., from microbiota) and the interconnectivity of individual pathways. The exclusion of non-human native reactions led to detached and poorly represented reaction networks and to loss of information. The consideration of connectivity between pathways led to better emphasis of certain central metabolites in the network; however, it occasionally overemphasized the hub compounds. We proposed and examined a penalization scheme to diminish the effect of such compounds in the pathway evaluation. In order to compare and assess the results between different methodologies, we also performed over-representation analysis of the same datasets. We believe that our findings will raise awareness on both the capabilities and shortcomings of the currently used pathway analysis practices in metabolomics. Additionally, it will provide insights on various methodologies and strategies that should be considered for the analysis and interpretation of metabolomics data. Full article

Aberrant translation, a characteristic feature of cancer, is regulated by the complex and sophisticated RNA binding proteins (RBPs) in the canonical translation machinery. N6-methyladenosine (m⁶A) modifications are the most abundant internal modifications in mRNAs mediated by methyltransferase-like 3 (METTL3). METTL3 is commonly aberrantly expressed in different tumors and affects the mRNA translation of many oncogenes or dysregulated tumor suppressor genes in a variety of ways. In this review, we discuss the critical roles of METTL3 in translation regulation and how METTL3 and m⁶A reader proteins in collaboration with RBPs within the canonical translation machinery promote aberrant translation in tumorigenesis, providing an overview of recent efforts aiming to ‘translate’ these results to the clinic. Full article

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren’s mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren’s binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren’s low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here, we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity. Full article

Cardiovascular diseases (CVDs) are the leading cause of death globally as well as the leading cause of mortality and morbidity in type 2 diabetes (T2D) patients. Results from large interventional studies have suggested hyperglycemia and poor glycemic control to be largely responsible for the development of CVDs. However, the association between hypoglycemia and cardiovascular events is also a key pathophysiological factor in the development of CVDs. Hypoglycemia is especially prevalent in T2D patients treated with oral sulfonylurea agents or exogenous insulin, increasing the susceptibility of this population to cardiovascular events. The adverse cardiovascular risk of hypoglycemia can persist even after the blood glucose levels have been normalized. Hypoglycemia may lead to vascular disease through mechanisms such as enhanced coagulation, oxidative stress, vascular inflammation, endothelial dysfunction, and platelet activation. In the following review, we summarize the evidence for the role of hypoglycemia in platelet activation and the subsequent effects this may have on the development of CVD. In addition, we review current evidence for the effectiveness of therapies in reducing the risk of CVDs. Full article

The expression patterns of IDO1 and PD-L2 have not been thoroughly investigated in benign lymphadenopathies. The aim with this study was to elucidate how IDO1 and PD-L2 are expressed in benign lymphadenopathies in patients with autoimmune diseases (AD) compared to patients without AD. Formalin-fixed paraffin-embedded lymph nodes from 22 patients with AD and 57 patients without AD were immunohistochemically stained to detect IDO1 and PD-L2. The material was previously stained with EBER in situ hybridization to detect cells harboring the Epstein–Barr virus (EBV). IDO1 and PD-L2 were generally expressed by leukocytes to low degrees, while follicular IDO1+ cells were very rare. IDO1+ cells in single germinal centers were detected in five patients, and there was a high co-occurrence of follicular EBV+ cells in these cases (three of five patients). There were also significant correlations between interfollicular EBV+ cells and interfollicular IDO1+ cells (Spearman rho = 0.32, p = 0.004) and follicular IDO1+ cells (Spearman rho = 0.34, p = 0.004). High or low amounts of IDO1+ or PD-L2+ cells were not statistically significantly associated with patients with AD. However, the lymphadenopathy with the highest amount of interfollicular IDO1+ cells, which was also the only lymphadenopathy in which endothelial cells expressed IDO1, was in a patient with sarcoidosis. This study further supports that the EBV induces the expression of IDO1 and our findings should be recognized by future studies on IDO1 and PD-L2 in inflammatory and malignant conditions. Full article

