Biochemistry

Generating atrial-like cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial for modeling and treating atrial-related diseases, such as atrial arrythmias including atrial fibrillations. However, it is essential to obtain a comprehensive understanding of the electrophysiological properties of these cells. The objective of the present study was to investigate the molecular, electrical, and biophysical properties of several ion channels, especially NaV1.5 channels, in atrial hiPSC cardiomyocytes. Atrial cardiomyocytes were obtained by the differentiation of hiPSCs treated with retinoic acid (RA). The quality of the atrial specification was assessed by qPCR, immunocytofluorescence, and western blotting. The electrophysiological properties of action potentials (APs), Ca2+ dynamics, K+ and Na+ currents were investigated using patch-clamp and optical mapping approaches. We evaluated mRNA transcript and protein expressions to show that atrial cardiomyocytes ex...

NG2 glia receive synaptic input from neurons, but the functional impact of this glial innervation is not well understood. In the developing cerebellum and somatosensory cortex the GABAergic input might regulate NG2 glia differentiation and myelination, and a switch from synaptic to extrasynaptic neuron–glia signaling was reported in the latter region. Myelination in the hippocampus is sparse, and most NG2 glia retain their phenotype throughout adulthood, raising the question of the properties and function of neuron-NG2 glia synapses in that brain region. Here, we compared spontaneous and evoked GABAA receptor-mediated currents of NG2 glia in juvenile and adult hippocampi of mice of either sex and assessed the mode of interneuron–glial signaling changes during development. With patch-clamp and pharmacological analyses, we found a decrease in innervation of hippocampal NG2 glia between postnatal days 10 and 60. At the adult stage, enhanced activation of extrasynaptic receptors occurre...

Protein misfolding and aggregation lead to amyloid generation that in turn may induce cell membrane disruption and leads to cell apoptosis. In an effort to prevent or treat amyloidogenesis, large number of studies has been paying attention on breakthrough of amyloid inhibitors. In the present work, we aim to access the effect of two drugs, that is, acetylsalicylic acid and 5-amino salicylic acid on insulin amyloids by using various biophysical, imaging, cell viability assay, and computational approaches. We established that both drugs reduce the turbidity, light scattering and fluorescence intensity of amyloid indicator dye thioflavin T. Premixing of drugs with insulin inhibited the nucleation phase and inhibitory potential was boosted by increasing the concentration of the drug. Moreover, addition of drugs at the studied concentrations attenuated the insulin fibril induced cytotoxicity in breast cancer cell line MDA-MB-231. Our results highlight the amino group of salicylic acid ex...

Numerous phenolic compounds have been reported in the last decade that have a good antioxidant property and interaction affinity towards mammalian serum albumins. In the present study, we have utilized human serum albumins as a model protein to examine their comparative interaction property with polyphenolic compound tannic acid (TA) by using various spectroscopic and calorimetric methods We have also monitored the esterase and antioxidant activity of mammalian serum albumins in the absence and presence of TA. The obtain results recommended that the TA have a good binding affinity (∼10(4) to 10(6)M(-1)) towards mammalian serum albumins and shows double sequential binding sites, which depends on the concentration of TA that induced the conformational alteration which responsible for the thermal stability of proteins. Binding affinity, structural transition and thermodynamic parameters were calculated from spectroscopic and calorimetric method reveals that non-covalent interaction cau...

To resolve the metabolite redox cycling associated with our earlier clinical compound 2, we carried out lead optimization of lead molecule 1. Compound 4 showed improved lipophilic ligand efficiency and demonstrated robust glucose lowering in diet-induced obese mice without a liability in predictive preclinical drug safety studies. Thus, it was selected as a clinical candidate and further studied in type 2 diabetic patients. Clinical data suggests no evidence of metabolite cycling, which is consistent with the preclinical profiling of metabolism.

A rat brain cDNA encoding for a novel protein with agmatinase activity was cloned and functionally expressed. The protein was expressed as a histidine-tagged fusion product with a molecular weight of about 63 kDa. Agmatine hydrolysis was strictly dependent on Mn 2+ ; K m and k cat values were 2.5 § 0.2 mM and 0.8 § 0.2 s ¡1 , respectively. The product putrescine was a linear competitive inhibitor (K i D 5 § 0.5 mM). The substrate speciWcity, metal ion requirement and pH optimum (9.5) coincide with those reported for Escherichia coli agmatinase, the best characterized of the agmatinases. However, as indicated by the k cat /K m (320 M ¡1 s ¡1 ), the recombinant protein was about 290-fold less eYcient than the bacterial enzyme. The deduced amino sequence revealed great diVerences with all known agmatinases, thus excluding the protein from the arginase family. It was, however, highly identical (>85%) to the predicted sequences for fragments of hypothetical or unnamed LIM domain-containing proteins. As a suggestion, the agmatinase activity is adscribed to a protein with an active site that promiscuously catalyze a reaction other than the one it evolved to catalyze.

Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for deliveri...

Although silicon nanoparticles dispersed in liquids are used in various applications ranging from bio-labeling to hydrogen production, their reactivities with their solvents and their catalytic properties remain still unexplored. Here, we discovered that, because of their surface structures and mechanical strain, silicon nanoparticles react strongly with their solvents and may act as catalysts for the dehydrogenation, at room temperature, of secondary alcohols (e.g. isopropanol) into ketones and hydrogen. This catalytic reaction was monitored by gas chromatography, pH measurements, mass spectroscopy and solid-state NMR. This discovery provides new understanding of the role played by silicon nanoparticles, and nanosilicon in general, in their reactivity in solvents in general as well as being candidates in catalysis.

