Prediction of permeability across intestinal cell monolayers for 219 disparate chemicals using in vitro experimental coefficients in a pH gradient system and in silico analyses by trivariate linear regressions and machine learning.

For medicines, the apparent membrane permeability coefficients (Papp) across human colorectal carcinoma cell line (Caco-2) monolayers under a pH gradient generally correlate with the fraction absorbed after oral intake. Furthermore, the in vitro Papp values of 29 industrial chemicals were found to have an inverse association with their reported no-observed effect levels for hepatotoxicity in rats. In the current study, we expanded our influx permeability predictions for the 90 previously investigated chemicals to both influx and efflux permeability predictions for 207 diverse primary compounds, along with those for 23 secondary compounds. Trivariate linear regression analysis found that the observed influx and efflux logPapp values determined by in vitro experiments significantly correlated with molecular weights and the octanol-water distribution coefficients at apical and basal pH levels (pH 6.0 and 7.4, respectively) (apical to basal, r = 0.76, n = 198; and basal to apical, r = 0.77, n = 202); the distribution coefficients were estimated in silico. Further, prediction accuracy was enhanced by applying a light gradient boosting machine learning system (LightGBM) to estimate influx and efflux logPapp values that incorporated 17 and 19 in silico chemical descriptors (r = 0.83-0.84, p < 0.001). The determination in vitro and/or prediction in silico of permeability coefficients across intestinal cell monolayers of a diverse range of industrial chemicals/food components/medicines could contribute to the safety evaluations of oral intakes of general chemicals in humans. Such new alternative methods could also reduce the need for animal testing during toxicity assessment.

Biodegradable Inorganic Nanoparticles for Cancer Theranostics: Insights into the Degradation Behavior.

Inorganic nanoparticles as a versatile nanoplatform have been broadly applied in the diagnosis and treatment of cancers due to their inherent superior physicochemical properties (including magnetic, thermal, optical, and catalytic performance) and excellent functions (e.g., imaging, targeted delivery, and controlled release of drugs) through surface functional modification or ingredient dopant. However, in practical biological applications, inorganic nanomaterials are relatively difficult to degrade and excrete, which induces a long residence time in living organisms and thus may cause adverse effects, such as inflammation and tissue cysts. Therefore, the development of biodegradable inorganic nanomaterials is of great significance for their biomedical application. This Review will focus on the recent advances of degradable inorganic nanoparticles for cancer theranostics with highlight on the degradation mechanism, aiming to offer an in-depth understanding of degradation behavior and related biomedical applications. Finally, key challenges and guidelines will be discussed to explore biodegradable inorganic nanomaterials with minimized toxicity issues, facilitating their potential clinical translation in cancer diagnosis and treatment.

Characterization of inorganic phosphate transport in the triple-negative breast cancer cell line, MDA-MB-231.

BACKGROUND: Recent studies demonstrate that interstitial inorganic phosphate is significantly elevated in the breast cancer microenvironment as compared to normal tissue. In addition it has been shown that breast cancer cells express high levels of the NaPi-IIb carrier (SLC34A2), suggesting that this carrier may play a role in breast cancer progression. However, the biochemical behavior of inorganic phosphate (Pi) transporter in this cancer type remains elusive. METHODS: In this work, we characterize the kinetic parameters of Pi transport in the aggressive human breast cancer cell line, MDA-MB-231, and correlated Pi transport with cell migration and adhesion. RESULTS: We determined the influence of sodium concentration, pH, metabolic inhibitors, as well as the affinity for inorganic phosphate in Pi transport. We observed that the inorganic phosphate is dependent on sodium transport (K0,5 value = 21.98 mM for NaCl). Furthermore, the transport is modulated by different pH values and increasing concentrations of Pi, following the Michaelis-Menten kinetics (K0,5 = 0.08 mM Pi). PFA, monensin, furosemide and ouabain inhibited Pi transport, cell migration and adhesion. CONCLUSIONS: Taken together, these results showed that the uptake of Pi in MDA-MB-231 cells is modulated by sodium and by regulatory mechanisms of intracellular sodium gradient. General Significance: Pi transport might be regarded as a potential target for therapy against tumor progression.

A computational method for selecting short peptide sequences for inorganic material binding.