Cardiorenal syndrome (CRS) is a complex, heterogeneous spectrum of symptoms that has kept cardiologists awake for decades. The heart failure (HF) population being burdened with multimorbidity poses diagnostic and therapeutic challenges even for experienced clinicians. Adding deteriorated renal function to the equation, which is one of the strongest predictors of adverse outcome, we measure ourselves against possibly the biggest problem in modern cardiology. With the rapid development of new renal assessment methods, we can treat CRS more effectively than ever. The presented review focuses on explaining the pathophysiology, recent advances and current practices of monitoring renal function in patients with acute CRS. Understanding the dynamic interaction between the heart and the kidney may improve patient care and support the selection of an effective and nephroprotective treatment strategy. Full article

The present study aims to assess the antioxidant and antiviral effectiveness of leaf extracts obtained from Olea europaea L. var. sativa and Olea europaea L. var. sylvestris. The total antioxidant activity was determined via both an ammonium phosphomolybdate assay and a nitric oxide radical inhibition assay. Both extracts showed reducing abilities in an in vitro system and in human HeLa cells. Indeed, after oxidative stress induction, we found that exposition to olive leaf extracts protects human HeLa cells from lipid peroxidation and increases the concentration of enzyme antioxidants such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase. Additionally, OESA treatment affects viral DNA accumulation more than OESY, probably due to the exclusive oleuropein content. In fact, subtoxic concentrations of oleuropein inhibit HSV-1 replication, stimulating the phosphorylation of PKR, c-FOS, and c-JUN proteins. These results provide new knowledge about the potential health benefits and mechanisms of action of oleuropein and oleuropein-rich extracts. Full article

White button mushroom (Agaricus bisporus (J.E. Lange) Imbach) is one of the widely consumed edible mushrooms. Indeed, A. bisporus fruiting bodies are a rich source of nutrients and bioactive molecules. In addition, several enzymes with biotechnological applications are found in A. bisporus (e.g., enzymes for lignocellulose degradation). Here, a novel ribotoxin-like protein (RL-P) from the edible mushroom A. bisporus was purified and characterized. This RL-P, named bisporitin, is a monomeric protein (17-kDa) exhibiting specific ribonucleolytic activity by releasing the α-fragment (hallmark of RL-Ps) when incubated with rabbit ribosomes. In addition, bisporitin shows magnesium-dependent endonuclease activity and displays a similar far-UV CD spectrum as ageritin, the prototype of RL-Ps, isolated from Cyclocybe aegerita fruiting bodies. Interestingly, bisporitin is the first member of RL-Ps to have noticeably lower thermal stability (T_m = 48.59 ± 0.98 °C) compared to RL-Ps isolated in other mushrooms (T_m > 70 °C). Finally, this protein is only partially hydrolyzed in an in vitro digestive system and does not produce adverse growing effects on eukaryotic cell lines. This evidence paves the way for future investigations on possible bioactivities of this RL-P in the digestive system. Full article