We propose a microfluidic platform where a dilution module to generate concentration gradient and a ladder-like fluidic network for a parallel synchronization of a droplet were integrated. The device consists of two layers: upper layer is for oil phase and lower layer for water phase. The desired concentration at the lower layer was achieved by means of controlled volumetric mixing ratios of two merging solution in a T-junction. By controlling channel length, fluidic resistances were adjusted. The water-in-oil droplets generated at lower layer were injected to the synchronization region where a top, middle, and bottom channel were interconnected among the three main channels. By a pressure difference caused from the droplets among main channels, the oil, carrier fluid, flows through the interconnection channels until the pressure in each channel was balanced automatically. The proposed device showed the feasibility of the synchronization of three droplets with 92% efficiency at the optimized flow rate condition. As the device enables passive synchronization of droplets with different concentrations, it has a potential to be integrated with a conventional droplet-based microfluidic platform for a reliability of mixing.

Background: A new Ultra performance liquid chromatographic analytical method was developed and validated for estimation of Quercetin in bulk powder. The reverse phase chromatographic elution using UPLC was carried out in gradient mode on C-18 column (Phenomenex Luna 5 µm, 150 mm X 4.6 mm) as stationary phase utilizing a mobile phase composed of Acetonitrile: 0.1 % formic acid buffer (50:50 v/v) with a flow rate of 1 ml/min and injection volume of 10µl.  The analysis was performed at temperature of 400C and detection of eluent was carried out using photo diode array at 371 nm. The newly developed Ultra performance liquid chromatography method was validated in terms of linearity and range, system suitability, specificity, precision, sensitivity, robustness, ruggedness and accuracy as per ICH guidelines. Results: The retention time of quercetin was found to be at 3.9 min with total run time of analysis is 7 minutes. The linearity was observed between the concentration ranges from 0.5 t...

Type 1 diabetes (T1D) is a chronic autoimmune disease caused by the immune-mediated reduction of β cells, resulting in lifelong dependence on exogenous insulin administration. We studied the impact of M1/M2 macrophages on the β cell level. We obtained the healthy state (absence of T1D) and unhealthy state (presence of T1D). We found that in the unhealthy state, β cell levels are decreased with a continuous alternative switch between M1 and M2 macrophages. However, in the healthy state, the β cell levels increase with a high level of M2 macrophages.

Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT‐29 Glc−/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT‐29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk‐cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl‐CoA synthesis is ...

To study the metabolic fate of L-histidine and histamine in rat isolated enterocytes, enterocytes were incubated in the presence of 0.1 mM L-[U-14C] histidine. At the rate of 11.1 ± 2.7 pmol/106 cells/30 min, the amino acid was incorporated into cellular proteins. 80 µM cycloheximide, i.e. a protein synthesis inhibitor, inhibited this incorporation by 70 ± 17%. L-histidine was used for cellular protein synthesis which depended on time and concentration. 0.1 mM L-[U-14C] histidine was little oxidized by intestinal cells, i.e. 0.12 ± 0.06 pmol/106 cells/30 min, and was not converted into histamine. When 10 mM histamine was added to the incubation medium, it completely inhibited the incorporation of 0.1 mM [1,4-14C] putrescine into isolated enterocytes. In enterocyte homogenates, this corresponded to inhibition by histamine of putrescine incorporation as catalyzed by transglutaminase activity. Since histamine incorporation into TCA-precipitable material derived from enterocyte homogena...

untreated rats (1). This basal NO production can be Liver cells can produce nitric oxide from L-argispectacularly increased in hepatocytes or Kupffer nine through either constitutive NO synthase or incells by in vivo or in vitro treatment with a combinaducible NO synthase (NOS) detected after in vivo or tion of cytokines and/or lipopolysaccharide (LPS) in vitro treatment with cytokines and/or lipopoly-(2 -7). Although NO can exert a beneficial effect (8saccharide (LPS). The effects of NO on liver cells 11), it has been proposed that when NO is produced are associated with protein synthesis and mitochonin excess it might be deleterious for liver physiology drial electron transfer inhibition. L-Arginine is also (6). To support this hypothesis, NO has been shown the precursor of L-ornithine and polyamines. The to decrease protein synthesis (7, 12), mitochondrial latter are considered to be protective in the liver in electron transfer (13, 14), and hepatocyte growth several experimental models. The aim of the present 1 Abbreviations used: NOS, nitric oxide synthase; LPS, lipopolythe cosubstrate for spermidine and spermine prosaccharide; BH 4 , 6-(R,S)-5,6,7,8-tetrahydro-L-biopterin; cNOS, constitutive NOS; iNOS, inducible NOS. duction (21), suggests that the liver is able to synthe-268

Butyrate and acetate are bacterial metabolites present in the large intestine lumen. Although butyrate is well known to inhibit the in vitro proliferation of human colon carcinoma cells in a process involving the hyperacetylation of specific nuclear histones, little is known about the possible link between butyrate metabolism and its growth‐inhibitory effect. In a previous study (Leschelle et al., 2000, Eur J Biochem 267: 6435–6442), we showed that butyrate accumulates and is metabolized in HT‐29 Glc−/+ cells without increasing oxygen consumption. In the present study, using the same cell line incubated with 14C‐labeled butyrate, we determined that a minor part of 14C from butyrate was recovered in nuclear histones. Unlike butyrate, acetate exerted no effect on cell growth but was a precursor for overall net histone acetylation. Although butyrate was able to increase the cellular AMP/ADP ratio, it did not affect the ATP cell content or the adenylate charge or the oxidation of endoge...