Discovering or designing biofunctionalized materials with improved quality highly depends on the ability to manipulate and control the peptide-inorganic interaction. Various peptides can be used as assemblers, synthesizers, and linkers in the material syntheses. In another context, specific and selective material-binding peptides can be used as recognition blocks in mining applications. In this study, we propose a new in silico method to select short 4-mer peptides with high affinity and selectivity for a given target material. This method is illustrated with the calcite (104) surface as an example, which has been experimentally validated. A calcite binding peptide can play an important role in our understanding of biomineralization. A practical aspect of calcite is a need for it to be selectively depressed in mining sites.

Genomic Analysis Unravels Reduced Inorganic Sulfur Compound Oxidation of Heterotrophic Acidophilic Acidicaldus sp. Strain DX-1.

Although reduced inorganic sulfur compound (RISC) oxidation in many chemolithoautotrophic sulfur oxidizers has been investigated in recent years, there is little information about RISC oxidation in heterotrophic acidophiles. In this study, Acidicaldus sp. strain DX-1, a heterotrophic sulfur-oxidizing acidophile, was isolated. Its genome was sequenced and then used for comparative genomics. Furthermore, real-time quantitative PCR was performed to identify the expression of genes involved in the RISC oxidation. Gene encoding thiosulfate: quinone oxidoreductase was present in Acidicaldus sp. strain DX-1, while no candidate genes with significant similarity to tetrathionate hydrolase were found. Additionally, there were genes encoding heterodisulfide reductase complex, which was proposed to play a crucial role in oxidizing cytoplasmic sulfur. Like many heterotrophic sulfur oxidizers, Acidicaldus sp. strain DX-1 had no genes encoding enzymes essential for the direct oxidation of sulfite. An indirect oxidation of sulfite via adenosine-5'-phosphosulfate was proposed in Acidicaldus strain DX-1. However, compared to other closely related bacteria Acidiphilium cryptum and Acidiphilium multivorum, which harbored the genes encoding Sox system, almost all of these genes were not detected in Acidicaldus sp. strain DX-1. This study might provide some references for the future study of RISC oxidation in heterotrophic sulfur-oxidizing acidophiles.

Enhancing aerobic biodegradation of 1,2-dibromoethane in groundwater using ethane or propane and inorganic nutrients.

1,2-Dibromoethane (ethylene dibromide; EDB) is a probable human carcinogen that was previously used as both a soil fumigant and a scavenger in leaded gasoline. EDB has been observed to persist in soils and groundwater, particularly under oxic conditions. The objective of this study was to evaluate options to enhance the aerobic degradation of EDB in groundwater, with a particular focus on possible in situ remediation strategies. Propane gas and ethane gas were observed to significantly stimulate the biodegradation of EDB in microcosms constructed with aquifer solids and groundwater from the FS-12 EDB plume at Joint Base Cape Cod (Cape Cod, MA), but only after inorganic nutrients were added. Ethene gas was also effective, but rates were appreciably slower than for ethane and propane. EDB was reduced to <0.02 µg/L, the Massachusetts state Maximum Contaminant Level (MCL), in microcosms that received ethane gas and inorganic nutrients. An enrichment culture (BE-3R) that grew on ethane or propane gas but not EDB was obtained from the site materials. The degradation of EDB by this culture was inhibited by acetylene gas, suggesting that degradation is catalyzed by a monooxygenase enzyme. The BE-3R culture was also observed to biodegrade 1,2-dichloroethane (DCA), a compound commonly used in conjunction with EDB as a lead scavenger in gasoline. The data suggest that addition of ethane or propane gas with inorganic nutrients may be a viable option to enhance degradation of EDB in groundwater aquifers to below current state or federal MCL values.

Temporomandibular Disorders in a Sample Population of the Brazilian Northeast

Temporomandibular disorder (TMD) is a common condition. This study is part of a research group and it investigated the prevalence of TMD and myofascial pain and its association with gender, age and socioeconomic class. The sample comprised 100 subjects, aged 15 to 70, users of the Family Health Units' services, in the city of Recife, PE, Brazil. The TMD degree was evaluated using the Research Diagnostic Criteria for TMD and socioeconomic class by the Economic Classification Criteria Brazil. Categorical variables were analyzed by chi-square test for proportions and Fisher's exact test for 2x2 tables, and binary logistic analysis to track the relationship between the independent and dependent variables. According to the results, 42% of the subjects had TMD and 14% myofascial pain. No statistically significant association could be found between TMD and gender or socioeconomic class, but it was found to have statistically significant association with age, and myofascial pain was associated with socioeconomic class. Considering that the results of the present study should be confirmed by further studies and the fact that this was a pilot study, the prevalence must be analyzed with caution.

