Search

Metal–organic frameworks (MOFs) have attracted extensive attention for the development of colorimetric detection methods due to their ease of modification and high density of active sites. However, most of the reported colorimetric sensors based on MOFs show only a single nanozyme activity. Herein, the bifunctional enzyme activities of a hexagonal prism Cu MOF with fumaric acid as the ligand (Cu FMA), namely laccase-like activity under alkaline conditions (pH = 8) and peroxidase-like activity under acidic conditions (pH = 4), were verified. The specificity of Cu FMA at different pH values may be due to the presence of the Cu+ active center introduced by the weak reducibility of FMA. At pH = 8, Cu+ active centers are beneficial for dissociating the H–O bonds of phenolic compounds for the laccase system. In contrast, the dissociation of H–O is weakened at pH = 4, which prompts the breaking of the O–O bonds of H2O2 as a Fenton-like reaction for the peroxidase system. Based on the dual enzyme activities, Cu FMA sensors exhibit outstanding detectability for epinephrine and glucose with linear ranges of 2.7–54.6 μM and 0.01–0.8 mM and detection limits of 1.1 μM and 2.28 × 10−7 M, respectively. The Cu FMA colorimetric sensor can be applied for detecting and measuring glucose and epinephrine in human serum samples. This work paves the way for Cu MOFs to be used as the basis for rational regulation of the activity of dual nanozymes and for multifunctional applications under completely independent conditions.

Significance Magnetic resonance imaging is hindered by inherently low sensitivity, which limits the method for the most part to observing water molecules in the body. Hyperpolarized molecules exhibit strongly enhanced MRI signals which opens the door for imaging low-concentration species in vivo. Biomolecules can be hyperpolarized and injected into a patient allowing for metabolism to be tracked in real time, greatly expanding the information available to the radiologist. Parahydrogen-induced polarization (PHIP) is a hyperpolarization method renowned for its low cost and accessibility, but is generally limited by low polarization levels, modest molecular concentrations, and contamination by polarization reagents. In this work we overcome these drawbacks in the production of PHIP-polarized [1-13C]fumarate, a biomarker of cell necrosis in metabolic 13C MRI., Hyperpolarized fumarate is a promising biosensor for carbon-13 magnetic resonance metabolic imaging. Such molecular imaging applications require nuclear hyperpolarization to attain sufficient signal strength. Dissolution dynamic nuclear polarization is the current state-of-the-art methodology for hyperpolarizing fumarate, but this is expensive and relatively slow. Alternatively, this important biomolecule can be hyperpolarized in a cheap and convenient manner using parahydrogen-induced polarization. However, this process requires a chemical reaction, and the resulting solutions are contaminated with the catalyst, unreacted reagents, and reaction side-product molecules, and are hence unsuitable for use in vivo. In this work we show that the hyperpolarized fumarate can be purified from these contaminants by acid precipitation as a pure solid, and later redissolved to a desired concentration in a clean aqueous solvent. Significant advances in the reaction conditions and reactor equipment allow for formation of hyperpolarized fumarate at 13C polarization levels of 30–45%.

For electrons to continuously enter and flow through the mitochondrial electron transport chain (ETC), they must ultimately land on a terminal electron acceptor (TEA), which is known to be oxygen in mammals. Paradoxically, we find that complex I and dihydroorotate dehydrogenase (DHODH) can still deposit electrons into the ETC when oxygen reduction is impeded. Cells lacking oxygen reduction accumulate ubiquinol, driving the succinate dehydrogenase (SDH) complex in reverse to enable electron deposition onto fumarate. Upon inhibition of oxygen reduction, fumarate reduction sustains DHODH and complex I activities. Mouse tissues display varying capacities to use fumarate as a TEA, most of which net reverse the SDH complex under hypoxia. Thus, we delineate a circuit of electron flow in the mammalian ETC that maintains mitochondrial functions under oxygen limitation.

Initiating and sustaining large-scale encapsulated ferrous fumarate double fortified salt interventions in the public distribution system in India poses several challenges that can be minimized by strengthening double fortified salt value chains., Key Findings Double fortified salt (DFS) interventions using encapsulated ferrous fumarate premix passed rigorous efficacy evaluations but faced challenges in showing impact at scale. Several technological, market-related, and policy barriers contributed to the reduced coverage and impact of the public-sector-led DFS interventions in India.Value chain analysis can be a useful method to identify and address demand and supply-side barriers to scaling up DFS in settings where the scale-up criteria are met. Key Implications Initiating and sustaining large-scale encapsulated ferrous fumarate DFS interventions in the public distribution system in India involve several challenges that can be minimized through strengthening DFS value chains. Critical interventions for addressing barriers in India's DFS value chain include— building an enabling institutional environment, demand creation through consumer awareness, strengthening institutional markets through public financing, managing cost and risks through public-private partnerships, and assuring quality during commercial scale-up.Considering the high price of DFS when introduced through private markets and the preference of a significant population to low-cost salt options, routing DFS through the public distribution system targeting low-income populations (that enables cost reduction through bulk purchase and subsidizes the product's retail price) is essential., Food fortification is a powerful strategy to reach large populations with multiple micronutrients added to a single food vehicle. The impact depends on the sustained provision and utilization of adequately fortified food by a large population (mainly in low-income and food-insecure settings). We apply a value chain (VC) analysis framework to diagnose and address the barriers to the uptake of encapsulated ferrous fumarate double fortified salt (DFS) distributed through public-sector-led DFS interventions in India. We adapt the VC requirements framework proposed by Henson and Humphrey to identify and categorize barriers along the DFS VC as technological, market-related, and policy-related. We conducted a desk review of published and unpublished literature on DFS and information available in the public domain, semi-structured interviews with VC stakeholders from the private sector, program data from implementing organizations, and participation in multistakeholder consultations on DFS. Major supply-side barriers were under-developed private markets, inconsistent demand from public markets, unpredictable returns-on-investments, and inadequate business incentives to invest in DFS. The product's weak consumer orientation, uncreated consumer demand, low awareness of fortified foods, inadequate nutrition signaling were significant demand-side barriers. Technological barriers related to the requirement of high-grade salt for DFS production and residual organoleptic property of mild discoloration of food. Policy barriers related to inadequate and irregular financing for distributing subsidized DFS through the public distribution system; insufficient policy support for risk-sharing and managing costs associated with fortification; and a weak institutional environment for sustaining DFS interventions. Building an enabling institutional environment, demand creation through consumer awareness, strengthening institutional markets through public financing, managing cost and risks through public-private partnerships, and assuring quality during commercial scale-up are critical interventions necessary to ensure impact at scale.