Rat enterocytes exposed to L-arginine in the absence of any other exogenous substrate were found to actively metabolize this cationic amino acid. L-Arginine was converted to L-citrulline either directly in a NADPH-sensitive manner thought to be coupled with the generation of NO, or indirectly through the sequence of reactions catalyzed by arginase and ornithine transcarbamylase. A large fraction of L-citrulline and L-ornithine generated from exogenous L-arginine was released in the incubation medium. The production of COz and (poly)amines from t-arginine occurred at rates 2 to 3 orders of magnitude lower than that characterizing the net uptake of the cationic amino acid, and this despite the fact that enterocytes were equipped to allow the interconversion of L-ornithine and L-glutamate. It is concluded that the oxidative catabolism of L-arginine in enterocytes is quantitatively negligible relative to its conversion to L-citrulline and t-ornithine.

Numerous data from both epidemiological and experimental origins indicate that some alimentary proteins and amino acids in supplements can modify the blood LDL cholesterol, HDL cholesterol and total cholesterol. After an initial approval of the health claim for soy protein consumption for the prevention of coronary heart disease, more recently it has been concluded from an overall analysis of literature that isolated soy protein with isoflavones only slightly decrease LDL and total cholesterol. Other plant extracts and also some proteins from animal origin have been reported to exert a lowering effect on blood cholesterol when compared with a reference protein (often casein). The underlying mechanisms are still little understood. Individual amino acids and mixture of amino acids have also been tested (mostly in animal studies) for their effects on cholesterol parameters and on cholesterol metabolism. Methionine, lysine, cystine, leucine, aspartate and glutamate have been tested individually and in combination in different models of either normo or hypercholesterolemic animals and found to be able to modify blood cholesterol and/or LDL cholesterol and/or HDL cholesterol. It is however not known if these results are relevant to human nutrition.

Eşime ve oğluma, vi vii ÖNSÖZ Yüksek lisans öğrenimim sırasında bana her türlü desteği sunan, değerli yorumlarıyla ve bilgi birikimleriyle beni en doğru şekilde yönlendiren değerli hocam Doç. Dr Mustafa Özcan"a, Ders ve tez aşamalarım sırasında bana tüm inkanları sunan, çalışmalarımı destekleyen Perkin Elmer Sağlık ve Çevre Bilimleri Ltd. Şti."ne ve değerli çalışma arkadaşlarıma, Tez ve ders aşamasında benden desteğini asla esirgemeyen sevgili arkadaşım Sesil Uzaşçı"ya, Yaşadığım tüm güzellikleri ve zorlukları paylaşan, sevgilerini yalnızca yanımdayken değil her anımda hissettiğim değerli dostlarıma, Her zaman yanımda olan canım annem, babam ve ağebeyime, bütün aileme ve beni ilk günden beri destekleyen sevgili hayat arkadaşım Emre Caner"e, Ailemize yeni katılacak olan oğlum Emir Caner"e, Teşekkür ederim. Hayat sizlerle güzel.. Sayenizde..

A new allelochemical (1), m.p. 282-284 ºC, m.f. C31H36O19, [M] + 712 (FABMS), has been isolated from methanolic extract of the seeds of Adenanthera pavonina Linn. alongwith two known compounds 2',4',7-trihydroxy isoflavone and isovitexin. The structure of a new compound was characterized as 3,5,7,3',4'-pentahydroxy flavone-3'-O-α-L-rhamnopyranosyl-(1→4)-O-α-L-arabinopyranosyl-(1→3)-O-β-D-xylopyranoside, by various colour reactions, spectral analysis and chemical degradations.

The dynamics of electron injection have been investigated in intramolecular i-motif conjugates possessing stilbenediether (Sd) and perylenediimide (PDI) chromophores separated by either four or six hemiprotonated cytosine C-C base pairs assembled with synthetic loops. Circular dichroism spectra are consistent with the formation of i-motif structures in the absence or presence of Sd and PDI chromophores. The fluorescence of the Sd chromophore is essentially completely quenched by neighboring C-C base pairs, consistent with their function as an electron donor and electron acceptor, respectively. However, the fluorescence of the PDI chromophore is only partially quenched. The dynamics of electron injection from singlet Sd into the i-motif and subsequent charge recombination has been determined by femtosecond transient absorption (fsTA) spectroscopy and compared with the results for electron injection and charge recombination in Sd-linked hairpins possessing cytosine-guanine (C-G) or 5-...

The dynamics and efficiency of photoinduced charge transport has been investigated in DNA capped hairpins possessing a stilbenedicarboxamide (Sa) hole donor and stilbenediether (Sd) hole acceptor separated by DNA G-quadruplex structures possessing 2-to-4 tetrads by means of femtosecond and nanosecond transient absorption spectroscopy with global analysis. The results for the quadruplex structures are compared with those for the corresponding duplex structures having G-C base pairs in place of the G-tetrads. Following photoinduced charge separation to form a contact radical ion pair, hole transport to form the Sa(-•)/Sd(+•) charge-separated state is slower but more efficient for the quadruplex vs duplex structures. Thus, the G-quadruplex serves as an effective conduit for positive charge rather than as a hole trap when inserted into a duplex, as previously postulated.

