With the debut of AlphaFold2, we now can get a highly-accurate view of a reasonable equilibrium tertiary structure of a protein molecule. Yet, a single-structure view is insufficient and does not account for the high structural plasticity of protein molecules. Obtaining a multi-structure view of a protein molecule continues to be an outstanding challenge in computational structural biology. In tandem with methods formulated under the umbrella of stochastic optimization, we are now seeing rapid advances in the capabilities of methods based on deep learning. In recent work, we advance the capability of these models to learn from experimentally-available tertiary structures of protein molecules of varying lengths. In this work, we elucidate the important role of the composition of the training dataset on the neural network’s ability to learn key local and distal patterns in tertiary structures. To make such patterns visible to the network, we utilize a contact map-based representation of protein tertiary structure. We show interesting relationships between data size, quality, and composition on the ability of latent variable models to learn key patterns of tertiary structure. In addition, we present a disentangled latent variable model which improves upon the state-of-the-art variable autoencoder-based model in key, physically-realistic structural patterns. We believe this work opens up further avenues of research on deep learning-based models for computing multi-structure views of protein molecules. Full article

Ageing is associated with reduced endothelium-derived nitric oxide (NO) production in the femoral artery of Sprague Dawley (SD) rats. In the current study, we examined endothelium-dependent relaxation (EDR) in the saphenous artery and its caudal branches. We used acetylcholine and the Proteinase-Activated receptor-2 (PAR2)-specific agonist (2fLIGRLO) with nitroarginine methylester (L-NAME) to assess EDR in two groups of male SD rats (age in weeks: young, 10–12; old, 27–29). Acetylcholine and 2fLIGRLO were potent NO-dependent relaxant agents in all arteries. For all arteries, EDR by acetylcholine decreased significantly in old compared to young SD rats. Interestingly, PAR2-induced EDR of proximal saphenous artery segments and caudal branches decreased significantly in old compared to young, but did not differ for the in-between middle and distal ends of the saphenous artery. L-NAME treatment increased subsequent contractions of proximal and middle segments of saphenous arteries by phenylephrine and U46619 in young, but not in old, SD rats. We conclude the SD saphenous artery and caudal branches exhibit regional characteristics that differ in response to specific EDR agonists, endothelial NO synthase inhibitor, and changes to endothelium function with increased age, which are, in part, attributed to decreased sensitivity of vascular smooth muscle to the gaseous transmitter NO. Full article

Dopamine (DA), the most abundant human brain catecholaminergic neurotransmitter, modulates key behavioral and neurological processes in young and senescent brains, including motricity, sleep, attention, emotion, learning and memory, and social and reward-seeking behaviors. The DA transporter (DAT) regulates transsynaptic DA levels, influencing all these processes. Compounds targeting DAT (e.g., cocaine and amphetamines) were historically used to shape mood and cognition, but these substances typically lead to severe negative side effects (tolerance, abuse, addiction, and dependence). DA/DAT signaling dysfunctions are associated with neuropsychiatric and progressive brain disorders, including Parkinson’s and Alzheimer diseases, drug addiction and dementia, resulting in devastating personal and familial concerns and high socioeconomic costs worldwide. The development of low-side-effect, new/selective medicaments with reduced abuse-liability and which ameliorate DA/DAT-related dysfunctions is therefore crucial in the fields of medicine and healthcare. Using the rat as experimental animal model, the present work describes the synthesis and pharmacological profile of (S)-MK-26, a new modafinil analogue with markedly improved potency and selectivity for DAT over parent drug. Ex vivo electrophysiology revealed significantly augmented hippocampal long-term synaptic potentiation upon acute, intraperitoneally delivered (S)-MK-26 treatment, whereas in vivo experiments in the hole-board test showed only lesser effects on reference memory performance in aged rats. However, in effort-related FR5/chow and PROG/chow feeding choice experiments, (S)-MK-26 treatment reversed the depression-like behavior induced by the dopamine-depleting drug tetrabenazine (TBZ) and increased the selection of high-effort alternatives. Moreover, in in vivo microdialysis experiments, (S)-MK-26 significantly increased extracellular DA levels in the prefrontal cortex and in nucleus accumbens core and shell. These studies highlight (S)-MK-26 as a potent enhancer of transsynaptic DA and promoter of synaptic plasticity, with predominant beneficial effects on effort-related behaviors, thus proposing therapeutic potentials for (S)-MK-26 in the treatment of low-effort exertion and motivational dysfunctions characteristic of depression and aging-related disorders. Full article

Synechococcus sp. PCC 11901 reportedly demonstrates the highest, most sustained growth of any known cyanobacterium under optimized conditions. Due to its recent discovery, our knowledge of its biology, including the factors underlying sustained, fast growth, is limited. Furthermore, tools specific for genetic manipulation of PCC 11901 are not established. Here, we demonstrate that PCC 11901 shows faster growth than other model cyanobacteria, including the fast-growing species Synechococcuselongatus UTEX 2973, under optimal growth conditions for UTEX 2973. Comparative genomics between PCC 11901 and Synechocystis sp. PCC 6803 reveal conservation of most metabolic pathways but PCC 11901 has a simplified electron transport chain and reduced light harvesting complex. This may underlie its superior light use, reduced photoinhibition, and higher photosynthetic and respiratory rates. To aid biotechnology applications, we developed a vitamin B₁₂ auxotrophic mutant but were unable to generate unmarked knockouts using two negative selectable markers, suggesting that recombinase- or CRISPR-based approaches may be required for repeated genetic manipulation. Overall, this study establishes PCC 11901 as one of the most promising species currently available for cyanobacterial biotechnology and provides a useful set of bioinformatics tools and strains for advancing this field, in addition to insights into the factors underlying its fast growth phenotype. Full article

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, classically associated with extensive loss of dopaminergic neurons of the substantia nigra pars compacta. The hallmark of the disease is the accumulation of pathogenic conformations of the presynaptic protein, α-synuclein (αSyn), and the formation of intraneuronal protein aggregate inclusions. Neurodegeneration of dopamine neurons leads to a prominent dopaminergic deficiency in the basal ganglia, responsible for motor disturbances. However, it is now recognized that the disease involves more widespread neuronal dysfunction, leading to early and late non-motor symptoms. The development of in vitro systems based on the differentiation of human-induced pluripotent stem cells provides us the unique opportunity to monitor alterations at the cellular and molecular level throughout the differentiation procedure and identify perturbations that occur early, even at the neuronal precursor stage. Here we aim to identify whether p.A53T-αSyn induced disturbances at the molecular level are already present in neural precursors. Towards this, we present data from transcriptomics analysis of control and p.A53T-αSyn NPCs showing altered expression in transcripts involved in axon guidance, adhesion, synaptogenesis, ion transport, and metabolism. The comparative analysis with the transcriptomics profile of p.A53T-αSyn neurons shows both distinct and overlapping pathways leading to neurodegeneration while meta-analysis with transcriptomics data from both neurodegenerative and neurodevelopmental disorders reveals that p.A53T-pathology has a significant overlap with the latter category. This is the first study showing that molecular dysregulation initiates early at the p.A53T-αSyn NPC level, suggesting that synucleinopathies may have a neurodevelopmental component. Full article