In recent decades, many efforts have been made to elucidate the genetic causes of non-syndromic cleft palate (nsCPO), a complex congenital disease caused by the interaction of several genetic and environmental factors. Since genome-wide association studies have evidenced a minor contribution of common polymorphisms in nsCPO inheritance, we used whole exome sequencing data to explore the role of ultra-rare variants in this study. In a cohort of 35 nsCPO cases and 38 controls, we performed a gene set enrichment analysis (GSEA) and a hypergeometric test for assessing significant overlap between genes implicated in nsCPO pathobiology and genes enriched in ultra-rare variants in our cohort. GSEA highlighted an enrichment of ultra-rare variants in genes principally belonging to cytoskeletal protein binding pathway (Probability Density Function corrected p-value = 1.57 × 10⁻⁴); protein-containing complex binding pathway (p-value = 1.06 × 10⁻²); cell adhesion molecule binding pathway (p-value = 1.24 × 10⁻²); ECM-receptor interaction pathway (p-value = 1.69 × 10⁻²); and in the Integrin signaling pathway (p-value = 1.28 × 10⁻²). Two genes implicated in nsCPO pathobiology, namely COL2A1 and GLI3, ranked among the genes (n = 34) with nominal enrichment in the ultra-rare variant collapsing analysis (Fisher’s exact test p-value < 0.05). These genes were also part of an independent list of genes highly relevant to nsCPO biology (n = 25). Significant overlap between the two sets of genes (hypergeometric test p-value = 5.86 × 10⁻³) indicated that enriched genes are likely to be implicated in physiological palate development and/or the pathological processes of oral clefting. In conclusion, ultra-rare variants collectively impinge on biological pathways crucial to nsCPO pathobiology and point to candidate genes that may contribute to the individual risk of disease. Sequencing can be an effective approach to identify candidate genes and pathways for nsCPO. Full article

Monoderm bacteria accumulate heme b via the coproporphyrin-dependent biosynthesis pathway. In the final step, in the presence of two molecules of H₂O₂, the propionate groups of coproheme at positions 2 and 4 are decarboxylated to form vinyl groups by coproheme decarboxylase (ChdC), in a stepwise process. Decarboxylation of propionate 2 produces an intermediate that rotates by 90° inside the protein pocket, bringing propionate 4 near the catalytic tyrosine, to allow the second decarboxylation step. The active site of ChdCs is stabilized by an extensive H-bond network involving water molecules, specific amino acid residues, and the propionate groups of the porphyrin. To evaluate the role of these H-bonds in the pocket stability and enzyme functionality, we characterized, via resonance Raman and electronic absorption spectroscopies, single and double mutants of the actinobacterial pathogen Corynebacterium diphtheriae ChdC complexed with coproheme and heme b. The selective elimination of the H-bond interactions between propionates 2, 4, 6, and 7 and the polar residues of the pocket allowed us to establish the role of each H-bond in the catalytic reaction and to follow the changes in the interactions from the substrate to the product. Full article

Astrocytic-secreted matricellular proteins have been shown to influence various aspects of synaptic function. More recently, they have been found altered in animal models of psychiatric disorders such as drug addiction. Hevin (also known as Sparc-like 1) is a matricellular protein highly expressed in the adult brain that has been implicated in resilience to stress, suggesting a role in motivated behaviors. To address the possible role of hevin in drug addiction, we quantified its expression in human postmortem brains and in animal models of alcohol abuse. Hevin mRNA and protein expression were analyzed in the postmortem human brain of subjects with an antemortem diagnosis of alcohol use disorder (AUD, n = 25) and controls (n = 25). All the studied brain regions (prefrontal cortex, hippocampus, caudate nucleus and cerebellum) in AUD subjects showed an increase in hevin levels either at mRNA or/and protein levels. To test if this alteration was the result of alcohol exposure or indicative of a susceptibility factor to alcohol consumption, mice were exposed to different regimens of intraperitoneal alcohol administration. Hevin protein expression was increased in the nucleus accumbens after withdrawal followed by a ethanol challenge. The role of hevin in AUD was determined using an RNA interference strategy to downregulate hevin expression in nucleus accumbens astrocytes, which led to increased ethanol consumption. Additionally, ethanol challenge after withdrawal increased hevin levels in blood plasma. Altogether, these results support a novel role for hevin in the neurobiology of AUD. Full article

