Mitigating cognitive deficits with teriflunomide: unraveling PI3K-modulated behavioral outcomes in mice.

BACKGROUND: Alzheimer's disease is a leading neurological disorder that gradually impairs memory and cognitive abilities, ultimately leading to the inability to perform even basic daily tasks. Teriflunomide is known to preserve neuronal activity and protect mitochondria in the brain slices exposed to oxidative stress. The current research was undertaken to investigate the teriflunomide's cognitive rescuing abilities against scopolamine-induced comorbid cognitive impairment and its influence on phosphatidylinositol-3-kinase (PI3K) inhibition-mediated behavior alteration in mice. METHODS: Swiss albino mice were divided into 7 groups; vehicle control, scopolamine, donepezil + scopolamine, teriflunomide (10 mg/kg) + scopolamine; teriflunomide (20 mg/kg) + scopolamine, LY294002 and LY294002 + teriflunomide (20 mg/kg). Mice underwent a nine-day protocol, receiving scopolamine injections (2 mg/kg) for the final three days to induce cognitive impairment. Donepezil, teriflunomide, and LY294002 treatments were given continuously for 9 days. MWM, Y-maze, OFT and rota-rod tests were conducted on days 7 and 9. On the last day, blood samples were collected for serum TNF-α analysis, after which the mice were sacrificed, and brain samples were harvested for oxidative stress analysis. RESULTS: Scopolamine administration for three consecutive days increased the time required to reach the platform in the MWM test, whereas, reduced the percentage of spontaneous alternations in the Y-maze, number of square crossing in OFT and retention time in the rota-rod test. In biochemical analysis, scopolamine downregulated the brain GSH level, whereas it upregulated the brain TBARS and serum TNF-α levels. Teriflunomide treatment effectively mitigated all the behavioral and biochemical alterations induced by scopolamine. Furthermore, LY294002 administration reduced the memory function and GSH level, whereas, uplifted the serum TNF-α levels. Teriflunomide abrogated the memory-impairing, GSH-lowering, and TNF-α-increasing effects of LY294002. CONCLUSION: Our results delineate that the improvement in memory, locomotion, and motor coordination might be attributed to the oxidative and inflammatory stress inhibitory potential of teriflunomide. Moreover, PI3K inhibition-induced memory impairment might be attributed to reduced GSH levels and increased TNF-α levels.

Enantiomer-Specific Cardiovascular Effects of the Ketone Body 3-Hydroxybutyrate.

BACKGROUND: The ketone body 3-hydroxybutyrate (3-OHB) increases cardiac output (CO) by 35% to 40% in healthy people and people with heart failure. The mechanisms underlying the effects of 3-OHB on myocardial contractility and loading conditions as well as the cardiovascular effects of its enantiomeric forms, D-3-OHB and L-3-OHB, remain undetermined. METHODS AND RESULTS: Three groups of 8 pigs each underwent a randomized, crossover study. The groups received 3-hour infusions of either D/L-3-OHB (racemic mixture), 100% L-3-OHB, 100% D-3-OHB, versus an isovolumic control. The animals were monitored with pulmonary artery catheter, left ventricle pressure-volume catheter, and arterial and coronary sinus blood samples. Myocardial biopsies were evaluated with high-resolution respirometry, coronary arteries with isometric myography, and myocardial kinetics with D-[11C]3-OHB and L-[11C]3-OHB positron emission tomography. All three 3-OHB infusions increased 3-OHB levels (P<0.001). D/L-3-OHB and L-3-OHB increased CO by 2.7 L/min (P<0.003). D-3-OHB increased CO nonsignificantly (P=0.2). Circulating 3-OHB levels correlated with CO for both enantiomers (P<0.001). The CO increase was mediated through arterial elastance (afterload) reduction, whereas contractility and preload were unchanged. Ex vivo, D- and L-3-OHB dilated coronary arteries equally. The mitochondrial respiratory capacity remained unaffected. The myocardial 3-OHB extraction increased only during the D- and D/L-3-OHB infusions. D-[11C]3-OHB showed rapid cardiac uptake and metabolism, whereas L-[11C]3-OHB demonstrated much slower pharmacokinetics. CONCLUSIONS: 3-OHB increased CO by reducing afterload. L-3-OHB exerted a stronger hemodynamic response than D-3-OHB due to higher circulating 3-OHB levels. There was a dissocitation between the myocardial metabolism and hemodynamic effects of the enantiomers, highlighting L-3-OHB as a potent cardiovascular agent with strong hemodynamic effects.