Human cytochrome P450 CYP3A4 is involved in the processing of more than 35% of current pharmaceuticals and therefore is responsible for multiple drug-drug interactions (DDI). In order to develop a method for the detection and prediction of the possible involvement of new drug candidates in CYP3A4-mediated DDI, we evaluated the application of midazolam (MDZ) as a probe substrate. MDZ is hydroxylated by CYP3A4 in two positions: 1-hydroxy MDZ formed at lower substrate concentrations, and up to 35% of 4-hydroxy MDZ at high concentrations. The ratio of the formation rates of these two products (the site of metabolism ratio, SOM) was used as a measure of allosteric heterotropic interactions caused by effector molecules using CYP3A4 incorporated in lipid nanodiscs. The extent of the changes in the SOM in the presence of effectors is determined by chemical structure and is concentration-dependent. MD simulations of CYP3A4 in the lipid bilayer suggest that experimental results can be explained by the movement of the F-F’ loop and concomitant changes in the shape and volume of the substrate-binding pocket. As a result of PGS binding at the allosteric site, several residues directly contacting MDZ move away from the substrate molecule, enabling the repositioning of the latter for minor product formation. Full article

Liposarcoma is a rare and heterogeneous soft tissue malignant tumor and has a significant impact on mortality with a poor prognosis. To date, there is no effective treatment for liposarcoma, whereas surgical resection is only the gold treatment with numerous adverse effects. Here we investigated whether tetrandrine inhibits the growth of SW872 human malignant liposarcoma cells. Of note, tetrandrine at 10 μM vastly inhibited growth and induced apoptosis, as evidenced by increased nuclear DNA fragmentation and sub-G1 population of SW872 cells. Mechanistically, treatment with tetrandrine led to activation of caspase-9/3 in SW872 cells, and z-VAD-fmk, a pan-caspase inhibitor, attenuated the tetrandrine-induced apoptosis and growth suppression in SW872 cells. In addition, tetrandrine treatment resulted in down-regulation of XIAP andSTAT-3 in SW872 cells, and importantly knockdown of STAT-3 caused a significant reduction of the cell survival. Tetrandrine also had abilities to up-regulate not only the expression of GRP78 and ATF-4 but also the phosphorylation of eIF-2α in SW872 cells. In summary, these results demonstrated that tetrandrine has strong growth-suppressive and apoptosis-inducing effects on SW872 cells, which are mediated through control of the intrinsic caspase pathway, down-regulation of XIAP and STAT-3, and triggering ER stress. Full article

Recent experimental studies suggest that ATP-driven molecular chaperones can stabilize protein substrates in their native structures out of thermal equilibrium. The mechanism of such non-equilibrium protein folding is an open question. Based on available structural and biochemical evidence, I propose here a unifying principle that underlies the conversion of chemical energy from ATP hydrolysis to the conformational free energy associated with protein folding and activation. I demonstrate that non-equilibrium folding requires the chaperones to break at least one of four symmetry conditions. The Hsp70 and Hsp90 chaperones each break a different subset of these symmetries and thus they use different mechanisms for non-equilibrium protein folding. I derive an upper bound on the non-equilibrium elevation of the native concentration, which implies that non-equilibrium folding only occurs in slow-folding proteins that adopt an unstable intermediate conformation in binding to ATP-driven chaperones. Contrary to the long-held view of Anfinsen’s hypothesis that proteins fold to their conformational free energy minima, my results predict that some proteins may fold into thermodynamically unstable native structures with the assistance of ATP-driven chaperones, and that the native structures of some chaperone-dependent proteins may be shaped by their chaperone-mediated folding pathways. Full article

Optic neuritis (ON), the most common ocular manifestation of multiple sclerosis, is an autoimmune inflammatory demyelinating disease also characterized by degeneration of retinal ganglion cells (RGCs) and their axons, which commonly leads to visual impairment despite attempted treatments. Although ON disease etiology is not known, changes in the redox system and exacerbated optic nerve inflammation play a major role in the pathogenesis of the disease. Silent information regulator 1 (sirtuin-1/SIRT1) is a ubiquitously expressed NAD⁺-dependent deacetylase, which functions to reduce/prevent both oxidative stress and inflammation in various tissues. Non-specific upregulation of SIRT1 by pharmacologic and genetic approaches attenuates RGC loss in experimental ON. Herein, we hypothesized that targeted expression of SIRT1 selectively in RGCs using an adeno-associated virus (AAV) vector as a delivery vehicle is an effective approach to reducing neurodegeneration and preserving vision in ON. We tested this hypothesis through intravitreal injection of AAV7m8.SNCG.SIRT1, an AAV2-derived vector optimized for highly efficient SIRT1 transgene transfer and protein expression into RGCs in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis that recapitulates optic neuritis RGC loss and axon demyelination. Our data show that EAE mice injected with a control vehicle exhibit progressive alteration of visual function reflected by decreasing optokinetic response (OKR) scores, whereas comparatively, AAV7m8.SNCG.SIRT1-injected EAE mice maintain higher OKR scores, suggesting that SIRT1 reduces the visual deficit imparted by EAE. Consistent with this, RGC survival determined by immunolabeling is increased and axon demyelination is decreased in the AAV7m8.SNCG.SIRT1 RGC-injected group of EAE mice compared to the mouse EAE counterpart injected with a vehicle or with control vector AAV7m8.SNCG.eGFP. However, immune cell infiltration of the optic nerve is not significantly different among all EAE groups of mice injected with either vehicle or AAV7m8.SNCG.SIRT1. We conclude that despite minimally affecting the inflammatory response in the optic nerve, AAV7m8-mediated SIRT1 transfer into RGCs has a neuroprotective potential against RGC loss, axon demyelination and vison deficits associated with EAE. Together, these data suggest that SIRT1 exerts direct effects on RGC survival and function. Full article

Peripheral nerve injuries pose a major clinical concern world-wide, and functional recovery after segmental peripheral nerve injury is often unsatisfactory, even in cases of autografting. Although it is well established that angiogenesis plays a pivotal role during nerve regeneration, the influence of lymphangiogenesis is strongly under-investigated. In this study, we analyzed the presence of lymphatic vasculature in healthy and regenerated murine peripheral nerves, revealing that nerve autografts contained increased numbers of lymphatic vessels after segmental damage. This led us to elucidate the interaction between lymphatic endothelial cells (LECs) and Schwann cells (SCs) in vitro. We show that SC and LEC secretomes did not influence the respective other cell types’ migration and proliferation in 2D scratch assay experiments. Furthermore, we successfully created lymphatic microvascular structures in SC-embedded 3D fibrin hydrogels, in the presence of supporting cells; whereas SCs seemed to exert anti-lymphangiogenic effects when cultured with LECs alone. Here, we describe, for the first time, increased lymphangiogenesis after peripheral nerve injury and repair. Furthermore, our findings indicate a potential lymph-repellent property of SCs, thereby providing a possible explanation for the lack of lymphatic vessels in the healthy endoneurium. Our results highlight the importance of elucidating the molecular mechanisms of SC–LEC interaction. Full article

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application. Full article

Chronic wounds are one of the most frequent complications that are associated with diabetes mellitus. The overproduction of reactive oxygen species (ROS) is a key factor in the delayed healing of a chronic wound. In the present work, we develop a novel in situ-forming silk sericin-based hydrogel (SSH) that is produced by a simple methodology using horseradish peroxidase (HRP) crosslinking as an advanced dressing for wound healing. The antioxidant and angiogenic effects were assessed in vitro and in vivo after in situ application using an excisional wound-healing model in a genetically-induced diabetic db/db mice and though the chick embryo choriollantoic membrane (CAM) assay, respectively. Wounds in diabetic db/db mice that were treated with SSH closed with reduced granulation tissue, decreased wound edge distance, and wound thickness, when compared to Tegaderm, a dressing that is commonly used in the clinic. The hydrogel also promoted a deposition of collagen fibers with smaller diameter which may have had a boost effect in re-epithelialization. SSH treatment slightly induced two important endogenous antioxidant defenses, superoxide dismutase and catalase. A CAM assay made it possible to observe that SSH led to an increase in the number of newly formed vessels without inducing an inflammatory reaction. The present hydrogel may result in a multi-purpose technology with angiogenic, antioxidant, and anti-inflammatory properties, while advancing efficient and organized tissue regeneration. Full article