Persistent pain can be managed with opioids, but their use is limited by the onset of tolerance. Ultramicronized N-palmitoylethanolamine (PEA) in vivo delays morphine tolerance with mechanisms that are still unclear. Since glial cells are involved in opioid tolerance and mast cells (MCs) are pivotal targets of PEA, we hypothesized that a potential mechanism by which PEA delays opioid tolerance might depend on the control of the crosstalk between these cells. Morphine treatment (30 mM, 30 min) significantly increased MC degranulation of RBL-2H3 cells, which was prevented by pre-treatment with PEA (100 mM, 18 h), as evaluated by β-hexosaminidase assay and histamine quantification. The impact of RBL-2H3 secretome on glial cells was studied. Six-hour incubation of astrocytes with control RBL-2H3-conditioned medium, and even more so co-incubation with morphine, enhanced CCL2, IL-1β, IL-6, Serpina3n, EAAT2 and GFAP mRNA levels. The response was significantly prevented by the secretome from PEA pre-treated RBL-2H3, except for GFAP, which was further upregulated, suggesting a selective modulation of glial signaling. In conclusion, ultramicronized PEA down-modulated both morphine-induced MC degranulation and the expression of inflammatory and pain-related genes from astrocytes challenged with RBL-2H3 medium, suggesting that PEA may delay morphine tolerance, regulating MC-astrocyte crosstalk. Full article

Melatonin, insulin, and Δ9-tetrahydrocannabinol (THC) have been shown to reverse cognitive deficits and attenuate neuropathologies in transgenic mouse models of Alzheimer’s disease (AD) when used individually. Here, we evaluated the therapeutic properties of long-term intranasal treatment with a novel nanoformulation containing melatonin, insulin, and THC in aged APPswe/PS1ΔE9 (APP/PS1) mice, a transgenic model of AD. Transgenic mice at the age of 12 months were intranasally administered with a new nanoformulation containing melatonin, insulin, and THC at doses of 0.04, 0.008, and 0.02 mg/kg, respectively, once daily for 3 months. The spatial memory of the mice was assessed using the radial arm water maze (RAWM) test before and after drug treatment. Brain tissues were collected at the end of the treatment period for the assessment of Aβ load, tauopathy state, and markers of mitochondrial function. The RAWM test revealed that the treatment with the melatonin–insulin–THC (MIT) nasal spray improved the spatial learning memory of APP/PS1 mice significantly. Results of protein analyses of brain homogenates indicated that MIT treatment significantly decreased the tau phosphorylation implicated in tau toxicity (p < 0.05) and the expression of CKMT1 associated with mitochondrial dysfunction. Moreover, MIT significantly decreased the expression of two mitochondrial fusion-related proteins, Mfn2 and Opa1 (p < 0.01 for both), while increasing the expression of a mitophagy regulator, Parkin, suggesting a compensatory enhancement of mitophagy due to MIT-promoted mitochondrial fusion. In conclusion, this study was the first to demonstrate the ability of an MIT nanoformulation to improve spatial memory in AD mice through its multi-targeting effects on Aβ production, tau phosphorylation, and mitochondrial dynamics. Thus, MIT may be a safe and effective therapeutic for AD. Full article

Pastures have become one of the most important sources of antibiotic resistance genes (ARGs) pollution, bringing risks to human health through the environment and the food that is grown there. Another significant source of food production is greenhouse horticulture, which is typically located near pastures. Through waterways, pasture-originated ARGs may transfer to the food in greenhouses. However, how these pasture-originated ARGs spread to nearby waterways and greenhouses has been much less investigated, while this may pose risks to humans through agricultural products. We analyzed 29 ARGs related to the most used antibiotics in livestock in the Netherlands at 16 locations in an agricultural area, representing pastures, greenhouses and lakes. We found that ARGs were prevalent in all surface waters surrounding pastures and greenhouses and showed a similar composition, with sulfonamide ARGs being dominant. This indicates that both pastures and greenhouses cause antibiotic resistance pressures on neighboring waters. However, lower pressures were found in relatively larger and isolated lakes, suggesting that a larger water body or a non-agricultural green buffer zone could help reducing ARG impacts from agricultural areas. We also observed a positive relationship between the concentrations of the class 1 integron (intl1 gene) —used as a proxy for horizontal gene transfer—and ARG concentration and composition. This supports that horizontal gene transfer might play a role in dispersing ARGs through landscapes. In contrast, none of the measured four abiotic factors (phosphate, nitrate, pH and dissolved oxygen) showed any impact on ARG concentrations. ARGs from different classes co-occurred, suggesting simultaneous use of different antibiotics. Our findings help to understand the spatial patterns of ARGs, specifically the impacts of ARGs from pastures and greenhouses on each other and on nearby waterways. In this way, this study guides management aiming at reducing ARGs′ risk to human health from agricultural products. Full article