In vitro degradation and antibacterial activity of bacterial cellulose deposited flax fabric reinforced with polylactic acid and polyhydroxybutyrate.

The objective of this study was to prepare biocomposites through the solution casting method followed by compression moulding in which bacterial cellulose (BC) deposited flax fabric (FF) produced through fermentation is coated with minimal amount of polylactic acid (PLA) and polyhydroxybutyrate (PHB). Biocomposites incorporated with 60 % of PLA or PHB (% w/w) show enhanced tensile strength. Cross-sectional morphology showed good superficial interaction of these biopolymers with fibres of FF thereby filling up the gaps present between the fibres. The tensile strength of biocomposites at 60 % PLA and 60 % PHB improved from 37.97 MPa (i.e., BC deposited FF produced in presence of honey) to 67.17 MPa and 56.26 MPa, respectively. Further, 0.25 % of nalidixic acid (NA) (% w/w) and 6 % of oleic acid (OA) (% w/w) incorporation into the biocomposites imparted prolonged antibacterial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The in vitro cytotoxicity of biocomposites was determined using L929 mouse fibroblast cells. The 3-(4,5-cime- thylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cytotoxicity tests showed that the PHB derived biocomposites along with antibacterial compounds in it were non-toxic. In vitro degradation of biocomposites was measured for up to 8 weeks in the mimicked physiological environment that showed a gradual rate of degradation over the period.

In vitro neuronal and glial response to magnetically stimulated piezoelectric poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV)/cobalt ferrite (CFO) microspheres.

Polymer biomaterials are being considered for tissue regeneration due to the possibility of resembling different extracellular matrix characteristics. However, most current scaffolds cannot respond to physical-chemical modifications of the cell microenvironment. Stimuli-responsive materials, such as electroactive smart polymers, are increasingly gaining attention once they can produce electrical potentials without external power supplies. The presence of piezoelectricity in human tissues like cartilage and bone highlights the importance of electrical stimulation in physiological conditions. Although poly(vinylidene fluoride) (PVDF) is one of the piezoelectric polymers with the highest piezoelectric response, it is not biodegradable. Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) is a promising copolymer of poly(hydroxybutyrate) (PHB) for tissue engineering and regeneration applications. It offers biodegradability, piezoelectric properties, biocompatibility, and bioactivity, making it a superior option to PVDF for biomedical purposes requiring biodegradability. Magnetoelectric polymer composites can be made by combining magnetostrictive particles and piezoelectric polymers to further tune their properties for tissue regeneration. These composites convert magnetic stimuli into electrical stimuli, generating local electrical potentials for various applications. Cobalt ferrites (CFO) and piezoelectric polymers have been combined and processed into different morphologies, maintaining biocompatibility for tissue engineering. The present work studied how PHBV/CFO microspheres affected neural and glial response in spinal cord cultures. It is expected that the electrical signals generated by these microspheres due to their magnetoelectric nature could aid in tissue regeneration and repair. PHBV/CFO microspheres were not cytotoxic and were able to impact neurite outgrowth and promote neuronal differentiation. Furthermore, PHBV/CFO microspheres led to microglia activation and induced the release of several bioactive molecules. Importantly, magnetically stimulated microspheres ameliorated cell viability after an in vitro ROS-induced lesion of spinal cord cultures, which suggests a beneficial effect on tissue regeneration and repair.

Magnetoactive Composite Conduits Based on Poly(3-hydroxybutyrate) and Magnetite Nanoparticles for Repair of Peripheral Nerve Injury.