Azobenzene-based photochromic lipids are valuable probes for the analysis of ion channel–lipid interactions. Rapid photoisomerization of these molecules enables the analysis of lipid gating kinetics and provides information on lipid sensing. Thermal relaxation of the metastable cis conformation to the trans conformation of azobenzene photolipids is rather slow in the dark and may be modified by ligand–protein interactions. Cis photolipid-induced changes in pure lipid membranes as visualized from the morphological response of giant unilamellar vesicles indicated that thermal cis–trans isomerization of both PhoDAG-1 and OptoDArG is essentially slow in the lipid bilayer environment. While the currents activated by cis PhoDAG remained stable upon termination of UV light exposure (dark, UV-OFF), cis OptoDArG-induced TRPC3/6/7 activity displayed a striking isoform-dependent exponential decay. The deactivation kinetics of cis OptoDArG-induced currents in the dark was sensitive to mutations in the L2 lipid coordination site of TRPC channels. We conclude that the binding of cis OptoDArG to TRPC channels promotes transition of cis OptoDArG to the trans conformation. This process is suggested to provide valuable information on DAG–ion channel interactions and may enable highly selective photopharmacological interventions. Full article

Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus safensis and expressed in Escherichia coli. The reduced molecular mass of BsXyn10 was 48 kDa upon SDS-PAGE. Bsxyn10 was optimally active at pH 7.0 and 60 °C, stable over a broad range of pH (5.0–8.0), and also revealed tolerance toward different modulators (metal cations, EDTA). The enzyme was active toward various xylans with no activity on the glucose-based polysaccharides. K_M, v_max, and k_cat for oat spelt xylan hydrolysis were found to be 1.96 g·L⁻¹, 58.6 μmole·min⁻¹·(mg protein)⁻¹, and 49 s⁻¹, respectively. Thermodynamic parameters for oat spelt xylan hydrolysis at 60 °C were ΔS* = −61.9 J·mol⁻¹·K⁻¹, ΔH* = 37.0 kJ·mol⁻¹ and ΔG* = 57.6 kJ·mol⁻¹. BsXyn10 retained high levels of activity at temperatures up to 60 °C. The thermodynamic parameters (ΔH*_D, ΔG*_D, ΔS*_D) for the thermal deactivation of BsXyn10 at a temperature range of 40–80 °C were: 192.5 ≤ ΔH*_D ≤ 192.8 kJ·mol⁻¹, 262.1 ≤ ΔS*_D ≤ 265.8 J·mol⁻¹·K⁻¹, and 99.9 ≤ ΔG*_D ≤ 109.6 kJ·mol⁻¹. The BsXyn10-treated oat spelt xylan manifested the catalytic release of xylooligosaccharides of 2–6 DP, suggesting that BsXyn10 represents a promising candidate biocatalyst appropriate for several biotechnological applications. Full article

Saturated fatty acids (SFAs) are considered to be detrimental to human health. One of the SFAs, myristic acid (MA), is known to exert a hypercholesterolemic effect in mice as well as humans. However, its effects on altering adipose tissue (AT) inflammation and systemic insulin resistance (IR) in obesity are still unclear. Here, we sought to determine the effects of a high fat (HF) diet supplemented with MA on obesity-associated metabolic disorders in mice. Wild-type C57BL/6 mice were fed a HF diet in the presence or absence of 3% MA for 12 weeks. Plasma lipids, plasma adipokines, AT inflammation, systemic IR, glucose homeostasis, and hepatic steatosis were assessed. The body weight and visceral adipose tissue (VAT) mass were significantly higher in mice receiving the HF+MA diet compared to HF diet-fed controls. Plasma total cholesterol levels were marginally increased in HF+MA-fed mice compared to controls. Fasting blood glucose was comparable between HF and HF+MA-fed mice. Interestingly, the plasma insulin and HOMA-IR index, a measure of insulin resistance, were significantly higher in HF+MA-fed mice compared to HF controls. Macrophage and inflammatory markers were significantly elevated in the AT and AT-derived stromal vascular cells upon MA feeding. Moreover, the level of circulating resistin, an adipokine promoting insulin resistance, was significantly higher in HF+MA-fed mice compared with HF controls. The insulin tolerance test revealed that the IR was higher in mice receiving the MA supplementation compared to HF controls. Moreover, the glucose tolerance test showed impairment in systemic glucose homeostasis in MA-fed mice. Analyses of liver samples showed a trend towards an increase in liver TG upon MA feeding. However, markers of oxidative stress and inflammation were reduced in the liver of mice fed an MA diet compared to controls. Taken together, our data suggest that chronic administration of MA in diet exacerbates obesity-associated insulin resistance and this effect is mediated in part, via increased AT inflammation and increased secretion of resistin. Full article

As responses of immortalized endothelial cells of the bovine retina (iBREC) to VEGF-A₁₆₅ depend on exposure time to the growth factor, we investigated changes evident after long-term treatment for nine days. The cell index of iBREC cultivated on gold electrodes—determined as a measure of permeability—was persistently reduced by exposure to the growth factor. Late after addition of VEGF-A₁₆₅ protein levels of claudin-1 and CD49e were significantly lower, those of CD29 significantly higher, and the plasmalemma vesicle associated protein was no longer detected. Nuclear levels of β-catenin were only elevated on day two. Extracellular levels of VEGF-A—measured by ELISA—were very low. Similar to the binding of the growth factor by brolucizumab, inhibition of VEGFR2 by tyrosine kinase inhibitors tivozanib or nintedanib led to complete, although transient, recovery of the low cell index when added early, though was inefficient when added three or six days later. Additional inhibition of other receptor tyrosine kinases by nintedanib was similarly unsuccessful, but additional blocking of c-kit by tivozanib led to sustained recovery of the low cell index, an effect observed only when the inhibitor was added early. From these data, we conclude that several days after the addition of VEGF-A₁₆₅ to iBREC, barrier dysfunction is mainly sustained by increased paracellular flow and impaired adhesion. Even more important, these changes are most likely no longer VEGF-A-controlled. Full article

Post-traumatic stress disorder (PTSD) is a trauma-related disorder. Platelet monoamine oxidase (MAO-B) is a peripheral biomarker associated with various symptoms in different psychopathologies, but its role in PTSD or different symptoms in PTSD is not clear. This study elucidated the association between platelet MAO-B activity and clinical symptoms occurring in PTSD. Platelet MAO-B activity was determined in 1053 male Caucasian subjects: 559 war veterans with PTSD (DSM-5 criteria), 62 combat exposed veterans who did not develop PTSD, and 432 non-combat exposed healthy controls. Clinical symptoms in PTSD were determined using CAPS and PANSS. Platelet MAO-B activity, controlled for the effect of smoking, was significantly increased in PTSD with severe versus mild and moderate traumatic symptoms, and was significantly decreased in PTSD subjects with severe versus mild positive, psychotic, and depressive symptoms. This finding was further confirmed with reduced platelet MAO-B activity in PTSD veterans with severe versus mild individual items of the PANSS-depressed, PANSS-psychotic, and PANSS-positive subscales. Altered platelet MAO-B activity, controlled for the possible confounders, was associated with the development and severity of different symptoms occurring in PTSD. These findings confirmed the role of platelet MAO-B activity as a peripheral marker of various psychopathological symptoms. Full article