Disfunção temporomandibular (DTM) é uma condição comum. Este estudo é parte de um grupo de pesquisa e investigou a prevalência de DTM e dor miofascial e suas associações com sexo, idade e classe socioeconômica. A amostra foi composta por 100 indivíduos, com idades entre 15 e 70 anos, usuários das Unidades de Saúde da Família, na cidade de Recife, PE. O grau de DTM foi avaliado usando os Critérios de Diagnósticos Científicos em DTM, e classe socioeconômica com o Critério de Classificação Econômica Brasil. As variáveis categóricas foram analisadas pelo teste do qui-quadrado para proporções e teste exato de Fisher para tabelas 2x2, e a análise logística binária para traçar a relação entre as variáveis independentes e dependentes. De acordo com os resultados, 42% dos indivíduos tinham DTM e 14% dor miofascial. Não houve associação estatisticamente significativa entre DTM e sexo ou classe socioeconômica, mas houve associação estatisticamente significativa com a idade e a dor miofascial foi associada com a classe socioeconômica. Considerando-se que os resultados do presente estudo devam ser confirmados em outros estudos e por causa de sua natureza piloto, a prevalência deve ser analisada com cautela.

Inorganic polyphosphates: biologically active biopolymers for biomedical applications.

Inorganic polyphosphate (polyP) is a widely occurring but only rarely investigated biopolymer which exists in both prokaryotic and eukaryotic organisms. Only in the last few years, this polymer has been identified to cause morphogenetic activity on cells involved in human bone formation. The calcium complex of polyP was found to display a dual effect on bone-forming osteoblasts and bone-resorbing osteoclasts. Exposure of these cells to polyP (Ca(2+) complex) elicits the expression of cytokines that promote the mineralization process by osteoblasts and suppress the differentiation of osteoclast precursor cells to the functionally active mature osteoclasts dissolving bone minerals. The effect of polyP on bone formation is associated with an increased release of the bone morphogenetic protein 2 (BMP-2), a key mediator that activates the anabolic processes leading to bone formation. In addition, polyP has been shown to act as a hemostatic regulator that displays various effects on blood coagulation and fibrinolysis and might play an important role in platelet-dependent proinflammatory and procoagulant disorders.

High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i)) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i) but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i) in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i), which could be cytotoxic because of its high affinity for Ca(2+), thereby interfering with Ca(2+) signaling.

Physiological conditions conducive to high cell density and high cyanophycin content in Ralstonia eutropha strain H16 possessing a KDPG aldolase gene-dependent addiction system.

The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.

Interactions between bicarbonate, potassium, and magnesium, and sulfur-dependent induction of luminescence in Vibrio fischeri.

In spite of its central importance in research efforts, the relationship between seawater compounds and bacterial luminescence has not previously been investigated in detail. Thus, in this study, we investigated the effect of cations (Na(+) , K(+) , NH(4) (+) , Mg(2+) , and Ca(2+) ) and anions (Cl(-) , HCO(3) (-) , CO(3) (2-) , and NO(3) (-) ) on the induction of both inorganic (sulfate, sulfite, and thiosulfate) and organic (L-cysteine and L-cystine) sulfur-dependent luminescence in Vibrio fischeri. We found that HCO(3) (-) (bicarbonate) and CO(3) (2-) (carbonate), in the form of various compounds, had a stimulatory effect on sulfur-dependent luminescence. The luminescence induced by bicarbonate was further promoted by the addition of magnesium. Potassium also increased sulfur-dependent luminescence when sulfate or thiosulfate was supplied as the sole sulfur source, but not when sulfite, L-cysteine, or L-cystine was supplied. The positive effect of potassium was accelerated by the addition of magnesium and/or calcium. Furthermore, the additional supply of magnesium improved the induction of sulfite- or L-cysteine-dependent luminescence, but not the l-cystine-dependent type. These results suggest that sulfur-dependent luminescence of V. fischeri under nutrient-starved conditions is mainly controlled by bicarbonate, carbonate, and potassium. In addition, our results indicate that an additional supply of magnesium is effective for increasing V. fischeri luminescence.