BACKGROUND AND AIMS NAFLD is a lipotoxic disease wherein hepatic steatosis and oxidative stress are key pathogenic features. However, whether free amino acids (FAAs) are associated with the oxidative stress response against lipotoxicity has yet to be determined. We hypothesized that an imbalance of FAAs aggravates hepatic steatosis by interfering with the oxidative stress sensor. APPROACH AND RESULTS C57BL/6 mouse immortalized hepatocytes, primary hepatocytes, and organoids were employed. Steatotic hepatocytes treated with oleic acid (OA) were cultured under FAA-modifying media based on the concentrations of FAAs in the hepatic portal blood of wild-type (WT) mice. As in vivo experiments, WT hepatocyte-specific Kelch-like ECH-associated protein 1 (Keap1) knockout mice (Keap1∆hepa ) and Cre- control mice (Keap1fx/fx ) were fed high-fat (HF) diets with modified amino acid content. The correlations were analyzed between the areas of lipid droplets (LDs) around central vein and plasma OA/FAA ratio in 61 patients with NAFLD. Mice fed an HF, Met-restricted, and tyrosine (Tyr)-deficient diet showed the NAFLD-like phenotype in which the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), triglyceride-rich VLDL, and fumarate were decreased in liver, but Keap1∆hepa ameliorated these phenomena. Reactive oxygen species and LDs induced by the deprivation of Met and Tyr were prevented in hepatic organoids generated from Keap1∆hepa . Dimethyl fumarate, an Nrf2 inducer, ameliorated the steatosis and increased the hepatic fumarate reduced by the deprivation of Met and Tyr in vitro. OA/Met or Tyr ratio in peripheral blood was associated with the hepatic steatosis in patients with NAFLD. CONCLUSIONS An imbalance between free fatty acids and Met and Tyr induces hepatic steatosis by disturbing the VLDL assembling through the Keap1-Nrf2 system.

Orally dosed drugs must dissolve in the gastrointestinal (GI) tract before being absorbed through the epithelial cell membrane. In vivo drug dissolution depends on the GI tract's physiological conditions such as pH, residence time, luminal buffers, intestinal motility, and transit and drug properties under fed and fasting conditions (Paixao, P. et al. Mol. Pharm. 2018 and Bermejo, et al. M. Mol. Pharm. 2018). The dissolution of an ionizable drug may benefit from manipulating in vivo variables such as the environmental pH using pH-modifying agents incorporated into the dosage form. A successful example is the use of such agents for dissolution enhancement of BCS class IIb (high-permeability, low-solubility, and weak base) drugs under high gastric pH due to the disease conditions or by co-administration of acid-reducing agents (i.e., proton pump inhibitors, H2-antagonists, and antacids). This study provides a rational approach for selecting pH modifiers to improve monobasic and dibasic drug compounds' dissolution rate and extent under high-gastric pH dissolution conditions, since the oral absorption of BCS class II drugs can be limited by either the solubility or the dissolution rate depending on the initial dose number. Betaine chloride, fumaric acid, and tartaric acid are examples of promising pH modifiers that can be included in oral dosage forms to enhance the rate and extent of monobasic and dibasic drug formulations. However, selection of a suitable pH modifier is dependent on the drug properties (e.g., solubility and pKa) and its interplay with the pH modifier pKa or pKas. As an example of this complex interaction, for basic drugs with high pKa and intrinsic solubility values and large doses, a polyprotic pH modifier can be expected to outperform a monoacid pH modifier. We have developed a hierarchical mass transport model to predict drug dissolution of formulations under varying pH conditions including high gastric pH. This model considers the effect of physical and chemical properties of the drug and pH modifiers such as pKa, solubility, and particle size distribution. This model also considers the impact of physiological conditions such as stomach emptying rate, stomach acid and buffer secretion, residence time in the GI tract, and aqueous luminal volume on drug dissolution. The predictions from this model are directly applicable to in vitro multi-compartment dissolution vessels and are validated by in vitro experiments in the gastrointestinal simulator. This model's predictions can serve as a potential data source to predict plasma concentrations for formulations containing pH modifiers administered under the high-gastric pH conditions. This analysis provides an improved formulation design procedure using pH modifiers by minimizing the experimental iterations under both in vitro and in vivo conditions.

Macrophage-mediated inflammation drives autoimmune and chronic inflammatory diseases. Treatment with anti-inflammatory agents can be an effective strategy to reduce this inflammation; however, high concentrations of these agents can have immune-dampening and other serious side effects. Synergistic combination of anti-inflammatory agents can mitigate dosing by requiring less drug. Multiple anti-inflammatory agents were evaluated in combination for synergistic inhibition of macrophage inflammation. The most potent synergy was observed between dexamethasone (DXM) and fumaric acid esters (e.g., monomethyl fumarate (MMF)). Furthermore, this combination was found to synergistically inhibit inflammatory nuclear factor κB (NF-κB) transcription factor activity. The optimal ratio for synergy was determined to be 1:1, and DXM and MMF were conjugated by esterification at this molar ratio. The DXM-MMF conjugate displayed improved inhibition of inflammation over the unconjugated combination in both murine and human macrophages. In the treatment of human donor monocyte-derived macrophages, the combination of DXM and MMF significantly inhibited inflammatory gene expression downstream of NF-κB and overall performed better than either agent alone. Further, the DXM-MMF conjugate significantly inhibited expression of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome-associated genes. The potent anti-inflammatory activity of the DXM-MMF conjugate in human macrophages indicates that it may have benefits in the treatment of autoimmune and inflammatory diseases.

Background Antiretrovirals, including tenofovir, can suppress human immunodeficiency virus (HIV) infection but cannot completely eradicate it. Patients with HIV infection are administered antiretroviral drugs over a long term; thus, managing consequent adverse drug reactions, such as renal dysfunction and bone mineral loss, is important. Currently, highly sensitive biomarkers that can detect adverse drug reactions early have not been well studied. Methods This single-center, prospective, observational study explored changes in the biomarkers of renal function, bone metabolism, and lipid profile before and after switching from tenofovir disoproxil fumarate (TDF) to tenofovir alafenamide (TAF) in patients with HIV infection. Results All 31 enrolled patients had been treated with antiretrovirals for more than 5 years. The rate of proteinuria decreased significantly after starting TAF-containing antiretroviral regimen. The urinary liver-type fatty acid binding protein (L-FABP)/creatinine ratio was significantly decreased at 3 and 6 months after switching to TAF compared with that before switching to TAF (− 0.5 μg/g Cr at 3 months, and − 0.8 μg/g Cr at 6 months; p p Conclusions Switching from TDF to TAF decreased the levels of renal and bone biomarkers, such as urinary L-FABP and NTx, but increased low density lipoprotein-cholesterol levels. Future studies should evaluate if these biomarkers, such as urinary L-FABP and NTx, truly detect serious adverse drug reactions early.

The macromolecule oligo(poly(ethylene glycol) fumarate) (OPF) exhibits promising attributes for creating suitable three-dimensional hydrogel environments to study cell behavior, deliver therapeutics, and serve as a degradable, nonfouling material. However, traditional synthesis techniques are time consuming, contain salt contaminants, and generate significant waste. These issues have been overcome with an alternative, one-pot approach that utilizes inert gas sparging. Departing from previous synthetic schemes that require acid scavengers, inert gas sparging removes byproducts in situ, eliminating significant filtration and postprocessing steps, while allowing a more uniform product. Characterized by nuclear magnetic resonance, gel permeation chromatography, and differential scanning calorimetry, nitrogen sparge synthesis yields an OPF product with greater polymer length than traditional acid scavenger synthesis methods. Furthermore, nitrogen-sparged OPF readily crosslinks using either ultraviolet or thermal initiator methods with or without the addition of short-chain diacrylate units, allowing for greater tunability in hydrogel properties with little to no cytotoxicity. Overall, inert gas sparging provides a longer chain and cleaner polymer product for hydrogel material studies while maintaining degradable characteristics. IMPACT STATEMENT: Using nitrogen sparging, we have demonstrated that oligo(poly(ethylene glycol) fumarate) (OPF) can be produced with decreased postprocessing, increased product purity, greater oligomerization, and cell viability. These properties lead to greater tunability in mechanical properties and a more versatile hydrogel for biomedical applications. The simplification of synthesis and elimination of impurities will expand the utility of OPF as a degradable hydrogel for cell culture, tissue engineering, regenerative medicine, and therapeutic delivery, among other applications.