Exogenous ganglioside GM1 has been reported to exert an immunomodulatory effect. We investigated the anti-inflammatory effect of GM1 ganglioside on endotoxin-induced uveitis (EIU) in rats and RAW 264.7 macrophages. Methods: EIU was induced in Lewis rats by administering a subcutaneous injection of lipopolysaccharide (LPS). GM1 was injected intraperitoneally for three consecutive days prior to the LPS injection. Twenty-four hours after the LPS injection, the integrity of the blood-aqueous barrier was evaluated by determining the protein concentration and number of infiltrating cells in the aqueous humor (AqH). Immunohistochemical and Western blot analyses of the iris-ciliary body (ICB) were performed to evaluate the effect of GM1 on the LPS-induced expression of cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1). The effect of GM1 on proinflammatory mediators and signaling cascades was examined in LPS-stimulated RAW 264.7 cells using Western blotting and immunofluorescence staining to further clarify the underlying anti-inflammatory mechanism. Results: GM1 significantly reduced the protein concentration and number of infiltrating cells in the AqH of rats with EIU. GM1 also decreased the LPS-induced expression of the ICAM-1 and COX-2 proteins in the ICB. In RAW 264.7 cells, GM1 inhibited the proinflammatory mediators induced by LPS, including inducible nitric oxide synthase (iNOS), COX-2, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and this inhibitory effect was potentially mediated by suppressing reactive oxygen species (ROS)-mediated activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). Conclusions: Based on this study, GM1 may be a potential anti-inflammatory agent for ocular inflammatory diseases. Full article

DYT-PRKRA (dystonia 16 or DYT-PRKRA) is caused by mutations in the PRKRA gene that encodes PACT, the protein activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR). PACT participates in several cellular pathways, of which its role as a PKR activator protein during integrated stress response (ISR) is the best characterized. Previously, we have established that the DYT-PRKRA mutations cause enhanced activation of PKR during ISR to sensitize DYT-PRKRA cells to apoptosis. In this study, we evaluate if the most prevalent substitution mutation reported in DYT-PRKRA patients alters PACT’s functional role in induction of type I IFNs via the retinoic acid-inducible gene I (RIG-I) signaling. Our results indicate that the P222L mutation augments PACT’s ability to induce IFN β in response to dsRNA and the basal expression of IFN β and IFN-stimulated genes (ISGs) is higher in DYT-PRKRA patient cells compared to cells from the unaffected controls. Additionally, IFN β and ISGs are also induced at higher levels in DYT-PRKRA cells in response to dsRNA. These results offer a new avenue for investigations directed towards understanding the underlying molecular pathomechanisms in DYT-PRKRA. Full article

Tumor-specific targeting with fluorescent probes can enhance contrast for identification of cancer during surgical resection and visualize otherwise invisible tumor margins. Nanobodies are the smallest naturally-occurring antigen-binding molecules with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated to an IR800 dye to target and label patient derived pancreatic cancer xenografts. After intravenous administration, the probe rapidly localized to the pancreatic cancer tumors within an hour and had a tumor-to-background ratio of 2.0 by 3 h. The fluorescence signal was durable over a prolonged period of time. With the rapid kinetics afforded by fluorescent nanobodies, both targeting and imaging can be performed on the same day as surgery. Full article

Preeclampsia (PE) is a severe pregnancy disorder with a pathophysiology not yet completely understood and without curative therapy. The histone modifications H3K4me³ and H3K9ac, as well as galectin-2 (Gal-2), are known to be decreased in PE. To gain a better understanding of the development of PE, the influence of Gal-2 on histone modification in trophoblasts and in syncytialisation was investigated. Immunohistochemical stains of 13 PE and 13 control placentas were correlated, followed by cell culture experiments. An analysis of H3K4me³ and H3K9ac was conducted, as well as cell fusion staining with E-cadherin and β-catenin—both after incubation with Gal-2. The expression of H3K4me³ and H3K9ac correlated significantly with the expression of Gal-2. Furthermore, we detected an increase in H3K4me³ and H3K9ac after the addition of Gal-2 to BeWo/HVT cells. Moreover, there was increased fusion of HVT cells after incubation with Gal-2. Gal-2 is associated with the histone modifications H3K4me³ and H3K9ac in trophoblasts. Furthermore, syncytialisation increased after incubation with Gal-2. Therefore, we postulate that Gal-2 stimulates syncytialisation, possibly mediated by H3K4me³ and H3K9ac. Since Gal-2, as well as H3K4me³ and H3K9ac, are decreased in PE, the induction of Gal-2 might be a promising therapeutic target. Full article

In order to identify factors involved in transcription of human snRNA genes and 3′ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with these human snRNA genes including RNA polymerase II (pol II), Cyclin-Dependent Kinase 7 (CDK7), Negative Elongation Factor (NELF), Suppressor of Ty 5 (SPT5), Mediator 23 (MED23) and several subunits of the Integrator Complex. Suppressor of Ty 6 (SPT6); Cyclin K, the partner of Cyclin-Dependent Kinase 12 (CDK12) and Cyclin-Dependent Kinase 13 (CDK13); and SWI/SNF chromatin remodelling complex-associated SWI/SNF-related, Matrix-associated, Regulator of Chromatin (SMRC) factors were also enriched. Several polyadenylation factors, including Cleavage and Polyadenylation Specificity Factor 1 (CPSF1), Cleavage Stimulation Factors 1 and 2 (CSTF1,and CSTF2) were enriched by U2 gene CAPTURE. We have already shown by chromatin immunoprecipitation (ChIP) that CSTF2—and Pcf11 and Ssu72, which are also polyadenylation factors—are associated with the human U1 and U2 genes. ChIP-seq and ChIP-qPCR confirm the association of SPT6, Cyclin K, and CDK12 with the U2 genes. In addition, knockdown of SPT6 causes loss of subunit 3 of the Integrator Complex (INTS3) from the U2 genes, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes and helped to identify a functional role for SPT6. Full article

The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated–mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated–mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator’s ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells. Full article

Hepatic steatosis and chronic hepatocyte damage ultimately lead to liver fibrosis. Key pathophysiological steps are the activation and transdifferentiation of hepatic stellate cells. We assessed the interplay between hepatocytes and hepatic stellate cells under normal and steatotic conditions. We hypothesized that hepatocyte-derived extracellular vesicles (EVs) modify the phenotype of stellate cells. By high speed centrifugation, EVs were isolated from conditioned media of the hepatocellular carcinoma cell line HepG2 under baseline conditions (C-EVs) or after induction of steatosis by linoleic and oleic acids for 24 h (FA-EVs). Migration of the human stellate cell line TWNT4 and of primary human stellate cells towards the respective EVs and sera of MAFLD patients were investigated using Boyden chambers. Phenotype alterations after incubation with EVs were determined by qRT-PCR, Western blotting and immunofluorescence staining. HepG2 cells released more EVs after treatment with fatty acids. Chemotactic migration of TWNT4 and primary hepatic stellate cells was increased, specifically towards FA-EVs. Prolonged incubation of TWNT4 cells with FA-EVs induced expression of proliferation markers and a myofibroblast-like phenotype. Though the expression of the collagen type 1 $\alpha$1 gene did not change after FA-EV treatment, expression of the myofibroblast markers, e.g., $\alpha$-smooth-muscle-cell actin and TIMP1, was significantly increased. We conclude that EVs from steatotic hepatocytes can influence the behavior, phenotypes and expression levels of remodeling markers of stellate cells and guides their directed migration. These findings imply EVs as operational, intercellular communicators in the pathophysiology of steatosis-associated liver fibrosis and might represent a novel diagnostic parameter and therapeutic target. Full article