Peripheral nerve injury poses a threat to the mobility and sensitivity of a nerve, thereby leading to permanent function loss due to the low regenerative capacity of mature neurons. To date, the most widely clinically applied approach to bridging nerve injuries is autologous nerve grafting, which faces challenges such as donor site morbidity, donor shortages, and the necessity of a second surgery. An effective therapeutic strategy is urgently needed worldwide to overcome the current limitations. Herein, a magnetic nerve guidance conduit (NGC) based on biocompatible biodegradable poly(3-hydroxybutyrate) (PHB) and 8 wt % of magnetite nanoparticles modified by citric acid (Fe3O4-CA) was fabricated by electrospinning. The crystalline structure of NGCs was studied by X-ray diffraction, which indicated an enlarged ß-phase of PHB in the composite conduit compared to a pure PHB conduit. Tensile tests revealed greater ductility of PHB/Fe3O4-CA: the composite conduit has Young's modulus of 221 ± 52 MPa and an elongation at break of 28.6 ± 2.9%, comparable to clinical materials. Saturation magnetization (σs) of Fe3O4-CA and PHB/Fe3O4-CA is 61.88 ± 0.29 and 7.44 ± 0.07 emu/g, respectively. The water contact angle of the PHB/Fe3O4-CA conduit is lower as compared to pure PHB, while surface free energy (σ) is significantly higher, which was attributed to higher surface roughness and an amorphous phase as well as possible PHB/Fe3O4-CA interface interactions. In vitro, the conduits supported the proliferation of rat mesenchymal stem cells (rMSCs) and SH-SY5Y cells in a low-frequency magnetic field (0.67 Hz, 68 mT). In vivo, the conduits were used to bridge damaged sciatic nerves in rats; pure PHB and composite PHB/Fe3O4-CA conduits did not cause acute inflammation and performed a barrier function, which promotes nerve regeneration. Thus, these conduits are promising as implants for the regeneration of peripheral nerves.

Comparing the efficacy in reducing brain injury of different neuroprotective agents following neonatal hypoxia-ischemia in newborn rats: a multi-drug randomized controlled screening trial.

Intrapartum hypoxia-ischemia leading to neonatal encephalopathy (NE) results in significant neonatal mortality and morbidity worldwide, with > 85% of cases occurring in low- and middle-income countries (LMIC). Therapeutic hypothermia (HT) is currently the only available safe and effective treatment of HIE in high-income countries (HIC); however, it has shown limited safety or efficacy in LMIC. Therefore, other therapies are urgently required. We aimed to compare the treatment effects of putative neuroprotective drug candidates following neonatal hypoxic-ischemic (HI) brain injury in an established P7 rat Vannucci model. We conducted the first multi-drug randomized controlled preclinical screening trial, investigating 25 potential therapeutic agents using a standardized experimental setting in which P7 rat pups were exposed to unilateral HI brain injury. The brains were analysed for unilateral hemispheric brain area loss after 7 days survival. Twenty animal experiments were performed. Eight of the 25 therapeutic agents significantly reduced brain area loss with the strongest treatment effect for Caffeine, Sonic Hedgehog Agonist (SAG) and Allopurinol, followed by Melatonin, Clemastine, ß-Hydroxybutyrate, Omegaven, and Iodide. The probability of efficacy was superior to that of HT for Caffeine, SAG, Allopurinol, Melatonin, Clemastine, ß-hydroxybutyrate, and Omegaven. We provide the results of the first systematic preclinical screening of potential neuroprotective treatments and present alternative single therapies that may be promising treatment options for HT in LMIC.

Teriflunomide as a therapeutic means for myelin repair.

BACKGROUND: Promotion of myelin repair in the context of demyelinating diseases such as multiple sclerosis (MS) still represents a clinical unmet need, given that this disease is not only characterized by autoimmune activities but also by impaired regeneration processes. Hence, this relates to replacement of lost oligodendrocytes and myelin sheaths-the primary targets of autoimmune attacks. Endogenous remyelination is mainly mediated via activation and differentiation of resident oligodendroglial precursor cells (OPCs), whereas its efficiency remains limited and declines with disease progression and aging. Teriflunomide has been approved as a first-line treatment for relapsing remitting MS. Beyond its role in acting via inhibition of de novo pyrimidine synthesis leading to a cytostatic effect on proliferating lymphocyte subsets, this study aims to uncover its potential to foster myelin repair. METHODS: Within the cuprizone mediated de-/remyelination model teriflunomide dependent effects on oligodendroglial homeostasis and maturation, related to cellular processes important for myelin repair were analyzed in vivo. Teriflunomide administration was performed either as pulse or continuously and markers specific for oligodendroglial maturation and mitochondrial integrity were examined by means of gene expression and immunohistochemical analyses. In addition, axon myelination was determined using electron microscopy. RESULTS: Both pulse and constant teriflunomide treatment efficiently boosted myelin repair activities in this model, leading to accelerated generation of oligodendrocytes and restoration of myelin sheaths. Moreover, teriflunomide restored mitochondrial integrity within oligodendroglial cells. CONCLUSIONS: The link between de novo pyrimidine synthesis inhibition, oligodendroglial rescue, and maintenance of mitochondrial homeostasis appears as a key for successful myelin repair and hence for protection of axons from degeneration.