Background: The tricarboxylic (TCA) acid cycle provides the energy needed for regulatory functions in the cardio-renal system. Recently, a genetic defect in the TCA cycle enzyme, fumarase hydratase, altered L-arginine metabolism and exacerbated hypertension in salt-sensitive rats. This study evaluated the effect of fumarate and its possible link to L-arginine metabolism in deoxycorticosterone (DOCA)-salt hypertension, a non-genetic model of hypertension.Method: Hypertension was induced with DOCA (25 mg/kg s.c, twice weekly) + 1% NaCL in uninephrectomised rats placed on fumarate (1 g/L, ad libitum). Blood pressure was measured in conscious rats via carotid cannulation. Biochemical and western blot analyses were carried out on kidney fractions.Results: Fumarate reduced mean blood pressure (198 ± 5 vs 167 ± 7 mmHg, p < .01), increased nitric oxide levels in the renal cortex (36.1 ± 2 vs 61.3 ± 4 nM/µg) and medulla (27.4 ± 1 vs 54.1 ± 2 nM/µg) of DOCA-salt rats (p < .01). Consistent with this, arginase activity was reduced (threefold) in the renal medulla but not cortex of DOCA-salt rats. Fumarate increased superoxide dismutase activity in the medulla (p < .001) of DOCA-hypertensive rats. However, catalase activity was exacerbated by fumarate in both renal cortex (4.5 ± 1 vs 11.2 ± 1) and medulla (3.7 ± 1 vs 16.3 ± 1 units/mg) of DOCA-salt rats (p < .001). Proteinuria (64.6%), kidney injury molecule-1 expression and kidney weight were reduced in DOCA-hypertensive rats treated with fumarate (p< .05). However, there was a paradoxical increase in TGF-β expression in fumarate-treated DOCA-salt rats. Conclusion: These data show that fumarate attenuated hypertension, renal injury and improved the redox state of the kidney in DOCA/salt hypertension by mechanisms involving selective reduction of L-arginine metabolism.

In this study, a light cross-linkable biocomposite scaffold based on a photo-cross-linkable poly (propylene fumarate) (PPF)-co-polycaprolactone (PCL) tri-block copolymer was synthesized and characterized. The developed biodegradable scaffold was further modified with β-tricalcium phosphate (β-TCP) bioceramic for bone tissue engineering applications. The developed biocomposite was characterized using H nuclear magnetic resonance and Fourier transform infrared spectroscopy. Moreover, the bioceramic particle size distribution and morphology were evaluated using Brunauer-Emmett-Teller method, X-ray diffraction, and scanning electron microscopy. The mechanical properties and biodegradation of the scaffolds were also evaluated. Cytotoxicity and mineralization assays were performed to analyze the biocompatibility and bioactivity capacity of the developed biocomposite. The characterization data confirmed the development of a biodegradable and photo-cross-linkable PCL-based biocomposite reinforced with β-TCP bioceramic. In vitro analyses demonstrated the biocompatibility and mineralization potential of the synthesized bioceramic. Altogether, the results of the present study suggest that the photo-cross-linkable PCL-PPF-PCL tri-block copolymer reinforced with β-TCP is a promising biocomposite for bone tissue engineering applications. According to the results, this newly synthesized material has a proper chemical composition for further clinically-relevant studies in tissue engineering.

Naftopidil (NAF), an α1-adrenoceptor antagonist, is administered as a treatment for benign prostatic hyperplasia; however, according to the Biopharmaceutical Classification System (BCS IV), it is a poorly-soluble drug that exhibits poor permeability. We aimed to increase the dissolution (%) of NAF by adding chitosan to a polymer-free formulation. Compared to the formulation prepared using Flivas®, at 60 min, the solid dispersion (SD) formulation containing NAF, fumaric acid, chitosan, and US2® in a 1:1:2:1 weight ratio improved the dissolution (%) of NAF in distilled water, pH 1.2 media, pH 4.0 and pH 6.8 buffers by 27.2-, 1.2-, 1.1- and 6.5-fold, respectively. The physicochemical properties of the SD1 formulation were also found to be altered, including its thermal properties, crystal intensity, and chemical interaction. As a result, the hydrogen bonding that occurs between NAF and fumaric acid was identified as a major factor in the increase in NAF dissolution (%). Further, chitosan was observed to contribute to the stability of NAF and SD1, which was assessed over a 3-month period. To our knowledge, this is the first study to employ a polymer-free system to improve the solubilization of NAF.

Copper (Cu) and iron (Fe) are essential for plant growth and are often in short supply under natural conditions. Molecular responses to simultaneous lack of both metals (-Cu-Fe) differ from those seen in the absence of either alone. Metabolome profiling of plant leaves previously revealed that fumarate levels fall under -Cu-Fe conditions. We employed lines lacking cytosolic FUMARASE2 (FUM2) activity to study the impact of constitutive suppression of cytosolic fumarate synthesis on plant growth under Cu and/or Fe deficiency. In fum2 mutants, photosynthesis and growth were less impaired under -Cu-Fe conditions than in wild-type (WT) seedlings. In particular, levels of photosynthetic proteins, chloroplast ultrastructure, amino acid profiles and redox state were less perturbed by simultaneous Cu-Fe deficiency in lines that cannot produce fumarate in the cytosol. Although cytosolic fumarate has been reported to promote acclimation of photosynthesis to low temperatures when metal supplies are adequate, the photosynthetic efficiency of fum2 lines grown under Cu-Fe deficiency in the cold was higher than in WT. Uptake and contents of Cu and Fe are similar in WT and fum2 plants under control and -Cu-Fe conditions, and lack of FUM2 does not alter the ability to sense metal deficiency, as indicated by marker gene expression. Collectively, we propose that reduced levels of cytosolic fumarate synthesis ultimately increase the availability of Fe for incorporation into metalloproteins.

A promising strategy that emerged in tissue engineering is to incorporate two-dimensional (2D) materials into polymer scaffolds, producing materials with desirable mechanical properties and surface chemistries, which also display broad biocompatibility. Black phosphorus (BP) is a 2D material that has sparked recent scientific interest due to its unique structure and electrochemical characteristics. In this study, BP nanosheets (BPNSs) were incorporated into a cross-linkable oligo[poly(ethylene glycol) fumarate] (OPF) hydrogel to produce a new nanocomposite for bone regeneration. BPNSs exhibited a controllable degradation rate coupled with the release of phosphate in vitro. MTS assay results together with live/dead images confirmed that the introduction of BPNSs into OPF hydrogels enhanced MC3T3-E1 cell proliferation. Moreover, the morphology parameters indicated better attachments of cells in the BPNSs containing group. Immunofluorescence images as well as intercellular ALP and OCN activities showed that adding a certain amount of BPNSs to OPF hydrogel could greatly improve differentiation of pre-osteoblasts on the hydrogel. Additionally, embedding black phosphorous into a neutral polymer network helped to control its cytotoxicity, with optimal cell growth observed at BP concentrations as high as 500 ppm. These results reinforced that the supplementation of OPF with BPNSs can increase the osteogenic capacity of polymer scaffolds for use in bone tissue engineering.