DNA-based molecular electronics will require charges to be transported from one site within a 2D or 3D architecture to another. While this has been shown previously in linear, π-stacked DNA sequences, the dynamics and efficiency of charge transport across a DNA three-way junction (3WJ) have yet to be determined. Here, we present an investigation of hole transport and trapping across a DNA-based three-way junction systems by a combination of femtosecond transient absorption spectroscopy and molecular dynamics simulations. Hole transport across the junction is proposed to be gated by conformational fluctuations in the ground state which bring the transiently-populated hole carrier nucleobases into better aligned geometries on the nanosecond timescale, thus modulating the - electronic coupling along the base pair sequence.

Materials. Aq 1 and PDI 2 chromophores were synthesized, purified, and characterized as previously described. Oligonucleotide synthesis was carried out using phosphoramidite chemistry on an Expedite 8909 synthesizer. For Aq and PDI monomers the coupling time was extending to 15 minutes. For PDI an additional wash cycle using dichloromethane was added to the protocol. The oligonucleotides were deprotected by incubating in 30% ammonium hydroxide at room temperature for 24 h, purified by RP-HPLC and characterized using MALDI-TOF spectroscopy. Thermal dissociation profiles were recorded on a Perkin Elmer Lambda 2 UV/VIS spectrometer with a Peltier Temperature Programmer. Circular dichroism (CD) spectra were recorded on a Jasco-J-815 CD spectrometer.

Chemicals. The synthesis of the dumbbell and hairpin was reported earlier. 1 All DNA samples and conjugates were dissolved in 10 mM phosphate buffer (pH=7.2) with 0.1 M NaCl. Concentrations were such that the absorbance varied between 0.3 and 1.0 at the pump wavelength of 265 nm. A melting temperature of 55 °C was determined for the hairpin from the first derivative of the temperature-dependent absorbance at 250 nm (Figure ). This value is slightly higher than the melting temperature of 52°C measured by McCullagh et al. 1 for a similar hairpin with 8 AT base pairs. The melting temperature of the dumbbell is much higher than for the hairpin and could not be precisely determined from our measurements (Figure ). Earlier, McCullagh et al. reported a melting temperature of 78°C for this dumbbell.

We report here the effect of replacing one or both of the purine or pyrimidine blocks of a diblock stilbene donor-acceptor capped hairpin with locked nucleic acid (LNA) bases on the dynamics and efficiency of hole transport. The structures of the DNA and LNA:DNA hybrids are tentatively assigned to B-or A-type structures on the basis of their circular dichroism spectra. Replacing the bases in either the A-block or the G-block of the diblock DNA hairpin with LNA bases results in a modest decrease in the base-to-base hopping rate constant and quantum yield for charge separation. Somewhat larger decreases are observed when all of the purine or pyrimidine bases are replaced by LNA bases.

Ascorbate is a major plant metabolite that plays crucial roles in various processes, from reactive oxygen scavenging to epigenetic regulation. However, to what extent and how ascorbate modulates metabolism is largely unknown. To address this, we investigated the consequences of chloroplastic and total cellular ascorbate-deficiencies by studying chloroplastic ascorbatetransporter pht4;4 mutant lines, and the ascorbate-deficient vtc2-4 mutant of Arabidopsis thaliana. Under regular growth conditions, both ascorbate-deficiencies caused minor alterations in photosynthesis, with no apparent signs of oxidative damage. In contrast, metabolomics analysis revealed a global and largely overlapping metabolome rewiring in both ascorbatedeficiencies, suggesting that chloroplastic ascorbate modulates plant metabolism. We observed significant alterations in amino acid metabolism, particularly in arginine metabolism, activation of nucleotide salvage pathways, and changes in secondary metabolism. In addition, proteomewide analysis of thermostability revealed that ascorbate may interact with enzymes involved in arginine metabolism, the Calvin-Benson cycle, and several photosynthetic electron transport components. Overall, our results suggest that, independently of oxidative stress, chloroplastic ascorbate interconnects and coordinates diverse metabolic pathways in vascular plants and thus acts as a regulatory hub.

Compound of 1- deoxynojirimycin (DNJ) in the mulberry leave extract (MLE) potentially hamper the hydrolysis process of various carbohydrates. This experiment aimed to study dynamic concentration of reductive sugar resulted from the hydrolysis of various carbohydrates by raw enzymes of the rumen liquid, with or without the addition of mulberry leave extract containing 1-deoxynojirimycin. Materials tested are maltose, sucrose, starch and cellulose.  The result of the experiment showed that the addition of mulberry leave extract on the media with the substrate of maltose hampered the activity of maltase.  It was concluded that MLE containing DNJ can be used as an agent of slow release mechanism of non-structural carbohydrates, especially maltose, in the rumen system

Bats are hosts to a large diversity of eukaryotic protozoan blood parasites that comprise species of Trypanosoma and different haemosporidian parasite taxa and bats have played an important role in the evolutionary history of both parasite groups. However, bats in several geographical areas have not been investigated, including in Burkina Faso, where no information about malaria parasites and trypanosomes of bats exists to date. In this study, we collected data on the prevalence and the phylogenetic relationships of protozoan blood parasites in nine different bat species in Burkina Faso. Hepatocystis parasites were detected in two species of epauletted fruit bats, and a relatively high diversity of trypanosome parasites was identified in five bat species. The phylogenetic analyses recovered the trypanosome parasites of the bat species Rhinolophus alcyone and Nycteris hispida as close relatives of T. livingstonei, the trypanosome infections in Scotophilus leucogaster as closely related to the species T. vespertilionis and the trypanosomes from Pipistrellus nanulus and Epomophorus gambianus might present the species T. dionisii. These findings of the first investigation in Burkina Faso present a first snapshot of the diversity of protozoan blood parasites in bats in this country.