Optimization of different process variables for the production of an indolizidine alkaloid, swainsonine from Metarhizium anisopliae.

Swainsonine is a polyhydroxylated indolizidine alkaloid having anticancer, antimetastatic, antiproliferative and immunomodulatory activities and also potential therapeutic applications against AIDS. In the present study, ten isolates of M. anisopliae were screened and enzyme assayed for the production of swainsonine in different media (Complex oatmeal, Czapekdox media with and without lysine (8% w/v) and Sabouraud dextrose broth (SDB)). Among these strains, ARSEF 1724 (UM8) was found to produce highest amount of swainsonine (1.34 µg/l) after 72 h of incubation under shake flask conditions at 180 rpm and 28 °C in complex oatmeal media. In order to maximize the yield of swainsonine the media composition including macro and micronutrients were optimized. The process variables including the chemical factors like carbon sources, nitrogen sources of both organic and inorganic nature and pH with constant inoculum size (1 × 10(8) spores/ml) were screened using classical one-factor-at-a-time (OFAT) approach to find their optimum levels. The present study shows that the nutrient requirement is specific for each strain of Metarhizium. Oatmeal extract (6%) was found to be the best supporting media along with nitrogen source, glucose (2%) as best carbon source and pH (~5) as the best for swainsonine production.

Inorganic-binding peptides as tools for surface quality control.

This paper highlights an innovative application of inorganic-binding peptides as quality control tools for detecting defects on inorganic surfaces of any shape. The approach involves attaching a fluorescent label to an inorganic-binding peptide and exploiting the peptide's high binding specificity to detect, by simple fluorescence microscopy, chemical composition defects of microm size and crystallographic state defects. Proof of concept was demonstrated by monitoring binding of a previously isolated ZnO-binding peptide to galvanized steel substrates. The approach was further validated for TiO(2) coatings and stainless steel, with two new, specific inorganic-binding peptides isolated by phage display.

Carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase: biological activities and selective toxicities.

Toxoplasma gondii adenosine kinase (EC 2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Several 6-benzylthioinosines have already been identified as subversive substrates of the T. gondii but not human adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not its host. In the present study, we report the testing of the metabolism of several carbocyclic 6-benzylthioinosines, as well as their efficacy as anti-toxoplasmic agents in cell culture. All the carbocyclic 6-benzylthioinosine analogues were metabolized to their 5'-monophosphate derivatives, albeit to different degrees. These results indicate that these compounds are not only ligands but also substrates of T. gondii adenosine kinase. All the carbocyclic 6-benzylthioinosine analogues showed a selective anti-toxoplasmic effect against wild type parasites, but not mutants lacking adenosine kinase. These results indicate that the oxygen atom of the sugar is not critical for substrate binding. The efficacy of these compounds varied with the position and nature of the substitution on their phenyl ring. Moreover, none of these analogues exhibited host toxicity. The best compounds were carbocyclic 6-(p-methylbenzylthio)inosine (IC(50)=11.9 microM), carbocyclic 6-(p-methoxybenzylthio)inosine (IC(50)=12.1 microM), and carbocyclic 6-(p-methoxycarbonylbenzylthio)inosine (IC(50)=12.8 microM). These compounds have about a 1.5-fold better efficacy relative to their corresponding 6-benzylthioinosine analogues (Rais et al., Biochem Pharmacol 2005;69:1409-19 [29]). The results further confirm that T. gondii adenosine kinase is an excellent target for chemotherapy and that carbocyclic 6-benzylthioinosines are potential anti-toxoplasmic agents.

Multidrug efflux transporters limit accumulation of inorganic, but not organic, mercury in sea urchin embryos.

Mercuric compounds are persistent global pollutants that accumulate in marine organisms and in humans who consume them. While the chemical cycles and speciation of mercury in the oceans are relatively well described, the cellular mechanisms that govern which forms of mercury accumulate in cells and why they persist are less understood. In this study we examined the role of multidrug efflux transport in the differential accumulation of inorganic (HgCl(2)) and organic (CH(3)HgCl) mercury in sea urchin (Strongylocentrotus purpuratus) embryos. We found that inhibition of MRP/ABCC-type transporters increases intracellular accumulation of inorganic mercury but had no effect on accumulation of organic mercury. Similarly, pharmacological inhibition of metal conjugating enzymes by ligands GST/GSH significantly increases this antimitotic potency of inorganic mercury, but had no effect on the potency of organic mercury. Our results point to MRP-mediated elimination of inorganic mercury conjugates as a cellular basis for differences in the accumulation and potency of the two major forms of mercury found in marine environments.