Background Tecfidera® (dimethyl fumarate [DMF]) is an approved product for the treatment of relapsing forms of multiple sclerosis. Monomethyl fumarate (MMF) is the only active metabolite of DMF and is responsible for its therapeutic efficacy. Objective The objective of this study was to determine whether two Bafiertam™ capsules each containing 95 mg of MMF is bioequivalent to one Tecfidera® capsule containing 240 mg of DMF, a prodrug of MMF. Methods This was a single-dose, open-label, randomized, two-way crossover study evaluating two treatments over two periods with a washout interval between treatments. Fifty healthy subjects were randomized to receive a single dose of the test drug MMF 190 mg as 2 × 95 mg delayed-release capsules or the reference drug DMF 240 mg as a 1 × 240-mg delayed-release capsule. Blood samples were obtained prior to dosing and at prespecified time points through 24 h post-dose to determine plasma concentrations of MMF. The pharmacokinetic parameters of MMF were calculated including maximum observed concentration, time to reach maximum observed concentration, apparent half-life of the drug in plasma, AUC0–t which is the area under the plasma concentration–time curve (AUC) from time zero (dosing time) to the last time point, t, with measurable analyte concentration, and AUC0–inf, which is AUC0–t plus the extrapolated AUC from time t to infinity. Results The geometric least-squares mean ratios (90% confidence interval) of the test drug MMF vs the reference drug DMF were 96.80% (92.18–101.64), 96.35% (91.81–101.12), and 104.84% (95.54–115.05) for AUC0–t, AUC0–inf, and maximum observed concentration, respectively. Two capsules of Bafiertam™ was safe and generally well tolerated. The most common adverse event for both products was flushing, 60% and 51%, for Bafiertam™ and Tecfidera®, respectively. Conclusions Based on the statistical analysis results of the pharmacokinetic parameters of MMF, a single oral dose of two Bafiertam™ DR 95 mg capsules is bioequivalent to a single oral dose of one Tecfidera® DR 240 mg capsule. Clinical Trial Registration This study was retrospectively registered with ClinicalTrials.gov (NCT04570670) on 30 September, 2020.

Significant advances have been made in unknown metabolite identification and expansion of the number of quantifiable metabolites in human plasma, serum, and whole blood using NMR spectroscopy. However, reliable quantitation of metabolites is still a challenge. A major bottleneck is the lack of a suitable internal standard that does not interact with the complex blood sample matrix and also does not overlap with metabolite peaks apart from exhibiting other favorable characteristics. With the goal of addressing this challenge, a comprehensive investigation of fumaric and maleic acids as potential internal standards was made along with a comparison with the conventional standards, TSP (trimethylsilylpropionic acid) and DSS (trimethylsilylpropanesulfonic acid). Both fumaric acid and maleic acid exhibited a surprisingly high performance with a quantitation error

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg2+ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l-malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.

Background Doravirine (DOR) is a nonnucleoside reverse-transcriptase inhibitor. In the phase 3 DRIVE-AHEAD trial in treatment-naive adults with human immunodeficiency virus type 1 (HIV-1) infection, DOR demonstrated noninferior efficacy compared with efavirenz (EFV) and superior profiles for neuropsychiatric tolerability and lipids at 48 weeks. We present data through week 96. Methods DRIVE-AHEAD is a phase 3, multicenter, double-blind, noninferiority trial in antiretroviral treatment-naive adults with HIV-1 RNA ≥1000 copies/mL. Participants were randomized to a daily fixed-dose tablet of DOR (100 mg), lamivudine (3TC; 300 mg) and tenofovir disoproxil fumarate (TDF; 300 mg) (DOR/3TC/TDF) or EFV (600 mg), emtricitabine (FTC; 200 mg) and TDF (300 mg) (EFV/FTC/TDF). The efficacy end point of interest at week 96 was the proportion of participants with HIV-1 RNA levels, Week 96 results support noninferior efficacy of doravirine (DOR)/3TC/ tenofovir disoproxil fumarate (TDF) compared with efavirenz (EFV)/ emtricitabine (FTC)/TDF established at week 48. DOR/3TC/TDF was well tolerated, with fewer neuropsychiatric and rash events than EFV/FTC/TDF.

(Pro)renin receptor [(P)RR] has multiple functions, but its regulation and role in the pathogenesis in glomerulonephritis (GN) are poorly defined. The aims of the present study were to determine the effects of direct renin inhibition (DRI) and demonstrate the role of (P)RR on the progression of crescentic GN. The anti-glomerular basement membrane nephritis rat model developed progressive proteinuria (83.64 ± 10.49 mg/day) and glomerular crescent formation (percent glomerular crescent: 62.1 ± 2.3%) accompanied by increased macrophage infiltration and glomerular expression of monocyte chemoattractant protein (MCP)-1, (P)RR, phospho-extracellular signal-regulated kinase (ERK)1/2, Wnt4, and active β-catenin. Treatment with DRI ameliorated proteinuria (20.33 ± 5.88 mg/day) and markedly reduced glomerular crescent formation (20.9 ± 2.6%), induction of macrophage infiltration, (P)RR, phospho-ERK1/2, Wnt4, and active β-catenin. Furthermore, primary cultured parietal epithelial cells stimulated by recombinant prorenin showed significant increases in cell proliferation. Notably, while the ERK1/2 inhibitor PD98059 or (P)RR-specific siRNA treatment abolished the elevation in cell proliferation, DRI treatment did not abrogate this elevation. Moreover, cultured mesangial cells showed an increase in prorenin-induced MCP-1 expression. Interestingly, (P)RR or Wnt4-specific siRNA treatment or the β-catenin antagonist XAV939 inhibited the elevation of MCP-1 expression, whereas DRI did not. These results suggest that (P)RR regulates glomerular crescent formation via the ERK1/2 signaling and Wnt/β-catenin pathways during the course of anti-glomerular basement membrane nephritis and that DRI mitigates the progression of crescentic GN through the reduction of (P)RR expression but not inhibition of prorenin binding to (P)RR.

An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a data set of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic reintroduction of active FH into HLRCC cell lines. This revealed a broad up-regulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new data set with prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell- and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation and a foundation for future efforts to exploit this knowledge for cancer therapy.

The activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription function has been implicated in the protection of neurodegenerative diseases. The cytoplasmic protein, Kelch-like ECH-associated protein 1 (Keap1), negatively regulates Nrf2. The Keap1-Nrf2 pathway is a potential therapeutic target for tackling free-radical damage. Dimethyl fumarate (DMF) is currently an approved drug for the treatment of relapsing multiple sclerosis. Recent studies showed that DMF modifies the reactive cysteines in the BTB domain of Keap1 and thus activates Nrf2 transcription function. Intriguingly, our crystal structure studies revealed that DMF also binds to the β-propeller domain (Keap1-DC) of Keap1. The crystal structure of the complex, refined to 1.54 Å resolution, revealed unexpected features: DMF binds (a) to the Nrf2-binding site (bottom region of Keap1-DC, site 1) with moderate interaction, and (b) to the top region of Keap1-DC, near to the blade II (site 2). The specificity of the binding 'site 2' was found to be unique to blade II of the β-propeller domain. The newly identified 'site 2' region in Keap1-DC may have a different functional role to regulate Nrf2. Moreover, the crystal structures of Keap1-DC in complex with the DMF analogs, including monoethyl fumarate, fumarate, and itaconate, also exhibited similar binding modes with Keap1-DC. Binding studies confirmed that DMF binds, in a nanomolar range, to the Keap1-DC region as well as the BTB domain of Keap1. Furthermore, the competitive binding assay in the presence of the Nrf2 peptide affirmed the direct binding of DMF at the Nrf2-binding region of Keap1-DC. Overall, our studies suggest that the drug molecule, DMF, binds at multiple sites of Keap1 and thus potentially activates Nrf2 function through covalent as well as the noncovalent mode of action, to combat oxidative stress. DATABASE: Structural data are available in RCSB-protein data bank database(s) under the accession numbers 6LRZ, 7C60, and 7C5E.

Objective We tested the hypothesis that chronic treatment with the direct renin inhibitor aliskiren improves vascular function in resistance and conduit arteries of type two diabetic and hypertensive patients. Method Sixteen patients with mild essential hypertension and with a previous diagnosis of noninsulin-dependent diabetes mellitus were included in the study. Patients were then randomized to aliskiren (150 mg once daily, n = 9), or ramipril (5 mg once daily, n = 7). Each patient underwent a biopsy of the subcutaneous tissue and small arteries were dissected and mounted on a pressurized micromyograph to evaluate endothelium dependent vasorelaxation in response to acetylcholine ± N omega-nitro-L-arginine methyl ester hydrochloride in vessels precontracted with norepinephrine. Endothelial function has been quantified also in large conduit arteries by flow-mediated dilation. Results A similar office blood pressure-lowering effect was observed with the two drugs, although changes in DBP were not statistically significant in the ramipril group. Aliskiren significantly improved endothelium-dependent relaxation in subcutaneous resistance arteries, as well as increased flow-mediated dilation in conduit arteries, whereas the effects induced by ramipril did not reach statistical significance. Only aliskiren significantly increased the expression of p1177-endothelial nitric oxide synthase in the endothelium. Both aliskiren and ramipril had a negligible effect on markers of oxidative stress. Conclusion Aliskiren restored endothelial function and induced a more prompt peripheral vasodilation in hypertensive and diabetic patients possibly through the increased production of nitric oxide via the enhanced expression and function of the active phosphorylated form of endothelial nitric oxide synthase.

26 p.-8 fig.-2 tab.-1 graph. abst.-+9 p. mat. supl.-10 fig.-2 tab., Common copathogenic factors, including oxidative stress and neuroinflammation, are found to play a vital role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Nowadays, owing to the multifactorial character of the diseases, no effective therapies are available, thus underlying the need for new strategies. Overexpression of the enzyme GSK-3β and downregulation of the Nrf2/ARE pathway are responsible for a decrease in antioxidant defense effects. These pieces of evidence underline the usefulness of dual GSK-3β inhibitors/Nrf2 inducers. In this regard, to design a dual modulator, the structures of a curcumin-based analogue, as GSK-3β inhibitor, and a diethyl fumarate fragment, as Nrf2 inducer, were combined. Among the hybrids, 5 and 6 proved to effectively inhibit GSK-3β, while 4 and 5 showed a marked ability to activate Nrf2 together to increase the neuronal resistance to oxidative stress. These last pieces of evidence translated into specific neuroprotective effects of 4 and 5 against PD pathological events including neurotoxicity elicited by α-synuclein aggregates and 6-hydroxydopamine. Hybrid 5 also showed neuroprotective effects in a C. elegans model of PD where the activation of GSK-3β is intimately involved in Nrf2 regulation. In summary, 5 emerged as an interesting multitarget derivative, valuable to be exploited in a multitarget PD perspective., This work was supported by the University of Bologna (RFO funds), MCIU (grant no. SAF2016-76693-R), and ISCiii CIBERNED (CB18/05/00040) and EDS, partially funded by the CNR project NUTR-AGE (FOE-2019, DSB.AD004.271).

Autologous grafts are the current gold standard of care for coronary artery bypass graft surgeries, but are limited by availability and plagued by high failure rates. Similarly, tissue engineering approaches to small diameter vascular grafts using naturally derived and synthetic materials fall short, largely due to inappropriate mechanical properties. Alternatively, decellularized extracellular matrix from tissue is biocompatible and has comparable strength to vessels, while poly(propylene fumarate) (PPF) has shown promising results for vascular grafts. This study investigates the integration of decellularized pericardial extracellular matrix (dECM) and PPF to create a biohybrid scaffold (dECM+PPF) suitable for use as a small diameter vascular graft. Our method to decellularize the ECM was efficient at removing DNA content and donor variability, while preserving protein composition. PPF was characterized and added to dECM, where it acted to preserve dECM against degradative effects of collagenase without disturbing the material's overall mechanics. A transport study showed that diffusion occurs across dECM+PPF without any effect from collagenase. The modulus of dECM+PPF matched that of human coronary arteries and saphenous veins. dECM+PPF demonstrated ample circumferential stress, burst pressure, and suture retention strength to survive in vivo. An in vivo study showed re-endothelialization and tissue growth. Overall, the dECM+PPF biohybrid presents a robust solution to overcome the limitations of the current methods of treatment for small diameter vascular grafts. STATEMENT OF SIGNIFICANCE: In creating a dECM+PPF biohybrid graft, we have observed phenomena that will have a lasting impact within the scientific community. First, we found that we can reduce donor variability through decellularization, a unique use of the decellularization process. Additionally, we coupled a natural material with a synthetic polymer to capitalize on the benefits of each: the cues provided to cells and the ability to easily tune material properties, respectively. This principle can be applied to other materials in a variety of applications. Finally, we created an off-the-shelf alternative to autologous grafts with a newly developed material that has yet to be utilized in any scaffolds. Furthermore, bovine pericardium has not been investigated as a small diameter vascular graft.

β-Alanine (β-Ala) is an important intermediate with numerous applications in food and feed additives, pharmaceuticals, polymeric materials, and electroplating industries. Its biological production routes that employ l-aspartate-α-decarboxylase (ADC) as the key enzyme are attractive. In this study, we developed an efficient and environmentally safe method for β-Ala production by co-expressing two different subtypes of ADC. A bacterial ADC from Bacillus subtilis (BSADC) and an insect ADC from Tribolium castaneum (TCADC) use pyruvoyl and pyridoxal-5′-phosphate (PLP) as cofactor, respectively. 3050 mM (271.5 g/L) β-Ala was achieved from l-aspartic acid by using the whole-cell biocatalyst co-expressing BSADC and TCADC, corresponding to a conversion rate of 92.4%. Meanwhile, one-pot synthesis of β-Ala from fumaric acid through using a tri-enzyme cascade route with two different subtypes of ADC and l-aspartase (AspA) from Escherichia coli was established. 2250 mM (200.3 g/L) β-Ala was obtained from fumaric acid with a conversion rate of 90.0%. This work proposes a novel strategy that improves β-Ala production in the decarboxylation pathway of l-aspartic acid.