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4-FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4-mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.

The NorA efflux pump of Staphylococcus aureus is known to play a major role in the development of resistance against quinolone drugs by reducing their concentration inside target pathogens. The objective of this study was to evaluate the ability of tannic acid to inhibit the gene expression of the NorA efflux pump in Staphylococcus aureus and to evaluate the in silico effect on the pump. Efflux pump inhibition was evaluated by fluorimetry. The checkerboard method evaluates the effect of the test substance in combination with an antimicrobial at different concentrations. To gene expression evaluation NorA the assay was performed using: a sub-inhibitory concentration preparation (MIC/4) of the antibiotic; a sub-inhibitory concentration preparation (MIC/4) of the antibiotic associated with tannic acid at a sub-inhibitory concentration (MIC/4). In this study, docking simulations were performed by the SWISSDOCK webserver. The ability of tannic acid to inhibit the NorA efflux pump can be related to both the ability to inhibit the gene expression of this protein, acting on signaling pathways involving the ArlRS membrane sensor. As well as acting directly through direct interaction with the NorA protein, as seen in the approach and in silico and in vitro per checkerboard method and fluorimetry of bromide accumulated in the cell. Staphylococcus aureus is the leading cause of human infectious diseases worldwide, ranging from superficial skin lesions to systemic and life-threatening infections such as osteomyelitis, endocarditis, pneumonia and septicemia. S. aureus virulence has been associated with the production of a large number of extracellular toxins, enzymes and cell-surface-associated proteins encoded by diverse genes 1 . The S. aureus NorA efflux pump, a member of the major facilitator superfamily (MFS), is known to play a major role in the development of resistance against quinolone drugs by reducing their concentration inside target pathogens. NorA is also a proton motive force which mediates the intrinsic resistance of many unrelated drugs, including the monocationic compound ethidium and the hydrophilic fluoroquinolone ciprofloxacin 2 . The NorA efflux pump present in S. aureus is expressed by the norA gene located in the Smal D fragment of the S. aureus chromosome 3 . The NorA efflux pump is characterized by the presence of 12 transmembrane segments and is related to the Bmr efflux pump present in Bacillus subtilis 5 . The NorA protein is predicted to have 388 amino acids with a molecular mass of 42,385 6 . The expression of MDR efflux pumps is usually tightly regulated by transcriptional regulators acting at different levels. Efflux pump genes are usually expressed at low levels, at least in the laboratory setting, with physiological high-level expression requiring the presence of an effector. Notably, antibiotics are not among the best efflux pump expression inducers, which suggests the extrusion of antibiotics currently in use for the treatment of infectious diseases is not always the pump's original function . Non-physiological, inherited high-level efflux

Canavan Disease (CD) is a leukodystrophy caused by homozygous null mutations in the gene encoding aspartoacylase (ASPA). ASPA-deficiency is characterized by severe psychomotor retardation, and excessive levels of the ASPA substrate Nacetylaspartate (NAA). ASPA is an oligodendrocyte marker and it is believed that CD has a central etiology. However, ASPA is also expressed by Schwann cells and ASPA-deficiency in the periphery might therefore contribute to the complex CD pathology. In this study, we assessed peripheral and central auditory function in the Aspa lacZ/lacZ rodent model of CD using auditory brainstem response (ABR). Increased ABR thresholds and the virtual loss of waveform peaks 4 and 5 from Aspa lacZ/lacZ mice, indicated altered central auditory processing in mutant mice compared with Aspa wt/wt controls and altered central auditory processing. Analysis of ABR latencies recorded from Aspa lacZ/lacZ mice revealed that the speed of nerve conduction was unchanged in the peripheral part of the auditory pathway, and impaired in the CNS. Histological analyses confirmed that ASPA was expressed in oligodendrocytes and Schwann cells of the auditory system. In keeping with our physiological results, the cellular organization of the cochlea, including the organ of Corti, was preserved and the spiral ganglion nerve fibres were normal in ASPA-deficient mice. In contrast, we detected substantial hypomyelination in the central auditory system of Aspa lacZ/lacZ mice. In summary, our data suggest that the lack of ASPA in the CNS is responsible for the observed hearing deficits, while ASPA-deficiency in the cochlear nerve fibres is tolerated both morphologically and functionally.

O carcinoma hepatocelular constitui uma das neoplasias malignas mais letais e com eficácia terapêutica limitada. Os extratos vegetais, ricos em metabólitos secundários, realizam funções benéficas ao organismo humano, como a redução da inflamação, dor, estresse oxidativo e da neoplasia. Assim, o objetivo deste estudo foi o de realizar a triagem fitoquímica (fenóis e flavonóides) do extrato do fruto de Brosimum gaudichaudii (EBG), avaliar in vitro a sua citotoxicidade (teste do MTT/células NIH/3T3), e suas atividades antioxidante (DPPH e Lipoperoxidação) e antineoplásica (teste do MTT/células HuH7.5). O EBG apresentou 48,02 ± 1,26 mg EAG/g de fenóis totais e 8 ± 0,92 mg EQ/g de flavonoides. No MTT, o EBG, de 100 a 1600 ug/mL, não diferiu do controle negativo, não promovendo citotoxicidade (p>0,05). O EBG, 600 µg/mL, apresentou uma atividade antioxidante de 79.80 ± 0.00% no teste do DPPH e de 88.01 ± 1.74% no teste de lipoperoxidação. Na avaliação anticancerígena em células de hepat...