On the bioprotective effects of 3-hydroxybutyrate: Thermodynamic study of binary 3HB-water systems.

Microorganisms must face various inconvenient conditions; therefore, they developed several approaches for protection. Such a strategy also involves the accumulation of compatible solutes, also called osmolytes. It has been proved that the monomer unit 3-hydroxybutyrate (3HB), which is present in sufficient concentration in poly(3-hydroxybutyrate) (PHB)-accumulating cells, serves as a chemical chaperone protecting enzymes against heat and oxidative stress and as a cryoprotectant for enzymes, bacterial cells, and yeast. The stress robustness of the cells is also strongly dependent on the behavior and state of intracellular water, especially during stress exposure. For a better understanding of the protective mechanism and effect of strongly hydrophilic 3HB in solutions at a wide range of temperatures, a binary phase diagram of system sodium 3HB (Na3HB)-water in equilibrium and the state diagrams showing the glass transitions in the system were constructed. To investigate the activity of water in various compositions of the Na3HB/water system, three experimental techniques have been used (dynamic water sorption analysis, water activity measurements, and sorption calorimetry). First, Na3HB proved its hydrophilic nature, which is very comparable with known compatible solutes (trehalose). Results of differential scanning calorimetry demonstrated that Na3HB is also highly effective in depressing the freezing point and generating a large amount of nonfrozen water (1.35 g of water per gram of Na3HB). Therefore, Na3HB represents a very effective cryoprotectant that can be widely used for numerous applications.

3-Hydroxybutyrate helps crayfish resistant to Vibrio parahaemolyticus infection in versatile ways.

The bacterial storage compound poly-ß-hydroxybutyrate (PHB) is a potential bio-control agent in aquaculture. It has been reported that PHB benefit to the survival and growth, and improve their immunity of aquatic animals. However, the cellular and molecular regulation mechanisms of PHB in immunity process remain unclear. This study investigated the immune mechanism of hemocytes regulated by Halomonas-PHB (PHB-HM) and PHB monomer 3-HB. Red claw crayfish Cherax quadricarinatus was used as the experimental animal in cytological study. Fluorescence microscopy and flow cytometry (FCM) analysis indicated that PHB-HM labeled with fluorescein isothiocyanate (FITC) could be engulfed by granulocytes (Gs) and semi-granulocytes (SGs) upon in vitro incubation. Transmission electron microscopy (TEM) further showed the ongoing degradation of PHB granules inside Gs and SGs after the injection of PHB-HM into crayfish sinus, but phagocytosis of PHB-HM by hyalinocyte (H) was not observed. Therefore, Gs and SGs are considered the main effector cells of cellular immunity induced by PHB-HM, and SGs likely played a particular important role in this process. To study the biosafety and molecular mechanism of PHB monomer 3-HB, hemocyte viability and expression of the related genes were determined after being exposed to 0-1 mg/mL of 3-HB, and Vibrio parahaemolyticus (VP) was used as the pathogenic bacterium. The results confirmed that 3-HB had no toxic effect on hemocytes by means of cell viability assay, and supplementation with 1 mg/mL of 3-HB suppressed the growth rate of VP in TSB medium. Moreover, injection of 3-HB into the blood sinus of crayfish remarkably improved the phagocytic rate of Gs and SGs on VP. Furthermore, transcriptome assay was designed to illuminate the molecular mechanism of 3-HB regulation using red swamp crayfish Procambarus clarkii as experimental animals. RNA-seq analysis and qRT-PCR verification revealed that the microtubule and cytoskeleton-related genes were high expressed 3 h after 3-HB injection, indicating both genes might involve in building up the innate immunity. In summary, bacterial storage PHB could be phagocytosed by main effector blood cells and likely to be degraded within the cells. 3-HB helped the crayfish resistant to pathogens through improving phagocytosis, suppressing the growth of pathogenic bacteria, and increasing the expression of microtubule-related genes. The findings in this work provide cytological and molecular evidence which will facilitate the application of PHB and 3-HB as immune-control agents in farming of aquatic animals.

