Synergistic effects of quercetin-loaded CoFe2O4@Liposomes regulate DNA damage and apoptosis in MCF-7 cancer cells: based on biophysical magnetic hyperthermia.

INTRODUCTION: Breast cancer (BC) is the most common malignancy in women globally. Significant progress has been made in developing structural nanoparticles (NPs) and formulations for targeted smart drug delivery (SDD) of pharmaceuticals, improving the precision of tumor cell targeting in therapy. SIGNIFICANCE: Magnetic hyperthermia (MHT) treatment using magneto-liposomes (MLs) has emerged as a promising adjuvant cancer therapy. METHODS: CoFe2O4 magnetic NPs (MNPs) were conjugated with nanoliposomes to form MLs, and the anticancer drug quercetin (Que) was loaded into MLs, forming Que-MLs composites for antitumor approach. The aim was to prepare Que-MLs for DD systems (DDS) under an alternating magnetic field (AMF), termed chemotherapy/hyperthermia (chemo-HT) techniques. The encapsulation efficiency (EE), drug loading capacity (DL), and drug release (DR) of Que and Que-MLs were evaluated. RESULTS: The results confirmed successful Que-loading on the surface of MLs, with an average diameter of 38 nm and efficient encapsulation into MLs (69%). In vitro, experimental results on MCF-7 breast cells using MHT showed high cytotoxic effects of novel Que-MLs on MCF-7 cells. Various analyses, including cytotoxicity, apoptosis, cell migration, western blotting, fluorescence imaging, and cell membrane internalization, were conducted. The Acridine Orange-ethidium bromide double fluorescence test identified 35% early and 55% late apoptosis resulting from Que-MLs under the chemo-HT group. TEM results indicated MCF-7 cell membrane internalization and digestion of Que-MLs, suggesting the presence of early endosome-like vesicles on the cytoplasmic periphery. CONCLUSIONS: Que-MLs exhibited multi-modal chemo-HT effects, displaying high toxicity against MCF-7 BC cells and showing promise as a potent cytotoxic agent for BC chemotherapy.

[Determination of cobalt in plasma blood samples by the ICP-MS method after oral intake of dietary supplements containing low doses of cobalt].

Salts of inorganic cobalt (Со) prevent the degradation of the alpha subunit of the hypoxia-inducible factor (HIF), imitating the state of hypoxia in the body and increasing the production of the endogenous hormone erythropoietin (EPO), and are used as doping substances that increase blood oxygen capacity and endurance, which give competitive advantages in sports. Currently, a large number of dietary supplements, including Co-containing ones, are offered on free sale. Their uncontrolled intake can affect not only the professional career of athletes, but also their health, due to the fact that this trace element and its salts are the strongest inorganic poisons and carcinogens. Despite this, their availability on the pharmaceutical market, a noticeable effect of erythropoiesis stimulation and a convenient oral form of administration lead to the need for their detection in modern doping control. The purpose of this research was to develop an approach to differentiate cobalt from vitamin B12, present in the body in its natural state, from the intake of cobalt salts by quantifying and comparing blood levels of vitamin B12 and total cobalt. Methods. The study involved 9 healthy volunteers (women and men) aged 25 to 45 years, leading an active lifestyle. Three of them took 2500 µg/day of cobalamin for 20 days (comparison group), three - dietary supplement containing cobalt asparaginate (100 µg/day in terms of pure cobalt), and the rest - dietary supplements with cobalt sulfate heptahydrate (100 µg/day in terms of pure cobalt) (administration groups) at the same time after meals. Blood samples were taken at baseline and on days 5, 9, 14 and 20. The concentrations of total cobalt in blood plasma samples of volunteers were measured by inductively coupled plasma mass-spectrometry (ICP-MS), the levels of cobalamin were determined on a Cobas 6000 immunochemical analyzer using the Elecsys Vitamin B12 II Assay ELISA kits. Results. It was found that oral intake of of cobalamin at a therapeutic dose significantly exceeding the recommended daily intake (3 µg), there was a regular slight increase in the blood concentration of total cobalt (1.1 times). At the same time intake of dietary supplements containing cobalt in the form of sulfate or asparaginate (about 100 µg per day in terms of pure cobalt) was accompanied by 4-6.7 fold increase in the concentration of total cobalt while unchanged vitamin B12 plasma concentration was observed. The detection of such changes can reliably indicate the use of prohibited salts and, of course, will be in demand for anti-doping control. Conclusion. Long-term monitoring of vitamin B12 and total cobalt levels, similar to hematological module of the Athlete Biological Passport program, will unambiguously detect possible abuse of cobalt salts and can be an additional evidence of the presence of these doping substances to other analytical methods, such as a combination of liquid chromatography and ICP-MS (LC-ICP-MS).

