Green Synthesis of Zinc Oxide Nanoparticles: Fortification for Rice Grain Yield and Nutrients Uptake Enhancement.

Applications of metal oxide nanoparticles in the agriculture sector are being extensively included as the materials are considered superior. In the present work, zinc oxide nanoparticle (ZnO NPs), with a developing fertilizer, is applied in the fortification of rice grain yield and nutrient uptake enhancement. To evaluate the role of ZnO NP, two field experiments were conducted during the 2018 and 2019 seasons. ZnO NPs were small, nearly spherical, and their sizes equal to 31.4 nm, as proved via the dynamic light scattering technique. ZnO NPs were applied as a fertilizer in different concentrations, varying between 20 and 60 mg/L as a foliar spray. The mixture of ZnSO4 and ZnO NP40 ameliorated yield component and nutrients (N, K, and Zn) uptake was enhanced compared to traditional ZnSO4 treatment. Nevertheless, the uptake of the phosphorous element (P) was adversely affected by the treatment of ZnO NPs. Thus, treatment via utilizing ZnO NPs as a foliar with a very small amount (40 ppm) with of basal ZnSO4 led to a good improvement in agronomic and physiological features; eventually, higher yield and nutrient-enriched rice grain were obtained.

Bisphenol A exposure disrupts aspartate transport in HepG2 cells.

The liver is the organ responsible for bisphenol A (BPA) metabolism, an environmental chemical agent. Exposure to this toxin is associated with liver abnormalities and dysfunction. An important role played by excitatory amino acid transporters (EAATs) of the slc1 gene family has been reported in liver injuries. To gain insight into a plausible effect of BPA exposure in the liver glutamate/aspartate transport, using the human hepatoblastoma cell line HepG2, we report a BPA-dependent dynamic regulation of SLC1A3 and SLC1A2. Through the use of radioactive [3 H]- d-aspartate uptake experiments and immunochemical approaches, we characterized time and dose-dependent regulation of the protein levels and function of these transporters after acute exposure to BPA. An increase in nuclear Yin Yang 1 was found. These results suggest an important involvement of the EAATs in liver physiology and its disruption after acute BPA exposure.

Long-lasting anti-bacterial activity and bacteriostatic mechanism of tea tree oil adsorbed on the amino-functionalized mesoporous silica-coated by PAA.

Tea tree oil (TTO) is an efficient natural antibacterial agent. However, the bacteriostatic effect of TTO does not prevail for a long period because of the volatile nature of the oil. Therefore, a novel sustained-release formulation of TTO should be developed for improving the applicability of TTO. Herein, the mesoporous silica was selected for constructing a carrier for TTO. Mesoporous silica is non-toxic, easy to modify and exhibited an adjustable pore size. First, the mesoporous silica was modified by an aminated silane coupling agent (NH2-MCM-41). Then, the polyacrylic acid (PAA) was bonded by electrostatic bonds (PAA-NH2-MCM-41), which imparted the sustained-release effect in the TTO, supported in the mesoporous silica channel (TTO/PAA-NH2-MCM-41). The prepared bacteriostatic agent can achieve long-term sustained-release properties. At room temperature (26 ℃), the release rate of TTO after 11 h release reached 50 %. However, the release rate of TTO from TTO/PAA-NH2-MCM-8 reached only 42 % after 24 h. Furthermore, the sustained release behavior of TTO/PAA-NH2-MCM-41 was consistent with the Korsmeyer-Peppas kinetic model. Compared to TTO, TTO/PAA-NH2-MCM-41 exhibited a stable and sustained bacteriostatic effect even after 50 days in a natural environment. The minimum inhibitory concentration (MIC) value of the TTO/PAA-NH2-MCM-41 against Escherichia coli (E. coli) was 0.37∼0.44 mg/mL. TTO altered the cell morphology of E. coli and broke the integrity of the cell membrane, leading to cell death.

Cancer cell membrane-coated mesoporous silica loaded with superparamagnetic ferroferric oxide and Paclitaxel for the combination of Chemo/Magnetocaloric therapy on MDA-MB-231 cells.