The definitive treatment for end-stage renal disease is kidney transplantation, which remains limited by organ availability and post-transplant complications. Alternatively, an implantable bioartificial kidney could address both problems while enhancing the quality and length of patient life. An implantable bioartificial kidney requires a bioreactor containing renal cells to replicate key native cell functions, such as water and solute reabsorption, and metabolic and endocrinologic functions. Here, we report a proof-of-concept implantable bioreactor containing silicon nanopore membranes to offer a level of immunoprotection to human renal epithelial cells. After implantation into pigs without systemic anticoagulation or immunosuppression therapy for 7 days, we show that cells maintain >90% viability and functionality, with normal or elevated transporter gene expression and vitamin D activation. Despite implantation into a xenograft model, we find that cells exhibit minimal damage,...

Major clinical challenges associated with islet transplantation for type 1 diabetes include shortage of donor organs, poor engraftment due to ischemia, and need for immunosuppressive medications. Semipermeable membrane capsules can immunoprotect transplanted islets by blocking passage of the host's immune components while providing exchange of glucose, insulin, and other small molecules. However, capsules-based diffusive transport often exacerbates ischemic injury to islets by reducing the rate of oxygen and nutrient transport. We previously reported the efficacy of a newly developed semipermeable ultrafiltration membrane, the silicon nanopore membrane (SNM) under convective-driven transport, in limiting the passage of pro-inflammatory cytokines while overcoming the mass transfer limitations associated with diffusion through nanometer-scale pores. In this study, we report that SNM-encapsulated mouse islets perfused in culture solution under convection outperformed those under diffusive conditions in terms of magnitude (1.49-fold increase in stimulation index and 3.86-fold decrease in shutdown index) and rate of insulin secretion (1.19-fold increase and 6.45-fold decrease during high and low glucose challenges), respectively. Moreover, SNM-encapsulated mouse islets under convection demonstrated rapid glucose-insulin sensing within a physiologically relevant time-scale while retaining healthy islet viability even under cytokine exposure. We conclude that encapsulation of islets with SNM under convection improves islet in vitro functionality. This approach may provide a novel strategy for islet transplantation in the clinical setting.

We developed a method to purify decorin core protein from tissue with the goal of preserving its native structure and biological function. Currently, most procedures rely on the use of denaturing reagents potentially altering the biological activity. Decorin was purified from corneal stromas without the use of detergents or chaotropic reagents. Proteoglycans isolated using anion exchange chromatography on Q-Sepharose were treated with chondroitinase ABC. Decorin was isolated by a second Q-Sepharose chromatography with affinity chromatographies on heparin-Sepharose and concanavalin A-Sepharose. SDS-PAGE revealed a 98.4% pure 44 kDa protein identified as decorin with a yield of 35 mg per 100 bovine corneas. Identification was confirmed by NanoESI and MALDI qTOF. The novel inclusion of 20% propylene glycol in extraction and column buffers resulted in recoveries of proteoglycans comparable with those observed with detergents and urea. Purified decorin did alter the rate of fibrillogenesis of type I collagen and inhibited the lateral fusion of collagen fibrils. It also bound to [ 125 I]TGF-b1 with an apparent K d of 40 nM. Circular dichroism spectroscopy of decorin displayed the spectra of a-helices and b-pleated sheets consistent with those obtained from recombinant decorin. Urea-induced unfolding was cooperative and reversible while thermal denaturation caused irreversible unfolding. Native decorin can be purified from tissue in quantity and quality for biophysical, biochemical, and biological assays.

In the normal feedback mechanism of injury and repair in the lung, fragmented heparan sulfate proteoglycans (HSPGs) from damaged extracellular matrix and cells are believed to interact with elastases to limit their activity. An imbalance in the HSPG-elastase response may play an important role in situations where uncontrolled lung injury leads to diseases such as emphysema. To gain insight into this complex process of heparin and heparan sulfate regulation of elastases, an experimental study was undertaken to resolve the mechanism and structural requirements of heparin inhibition of human neutrophil elastase (HNE). Kinetic analyses were completed using in vitro assays with synthetic and insoluble elastin substrates in the presence of HNE and various heparin preparations (14-15 kDa; 17-19 kDa), heparin-derived oligosaccharides (4-22 saccharides), and chemically modified heparins (2-O-, 6-O-, O-, and N-desulfated). Results showed that heparin inhibits HNE by a tight-binding, hyperbolic, competitive mechanism, contrary to previous reports in the literature. A minimum length of at least 12-14 saccharides is required for inhibition, after which inhibitory activity increases with chain length (or molecular mass). Although all N-and O-sulfate groups contribute to inhibition, 2-O-sulfate groups are less critical than either N-or 6-O-sulfate groups, indicating that inhibitory activity is dependent upon the heparin fine structure. Molecular-docking simulations support the kinetic results and provide a plausible model for the size requirement, whereby positively charged, clamp-like regions at the ends of the interdomain crevice (elastase fold) are used by heparin to bridge the active site and inhibit activity.