Cobalt chloride-simulated hypoxia elongates primary cilia in immortalized human retina pigment epithelial-1 cells.

Primary cilia are microtubule-based organelles that are involved in sensing micro-environmental cues and regulating cellular homeostasis via triggering signaling cascades. Hypoxia is one of the most common environmental stresses that organ and tissue cells may often encounter during embryogenesis, cell differentiation, infection, inflammation, injury, cerebral and cardiac ischemia, or tumorigenesis. Although hypoxia has been reported to promote or inhibit primary ciliogenesis in different tissues or cultured cell lines, the role of hypoxia in ciliogenesis is controversial and still unclear. Here we investigated the primary cilia change under cobalt chloride (CoCl2)-simulated hypoxia in immortalized human retina pigment epithelial-1 (hTERT RPE-1) cells. We found CoCl2 treatment elongated primary cilia in a time- and dose-dependent manner. The prolonged cilia recovered back to near normal length when CoCl2 was washed out from the cell culture medium. Under CoCl2-simulated hypoxia, the protein expression levels of HIF-1/2α and acetylated-α-tubulin (cilia marker) were increased, while the protein expression level of Rabaptin-5 is decreased during hypoxia. Taken together, our results suggest that hypoxia may elongate primary cilia by downregulating Rabaptin-5 involved endocytosis. The coordination between endocytosis and ciliogenesis may be utilized by cells to adapt to hypoxia.

Cobalt chloride-stimulated hypoxia promotes the proliferation of cholesteatoma keratinocytes via the PI3K/Akt signaling pathway.

Herein, we purposed to explore whether hypoxia triggers proliferation of cholesteatoma keratinocytes via the PI3K-Akt signaling cascade. Cells were inoculated with different concentration of CoCl2. The proliferation and cellular HIF-1α, p-PDK1 and p­Akt expression levels of cholesteatoma keratinocytes were assessed in vitro. Hypoxia escalated cell proliferation via upregulating p-PDK1 and p­Akt expressions. Specific inhibitor of the PI3K-Akt signaling cascade, LY294002 markedly inhibited the expression of p­Akt and significantly reduces the hypoxia­induced proliferation of cholesteatoma keratinocytes. Our data provides research evidence confirming that hypoxia participates in the onset and progress of cholesteatoma.

Carcinogenic hazard assessment of cobalt-containing alloys in medical devices: Review of in vivo studies.

Cobalt (Co) alloys have been used for over seven decades in a wide range of medical devices, including, but not limited to, hip and knee implants, surgical tools, and vascular stents, due to their favorable biocompatibility, durability, and mechanical properties. A recent regulatory hazard classification review by the European Chemicals Agency (ECHA) resulted in the classification of metallic Co as a Class 1B Carcinogen (presumed to have carcinogenic potential for humans), primarily based on inhalation rodent carcinogenicity studies with pure metallic Co. The ECHA review did not specifically consider the carcinogenicity hazard potential of forms or routes of Co that are relevant for medical devices. The purpose of this review is to present a comprehensive assessment of the available in vivo preclinical data on the carcinogenic hazard potential of exposure to Co-containing alloys (CoCA) in medical devices by relevant routes. In vivo data were reviewed from 33 preclinical studies that examined the impact of Co exposure on local and systemic tumor incidence in rats, mice, guinea pigs, and hamsters. Across these studies, there was no significant increase of local or systemic tumors in studies relevant for medical devices. Taken together, the relevant in vivo data led to the conclusion that CoCA in medical devices are not a carcinogenic hazard in available in vivo models. While specific patient and implant factors cannot be fully replicated using in vivo models, the available in vivo preclinical data support that CoCA in medical devices are unlikely a carcinogenic hazard to patients.