To effectively inhibit the growth of breast cancer cells (MDA-MB-231 cells) by the combination method of chemotherapy and magnetic hyperthermia, we fabricated a biomimetic drug delivery (CSiFePNs) system composed of mesoporous silica nanoparticles (MSNs) containing superparamagnetic ferroferric oxide and Paclitaxel (PTX) coated with MDA-MB-231 cell membranes (CMs). In the in vitro cytotoxicity tests, the MDA-MB-231 cells incubated with CSiFePNs obtained IC50 value of 0.8 µgL-1, 3.5-fold higher than that of SiFePNs. The combination method of chemotherapy and magnetic hyperthermia can effectively inhibit the growth of MDA-MB-231 cells.

[Ion transporters in the lungs. Use as therapeutic targets]. / Transportadores de iones en pulmón. Uso como dianas terapéuticas.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

PmtA functions as a ferrous iron and cobalt efflux pump in Streptococcus suis.

Transition metals are nutrients essential for life. However, an excess of metals can be toxic to cells, and host-imposed metal toxicity is an important mechanism for controlling bacterial infection. Accordingly, bacteria have evolved metal efflux systems to maintain metal homeostasis. Here, we established that PmtA functions as a ferrous iron [Fe(II)] and cobalt [Co(II)] efflux pump in Streptococcus suis, an emerging zoonotic pathogen responsible for severe infections in both humans and pigs. pmtA expression is induced by Fe(II), Co(II), and nickel [Ni(II)], whereas PmtA protects S. suis against Fe(II) and ferric iron [Fe(III)]-induced bactericidal effect, as well as Co(II) and zinc [Zn(II)]-induced bacteriostatic effect. In the presence of elevated concentrations of Fe(II) and Co(II), ΔpmtA accumulates high levels of intracellular iron and cobalt, respectively. ΔpmtA is also more sensitive to streptonigrin, a Fe(II)-activated antibiotic. Furthermore, growth defects of ΔpmtA under Fe(II) or Co(II) excess conditions can be alleviated by manganese [Mn(II)] supplementation. Finally, PmtA plays a role in tolerance to H2O2-induced oxidative stress, yet is not involved in the virulence of S. suis in mice. Together, these data demonstrate that S. suis PmtA acts as a Fe(II) and Co(II) efflux pump, and contributes to oxidative stress resistance.

Transportadores de iones en pulmón: Uso como dianas terapéuticas / Ion transporters in the lungs. Use as therapeutic targets

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

The median eminence as the hypothalamic area involved in rapid transfer of glucose to the brain: functional and cellular mechanisms.

Our data proposes that glucose is transferred directly to the cerebrospinal fluid (CSF) of the hypothalamic ventricular cavity through a rapid "fast-track-type mechanism" that would efficiently stimulate the glucosensing areas. This mechanism would occur at the level of the median eminence (ME), a periventricular hypothalamic zone with no blood-brain barrier. This "fast-track" mechanism would involve specific glial cells of the ME known as ß2 tanycytes that could function as "inverted enterocytes," expressing low-affinity glucose transporters GLUT2 and GLUT6 in order to rapidly transfer glucose to the CSF. Due to the large size of tanycytes, the presence of a high concentration of mitochondria and the expression of low-affinity glucose transporters, it would be expected that these cells accumulate glucose in the endoplasmic reticulum (ER) by sequestering glucose-6-phosphate (G-6-P), in a similar way to that recently demonstrated in astrocytes. Glucose could diffuse through the cells by micrometric distances to be released in the apical region of ß2 tanycytes, towards the CSF. Through this mechanism, levels of glucose would increase inside the hypothalamus, stimulating glucosensing mechanisms quickly and efficiently. KEY MESSAGES: • Glucose diffuses through the median eminence cells (ß2 tanycytes), towards the hypothalamic CSF. • Glucose is transferred through a rapid "fast-track-type mechanism" via GLUT2 and GLUT6. • Through this mechanism, hypothalamic glucose levels increase, stimulating glucosensing.

Caffeine-free hawk tea lowers cholesterol by reducing free cholesterol uptake and the production of very-low-density lipoprotein.