Proteoglycans, once thought to primarily serve as structural components of extracellular matrix, are now being focused on for their role in tissue and cell regulation, particularly angiogenesis. Many growth factors, notably the fibroblast growth family ͑FGF͒ which now numbers 19 members, bind to heparin and heparan sulfate proteoglycans and this binding has been shown to have a significant impact on the availability and activity of these growth factors. Proteoglycans can serve as both temporal and spatial regulators and effective inhibitor design may depend on disruption of these interactions. We have developed a simple assay for evaluating small inhibitors of proteoglycan-ligand binding. The assay is based on cell-free incubation of the reactants and filtration across a cationic membrane. Conditions were established that allow one to semiquantitatively determine binding constants for both direct proteoglycan as well as soluble inhibitor affinity. The assay has been demonstrated using a model heparan sulfate proteoglycan preparation ͑perlecan from cultured bovine endothelial cells͒ and FGF-2. Protamine sulfate, sucrose octasulfate, and heparin were analyzed as model inhibitor molecules. This type of assay may have wide application as a fast and easy screening tool for small potential agonists and antagonists of proteoglycan-protein interactions.

Background. At the present time, COVID-19 vaccines are at the testing stage, and an effective treatment for COVID-19 incorporating appropriate safety measures remains the most significant obstacle to be overcome. A strategic countermeasure is, therefore, urgently required. Aim. This study aims to evaluate the efficacy and safety of a combination of lopinavir/ritonavir-azithromycin, lopinavir/ritonavir-doxycycline, and azithromycin-hydroxychloroquine used to treat patients with mild to moderate COVID-19 infections. Setting and Design. This study was conducted at four different clinical study sites in Indonesia. The subjects gave informed consent for their participation and were confirmed as being COVID-19-positive by means of an RT-PCR test. The present study constituted a randomized, double-blind, and multicenter clinical study of patients diagnosed with mild to moderate COVID-19 infection. Materials and Methods. Six treatment groups participated in this study: a Control group admin...

Vector-borne diseases emergence, particularly malaria, present a significant public health challenge worldwide. Anophelines are predominant malaria vectors, with varied distribution, and influenced by environment and climate. This study, in Ghana, modelled environmental suitability for Anopheles stephensi, a potential vector that may threaten advances in malaria and vector control. Understanding this vector's distribution and dynamics ensures effective malaria and vector control programmes implementation. We explored the MaxEnt ecological modelling method to forecast An. stephensi's potential hotspots and niches. We analysed environmental and climatic variables to predict spatial distribution and ecological niches of An. stephensi with a spatial resolution of approximately 5 km 2 . Analysing geospatial and species occurrence data, we identified optimal environmental conditions and important factors for its presence. The model's most important variables guided hotspot prediction across several ecological zones aside from urban and peri-urban regions. Considering the vector's complex bionomics, these areas provide varying and adaptable conditions for the vector to colonise and establish. This is shown by the AUC = 0.943 prediction accuracy of the model, which is considered excellent. Based on our predictions, this vector species would thrive in the Greater Accra, Ashanti Central, Upper East, Northern, and North East regions. Forecasting its environmental suitability by ecological niche modelling supports proactive surveillance and focused malaria management strategies. Public health officials can act to reduce the risk of malaria transmission by identifying areas where mosquitoes may breed, which will ultimately improve health outcomes and disease control. Malaria is a potentially fatal infection that is transmitted to people through the bites of infected female Anopheles mosquitoes. Nearly half of the world's population was at risk of malaria in 2021 1 . COVID-related interruptions increased malaria incidence and fatalities at the pandemic's peak (2020-2021) 2 . Sub-Saharan Africa has the highest global malaria mortality and morbidity rates. As of 2021, the WHO African Region recorded approximately 95%, of all reported malaria cases and 96% of deaths . Despite significant efforts to scale up vector control strategies, including the use of Long-Lasting Insecticidal Nets (LLINs) and Indoor Residual Spraying (IRS), this high rate of malaria persists. This is a direct effect of the continent's highly efficient Anopheles vector species, Anopheles gambiae complex 4 . Anopheles stephensi species, an Asian malaria vector, has become an invasive species in the Horn of Africa (HOA) in recent years . It was initially discovered on the African continent in 2012 in a Djibouti seaport, then