The deposition of dense fibril plaques represents the pathological hallmark for a multitude of human disorders, including many neurodegenerative diseases. Fibril plaques are predominately composed of amyloid fibrils, characterized by their underlying cross beta-sheet architecture. Research into the mechanisms of amyloid formation has mostly focused on characterizing and modeling the growth of individual fibrils and associated oligomers from their monomeric precursors. Much less is known about the mechanisms causing individual fibrils to assemble into ordered fibrillar suprastructures. Elucidating the mechanisms regulating this “secondary” self-assembly into distinct suprastructures is important for understanding how individual protein fibrils form the prominent macroscopic plaques observed in disease. Whether and how amyloid fibrils assemble into either 2D or 3D supramolecular structures also relates to ongoing efforts on using amyloid fibrils as substrates or scaffolds for self-assembling functional biomaterials. Here, we investigated the conditions under which preformed amyloid fibrils of a lysozyme assemble into larger superstructures as a function of charge screening or pH. Fibrils either assembled into three-dimensional gel clusters or two-dimensional fibril sheets. The latter displayed optical birefringence, diagnostic of amyloid plaques. We presume that pH and salt modulate fibril charge repulsion, which allows anisotropic fibril–fibril attraction to emerge and drive the transition from 3D to 2D fibril self-assembly. Full article

Acid-sensing ion channels (ASICs) are proton-gated, voltage-independent sodium channels widely expressed throughout the central and peripheral nervous systems. They are involved in synaptic plasticity, learning/memory, fear conditioning and pain. Zinc, an important trace metal in the body, contributes to numerous physiological functions, with neurotransmission being of note. Zinc has been implicated in the modulation of ASICs by binding to specific sites on these channels and exerting either stimulatory or inhibitory effects depending on the ASIC subtype. ASICs have been linked to several neurological and psychological disorders, such as Alzheimer’s disease, Parkinson’s disease, ischemic stroke, epilepsy and cocaine addiction. Different ASIC isoforms contribute to the persistence of each of these neurological and psychological disorders. It is critical to understand how various zinc concentrations can modulate specific ASIC subtypes and how zinc regulation of ASICs can contribute to neurological and psychological diseases. This review elucidates zinc’s structural interactions with ASICs and discusses the potential therapeutic implications zinc may have on neurological and psychological diseases through targeting ASICs. Full article