Medicinal plants show important therapeutic value in chronic disease treatment. However, due to their diverse ingredients and complex biological effects, the molecular mechanisms of medicinal plants are yet to be explored. By means of several high-throughput platforms, here we show hawk tea extract (HTE) inhibits Niemann-Pick C1-like 1 (NPC1L1)-mediated free cholesterol uptake, thereby inducing the transcription of low-density lipoprotein receptor (LDLR) downstream of the sterol response element binding protein 2 (SREBP2) pathway. Meanwhile, HTE suppresses hepatocyte nuclear factor 4α (HNF4α)-mediated transcription of microsomal triglyceride transfer protein (MTP) and apolipoprotein B (APOB), thereby decreasing the production of very-low-density lipoprotein. The catechin EGCG ((-)-epigallocatechin gallate) and the flavonoids kaempferol and quercetin are identified as the bioactive components responsible for the effects on the NPC1L1-SREBP2-LDLR axis and HNF4α-MTP/APOB axis, respectively. Overall, hawk tea works as a previously unrecognized cholesterol-lowering agent in a multi-target and multi-component manner.

Sugarcane apoplast fluid modulates the global transcriptional profile of the diazotrophic bacteria Paraburkholderia tropica strain Ppe8.

The stalk apoplast fluid of sugarcane contains different sugars, organic acids and amino acids that may supply the demand for carbohydrates by endophytic bacteria including diazotrophs P. tropica (syn. B. tropica) strain Ppe8, isolated from sugarcane, is part of the bacterial consortium recommended as inoculant to sugarcane. However, little information has been accumulated regarding this plant-bacterium interaction considering that it colonizes internal sugarcane tissues. Here, we made use of the RNA-Seq transcriptomic analysis to study the influence of sugarcane stalk apoplast fluid on Ppe8 gene expression. The bacterium was grown in JMV liquid medium (100 ml), divided equally and then supplemented with 50 ml of fresh JMV medium or 50 ml of apoplast fluid extracted from sugarcane variety RB867515. Total RNA was extracted 2 hours later, the rRNAs were depleted and mRNAs used to construct libraries to sequence the fragments using Ion Torrent technology. The mapping and statistical analysis were carried out with CLC Genomics Workbench software. The RNA-seq data was validated by RT-qPCR using the reference genes fliP1, paaF, and groL. The data analysis showed that 544 genes were repressed and 153 genes were induced in the presence of apoplast fluid. Genes that induce plant defense responses, genes related to chemotaxis and movements were repressed in the presence of apoplast fluid, indicating that strain Ppe8 recognizes the apoplast fluid as a plant component. The expression of genes involved in bacterial metabolism was regulated (up and down), suggesting that the metabolism of strain Ppe8 is modulated by the apoplast fluid. These results suggest that Ppe8 alters its gene expression pattern in the presence of apoplast fluid mainly in order to use compounds present in the fluid as well as to avoid the induction of plant defense mechanisms. This is a pioneer study showing the role played by the sugarcane apoplast fluid on the global modulation of genes in P. tropica strain Ppe8.

Monosodium Glutamate Reduces 68Ga-PSMA-11 Uptake in Salivary Glands and Kidneys in a Preclinical Prostate Cancer Model.

We evaluated the ability of monosodium glutamate (MSG) to reduce salivary and kidney uptake of a prostate-specific membrane antigen (PSMA) radioligand without affecting tumor uptake. Methods: LNCaP tumor-bearing mice were intraperitoneally injected with MSG (657, 329, or 164 mg/kg) or phosphate-buffered saline (PBS). Fifteen minutes later, the mice were intravenously administered 68Ga-PSMA-11. PET/CT imaging and biodistribution studies were performed 1 h after administration. Results: Tumor uptake (percentage injected dose per gram [%ID]) was not statistically different between groups, at 8.42 ± 1.40 %ID in the 657 mg/kg group, 7.19 ± 0.86 %ID in the 329 mg/kg group, 8.20 ± 2.44 %ID in the 164 mg/kg group, and 8.67 ± 1.97 %ID in the PBS group. Kidney uptake was significantly lower in the 657 mg/kg group (85.8 ± 24.2 %ID) than in the 329 mg/kg (159 ± 26.2 %ID), 164 mg/kg (211 ± 27.4 %ID), and PBS groups (182 ± 33.5 %ID) (P < 0.001). Salivary gland uptake was lower in the 657 mg/kg (3.72 ± 2.12 %ID) and 329 mg/kg (5.74 ± 0.62 %ID) groups than in the PBS group (10.04 ± 2.52 %ID) (P < 0.01). Conclusion: MSG decreased salivary and kidney uptake of 68Ga-PSMA-11 in a dose-dependent manner, whereas tumor uptake was unaffected.