Vector-borne diseases emergence, particularly malaria, present a significant public health challenge worldwide. Anophelines are predominant malaria vectors, with varied distribution, and influenced by environment and climate. This study, in Ghana, modelled environmental suitability for Anopheles stephensi, a potential vector that may threaten advances in malaria and vector control. Understanding this vector's distribution and dynamics ensures effective malaria and vector control programmes implementation. We explored the MaxEnt ecological modelling method to forecast An. stephensi's potential hotspots and niches. We analysed environmental and climatic variables to predict spatial distribution and ecological niches of An. stephensi with a spatial resolution of approximately 5 km 2 . Analysing geospatial and species occurrence data, we identified optimal environmental conditions and important factors for its presence. The model's most important variables guided hotspot prediction across several ecological zones aside from urban and peri-urban regions. Considering the vector's complex bionomics, these areas provide varying and adaptable conditions for the vector to colonise and establish. This is shown by the AUC = 0.943 prediction accuracy of the model, which is considered excellent. Based on our predictions, this vector species would thrive in the Greater Accra, Ashanti Central, Upper East, Northern, and North East regions. Forecasting its environmental suitability by ecological niche modelling supports proactive surveillance and focused malaria management strategies. Public health officials can act to reduce the risk of malaria transmission by identifying areas where mosquitoes may breed, which will ultimately improve health outcomes and disease control. Malaria is a potentially fatal infection that is transmitted to people through the bites of infected female Anopheles mosquitoes. Nearly half of the world's population was at risk of malaria in 2021 1 . COVID-related interruptions increased malaria incidence and fatalities at the pandemic's peak (2020-2021) 2 . Sub-Saharan Africa has the highest global malaria mortality and morbidity rates. As of 2021, the WHO African Region recorded approximately 95%, of all reported malaria cases and 96% of deaths . Despite significant efforts to scale up vector control strategies, including the use of Long-Lasting Insecticidal Nets (LLINs) and Indoor Residual Spraying (IRS), this high rate of malaria persists. This is a direct effect of the continent's highly efficient Anopheles vector species, Anopheles gambiae complex 4 . Anopheles stephensi species, an Asian malaria vector, has become an invasive species in the Horn of Africa (HOA) in recent years . It was initially discovered on the African continent in 2012 in a Djibouti seaport, then

ABSTRACTPotassium bromate (KBrO3), an environmental pollutant, is a well‐known human carcinogen and a potent nephrotoxic agent. Currently, natural products have built a well‐recognized role in the management of many diseases induced by pollutants. As potent natural sources of bioactive compounds, marine algae have been demonstrated to be rich in novel secondary metabolites with a broad range of biological functions. In this study, adults male mice were orally treated for 15 days with KBrO3 (0.5 g/L) associated or not with extract of Alsidium corallinum, a red Mediterranean alga. In vitro study demonstrated that algal extract has antioxidant efficacy attributable to the presence of flavonoids and polyphenols. Among these, Liquid chromatography–mass spectrometry analysis showed A. corallinum is rich in kaempferol, apigenin, catechin, and quercetin flavonoids. In vivo study showed that supplementation with the alga significantly prevented KBrO3‐induced nephrotoxicity as indicated by pl...

Cellular adhesion plays an important role in numerous fundamental physiological and pathological processes. Its measurement is relatively complex, requires sophisticated equipment, and, in most cases, cannot be carried out without breaking the links between the studied cell and its target. In this contribution, we propose a novel, nanomotion-based, technique that overcomes these drawbacks. The applied force is generated by the studied cell itself (nanomotion), whereas cellular movements are detected by traditional optical microscopy and dedicated software. The measurement is non-destructive, single-cell sensitive, and permits following the evolution of the adhesion as a function of time. We applied the technique on different strains of the fungal pathogen Candida albicans on a fibronectin-coated surface. We demonstrated that this novel approach can significantly simplify, accelerate, and make more affordable living cells–substrate adhesion measurements.

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of ␤-sheets. The binding of N-Flo1p to D-mannose, ␣-methyl-D-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono-and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for lowmolecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level.

A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour. Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but comparable analyses have yet to be performed in an animal. Here we describe the use of RNA interference to inhibit the function of ,86% of the 19,427 predicted genes of C. elegans. We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated with a phenotype. We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in these regions tend to share transcriptional profiles. Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate systematic analyses of the connections among gene sequence, chromosomal location and gene function in C. elegans. The ability to inactivate a target gene transiently by RNAi 1 has greatly accelerated the analysis of loss-of-function phenotypes in C. elegans and other organisms. Although several large-scale RNAibased screens have been used to study gene function in C. elegans 2-4 , in total only about a third of the predicted genes have been analysed so far. Genome-wide RNAi analyses would not only provide a key resource for studying gene function in C. elegans but should also address important issues in functional genomics, such as the global organization of gene functions in a metazoan genome. In addition, because more than half of the genes in C. elegans have a human homologue, this kind of functional analysis in the worm should provide insights into human gene function.

Auricularia auricula-judae is a widely cultivated mushroom species known for its edible and medicinal properties. Polysaccharides have been the focus of research because of their potential bioactivities; nonetheless, the structural complexity and molecular weight have hindered a complete understanding of their bioactivities. In this study, AP-1 polysaccharide was isolated from A. auricula-judae and subjected to ultrasonic degradation at different time points to improve their anti-inflammatory effects. The results showed that when AP-1 was degraded for 9 min (AP-2) and 20 min (AP-3), the NO inhibition rate was significantly increased in LPSstimulated RAW 264.7 cells. The structural and physiochemical properties of native and degraded polysaccharides were analyzed, and it was found that the degradation process significantly reduced molecular weight and altered the particle size, viscosity, crystallinity, and helical structure. Furthermore, native and degraded polysaccharides (AP-1, AP-2, and AP-3) anti-inflammatory effects were investigated in the DSS-induced colitis mouse model. Degraded polysaccharides resulted in significant improvements, including recovery from weight loss, reduced disease activity, shortened colon length, and decreased inflammation, while AP-3 showed the most promising effects. Gut microbiota 16S rRNA sequencing revealed that AP-3 potentially increases healthy gut microbiota and inhibits unhealthy gut microbiota. Overall, this study demonstrates that ultrasonic degradation could be a great technique to modify polysaccharides' MW and physiochemical properties to improve antiinflammatory and gut microbiota regulatory effects.