It has been suggested that aldosterone breakthrough during treatment with a type 1 angiotensin II receptor (AT1R) blocker (ARB) may be an important risk factor for the progression of renal and cardiovascular disease. We examined whether the direct renin inhibitor, aliskiren caused aldosterone breakthrough in angiotensin II (Ang II)-dependent hypertensive mice. The effect of combination therapy with aliskiren and eplerenone was compared with that of therapy using renin–angiotensin system (RAS) blockade. Tsukuba hypertensive mice were treated for 12 weeks with aliskiren (30 mg/kg/day, i.p), candesartan (5 mg/kg/day, p.o), eplerenone (100 mg/kg/day, p.o) aliskiren and candesartan, aliskiren and eplerenone or candesartan and eplerenone. Blood pressure, urinary aldosterone and angiotensinogen (AGTN) excretion; plasma endothelin-1 concentration; kidney weight; urinary albumin excretion (UAE); glomerular injury; and renal messenger RNA (mRNA) levels for transforming growth factor (TGF)-β1, plasminogen activator inhibitor (PAI)-1, angiotensin-converting enzyme (ACE) and AT1R were measured. Combination therapy with aliskiren and candesartan caused a further decrease in blood pressure (p

C/EBP homologous protein (CHOP) is a transcription factor that is elevated in adipose tissue across many models of diabetes and metabolic stress. Although increased CHOP levels are associated with the terminal response to endoplasmic reticulum stress and apoptosis, there is no evidence for CHOP mediated apoptosis in the adipose tissue during diabetes. CHOP protein levels increase in parallel with protein succination, a fumarate derived cysteine modification, in the adipocyte during metabolic stress. We investigated the factors contributing to sustained CHOP proteins levels in the adipocyte, with an emphasis on the regulation of CHOP protein turnover by metabolite-driven modification of Keap1 cysteines. CHOP protein stability was investigated in conditions of nutrient stress due to high glucose or elevated fumarate (fumarase knockdown model); where cysteine succination is specifically elevated. CHOP protein turnover is significantly reduced in models of elevated glucose and fumarate with a ~30% increase in CHOP stability (p > 0.01), in part due to decreased CHOP phosphorylation. Sustained CHOP levels occur in parallel with elevated heme-oxygenase-1, a production of increased Nrf2 transcriptional activity and Keap1 modification. While Keap1 is directly succinated in the presence of excess fumarate derived from genetic knockdown of fumarase (fumarate levels are elevated >20-fold), it is the oxidative modification of Keap1 that predominates in adipocytes matured in high glucose (fumarate increases 4–5 fold). Elevated fumarate indirectly regulates CHOP stability through the induction of oxidative stress. The antioxidant N-acetylcysteine (NAC) reduces fumarate levels, protein succination and CHOP levels in adipocytes matured in high glucose. Elevated CHOP does not contribute elevated apoptosis in adipocytes, but plays a redox-dependent role in decreasing the adipocyte secretion of interleukin-13, an anti-inflammatory chemokine. NAC treatment restores adipocyte IL-13 secretion, confirming the redox-dependent regulation of a potent anti-inflammatory eotaxin. This study demonstrates that physiological increases in the metabolite fumarate during high glucose exposure contributes to the presence of oxidative stress and sustained CHOP levels in the adipocyte during diabetes. The results reveal a novel metabolic link between mitochondrial metabolic stress and reduced anti-inflammatory adipocyte signaling as a consequence of reduced CHOP protein turnover.

Background Diroximel fumarate (DRF) is a novel oral fumarate approved in the USA for relapsing forms of multiple sclerosis. DRF is converted to monomethyl fumarate, the pharmacologically active metabolite of dimethyl fumarate (DMF). DRF 462 mg and DMF 240 mg produce bioequivalent exposure of monomethyl fumarate and are therefore expected to have similar efficacy/safety profiles; the distinct chemical structure of DRF may contribute to its tolerability profile. Objectives The objective of this study was to compare the gastrointestinal tolerability of DRF and DMF over 5 weeks in patients with relapsing–remitting multiple sclerosis. Methods EVOLVE-MS-2 was a phase III, randomized, double-blind, head-to-head, 5-week study evaluating the gastrointestinal tolerability of DRF 462 mg vs DMF 240 mg, administered twice daily in patients with relapsing–remitting multiple sclerosis, using two self-administered gastrointestinal symptom scales: Individual Gastrointestinal Symptom and Impact Scale (IGISIS) and Global Gastrointestinal Symptom and Impact Scale (GGISIS). The primary endpoint was the number of days with an IGISIS intensity score ≥ 2 relative to exposure. Other endpoints included the degree of gastrointestinal symptom severity measured by IGISIS/GGISIS and assessment of safety/tolerability. Results DRF-treated patients experienced a statistically significant reduction (46%) in the number of days with an IGISIS symptom intensity score ≥ 2 compared with DMF-treated patients (rate ratio [95% confidence interval]: 0.54 [0.39–0.75]; p = 0.0003). Lower rates of gastrointestinal adverse events (including diarrhea, nausea, vomiting, and abdominal pain) were observed with DRF than DMF (34.8% vs 49.0%). Fewer patients discontinued DRF than DMF because of adverse events (1.6% vs 5.6%) and gastrointestinal adverse events (0.8% vs 4.8%). Conclusions DRF demonstrated an improved gastrointestinal tolerability profile compared with DMF, with less severe gastrointestinal events and fewer days of self-assessed gastrointestinal symptoms, fewer gastrointestinal adverse events, and lower discontinuation rates because of gastrointestinal adverse events. Clinical Trials Registration ClinicalTrials.gov (NCT03093324). Electronic supplementary material The online version of this article (10.1007/s40263-020-00700-0) contains supplementary material, which is available to authorized users.

Megalin is an endocytic receptor contributing to protein reabsorption. Impaired expression or trafficking of megalin increases urinary renin and allowed the detection of prorenin, which normally is absent in urine. Here, we investigated (pro)renin uptake by megalin, using both conditionally immortalized proximal tubule epithelial cells and Brown Norway Rat yolk sac cells (BN16). To distinguish binding and internalization, cells were incubated with recombinant human (pro)renin at 4°C and 37°C, respectively. (Pro)renin levels were assessed by immunoradiometric assay. At 4°C, BN16 cells bound 3× more prorenin than renin, suggestive for a higher affinity of prorenin. Similarly, at 37°C, prorenin accumulated at 3- to 4-fold higher levels than renin in BN16 cells. Consequently, depletion of medium prorenin (but not renin) content occurred after 24 hours. No such differences were observed in conditionally immortalized proximal tubule epithelial cells, and M6P (mannose-6-phosphate) greatly reduced conditionally immortalized proximal tubule epithelial cells (pro)renin uptake, suggesting that these cells accumulate (pro)renin largely via M6P receptors. M6P did not affect (pro)renin uptake in BN16 cells. Yet, inhibiting megalin expression with siRNA greatly reduced (pro)renin binding and internalization by BN16 cells. Furthermore, treating BN16 cells with albumin, an endogenous ligand of megalin, also decreased binding and internalization of (pro)renin, while deleting the (pro)renin receptor affected the latter only. Exposing prorenin’s prosegment with the renin inhibitor aliskiren dramatically increased prorenin binding, while after prosegment cleavage with trypsin prorenin binding was identical to that of renin. In conclusion, megalin might function as an endocytic receptor for (pro)renin and displays a preference for prorenin. Megalin-mediated endocytosis requires the (pro)renin receptor.