Cholesterol efflux is a major atheroprotective function of high-density lipoproteins (HDLs) which removes cholesterol from the foam cells of lipid-rich plaques in Type 2 diabetes. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin phosphate increases plasma glucagon-like peptide-1 (GLP-1) concentrations and is used to treat Type 2 diabetes. GLP-1 plays an important role in regulating insulin secretion and expression via the GLP-1 receptor (GLP-1R), which is expressed in pancreatic islets as well as freshly isolated human monocytes and THP-1 cells. Here, we identified a direct role of GLP-1 and DPP-4 inhibition in HDL function. Cholesterol efflux was measured in cultivated phorbol 12-myristate 13-acetate-treated THP-1 cells radiolabeled with ³H-cholesterol and stimulated with liver X receptor/retinoid X receptor agonists. Contrary to vildagliptin, sitagliptin phosphate together with GLP-1 significantly (p < 0.01) elevated apolipoprotein (apo)A1-mediated cholesterol efflux in a dose-dependent manner. The sitagliptin-induced increase in cholesterol efflux did not occur in the absence of GLP-1. In contrast, adenosine triphosphate-binding cassette transporter A1 (ABCA1) mRNA and protein expressions in the whole cell fraction were not changed by sitagliptin in the presence of GLP-1, although sitagliptin treatment significantly increased ABCA1 protein expression in the membrane fraction. Furthermore, the sitagliptin-induced, elevated efflux in the presence of GLP-1 was significantly decreased by a GLP-1R antagonist, an effect that was not observed with a protein kinase A inhibitor. To our knowledge, the present study reports for the first time that sitagliptin elevates cholesterol efflux in cultivated macrophages and may exert anti-atherosclerotic actions that are independent of improvements in glucose metabolism. Our results suggest that sitagliptin enhances HDL function by inducing a de novo HDL synthesis via cholesterol efflux. Full article

Salvia bulleyana is a plant native to the Chinese Yunnan Province. This species has been used in traditional Chinese medicine as a substitute for Danshen (the roots of Salvia miltiorrhiza). The aim of our study was to establish an effective system for propagating S. bulleyana shoots to obtain large amounts of material rich in bioactive compounds. Phytohormones were used to regulate shoot growth and regeneration potential and influence plant secondary metabolism. The shoot tips were incubated on a Murashige and Skoog agar medium supplemented with 0.1 or 0.5 mg/L IAA (indole-3-acetic acid) and the cytokinins benzylaminopurine (BAP), meta-topoline (M-T), 6-benzylaminopurine riboside (RBAP), N-benzyl-9-(2-tetrahydropyranyl)-adenine (BPA) or kinetin, (K) at concentrations of 0.5, 1 or 2 mg/L. It was observed that the type and concentration of growth regulator significantly influenced the regeneration potential of S. bulleyana shoots. The highest multiplication rate was obtained when 0.1 mg/L IAA and 2 mg/L BPA were used. Under these conditions, 100% of shoot tips formed buds and almost seven buds/shoot per explant were obtained after five weeks. Meanwhile, the highest biomass was found for shoots growing on a medium supplemented with 0.1 mg/L IAA and 1 mg/L M-T: 1.2 g of fresh weight and 0.17 g of dry weight. However, a medium with 0.1 mg/L IAA and 2 mg/L RBAP was most favorable for bioactive phenolic acid content, with a total polyphenol level (37.7 mg/g dw) 4.5 times higher than in shoots grown on medium without growth regulators (8.23 mg/g dw). Finally, optimal conditions were selected by TOPSIS (technique for order of preference by similarity to the ideal solution); the culture of S. bulleyana grown on an MS medium containing 0.1 mg/L IAA and 1 mg/L M-T was found to be the most efficient for polyphenol accumulation and can be used for the production of medicinally relevant compounds. Full article

Mitochondria are widely considered the “power hub” of the cell because of their pivotal roles in energy metabolism and oxidative phosphorylation. However, beyond the production of ATP, which is the major source of chemical energy supply in eukaryotes, mitochondria are also central to calcium homeostasis, reactive oxygen species (ROS) balance, and cell apoptosis. The mitochondria also perform crucial multifaceted roles in biosynthetic pathways, serving as an important source of building blocks for the biosynthesis of fatty acid, cholesterol, amino acid, glucose, and heme. Since mitochondria play multiple vital roles in the cell, it is not surprising that disruption of mitochondrial function has been linked to a myriad of diseases, including neurodegenerative diseases, cancer, and metabolic disorders. In this review, we discuss the key physiological and pathological functions of mitochondria and present bioactive compounds with protective effects on the mitochondria and their mechanisms of action. We highlight promising compounds and existing difficulties limiting the therapeutic use of these compounds and potential solutions. We also provide insights and perspectives into future research windows on mitochondrial modulators. Full article