5-hydroxyfurfural (5HMF), an allosteric effector of hemoglobin (Hb) with an ability to increase Hb affinity for oxygen has been studied extensively for its antisickling effect in vitro and in vivo, and in humans for the treatment of sickle cell disease (SCD). One of the downstream pathophysiologies of SCD is nitric oxide (NO) deficiency, therefore increasing NO (bio)availability is known to mitigate the severity of SCD symptoms. We report the synthesis of an NO-releasing prodrug of 5HMF (5HMF-NO), which in vivo, is expected to be bio-transformed into 5HMF and NO, with concomitant therapeutic activities. In vitro studies showed that when incubated with whole blood, 5HMF-NO releases NO, as anticipated. When incubated with sickle blood, 5HMF-NO formed Schiff base adduct with Hb, increased Hb affinity for oxygen, and prevented hypoxia-induced erythrocyte sickling, which at 1 mM concentration were 16%, 10% and 27%, respectively, compared to 21%, 18% and 21% for 5HMF. Crystal structures of 5HMF-NO with Hb showed 5HMF-NO bound to unliganded (deoxygenated) Hb, while the hydrolyzed product, 5HMF bound to liganded (carbonmonoxy-ligated) Hb. Our findings from this proof-of-concept study suggest that the incorporation of NO donor group to 5HMF and analogous molecules could be a novel beneficial strategy to treat SCD and warrants further detailed in vivo studies. Full article

Flory’s random coil model assumes that conformational fluctuations of amino acid residues in unfolded poly(oligo)peptides and proteins are uncorrelated (isolated pair hypothesis, IPH). This implies that conformational energies, entropies and solvation free energies are all additive. Nearly 25 years ago, analyses of coil libraries cast some doubt on this notion, in that they revealed that aromatic, but also β-branched side chains, could change the ³J(H^NH^Cα) coupling of their neighbors. Since then, multiple bioinformatical, computational and experimental studies have revealed that conformational propensities of amino acids in unfolded peptides and proteins depend on their nearest neighbors. We used recently reported and newly obtained Ramachandran plots of tetra- and pentapeptides with non-terminal homo- and heterosequences of amino acid residues to quantitatively determine nearest neighbor coupling between them with a Ising type model. Results reveal that, depending on the choice of amino acid residue pairs, nearest neighbor interactions either stabilize or destabilize pairs of polyproline II and β-strand conformations. This leads to a redistribution of population between these conformations and a reduction in conformational entropy. Interactions between residues in polyproline II and turn(helix)-forming conformations seem to be cooperative in most cases, but the respective interaction parameters are subject to large statistical errors. Full article

Gut microbes have been recognized to convert human bile acids by deconjugation, dehydroxylation, dehydrogenation, and epimerization of the cholesterol core, but the ability to re-conjugate them with amino acids as an additional conversion has been recently described. These new bile acids are known as microbially conjugated bile acids (MCBAs). The aim of this study was to evaluate the MCBAs diversity produced by the gut microbiota through a metabolomics approach. In this study, fresh fecal samples from healthy donors were evaluated to explore the re-conjugation of chenodeoxycholic and 3-oxo-chenodeoxycholic acids by the human gut microbiota. No significant differences were found between the conversion trend of both BAs incubations. The in vitro results showed a clear trend to first accumulate the epimer isoursochenodeoxycholic acid and the dehydroxylated lithocholic acid derivatives in samples incubated with chenodeoxycholic and 3-oxo-chenodeoxycholic acid. They also showed a strong trend for the production of microbially conjugated dehydroxylated bile acids instead of chenodeoxycholic backbone conjugates. Different molecules and isomers of MCBAs were identified, and the new ones, valolithocholate ester and leucolithocholate ester, were identified and confirmed by MS/MS. These results document the gut microbiota’s capability to produce esters of MCBAs on hydroxyls of the sterol backbone in addition to amides at the C24 acyl site. This study opens a new perspective to study the BAs diversity produced by the human gut microbiota. Full article

Background: Alcohol-related brain degeneration is linked to cognitive-motor deficits and impaired signaling through insulin/insulin-like growth factor type 1 (IGF-1)-Akt pathways that regulate cell survival, plasticity, metabolism, and homeostasis. In addition, ethanol inhibits Aspartyl-asparaginyl-β-hydroxylase (ASPH), a downstream target of insulin/IGF-1-Akt signaling and an activator of Notch networks. Previous studies have suggested that early treatment with insulin sensitizers or dietary soy could reduce or prevent the long-term adverse effects of chronic ethanol feeding. Objective: The goal of this study was to assess the effects of substituting soy isolate for casein to prevent or reduce ethanol’s adverse effects on brain structure and function. Methods: Young adolescent male and female Long Evans were used in a 4-way model as follows: Control + Casein; Ethanol + Casein; Control + Soy; Ethanol + Soy; Control = 0% ethanol; Ethanol = 26% ethanol (caloric). Rats were fed isocaloric diets from 4 to 11 weeks of age. During the final experimental week, the Morris Water maze test was used to assess spatial learning (4 consecutive days), after which the brains were harvested to measure the temporal lobe expression of the total phospho-Akt pathway and downstream target proteins using multiplex bead-based enzyme-linked immunosorbent assays (ELISAs) and duplex ELISAs. Results: Ethanol inhibited spatial learning and reduced brain weight, insulin signaling through Akt, and the expression of ASPH when standard casein was provided as the protein source. The substitution of soy isolate for casein largely abrogated the adverse effects of chronic ethanol feeding. In contrast, Notch signaling protein expression was minimally altered by ethanol or soy isolate. Conclusions: These novel findings suggest that the insulin sensitizer properties of soy isolate may prevent some of the adverse effects that chronic ethanol exposure has on neurobehavioral function and insulin-regulated metabolic pathways in adolescent brains. Full article

Cell cryopreservation is an essential part of the biotechnology, food, and health care industries. There is a need to develop more effective, less toxic cryoprotective agents (CPAs) and methods, especially for mammalian cells. We investigated the impact of an insect antifreeze protein from Anatolica polita (ApAFP752) on mammalian cell cryopreservation using the human embryonic kidney cell line HEK 293T. An enhanced green fluorescent protein (EGFP)-tagged antifreeze protein, EGFP–ApAFP752, was transfected into the cells and the GFP was used to determine the efficiency of transfection. AFP was assessed for its cryoprotective effects intra- and extracellularly and both simultaneously at different concentrations with and without dimethyl sulfoxide (DMSO) at different concentrations. Comparisons were made to DMSO or medium alone. Cells were cryopreserved at −196 °C for ≥4 weeks. Upon thawing, cellular viability was determined using trypan blue, cellular damage was assessed by lactate dehydrogenase (LDH) assay, and cellular metabolism was measured using a metabolic activity assay (MTS). The use of this AFP significantly improved cryopreserved cell survival when used with DMSO intracellularly. Extracellular AFP also significantly improved cell survival when included in the DMSO freezing medium. Intra- and extracellular AFP used together demonstrated the most significantly increased cryoprotection compared to DMSO alone. These findings present a potential method to improve the viability of cryopreserved mammalian cells. Full article

The proto-oncogene MDM2 is frequently amplified in many human cancers and its overexpression is clinically associated with a poor prognosis. The oncogenic activity of MDM2 is demonstrated by its negative regulation of tumor suppressor p53 and the substrate proteins involved in DNA repair, cell cycle control, and apoptosis pathways. Thus, inhibition of MDM2 activity has been pursued as an attractive direction for the development of anti-cancer therapeutics. Virtual screening was performed using the crystal structure of the MDM2-MDMX RING domain dimer against a natural product library and identified a biflavonoid Hinokiflavone as a promising candidate compound targeting MDM2. Hinokiflavone was shown to bind the MDM2-MDMX RING domain and inhibit MDM2-mediated ubiquitination in vitro. Hinokiflavone treatment resulted in the downregulation of MDM2 and MDMX and induction of apoptosis in various cancer cell lines. Hinokiflavone demonstrated p53-dependent and -independent tumor-suppressive activity. This report provides biochemical and cellular evidence demonstrating the anti-cancer effects of Hinokiflavone through targeting the MDM2-MDMX RING domain. Full article

Arrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the β2-adernergic receptor (β2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognition. Full article

Our aim was to evaluate the expression of biomarkers, CD44v6, CD147, EGFR, p53, p63, p73, p16, and podoplanin in oral leukoplakias (OL) and to assess their potential for prediction of malignant transformation (MT). We analyzed the expression of CD44v6, CD147, EGFR, p53, p63, p73, p16, and podoplanin by immunohistochemistry in 52 OL, comprised of 41 low-grade (LG) dysplasia and 11 high-grade (HG) cases. Twelve healthy normal tissues (NT) were also included. Univariate and multivariate analysis were performed to evaluate any association with MT. Variable expression among the studied markers was observed, with a significant increase of high expression from NT to LG and HG cases in CD44v6 (p = 0.002), P53 (p = 0.002), P73 (p = 0.043), and podoplanin (p < 0.001). In multivariate analysis, cases with high podoplanin score showed a significant increased risk of MT (HR of 10.148 (95% CI of 1.503–68.532; p = 0.017). Furthermore, podoplanin combined with binary dysplasia grade obtained a HR of 10.238 (95% CI of 2.06–50.889; p = 0.004). To conclude, CD44v6, p53, p73, and podoplanin showed an increasing expression along the natural history of oral carcinogenesis. Podoplanin expression independently or combined with dysplasia grade could be useful predictive markers of MT in OL. Full article

Intermittent jumping force is an operational atomic-force microscopy mode that produces simultaneous topography and tip-sample maximum-adhesion images based on force spectroscopy. In this work, the operation conditions have been implemented scanning in a repulsive regime and applying very low forces, thus avoiding unspecific tip-sample forces. Remarkably, adhesion images give only specific rupture events, becoming qualitative and quantitative molecular recognition maps obtained at reasonably fast rates, which is a great advantage compared to the force–volume modes. This procedure has been used to go further in discriminating between two similar protein molecules, avidin and streptavidin, in hybrid samples. The adhesion maps generated scanning with biotinylated probes showed features identified as avidin molecules, in the range of 40–80 pN; meanwhile, streptavidin molecules rendered 120–170 pN at the selected working conditions. The gathered results evidence that repulsive jumping force mode applying very small forces allows the identification of biomolecules through the specific rupture forces of the complexes and could serve to identify receptors on membranes or samples or be applied to design ultrasensitive detection technologies. Full article

Endocrine disruptors (EDs) are chemical substances capable of affecting endocrine system functioning and interfering with organ morphogenesis and physiological functions. The development and regeneration of bone tissues have a complex hormonal regulation, and therefore, bone tissue cells can be considered potential targets for endocrine disruptors. In that regard, the aim of this research was to investigate the impact of ED exposure on the inflammatory response and oxidative stress in an experimental model of collagen-induced arthritis (CIA). Arthritis was induced by an emulsion of type II collagen (CII) and complete Freund’s adjuvant, which was administered intradermally on days 0 and 21. Mice from day 21 to day 35 received the following EDs by oral gavage: cypermethrin (CP), diethyl phthalate (DEP), vinclozolin (VCZ), 17α-ethinylestradiol (EE), perfluorooctanesulfonic acid (PFOS) and atrazine (ATR). ED exposure caused worsening of clinical signs (erythema and edema in the hind paws), histological and radiographic changes, as well as behavioral deficits, induced by CII injections. Furthermore, ED exposure significantly increased the degree of inflammation and oxidative damage induced by arthritis; this upregulation was more evident after exposure to ATR than to other EDs. The results from our study suggest that exposure to EDs may play a deleterious role in the progression of RA; therefore, exposure to EDs should be limited. Full article

Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties. Full article

There are several links between insulin resistance and neurodegenerative disorders such as Parkinson’s disease. However, the direct influence of insulin signaling on abnormal α-synuclein accumulation—a hallmark of Parkinson’s disease—remains poorly explored. To our best knowledge, this work is the first attempt to investigate the direct effects of insulin signaling on pathological α-synuclein accumulation induced by the addition of α-synuclein preformed fibrils in primary dopaminergic neurons. We found that modifying insulin signaling through (1) insulin receptor inhibitor GSK1904529A, (2) SHIP2 inhibitor AS1949490 or (3) PTEN inhibitor VO-OHpic failed to significantly affect α-synuclein aggregation in dopaminergic neurons, in contrast to the aggregation-reducing effects observed after the addition of glial cell line-derived neurotrophic factor. Subsequently, we tested different media formulations, with and without insulin. Again, removal of insulin from cell culturing media showed no effect on α-synuclein accumulation. We observed, however, a reduced α-synuclein aggregation in neurons cultured in neurobasal medium with a B27 supplement, regardless of the presence of insulin, in contrast to DMEM/F12 medium with an N2 supplement. The effects of culture conditions were present only in dopaminergic but not in primary cortical or hippocampal cells, indicating the unique sensitivity of the former. Altogether, our data contravene the direct involvement of insulin signaling in the modulation of α-synuclein aggregation in dopamine neurons. Moreover, we show that the choice of culturing media can significantly affect preformed fibril-induced α-synuclein phosphorylation in a primary dopaminergic cell culture. Full article

Protein kinases are major drug targets. Most kinase inhibitors are directed against the adenosine triphosphate (ATP) cofactor binding site, which is largely conserved across the human kinome. Hence, such kinase inhibitors are often thought to be promiscuous. However, experimental evidence and activity data for publicly available kinase inhibitors indicate that this is not generally the case. We have investigated whether inhibitors of closely related human kinases with single- or multi-kinase activity can be differentiated on the basis of chemical structure. Therefore, a test system consisting of two distinct kinase triplets has been devised for which inhibitors with reported triple-kinase activities and corresponding single-kinase activities were assembled. Machine learning models derived on the basis of chemical structure distinguished between these multi- and single-kinase inhibitors with high accuracy. A model-independent explanatory approach was applied to identify structural features determining accurate predictions. For both kinase triplets, the analysis revealed decisive features contained in multi-kinase inhibitors. These features were found to be absent in corresponding single-kinase inhibitors, thus providing a rationale for successful machine learning. Mapping of features determining accurate predictions revealed that they formed coherent and chemically meaningful substructures that were characteristic of multi-kinase inhibitors compared with single-kinase inhibitors. Full article

Intercellular communication between monocytes/macrophages and cells involved in tissue regeneration, such as mesenchymal stromal cells (MSCs) and primary tissue cells, is essential for tissue regeneration and recovery of homeostasis. Typically, in the final phase of the inflammation-resolving process, this intercellular communication drives an anti-inflammatory immunomodulatory response. To obtain a safe and effective treatment to counteract the cytokine storm associated with a disproportionate immune response to severe infections, including that associated with COVID-19, by means of naturally balanced immunomodulation, our group has standardized the production under GMP-like conditions of a secretome by coculture of macrophages and MSCs. To characterize this proteome, we determined the expression of molecules related to cellular immune response and tissue regeneration, as well as its possible toxicity and anti-inflammatory potency. The results show a specific molecular pattern of interaction between the two cell types studied, with an anti-inflammatory and regenerative profile. In addition, the secretome is not toxic by itself on human PBMC or on THP-1 monocytes and prevents lipopolysaccharide (LPS)-induced growth effects on those cell types. Finally, PRS CK STORM prevents LPS-induced TNF-A and IL-1Β secretion from PBMC and from THP-1 cells at the same level as hydrocortisone, demonstrating its anti-inflammatory potency. Full article