Aim: We recently proposed miR-142-3p as a molecular player in inflammatory synaptopathy, a new pathogenic hallmark of multiple sclerosis (MS) and of its mouse model experimental autoimmune encephalomyelitis (EAE), that leads to neuronal loss independently of demyelination. MiR-142-3p seems to be unique among potential biomarker candidates in MS, since it is an inflammatory miRNA playing a dual role in the immune and central nervous systems. Here, we aimed to verify the impact of miR-142-3p circulating in the cerebrospinal fluid (CSF) of MS patients on clinical parameters, neuronal excitability and its potential interaction with disease modifying therapies (DMTs). Methods and Results: In a cohort of 151 MS patients, we found positive correlations between CSF miR-142-3p levels and clinical progression, IL-1β signalling as well as synaptic excitability measured by transcranial magnetic stimulation. Furthermore, therapy response of patients with ‘low miR-142-3p’ to dimethyl fumarate (DMF), an established disease-modifying treatment (DMT), was superior to that of patients with ‘high miR-142-3p’ levels. Accordingly, the EAE clinical course of heterozygous miR-142 mice was ameliorated by peripheral DMF treatment with a greater impact relative to their wild type littermates. In addition, a central protective effect of this drug was observed following intracerebroventricular and ex vivo acute treatments of EAE wild type mice, showing a rescue of miR-142-3p-dependent glutamatergic alterations. By means of electrophysiology, molecular and biochemical analysis, we suggest miR-142-3p as a molecular target of DMF. Conclusion: MiR-142-3p is a novel and potential negative prognostic CSF marker of MS and a promising tool for identifying personalised therapies.

To investigate the effects of dietary fumaric acid (FUA) on performance, carcasses, nutrient digestibility, blood metabolites, digestive enzymes, and cecal microbiota in Japanese quail chicks. Three hundred unsexed Japanese quail (1-wk-old) were randomly assigned to 5 groups. Supplementation of FUA in the diet of Japanese quail chicks exhibited a significant improvement in growth performance through the different experimental periods studied compared with those receiving unsupplemented one. The digestibility of crude protein (CP) and metabolizable energy (ME) were improved with 10 and 15 g/kg FUA, respectively. Apart from lipase enzyme, birds fed 5 and 15 g/kg FUA recorded higher activity of amylase. There were no significant changes among experimental groups on the relative weights of carcass, gizzard, heart, and dressing. Dietary supplementation of FUA at different levels (P> 0.05) increased total protein (TP) and globulin (GLB) concentrations and A/G % compared with control group. A significant (P < 0.01) decrease in plasma low density lipoprotein (LDL) and total cholesterol (TC) levels and increase in high density lipoprotein (HDL) concentrations were observed in chicks fed with FUA containing diets. Immunoglobulin G (IgG) (P = 0.0026) and M (IgM) (P = 0.0007) levels were greater in groups treated with either 10 or 15 g FUA/kg diet. A significant increase in plasma Ca concentration was noticed in chicks received 15 g FUA/ kg compared with the other groups. Quail chicks received diets containing FUA at different levels exhibited reduced cecal count of coliform, E. coli, and Salmonella as compared with control group. In conclusion, supplementation of fumaric acid (especially 15 g/kg diet) in quail chick diets improved their growth, digestibility of nutrients, immune response, antioxidant status, digestive enzyme, and intestinal health.

Experimental studies on the reactive extraction of fumaric acid with Amberlite LA-2 from Rhizopus oryzae suspensions using three solvents with different dielectric constants varying from 9.08 to 1.90 (dichloromethane, n-butyl acetate, and n-heptane, respectively) underlined the particular behavior of the extraction system in the presence of fungal biomass. The interfacial mass flow of the reaction product was found to be significantly affected by the biomass, due mainly to its adsorption onto the phase separation interface, this leading to the appearance of a physical barrier against the solute’s transfer. However, the magnitude of the adsorption phenomenon was found to depend on Rhizopus oryzae’s affinity for the solvent phase, which increased significantly from dichloromethane to n-heptane. The negative influence of the biomass on the interfacial mass transfer can be partially counteracted by adding 1-octanol into the organic phase, improving the solvent’s ability to solve the fumaric acid–Amberlite LA-2 complex and simplifying the reactive extraction mechanism, effects that were found to be more important for low-polar solvents. Consequently, for the same mixing intensity, the maximum amplification factor was reached for n-heptane, its value being almost 5–6 times higher than that obtained for dichloromethane and over 2 times higher than that obtained with n-butyl acetate.

Bioorthogonal reactions are rapid, specific and high yield reactions that can be performed in in vivo microenvironments or simulated microenvironments. At present, the main biorthogonal reactions include Staudinger ligation, copper-catalyzed azide alkyne cycloaddition, strain-promoted [3 + 2] reaction, tetrazine ligation, metal-catalyzed coupling reaction and photo-induced biorthogonal reactions. To date, many reviews have reported that bioorthogonal reactions have been used widely as a powerful tool in the field of life sciences, such as in target recognition, drug discovery, drug activation, omics research, visualization of life processes or exogenous bacterial infection processes, signal transduction pathway research, chemical reaction dynamics analysis, disease diagnosis and treatment. In contrast, to date, few studies have investigated the application of bioorthogonal reactions in the analysis of biomacromolecules in vivo. Therefore, the application of bioorthogonal reactions in the analysis of proteins, nucleic acids, metabolites, enzyme activities and other endogenous molecules, and the determination of disease-related targets is reviewed. In addition, this review discusses the future development opportunities and challenges of biorthogonal reactions. This review presents an overview of recent advances for application in biomolecular analysis and disease diagnosis, with a focus on proteins, metabolites and RNA detection.

The male factor is responsible for infertility in about 35–40% of all cases. Idiopathic oligo- and/or astheno- and/or therato-zoospermia is one of the most common male fertility disorders and remains a significant therapeutic challenge. The primary cause of idiopathic male infertility remains unknown but seems to be associated with oxidative stress. Objective: The use of antioxidative formulation to improve qualitative and quantitative deficiencies in the male gametes. In total, 78 subjects were treated with a combination of 1,725 mg L-carnitine fumarate, 500 mg acetyl-L-carnitine, 90 mg vitamin C, 20 mg coenzyme Q10, 10 mg zinc, 200 µg folic acid, 50 µg selenium, and 1.5 µg vitamin B12 (Proxeed® Plus, Sigma-Tau, Italy) for 6 months; the preparation was taken twice daily from the time idiopathic infertility was diagnosed. Basic seminal parameters were evaluated by a European Society of Human Reproduction and Embryology (ESHRE) -certified embryologist following the fifth edition of the World Health Organisation (2010) guidelines at three time points: at baseline and 3 and 6 months of treatment. Improvements in semen parameters (differing in terms of dynamics) were evident at 3 months and gradually improved over the 6 months of treatment. Each parameter: sperm concentration, total sperm count, sperm total and progressive motility improved significantly after treatment except for the percentage of sperm of abnormal morphology and ejaculate volume. Proxeed Plus was effective for patients with idiopathic infertility; however, a long treatment period is needed to achieve optimal results.