Suitable test concentration of cobalt and concomitant reactivity to nickel and chromium: A multicentre study from the Swedish Contact Dermatitis Research Group.

BACKGROUND: In Sweden, cobalt chloride 0.5% has been included in the baseline series since the mid-1980s. A recent study from Stockholm showed that cobalt chloride 1% petrolatum (pet.) was more suitable than 0.5%. Cobalt chloride at 1.0% has been patch tested for decades in many European countries and around the world. OBJECTIVES: To study the suitability of patch testing to cobalt 1.0% vs 0.5% and to analyze the co-occurrence of allergy to cobalt, chromium, and nickel. RESULTS: Contact allergy to cobalt was shown in 90 patients (6.6%). Eighty (5.9%) patients tested positive to cobalt 1.0%. Thirty-seven of the 90 patients (41.1%) with cobalt allergy were missed by cobalt 0.5% and 10 (0.7%) were missed by cobalt 1.0% (P < .001). No case of patch test sensitization was reported. Allergy to chromium was seen in 2.6% and allergy to nickel in 13.3%. Solitary allergy to cobalt without nickel allergy was shown in 61.1% of cobalt-positive individuals. Female patients had larger proportions of positive reactions to cobalt (P = .036) and nickel (P < .001) than males. CONCLUSION: The results speak in favor of replacing cobalt chloride 0.5% with cobalt chloride 1.0% pet. in the Swedish baseline series, which will be done 2021.

Distinct Concentration-Dependent Molecular Pathways Regulate Bone Cell Responses to Cobalt and Chromium Exposure from Joint Replacement Prostheses.

Systemic cobalt (Co) and chromium (Cr) concentrations may be elevated in patients with metal joint replacement prostheses. Several studies have highlighted the detrimental effects of this exposure on bone cells in vitro, but the underlying mechanisms remain unclear. In this study, we use whole-genome microarrays to comprehensively assess gene expression in primary human osteoblasts, osteoclast precursors and mature resorbing osteoclasts following exposure to clinically relevant circulating versus local periprosthetic tissue concentrations of Co2+ and Cr3+ ions and CoCr nanoparticles. We also describe the gene expression response in osteoblasts on routinely used prosthesis surfaces in the presence of metal exposure. Our results suggest that systemic levels of metal exposure have no effect on osteoblasts, and primarily inhibit osteoclast differentiation and function via altering the focal adhesion and extracellular matrix interaction pathways. In contrast, periprosthetic levels of metal exposure inhibit both osteoblast and osteoclast activity by altering HIF-1α signaling and endocytic/cytoskeletal genes respectively, as well as increasing inflammatory signaling with mechanistic implications for adverse reactions to metal debris. Furthermore, we identify gene clusters and KEGG pathways for which the expression correlates with increasing Co2+:Cr3+ concentrations, and has the potential to serve as early markers of metal toxicity. Finally, our study provides a molecular basis for the improved clinical outcomes for hydroxyapatite-coated prostheses that elicit a pro-survival osteogenic gene signature compared to grit-blasted and plasma-sprayed titanium-coated surfaces in the presence of metal exposure.

Real-Time Monitoring the Effect of Cytopathic Hypoxia on Retinal Pigment Epithelial Barrier Functionality Using Electric Cell-Substrate Impedance Sensing (ECIS) Biosensor Technology.