The novel sustained 3-hydroxybutyrate donor poly-D-3-hydroxybutyric acid prevents inflammatory bowel disease through upregulation of regulatory T-cells.

Inflammatory bowel disease (IBD) is a chronic persistent intestinal disorder, with ulcerative colitis and Crohn's disease being the most common. However, the physio-pathological development of IBD is still unknown. Therefore, research on the etiology and treatment of IBD has been conducted using a variety of approaches. Short-chain fatty acids such as 3-hydroxybutyrate (3-HB) are known to have various physiological activities. In particular, the production of 3-HB by the intestinal microflora is associated with the suppression of various inflammatory diseases. In this study, we investigated whether poly-D-3-hydroxybutyric acid (PHB), a polyester of 3-HB, is degraded by intestinal microbiota and works as a slow-release agent of 3-HB. Further, we examined whether PHB suppresses the pathogenesis of IBD models. As long as a PHB diet increased 3-HB concentrations in the feces and blood, PHB suppressed weight loss and histological inflammation in a dextran sulfate sodium-induced IBD model. Furthermore, PHB increased the accumulation of regulatory T cells in the rectum without affecting T cells in the spleen. These results indicate that PHB has potential applications in treating diseases related to the intestinal microbiota as a sustained 3-HB donor. We show for the first time that biodegradable polyester exhibits intestinal bacteria-mediated bioactivity toward IBD. The use of bioplastics, which are essential materials for sustainable social development, represents a novel approach to diseases related to dysbiosis, including IBD.

Electrospun Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate)/Olive Leaf Extract Fiber Mesh as Prospective Bio-Based Scaffold for Wound Healing.

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters synthesized by various microorganisms. Due to their biocompatibility and biodegradation, PHAs have been proposed for biomedical applications, including tissue engineering scaffolds. Olive leaf extract (OLE) can be obtained from agri-food biowaste and is a source of polyphenols with remarkable antioxidant properties. This study aimed at incorporating OLE inside poly(hydroxybutyrate-co-hydroxyvalerate) (PHBHV) fibers via electrospinning to obtain bioactive bio-based blends that are useful in wound healing. PHBHV/OLE electrospun fibers with a size of 1.29 ± 0.34 µm were obtained. Fourier transform infrared chemical analysis showed a uniform surface distribution of hydrophilic -OH groups, confirming the presence of OLE in the electrospun fibers. The main OLE phenols were released from the fibers within 6 days. The biodegradation of the scaffolds in phosphate buffered saline was investigated, demonstrating an adequate stability in the presence of metalloproteinase 9 (MMP-9), an enzyme produced in chronic wounds. The scaffolds were preliminarily tested in vitro with HFFF2 fibroblasts and HaCaT keratinocytes, suggesting adequate cytocompatibility. PHBHV/OLE fiber meshes hold promising features for wound healing, including the treatment of ulcers, due to the long period of durability in an inflamed tissue environment and adequate cytocompatibility.

Biodegradable and Electrically Conductive Melanin-Poly (3-Hydroxybutyrate) 3D Fibrous Scaffolds for Neural Tissue Engineering Applications.