How can 50 years of solute transport data in articular cartilage inform the design of arthritis therapeutics?

OBJECTIVE: For the last half century, transport of nutrients and therapeutics in articular cartilage has been studied with various in vitro systems that attempt to model in vivo conditions. However, experimental technique, tissue species, and tissue storage condition (fresh/frozen) vary widely and there is debate on the most appropriate model system. Additionally, there is still no clear overarching framework with which to predict solute transport properties based on molecular characteristics. This review aims to develop such a framework, and to assess whether experimental procedure affects trends in transport data. METHODS: Solute data from 31 published papers that investigated transport in healthy articular cartilage were obtained and analyzed for trends. RESULTS: Here, we show that diffusivity of spherical and globular solutes in cartilage can be predicted by molecular weight (MW) and hydrodynamic radius via a power-law relationship. This relationship is robust for many solutes, spanning 5 orders of magnitude in MW and was not affected by variations in cartilage species, age, condition (fresh/frozen), and experimental technique. Traditional models of transport in porous media exhibited mixed effectiveness at predicting diffusivity in cartilage, but were good in predicting solute partition coefficient. CONCLUSION: Ultimately, these robust relationships can be used to accurately predict and improve transport of solutes in adult human cartilage and enable the development of better optimized arthritis therapeutics.

Nano-delivery of trace minerals for marine fish larvae: influence on skeletal ossification, and the expression of genes involved in intestinal transport of minerals, osteoblast differentiation, and oxidative stress response.

Currently, the larviculture of many marine fish species with small-sized larvae depends for a short time after hatching, on the supply of high-quality live zooplankton to ensure high survival and growth rates. During the last few decades, the research community has made great efforts to develop artificial diets, which can completely substitute live prey. However, studies aimed at determining optimal levels of minerals in marine larvae compound feeds and the potential of novel delivery vectors for mineral acquisition has only very recently begun. Recently, the agro-food industry has developed several nano-delivery systems, which could be used for animal feed, too. Delivery through nano-encapsulation of minerals and feed additives would protect the bioactive molecules during feed manufacturing and fish feeding and allow an efficient acquisition of active substances into biological system. The idea is that dietary minerals in the form of nanoparticles may enter cells more easily than their larger counterparts enter and thus speed up their assimilation in fish. Accordingly, we evaluated the efficacy of early weaning diets fortified with organic, inorganic, or nanoparticle forms of trace minerals (Se, Zn, and Mn) in gilthead seabream (Sparus aurata) larvae. We tested four experimental diets: a trace mineral-deficient control diet, and three diets supplemented with different forms of trace minerals. At the end of the feeding trial, larvae growth performance and ossification, and the level of expression of six target genes (SLC11A2ß, dmt1, BMP2, OC, SOD, GPX), were evaluated. Our data demonstrated that weaning diets supplemented with Mn, Se, and Zn in amino acid-chelated (organic) or nanoparticle form were more effective than diets supplemented with inorganic form of minerals to promote bone mineralization, and prevent skeletal anomalies in seabream larvae. Furthermore, nanometals markedly improved larval stress resistance in comparison to inorganic minerals and upregulated mRNA copy number of OC gene. The expression of this gene was strongly correlated with mineralization degree, thus confirming its potency as a good marker of bone mineralization in gilthead seabream larvae.

Efficient clearance of Aß protofibrils in AßPP-transgenic mice treated with a brain-penetrating bifunctional antibody.