The development of antimicrobial agents against multidrug-resistant bacteria is an important medical challenge. Antimicrobial peptides (AMPs), human cathelicidin LL-37 and its derivative OP-145, possess a potent antimicrobial activity and were under consideration for clinical trials. In order to overcome some of the challenges to their therapeutic potential, a very promising AMP, SAAP-148 was designed. Here, we studied the mode of action of highly cationic SAAP-148 in comparison with OP-145 on membranes of Enterococcus hirae at both cellular and molecular levels using model membranes composed of major constituents of enterococcal membranes, that is, anionic phosphatidylglycerol (PG) and cardiolipin (CL). In all assays used, SAAP-148 was consistently more efficient than OP-145, but both peptides displayed pronounced time and concentration dependences in killing bacteria and performing at the membrane. At cellular level, Nile Red-staining of enterococcal membranes showed abnormalities and cell shrinkage, which is also reflected in depolarization and permeabilization of E. hirae membranes. At the molecular level, both peptides abolished the thermotropic phase transition and induced disruption of PG/CL. Interestingly, the membrane was disrupted before the peptides neutralized the negative surface charge of PG/CL. Our results demonstrate that SAAP-148, which kills bacteria at a significantly lower concentration than OP-145, shows stronger effects on membranes at the cellular and molecular levels. Full article

Cocaine use disorder has been reported to cause transgenerational effects. However, due to the lack of standardized biomarkers, the effects of cocaine use during pregnancy on postnatal development and long-term neurobiological and behavioral outcomes have not been investigated thoroughly. Therefore, in this study, we examined extracellular vesicles (EVs) in adult (~12 years old) female and male rhesus monkeys prenatally exposed to cocaine (n = 11) and controls (n = 9). EVs were isolated from the cerebrospinal fluid (CSF) and characterized for the surface expression of specific tetraspanins, concentration (particles/mL), size distribution, and cargo proteins by mass spectrometry (MS). Transmission electron microscopy following immunogold labeling for tetraspanins (CD63, CD9, and CD81) confirmed the successful isolation of EVs. Nanoparticle tracking analyses showed that the majority of the particles were <200 nm in size, suggesting an enrichment for small EVs (sEV). Interestingly, the prenatally cocaine-exposed group showed ~54% less EV concentration in CSF compared to the control group. For each group, MS analyses identified a number of proteins loaded in CSF-EVs, many of which are commonly listed in the ExoCarta database. Ingenuity pathway analysis (IPA) demonstrated the association of cargo EV proteins with canonical pathways, diseases and disorders, upstream regulators, and top enriched network. Lastly, significantly altered proteins between groups were similarly characterized by IPA, suggesting that prenatal cocaine exposure could be potentially associated with long-term neuroinflammation and risk for neurodegenerative diseases. Overall, these results indicate that CSF-EVs could potentially serve as biomarkers to assess the transgenerational adverse effects due to prenatal cocaine exposure. Full article

In recent years, researchers have leveraged the ubiquitin-proteasome system (UPS) to induce selective degradation of proteins by E3 ubiquitin ligases, which has great potential as novel therapeutics for human diseases, including cancer and neurodegenerative disorders. However, despite extensive efforts, only a handful of ~600 human E3 ligases were utilized, and numerous protein–protein interaction surfaces on E3 ligases were not explored. To tackle these problems, we leveraged a structure-based protein engineering technology to develop a multi-domain fusion protein bringing functional E3 ligases to the proximity of a target protein to trigger its proteasomal degradation, which we termed Ubiquitin Variant Induced Proximity (UbVIP). We first generated non-inhibitory synthetic UbV binders for a selected group of human E3 ligases. With these UbVs employed as E3 ligase engagers, we designed a library of UbVIPs targeting a DNA damage response protein 53BP1. We observed that two UbVIPs recruiting RFWD3 and NEDD4L could effectively induce proteasome degradation of 53BP1 in human cell lines. This provides a proof-of-principle that UbVs can act as a means of targeted degradation for nucleus-localized proteins. Our work demonstrated that UbV technology is suitable to develop protein-based molecules for targeted degradation and can help identify novel E3 ligases for future therapeutic development. Full article

Melanocortin Receptor Accessory Protein 2 (MRAP2) modulates the trafficking and signal transduction of several G-protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, such as Prokineticin receptors (PKRs). They bind the endogenous ligand prokineticin 2 (PK2), a novel adipokine that has an anorexic effect and modulates thermoregulation and energy homeostasis. In the present work, we used biochemical techniques to analyze the mechanism of interaction of MRAP2 with PKR2 and we identified the specific amino acid regions involved in the complex formation. Our results indicate that MRAP2 likely binds to the N-terminal region of PKR2, preventing glycosylation and consequently the correct receptor localization. We also identified a C-terminal region of MRAP2 that is critical for the interaction with PKR2. Consequently, we analyzed the role of the prokineticin transduction system in the regulation of MRAP2 expression in tissues involved in the control of food intake: at the central level, in hypothalamic explants, and at the peripheral level, in adipocytes. We demonstrated the modulation of MRAP2 expression by the prokineticin transduction system. Full article

For single-molecule studies in solution, very small concentrations of dye-labelled molecules are employed in order to achieve single-molecule sensitivity. In typical studies with confocal microscopes, often concentrations in the pico-molar regime are required. For various applications that make use of single-molecule Förster resonance energy transfer (smFRET) or two-color coincidence detection (TCCD), the molecule concentration must be set explicitly to targeted values and furthermore needs to be stable over a period of several hours. As a consequence, specific demands must be imposed on the surface passivation of the cover slides during the measurements. The aim of having only one molecule in the detection volume at the time is not only affected by the absolute molecule concentration, but also by the rate of diffusion. Therefore, we discuss approaches to control and to measure absolute molecule concentrations. Furthermore, we introduce an approach to calculate the probability of chance coincidence events and demonstrate that measurements with challenging smFRET samples require a strict limit of maximal sample concentrations in order to produce meaningful results. Full article

Parkinson’s disease (PD) is associated with motor and non-motor symptoms and characterized by aggregates of alpha-synuclein (αSyn). Naturally occurring antibodies (nAbs) are part of the innate immune system, produced without prior contact to their specific antigen, and polyreactive. The abundance of nAbs against αSyn is altered in patients with PD. In this work, we biophysically characterized nAbs against αSyn (nAbs-αSyn) and determined their biological effects. nAbs-αSyn were isolated from commercial intravenous immunoglobulins using column affinity purification. Biophysical properties were characterized using a battery of established in vitro assays. Biological effects were characterized in HEK293T cells transiently transfected with fluorescently tagged αSyn. Specific binding of nAbs-αSyn to monomeric αSyn was demonstrated by Dot blot, ELISA, and Surface Plasmon Resonance. nAbs-αSyn did not affect viability of HEK293T cells as reported by Cell Titer Blue and LDH Assays. nAbs-αSyn inhibited fibrillation of αSyn reported by the Thioflavin T aggregation assay. Altered fibril formation was confirmed with atomic force microscopy. In cells transfected with EGFP-tagged αSyn we observed reduced formation of aggresomes, perinuclear accumulations of αSyn aggregates. The results demonstrate that serum of healthy individuals contains nAbs that specifically bind αSyn and inhibit aggregation of αSyn in vitro. The addition of nAbs-αSyn to cultured cells affects intracellular αSyn aggregates. These findings help understanding the role of the innate immune systems for the pathogenesis of PD and suggest that systemic αSyn binding agents could potentially affect neuronal αSyn pathology. Full article