Due to the severity of peripheral nerve injuries (PNI) and spinal cord injuries (SCI), treatment options for patients are limited. In this context, biomaterials designed to promote regeneration and reinstate the lost function are being explored. Such biomaterials should be able to mimic the biological, chemical, and physical cues of the extracellular matrix for maximum effectiveness as therapeutic agents. Development of biomaterials with desirable physical, chemical, and electrical properties, however, has proven challenging. Here a novel biomaterial formulation achieved by blending the pigment melanin and the natural polymer Poly-3-hydroxybutyrate (PHB) is proposed. Physio-chemical measurements of electrospun fibers reveal a feature rich surface nano-topography, a semiconducting-nature, and brain-tissue-like poroviscoelastic properties. Resulting fibers improve cell adhesion and growth of mouse sensory and motor neurons, without any observable toxicity. Further, the presence of polar functional groups positively affect the kinetics of fibers degradation at a pH (≈7.4) comparable to that of body fluids. Thus, melanin-PHB blended scaffolds are found to be physio-chemically, electrically, and biologically compatible with neural tissues and could be used as a regenerative modality for neural tissue injuries. A biomaterial for scaffolds intended to promote regeneration of nerve tissue after injury is developed. This biomaterial, obtained by mixing the pigment melanin and the natural polymer PHB, is biodegradable, electrically conductive, and beneficial to the growth of motor and sensory neurons. Thus, it is believed that this biomaterial can be used in the context of healthcare applications.

Structural and Biochemical Basis for Development of Diketo Acid Inhibitors Targeting the Cap-Snatching Endonuclease of the Ebinur Lake Virus (Order: Bunyavirales).

The Bunyavirales contain many important human pathogens that lack an antiviral therapy. The cap-snatching endonuclease (EN) of segmented negative-strand RNA viruses is an attractive target for broad-spectrum antivirals due to its essential role in initiating viral transcription. L-742,001, a previously reported diketo acid inhibitor against influenza virus EN, demonstrated potent EN inhibition and antiviral activity on various bunyaviruses. However, the precise inhibitory mechanism of the compound is still poorly understood. We recently characterized a highly active EN from Ebinur Lake virus (EBIV), a newly identified member of the Orthobunyavirus genus, and obtained its high-resolution structures, paving the way for structure-guided inhibitor development. Here, nine L-742,001 derivatives were designed and synthesized de novo, and their structure-activity relationship with EBIV EN was studied. In vitro biochemical data showed that the compounds inhibited the EBIV EN activity with different levels and could be divided into three categories. Five representative compounds were selected for further cell-based antiviral assay, and the results largely agreed with those of the EN assays. Furthermore, the precise binding modes of L-742,001 and its derivatives in EN were revealed by determining the high-resolution crystal structures of EN-inhibitor complexes, which suggested that the p-chlorobenzene is essential for the inhibitory activity and the flexible phenyl has the greatest exploration potential. This study provides an important basis for the structure-based design and optimization of inhibitors targeting EN of segmented negative-strand RNA viruses. IMPORTANCE The Bunyavirales contain many important human pathogens such as Crimean-Congo hemorrhagic fever virus and Lassa virus that pose serious threats to public health; however, currently there are no specific antiviral drugs against these viruses. The diketo acid inhibitor L-742,001 is a potential drug as it inactivates the cap-snatching endonuclease (EN) encoded by bunyaviruses. Here, we designed and synthesized nine L-742,001 derivatives and assessed the structure-activity relationship using EN of the newly identified Ebinur Lake virus (EBIV) as a research model. Our results revealed that the p-chlorobenzene of this broad-spectrum EN inhibitor is crucial for the inhibitory activity and the flexible phenyl "arm" has the best potential for further optimization. As cap-snatching ENs are present not only in bunyaviruses but also in influenza viruses, our data provide important guidelines for the development of novel and more potent diketo acid-based antiviral drugs against those viruses.

Teriflunomide shifts the astrocytic bioenergetic profile from oxidative metabolism to glycolysis and attenuates TNFα-induced inflammatory responses.