BACKGROUND: Amyloid-ß (Aß) immunotherapy is one of the most promising disease-modifying strategies for Alzheimer's disease (AD). Despite recent progress targeting aggregated forms of Aß, low antibody brain penetrance remains a challenge. In the present study, we used transferrin receptor (TfR)-mediated transcytosis to facilitate brain uptake of our previously developed Aß protofibril-selective mAb158, with the aim of increasing the efficacy of immunotherapy directed toward soluble Aß protofibrils. METHODS: Aß protein precursor (AßPP)-transgenic mice (tg-ArcSwe) were given a single dose of mAb158, modified for TfR-mediated transcytosis (RmAb158-scFv8D3), in comparison with an equimolar dose or a tenfold higher dose of unmodified recombinant mAb158 (RmAb158). Soluble Aß protofibrils and total Aß in the brain were measured by enzyme-linked immunosorbent assay (ELISA). Brain distribution of radiolabeled antibodies was visualized by positron emission tomography (PET) and ex vivo autoradiography. RESULTS: ELISA analysis of Tris-buffered saline brain extracts demonstrated a 40% reduction of soluble Aß protofibrils in both RmAb158-scFv8D3- and high-dose RmAb158-treated mice, whereas there was no Aß protofibril reduction in mice treated with a low dose of RmAb158. Further, ex vivo autoradiography and PET imaging revealed different brain distribution patterns of RmAb158-scFv8D3 and RmAb158, suggesting that these antibodies may affect Aß levels by different mechanisms. CONCLUSIONS: With a combination of biochemical and imaging analyses, this study demonstrates that antibodies engineered to be transported across the blood-brain barrier can be used to increase the efficacy of Aß immunotherapy. This strategy may allow for decreased antibody doses and thereby reduced side effects and treatment costs.

A multi-center preclinical study of gadoxetate DCE-MRI in rats as a biomarker of drug induced inhibition of liver transporter function.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and transplantation. DILI can be the result of impaired hepatobiliary transporters, with altered bile formation, flow, and subsequent cholestasis. We used gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), combined with pharmacokinetic modelling, to measure hepatobiliary transporter function in vivo in rats. The sensitivity and robustness of the method was tested by evaluating the effect of a clinical dose of the antibiotic rifampicin in four different preclinical imaging centers. The mean gadoxetate uptake rate constant for the vehicle groups at all centers was 39.3 +/- 3.4 s-1 (n = 23) and 11.7 +/- 1.3 s-1 (n = 20) for the rifampicin groups. The mean gadoxetate efflux rate constant for the vehicle groups was 1.53 +/- 0.08 s-1 (n = 23) and for the rifampicin treated groups was 0.94 +/- 0.08 s-1 (n = 20). Both the uptake and excretion transporters of gadoxetate were statistically significantly inhibited by the clinical dose of rifampicin at all centers and the size of this treatment group effect was consistent across the centers. Gadoxetate is a clinically approved MRI contrast agent, so this method is readily transferable to the clinic. CONCLUSION: Rate constants of gadoxetate uptake and excretion are sensitive and robust biomarkers to detect early changes in hepatobiliary transporter function in vivo in rats prior to established biomarkers of liver toxicity.

Formate rescues neural tube defects caused by mutations in Slc25a32.

Periconceptional folic acid (FA) supplementation significantly reduces the prevalence of neural tube defects (NTDs). Unfortunately, some NTDs are FA resistant, and as such, NTDs remain a global public health concern. Previous studies have identified SLC25A32 as a mitochondrial folate transporter (MFT), which is capable of transferring tetrahydrofolate (THF) from cellular cytoplasm to the mitochondria in vitro. Herein, we show that gene trap inactivation of Slc25a32 (Mft) in mice induces NTDs that are folate (5-methyltetrahydrofolate, 5-mTHF) resistant yet are preventable by formate supplementation. Slc25a32gt/gt embryos die in utero with 100% penetrant cranial NTDs. 5-mTHF supplementation failed to promote normal neural tube closure (NTC) in mutant embryos, while formate supplementation enabled the majority (78%) of knockout embryos to complete NTC. A parallel genetic study in human subjects with NTDs identified biallelic loss of function SLC25A32 variants in a cranial NTD case. These data demonstrate that the loss of functional Slc25a32 results in cranial NTDs in mice and has also been observed in a human NTD patient.

First-in-Class Combination Therapy of a Copper(II) Metallo-Phosphorus Dendrimer with Cytotoxic Agents.

The metallo-phosphorus dendrimer 1G3-Cu (generation 3 dendrimer bearing 48 conjugated copper(II) on its surface) has antiproliferative activity related to its capacity to activate Bax translocation. In the present study, we evaluate the activity of an association of 1G3-Cu with 5 cytotoxic agents used in chemotherapy having different modes of action. Data show no additive effect with camptothecin and cisplatin, additivity with paclitaxel and MG132, and synergy with doxorubicin. Results suggest that the multivalent Cu-conjugated dendrimer 1G3-Cu (activator of Bax translocation) plays an important role in boosting the clinical impact of Bax accumulation stimulated by the proteasome inhibitor MG132, antimitotic taxanes, and the topo II inhibitor doxorubicin.