Hyperglycaemia and its resulting glucotoxicity are among the most prominent hallmarks of diabetes mellitus (DM) development. Persistent hyperglycaemia further leads to oxidative stress via mitochondrial dysfunction and subsequent ER stress onset, while associated hyperlipidaemia triggers the adipose tissue to secrete pro-inflammatory cytokines. In this study, the effect of calystegines has been investigated in an experimental model of hyperglycaemia induced on human ASCs cells. Different cellular pathways including apoptosis, oxidative and ER stress, inflammation as well as Pi3K/AKT/mTOR metabolic-associated axis have been evaluated by means on RT-qPCR, western blot, and flow cytometry techniques. Treatment of HuASCs cells with calystegines strongly promoted the hyperglycaemic cells survival and significantly diminished oxidative stress, mitochondrial dynamics failure and ER stress, while improving the endogenous cellular antioxidant defenses. Interestingly, nortropane alkaloids efficiently prevented the hyperglycaemia-mediated inflammatory response, as evidenced by the regulation of the pro- and anti-inflammatory response in HuASCs cells. Finally, we evidenced that calystegines may exert their protective effect on HuASCs cells metabolic functions through the restoration of the defective PI3K/AKT/mTOR pathway. Overall, the present investigation demonstrated that calystegines possess important abilities to protect HuASCs against hyperglycaemia-induced cellular dysfunction, and it evidenced that the observed effects are associated to the promotion of PI3K/AKT/mTOR pathway. Full article

Aldose reductase (ALR2) is the enzyme in charge of developing cellular toxicity caused by diabetic hyperglycemia, which in turn leads to the generation of reactive oxygen species triggering oxidative stress. Therefore, inhibiting ALR2 while pursuing a concomitant anti-oxidant activity through dual-acting agents is now recognized as the gold standard treatment for preventing or at least delaying the progression of diabetic complications. Herein we describe a novel series of (E)-benzaldehyde O-benzyl oximes 6a–e, 7a–e, 8a–e, and 9–11 as ALR2 inhibitors endowed with anti-oxidant properties. Inspired by the natural products, the synthesized derivatives are characterized by a different polyhydroxy substitution pattern on their benzaldehyde fragment, which proved crucial for both the enzyme inhibitory activity and the anti-oxidant capacity. Derivatives (E)-2,3,4-trihydroxybenzaldehyde O-(3-methoxybenzyl) oxime (7b) and (E)-2,3,4-trihydroxybenzaldehyde O-(4-methoxybenzyl) oxime (8b) turned out to be the most effective dual-acting products, proving to combine the best ALR2 inhibitory properties with significant anti-oxidant efficacy. Full article

Cell adhesion molecule L1 is a cell surface glycoprotein that promotes neuronal cell migration, fosters regeneration after spinal cord injury and ameliorates the consequences of neuronal degeneration in mouse and zebrafish models. Counter-indicative features of L1 were found in tumor progression: the more L1 is expressed, the more tumor cells migrate and increase their metastatic potential. L1′s metastatic potential is further evidenced by its promotion of epithelial–mesenchymal transition, endothelial cell transcytosis and resistance to chemo- and radiotherapy. These unfortunate features are indicated by observations that cells that normally do not express L1 are induced to express it when becoming malignant. With the aim to ameliorate the devastating functions of L1 in tumors, we designed an alternative approach to counteract tumor cell migration. Libraries of small organic compounds were screened using the ELISA competition approach similar to the one that we used for identifying L1 agonistic mimetics. Whereas in the former approach, a function-triggering monoclonal antibody was used for screening libraries, we here used the function-inhibiting monoclonal antibody 324 that reduces the migration of neurons. We now show that the L1 antagonistic mimetics anagrelide, 2-hydroxy-5-fluoropyrimidine and mestranol inhibit the migration of cultured tumor cells in an L1-dependent manner, raising hopes for therapy. Full article

One of the most desirable properties that biomaterials designed for tissue engineering or drug delivery applications should fulfill is biodegradation and resorption without toxicity. Therefore, there is an increasing interest in the development of biomaterials able to be enzymatically degraded once implanted at the injury site or once delivered to the target organ. In this paper, we demonstrate the protease sensitivity of self-assembling amphiphilic peptides, in particular, RAD16-I (AcN-RADARADARADARADA-CONH₂), which contains four potential cleavage sites for trypsin. We detected that when subjected to thermal denaturation, the peptide secondary structure suffers a transition from β-sheet to random coil. We also used Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) to detect the proteolytic breakdown products of samples subjected to incubation with trypsin as well as atomic force microscopy (AFM) to visualize the effect of the degradation on the nanofiber scaffold. Interestingly, thermally treated samples had a higher extent of degradation than non-denatured samples, suggesting that the transition from β-sheet to random coil leaves the cleavage sites accessible and susceptible to protease degradation. These results indicate that the self-assembling peptide can be reduced to short peptide sequences and, subsequently, degraded to single amino acids, constituting a group of naturally biodegradable materials optimal for their application in tissue engineering and regenerative medicine. Full article

Passive immunotherapy is a very promising approach for the treatment of Alzheimer’s disease (AD). Among the different antibodies under development, those targeting post-translationally modified Aβ peptides might combine efficient reduction in beta-amyloid accompanied by lower sequestration in peripheral compartments and thus anticipated and reduced treatment-related side effects. In that regard, we recently demonstrated that the antibody-mediated targeting of isoD7-modified Aβ peptides leads to the attenuation of AD-like amyloid pathology in 5xFAD mice. In order to assess novel strategies to enhance the efficacy of passive vaccination approaches, we investigated the role of CD33 for Aβ phagocytosis in transgenic mice treated with an isoD7-Aβ antibody. We crossbred 5xFAD transgenic mice with CD33 knock out (CD33KO) mice and compared the amyloid pathology in the different genotypes of the crossbreds. The knockout of CD33 in 5xFAD mice leads to a significant reduction in Aβ plaques and concomitant rescue of behavioral deficits. Passive immunotherapy of 5xFAD/CD33KO showed a significant increase in plaque-surrounding microglia compared to 5xFAD treated with the antibody. Additionally, we observed a stronger lowering of Aβ plaque load after passive immunotherapy in 5xFAD/CD33KO mice. The data suggest an additive effect of passive immunotherapy and CD33KO in terms of lowering Aβ pathology. Hence, a combination of CD33 antagonists and monoclonal antibodies might represent a strategy to enhance efficacy of passive immunotherapy in AD. Full article

IMGT^®, the international ImMunoGeneTics information system^®, created in 1989, by Marie-Paule Lefranc (Université de Montpellier and CNRS), marked the advent of immunoinformatics, a new science which emerged at the interface between immunogenetics and bioinformatics for the study of the adaptive immune responses. IMGT^® is based on a standardized nomenclature of the immunoglobulin (IG) and T cell receptor (TR) genes and alleles from fish to humans and on the IMGT unique numbering for the variable (V) and constant (C) domains of the immunoglobulin superfamily (IgSF) of vertebrates and invertebrates, and for the groove (G) domain of the major histocompatibility (MH) and MH superfamily (MhSF) proteins. IMGT^® comprises 7 databases, 17 tools and more than 25,000 pages of web resources for sequences, genes and structures, based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts. IMGT^® reference directories are used for the analysis of the NGS high-throughput expressed IG and TR repertoires (natural, synthetic and/or bioengineered) and for bridging sequences, two-dimensional (2D) and three-dimensional (3D) structures. This manuscript focuses on the IMGT^®Homo sapiens IG and TR loci, gene order, copy number variation (CNV) and haplotypes new concepts, as a paradigm for jawed vertebrates genome assemblies. Full article