Astrocytes utilize both glycolytic and mitochondrial pathways to power cellular processes that are vital to maintaining normal CNS functions. These cells also mount inflammatory and acute phase reactive programs in response to diverse stimuli. While the metabolic functions of astrocytes under homeostatic conditions are well-studied, the role of cellular bioenergetics in astrocyte reactivity is poorly understood. Teriflunomide exerts immunomodulatory effects in diseases such as multiple sclerosis by metabolically reprogramming lymphocytes and myeloid cells. We hypothesized that teriflunomide would constrain astrocytic inflammatory responses. Purified murine astrocytes were grown under serum-free conditions to prevent acquisition of a spontaneous reactive state. Stimulation with TNFα activated NFκB and increased secretion of Lcn2. TNFα stimulation increased basal respiration, maximal respiration, and ATP production in astrocytes, as assessed by oxygen consumption rate. TNFα also increased glycolytic reserve and glycolytic capacity of astrocytes but did not change the basal glycolytic rate, as assessed by measuring the extracellular acidification rate. TNFα specifically increased mitochondrial ATP production and secretion of Lcn2 required ATP generated by oxidative phosphorylation. Inhibition of dihydroorotate dehydrogenase via teriflunomide transiently increased both oxidative phosphorylation and glycolysis in quiescent astrocytes, but only the increased glycolytic ATP production was sustained over time, resulting in a bias away from mitochondrial ATP production even at doses down to 1 µM. Preconditioning with teriflunomide prevented the TNFα-induced skew toward oxidative phosphorylation, reduced mitochondrial ATP production, and reduced astrocytic inflammatory responses, suggesting that this drug may limit neuroinflammation by acting as a metabolomodulator.

Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5-7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

Teriflunomide Preserves Neuronal Activity and Protects Mitochondria in Brain Slices Exposed to Oxidative Stress.

Teriflunomide (TFN) limits relapses in relapsing-remitting multiple sclerosis (RRMS) by reducing lymphocytic proliferation through the inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) and the subsequent modulation of de novo pyrimidine synthesis. Alterations of mitochondrial function as a consequence of oxidative stress have been reported during neuroinflammation. Previously, we showed that TFN prevents alterations of mitochondrial motility caused by oxidative stress in peripheral axons. Here, we aimed to validate TFN effects on mitochondria and neuronal activity in hippocampal brain slices, in which cellular distribution and synaptic circuits are largely preserved. TFN effects on metabolism and neuronal activity were investigated by assessing oxygen partial pressure and local field potential in acute slices. Additionally, we imaged mitochondria in brain slices from the transgenic Thy1-CFP/COX8A)S2Lich/J (mitoCFP) mice using two-photon microscopy. Although TFN could not prevent oxidative stress-related depletion of ATP, it preserved oxygen consumption and neuronal activity in CNS tissue during oxidative stress. Furthermore, TFN prevented mitochondrial shortening and fragmentation of puncta-shaped and network mitochondria during oxidative stress. Regarding motility, TFN accentuated the decrease in mitochondrial displacement and increase in speed observed during oxidative stress. Importantly, these effects were not associated with neuronal viability and did not lead to axonal damage. In conclusion, during conditions of oxidative stress, TFN preserves the functionality of neurons and prevents morphological and motility alterations of mitochondria.

Antitumor bioactivity and gut microbiota modulation of polyhydroxybutyrate (PHB) in a rat animal model for colorectal cancer.

Polyhydroxybutyrate (PHB) is a non-toxic polyhydroxyalkanoate polymer produced by several microorganisms, widely used as a biological substitute for plastics derived from fossil hydrocarbons. In this work, PHB polymer has been tested in an animal model for colorectal cancer. In the animal model, PHB has been able to reduce the number of polyps by 48,1%, and the tumoral extension area by 58,1%. Also, PHB induces a selective increase in beneficial gut bacterial taxons in this animal model, and a selective reduction in pro-inflammatory taxons, demonstrating its value as a nutraceutical compound. This antitumor effect is caused by gut production of 3-hydroxybutyrate and butyrate. In this animal model, 3-hydroxybutyrate is also observed in plasma and in brain tissue, after PHB consumption, making PHB supplementation interesting as a bioactive compound in other extraintestinal conditions, as 3-hydroxybutyrate has been reported to enhance brain and cognitive function, cardiac performance, appetite suppression and diabetes. Therefore, PHB could be postulated as an interesting non-polysaccharide antitumor prebiotic, paving the way towards its future use in functional foods.

Design, Synthesis, and Biological Evaluation of a Novel Series of Teriflunomide Derivatives as Potent Human Dihydroorotate Dehydrogenase Inhibitors for Malignancy Treatment.