The influence of cellular senescence on intracellular vitamin C transport, accumulation, and function.

In humans, vitamin C (VC) accumulates at higher concentrations in cells than in plasma, and this intracellular accumulation appears critical to several important physiological functions. However, although VC accumulation decreases in the elderly, the influence of cellular senescence on the transport, accumulation, and function of VC is poorly understood. In this study, we investigated the effects of supplementation with both ascorbic acid (AsA) and dehydroascorbic acid (DehAsA) on the uptake and accumulation of VC, AsA, and DehAsA into cells and the effect of AsA on the levels of intracellular reactive oxygen species (ROS) in human fibroblast TIG-1 cells. We also assessed how that supplementation affected senescence-associated changes in intracellular VC transport and accumulation. AsA supplementation significantly increased intracellular levels of AsA, DehAsA, and total VC (i.e., reduced AsA plus oxidized DehAsA) in senescent cells compared with young cells. DehAsA supplementation also significantly increased intracellular AsA and total VC levels in senescent cells, but not DehAsA, and the increases were less than after adding AsA. Among the molecules related to VC accumulation, the mRNA and protein expressions of sodium-dependent VC transporter 2 (SLC23A2) were increased in senescent cells. Furthermore, intracellular peroxide and superoxide anion levels were higher in senescent cells, with AsA supplementation markedly attenuating spontaneous intracellular peroxide accumulation. Overall, our results therefore suggest that VC transport and accumulation improved in senescent human fibroblast TIG-1 cells due to the adaptive upregulation of sodium-dependent VC transporter 2 in response to increased ROS levels. We conclude that adequate supplementation with AsA can effectively mitigate senescence-associated intracellular ROS.

Involvement of bilitranslocase and beta-glucuronidase in the vascular protection by hydroxytyrosol and its glucuronide metabolites in oxidative stress conditions.

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because it is extensively metabolized into glucuronides (GCs). The aim of this study was to test HT and GC vasculoprotective effects and the underlying mechanisms using aorta rings from 8-week-old male Wistar rats. In the absence of oxidative stress, incubation with 100 µM HT or GC for 5 min did not exert any vasorelaxing effect and did not influence the vascular function. Conversely, in condition of oxidative stress [upon incubation with 500 µM tert-butylhydroperoxide (t-BHP) for 30 min], preincubation with HT or GC improved acetylcholine-induced vasorelaxation compared with untreated samples (no t-BHP). This protective effect was lost for GC, but not for HT, when a washing step (15 min) was introduced between preincubation with HT or GC and t-BHP addition, suggesting that only HT enters the cells. In agreement, bilitranslocase inhibition with 100 µM phenylmethanesulfonyl fluoride for 20 min reduced significantly HT, but not GC, effect on the vascular function upon stress induction. Moreover, GC protective effect (improvement of endothelium-dependent relaxation in response to acetylcholine) in oxidative stress conditions was reduced by preincubation of aorta rings with 300 µM D-saccharolactone to inhibit ß-glucuronidase, which can deconjugate polyphenols. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that, in conditions of oxidative stress, GC can be deconjugated into HT that is transported through the cell membrane by bilitranslocase to protect vascular function.

Chemical Modulation of the Human Oligopeptide Transporter 1, hPepT1.

In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is located across the apical membranes of the small intestine and kidney, where it serves as a high-capacity low-affinity transporter of a broad range of di- and tripeptides. hPepT1 is also overexpressed in the colon of inflammatory bowel disease (IBD) patients, where it mediates the transport of harmful peptides of bacterial origin. Therefore, hPepT1 is a drug target for prodrug substrates interacting with intracellular proteins or inhibitors blocking the transport of toxic bacterial products. In this study, we construct multiple structural models of hPepT1 representing different conformational states that occur during transport and inhibition. We then identify and characterize five ligands of hPepT1 using computational methods, such as virtual screening and QM-polarized ligand docking (QPLD), and experimental testing with uptake kinetic measurements and electrophysiological assays. Our results improve our understanding of the substrate and inhibitor specificity of hPepT1. Furthermore, the newly discovered ligands exhibit unique chemotypes, providing a framework for developing tool compounds with optimal intestinal absorption as well as future IBD therapeutics against this emerging drug target.