Angiogenesis in calcium phosphate scaffolds by inorganic copper ion release.

Angiogenesis in a tissue-engineered device may be induced by incorporating growth factors (e.g., vascular endothelial growth factor [VEGF]), genetically modified cells, and=or vascular cells. It represents an important process during the formation and repair of tissue and is essential for nourishment and supply of reparative and immunological cells. Inorganic angiogenic factors, such as copper ions, are therefore of interest in the fields of regenerative medicine and tissue engineering due to their low cost, higher stability, and potentially greater safety compared with recombinant proteins or genetic engineering approaches. The purpose of this study was to compare tissue responses to 3D printed macroporous bioceramic scaffolds implanted in mice that had been loaded with either VEGF or copper sulfate. These factors were spatially localized at the end of a single macropore some 7 mm from the surface of the scaffold. Controls without angiogenic factors exhibited only poor tissue growth within the blocks; in contrast, low doses of copper sulfate led to the formation of microvessels oriented along the macropore axis. Further, wound tissue ingrowth was particularly sensitive to the quantity of copper sulfate and was enhanced at specific concentrations or in combination with VEGF. The potential to accelerate and guide angiogenesis and wound healing by copper ion release without the expense of inductive protein(s) is highly attractive in the area of tissue-engineered bone and offers significant future potential in the field of regenerative biomaterials.

Thiophenecarboxylate suppressor of cyclic nucleotides discovered in a small-molecule screen blocks toxin-induced intestinal fluid secretion.

We carried out a "pathway" screen of 50,000 small molecules to identify novel modulators of cAMP signaling. One class of compounds, the 2-(acylamino)-3-thiophenecarboxylates, strongly suppressed cAMP and cGMP in multiple cell lines in response to different agonists acting on G-protein-coupled receptors, adenylyl cyclase, and guanylyl cyclase. The best compounds from structure-activity analysis of 124 analogs, including several synthesized chiral analogs, had and IC(50) of <5 microM for suppression of agonist-induced cAMP and cGMP elevation. Measurements of cAMP, cGMP, and downstream signaling in response to various activators/inhibitors suggested that the 2-(acylamino)-3-thiophenecarboxylates function as nonselective phosphodiesterase activators, although it was not determined whether their action on phosphodiesterases is direct or indirect. The 2-(acylamino)-3-thiophenecarboxylates suppressed CFTR-mediated Cl(-) current in T84 colonic cells in response to cholera and Escherichia coli (STa) toxins, and prevented intestinal fluid accumulation in a closed-loop mouse model of secretory diarrhea. They also prevented cyst growth in an in vitro renal epithelial cell model of polycystic kidney disease. The 2-(acylamino)-3-thiophenecarboxylates represent the first small-molecule cyclic nucleotide suppressors, whose potential therapeutic indications include secretory diarrheas, polycystic kidney disease, and growth inhibition of cAMP-dependent tumors.

Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae.

The cells of Saccharomyces cerevisiae accumulate inorganic polyphosphate (polyP) when reinoculated on a phosphate-containing medium after phosphorus starvation. Total polyP accumulation was similar at cultivation on both glucose and ethanol. Five separate fractions of polyP: acid-soluble fraction polyP1, salt-soluble fraction polyP2, weakly alkali-soluble fraction polyP3, alkali-soluble fraction polyP4, and polyP5, have been obtained from the cells grown on glucose and ethanol under phosphate overplus. The dynamics of polyP fractions depend on a carbon source. The accumulation rates for fractions polyP2 and polyP4 were independent of the carbon source. The accumulation rates of polyP1 and polyP3 were higher on glucose, while fraction polyP5 accumulated faster on ethanol. As to the maximal polyP levels, they were independent of the carbon source for fractions polyP2, polyP3, and polyP4. The maximal level of fraction polyP1 was higher on glucose than on ethanol, but the level of fraction polyP5 was higher on ethanol. It was assumed that accumulation of separate polyP fractions has a metabolic interrelation with different energy-providing pathways. The polyphosphate nature of fraction polyP5 was demonstrated for the first time by (31)P nuclear magnetic resonance spectroscopy, enzymatic assay, and electrophoresis.