Human dihydroorotate dehydrogenase (hDHODH), as the fourth and rate-limiting enzyme of the de novo pyrimidine synthesis pathway, is regarded as an attractive target for malignancy therapy. In the present study, a novel series of teriflunomide derivatives were designed, synthesized, and evaluated as hDHODH inhibitors. 13t was the optimal compound with promising enzymatic activity (IC50 = 16.0 nM), potent antiproliferative activity against human lymphoma Raji cells (IC50 = 7.7 nM), and excellent aqueous solubility (20.1 mg/mL). Mechanistically, 13t directly inhibited hDHODH and induced cell cycle S-phase arrest in Raji cells. The acute toxicity assay indicated a favorable safety profile of 13t. Notably, 13t displayed significant tumor growth inhibition activity with a tumor growth inhibition (TGI) rate of 81.4% at 30 mg/kg in a Raji xenograft model. Together, 13t is a promising inhibitor of hDHODH and a preclinical candidate for antitumor therapy, especially for lymphoma.

Aligned silk fibroin/poly-3-hydroxybutyrate nanofibrous scaffolds seeded with adipose-derived stem cells for tendon tissue engineering.

In this work we investigated tenogenic differentiation of adipose-derived mesenchymal stem cells (AdMSCs), which were seeded onto silk fibroin/poly-3-hydroxybutyrate (SF/P3HB) scaffolds with aligned topography, and high mechanical strength. The electrospinning process was optimized by using the response surface method (RSM) and SF/P3HB nanofibrous matrices with a total polymer concentration of 5% (SF: PHB = 3: 1), flow rate 1 mL/h, collector rotation speed 2000 rpm, applied voltage 14 kV, and collector distance 25 cm were obtained. The average fiber diameter was 699 ± 203 nm and 80% of the nanofibers were aligned within the ±15o range. SF reinforcement reduced the crystallinity of P3HB, and the elastic modulus was found to be 197.0 ± 7.7 MPa. The scaffolds showed bacteriostatic effect. A 21-day of cell culture study was performed with rat rAdMSCs in the absence and presence of tenogenic differentiation factor-5 (GDF-5). The results demonstrated that SF/P3HB scaffolds allow the cells to proliferate and differentiate to the tenocytes. However, no significant effect of GDF-5 on the differentiation of cells was observed. These findings indicated that our aligned SF/P3HB scaffolds have a significant potential to be used for tendon tissue engineering.

Teriflunomide Promotes Oligodendroglial 8,9-Unsaturated Sterol Accumulation and CNS Remyelination.

BACKGROUND AND OBJECTIVES: To test whether low concentrations of teriflunomide (TF) could promote remyelination, we investigate the effect of TF on oligodendrocyte in culture and on remyelination in vivo in 2 demyelinating models. METHODS: The effect of TF on oligodendrocyte precursor cell (OPC) proliferation and differentiation was assessed in vitro in glial cultures derived from neonatal mice and confirmed on fluorescence-activated cell sorting-sorted adult OPCs. The levels of the 8,9-unsaturated sterols lanosterol and zymosterol were quantified in TF- and sham-treated cultures. In vivo, TF was administered orally, and remyelination was assessed both in myelin basic protein-GFP-nitroreductase (Mbp:GFP-NTR) transgenic Xenopus laevis demyelinated by metronidazole and in adult mice demyelinated by lysolecithin. RESULTS: In cultures, low concentrations of TF down to 10 nM decreased OPC proliferation and increased their differentiation, an effect that was also detected on adult OPCs. Oligodendrocyte differentiation induced by TF was abrogated by the oxidosqualene cyclase inhibitor Ro 48-8071 and was mediated by the accumulation of zymosterol. In the demyelinated tadpole, TF enhanced the regeneration of mature oligodendrocytes up to 2.5-fold. In the mouse demyelinated spinal cord, TF promoted the differentiation of newly generated oligodendrocytes by a factor of 1.7-fold and significantly increased remyelination. DISCUSSION: TF enhances zymosterol accumulation in oligodendrocytes and CNS myelin repair, a beneficial off-target effect that should be investigated in patients with multiple sclerosis.