Thiol-containing nucleophiles such as cysteine react spontaneously with the citric acid cycle intermediate fumarate to form S-(2-succino)-adducts. In Bacillus subtilis, a salvaging pathway encoded by the yxe operon has recently been identified for the detoxification and exploitation of these compounds as sulfur sources. This route involves acetylation of S-(2-succino)cysteine to N-acetyl-2-succinocysteine, which is presumably converted to oxaloacetate and N-acetylcysteine, before a final deacetylation step affords cysteine. The critical oxidative cleavage of the C-S bond of N-acetyl-S-(2-succino)cysteine was proposed to depend on the predicted flavoprotein monooxygenase YxeK. Here, we characterize YxeK and verify its role in S-(2-succino)-adduct detoxification and sulfur metabolism. Detailed biochemical and mechanistic investigation of YxeK including 18 O-isotope-labeling experiments, homology modeling, substrate specificity tests, site-directed mutagenesis, and (pre-)steady-state kinetics provides insight into the enzyme's mechanism of action, which may involve a noncanonical flavin-N5-peroxide species for C-S bond oxygenolysis.

Chinese hamster ovary cell constructs expressing either the β 1-, β 2- or β 3-adrenergic receptor (AR) were used to determine whether a novel β-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific β-AR subtype. EC50 values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the β 3-AR subtype, with an EC50 of 6 × 10–9 M, with no detectible agonistic activity at the β 2-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the β-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for β 1-, β2-, and β 3-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; β-AR pan-agonist), dobutamine hydrochloride (DOB; specific β 1-AA), salbutamol sulfate (SAL; specific β 2-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific β 3-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for β 1-, β 2-, and β 3-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant β-AR mRNA in both adipose tissues was the β 2-AR (P < 0.05), with the β 1- and β 3-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these β-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the β 1- and β 2-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to β-adrenergic ligands, especially those that are agonists at the β 1- and β3-receptor subtypes. The minimal mRNA expression of the β 1- and β 3 subtypes in i.m. adipose tissue likely limits the response potential to agonists for these β-AR subtypes.

The study was designed to determine the impact of acute toxicity of fumaronitrile exposure through tissue damaging, oxidative stress enzymes and histopathological studies in gills, liver and muscle cells of freshwater tilapia fish (Oreochromis mossambicus). In gill, liver, and muscle cells, biochemical indicators such as tissue damage enzymes (Acid Phosphatase (ACP), Alkaline Phosphatase (ALP), and Lactate Dehydrogenase (LDH)) and antioxidative enzymes (Superoxide Dismutase (SOD); Catalase (CAT); Glutathione-S-transferase (GST); Reduced Glutathione (GSH); Glutamate oxaloacetate transaminase (GOT) and Glutamate pyruvate transaminase (GPT) were quantified in the time interval of 30, 60 and 90 days exposure to the fumaronitrile. After 90 days, under 6 ppb exposure conditions, the acid phosphatase (ACP) levels of fish increased significantly in the gills (3.439 μmol/mg protein/min), liver (1.743 μmol/mg protein/min), and muscles (2.158 μmol/mg protein/min). After 90 days of exposure to the same concentration and days, ALP activity increased significantly in gills (4.354 μmol/mg protein/min) and liver (1.754 μmol/mg protein/min), but muscle cells had a little decrease in ALP activity (2.158 μmol/mg protein/min). The LDH concentration in gills following treatment with fumaronitrile over a period of 0–90 days was 3.573 > 3.521 > 2.245 μmol/mg protein/min over 30 > 60 > 90 days. However, at the same dose and treatment duration, a greater LDH level of 0.499 μmol/mg protein/min was found in liver and muscle cells. Histopathological abnormalities in the gills, liver, and muscle cells of treated fish were also examined, indicating that fumaronitrile treatment generated the most severe histological changes. The current study reveals that fumaronitrile exposure has an effect on Oreochromis mossambicus survival, explaining and emphasising the risk associated with this POP exposure to ecosystems and human populations.

Analytical procedure development for quantifying 10 impurities in Tenofovir Alafenamide Fumarate (TAF) tablets was a challenge for analytical and formulation researchers. The aim of this paper was to develop a robust, regulatory-flexible, application-specific Ultra Performance Liquid Chromatography (UPLC) analytical procedure using the Analytical Lifecycle Management (ALM) and the Analytical Quality by Design (AQbD) for the estimation of the TAF tablets. In this work, the Analytical Target Profile (ATP) for the analytical procedure and the Critical Analytical Attributes (CAAs) were identified. Through the risk assessment studies, the high-risk analytical conditions were found, and they were screened and optimized by the Design of Experiment (DoE) to obtain the Design Space (DS) and identify the working point. The prediction intervals were used to examine the robustness of the analytical procedure. And the procedure performance qualification and the continued procedure performance verification were used to ensure routine application of analytical procedure. Finally, the 10 impurities were separated within 20 min by UPLC. The success of this study demonstrates the usefulness of using ALM and AQbD for analytical procedure development and provides a reference for the analytical procedure development for other drugs.

BACKGROUND The objective of the study was to evaluate the physicochemical characteristics and shelf-life of corn tortilla stored at room temperature (25 °C) using aw modifiers (propylene glycol and glycerol) and pH modifiers (fumaric acid and sodium benzoate) as preservatives combined with calcium hydroxide. Detection thresholds were used to determine the maximum preservative concentration and calcium hydroxide. Physicochemical characterization and sensory evaluation were used to determine the stability and sensory shelf-life of tortillas. RESULTS Control, calcium hydroxide, calcium hydroxide + fumaric acid, calcium hydroxide + glycerol, calcium hydroxide + propylene glycol, and calcium hydroxide + sodium benzoate treatments had rounded half-lives of 1, 2, 1, 2, 2, and 2 days respectively. Glycerol combined with calcium hydroxide resulted in tortillas with lower pH variations over time. Mold presence was the critical attribute causing tortilla rejection. CONCLUSION The use of aw modifiers had a better effect in preserving corn tortilla, as the concentration of pH modifiers at detection threshold levels was not able to reach an optimum performance when combined with calcium hydroxide. © 2021 Society of Chemical Industry.

Fumaric acid is an additive allowed by the Codex Alimentarius and under evaluation by the International Organisation of Vine and Wine (OIV) that can be used for wine acidification but also to inhibit malolactic fermentation (MLF). The use of 300–900 mg/L of fumaric acid can inhibit MLF in red wines decreasing pH by 0.2 units or more depending on the buffer capacity. When MLF was running with populations of either 7 or 8 log CFU/mL strain alpha (Oenococcus oeni) the application of 600 mg/L of fumaric acid stopped the process for more than 50 days and cells were undetected in specific media. In triangular tastings, fumaric acid was not detected at 300–600 mg/L (p < .05). In subsequent preference tests, some tasters perceived more acidity and body. Fumaric acid is a useful technological additive to improve wine microbiological stability and freshness, also allowing reduction of SO2 levels. Trujillo San Isidro