Disruption of retinal pigment epithelial (RPE barrier integrity is a hallmark feature of various retinal blinding diseases, including diabetic macular edema and age-related macular degeneration, but the underlying causes and pathophysiology are not completely well-defined. One of the most conserved phenomena in biology is the progressive decline in mitochondrial function with aging leading to cytopathic hypoxia, where cells are unable to use oxygen for energy production. Therefore, this study aimed to thoroughly investigate the role of cytopathic hypoxia in compromising the barrier functionality of RPE cells. We used Electric Cell-Substrate Impedance Sensing (ECIS) system to monitor precisely in real time the barrier integrity of RPE cell line (ARPE-19) after treatment with various concentrations of cytopathic hypoxia-inducing agent, Cobalt(II) chloride (CoCl2). We further investigated how the resistance across ARPE-19 cells changes across three separate parameters: Rb (the electrical resistance between ARPE-19 cells), α (the resistance between the ARPE-19 and its substrate), and Cm (the capacitance of the ARPE-19 cell membrane). The viability of the ARPE-19 cells and mitochondrial bioenergetics were quantified with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and seahorse technology, respectively. ECIS measurement showed that CoCl2 reduced the total impedance of ARPE-19 cells in a dose dependent manner across all tested frequencies. Specifically, the ECIS program's modelling demonstrated that CoCl2 affected Rb as it begins to drastically decrease earlier than α or Cm, although ARPE-19 cells' viability was not compromised. Using seahorse technology, all three concentrations of CoCl2 significantly impaired basal, maximal, and ATP-linked respirations of ARPE-19 cells but did not affect proton leak and non-mitochondrial bioenergetic. Concordantly, the expression of a major paracellular tight junction protein (ZO-1) was reduced significantly with CoCl2-treatment in a dose-dependent manner. Our data demonstrate that the ARPE-19 cells have distinct dielectric properties in response to cytopathic hypoxia in which disruption of barrier integrity between ARPE-19 cells precedes any changes in cells' viability, cell-substrate contacts, and cell membrane permeability. Such differences can be used in screening of selective agents that improve the assembly of RPE tight junction without compromising other RPE barrier parameters.

Development and Assessment of Nano-Technologies for Cancer Treatment: Cytotoxicity and Hyperthermia Laboratory Studies.

Cancer treatment by magnetic hyperthermia offers numerous advantages, but for practical applications many variables still need to be adjusted before developing a controlled and reproducible cancer treatment that is bio-compatible (non-damaging) to healthy cells. In this work, Fe3O4 and CoFe2O4 were synthesized and systematically studied for the development of efficient therapeutic agents for applications in hyperthermia. The biocompatibility of the materials was further evaluated using HepG2 cells as biological model. Colorimetric and microscopic techniques were used to evaluate the interaction of magnetic nano-materials (MNMs) and HepG2 cells. Finally, the behavior of MNMs was evaluated under the influence of an alternating magnetic field (AMF), observing a more efficient temperature increment for CoFe2O4, a desirable behavior for biomedical applications since lower doses and shorter expositions to alternating magnetic field might be required.

A Cobalt Schiff-Base Complex as a Putative Therapeutic for Azide Poisoning.

There is presently no antidote available to treat azide poisoning. Here, the Schiff-base compound Co(II)-2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1(17)2,11,13,15-pentaenyl dibromide (Co(II)N4[11.3.1]) is investigated to determine if it has the capability to antagonize azide toxicity through a decorporation mechanism. The stopped-flow kinetics of azide binding to Co(II)N4[11.3.1] in the absence of oxygen exhibited three experimentally observable phases: I (fast); II (intermediate); and III (slow). The intermediate phase II accounted for ∼70% of the overall absorbance changes, representing the major process observed, with second-order rate constants of 29 (±4) M-1 s-1 at 25 °C and 70 (±10) M-1 s-1 at 37 °C. The data demonstrated pH independence of the reaction around neutrality, suggesting the unprotonated azide anion to be the attacking species. The binding of azide to Co(II)N4[11.3.1] appears to have a complicated mechanism leading to less than ideal antidotal capability; nonetheless, this cobalt complex does protect against azide intoxication. Administration of Co(II)N4[11.3.1] at 5 min post sodium azide injection (ip) to mice resulted in a substantial decrease of righting-recovery times, 12 (±4) min, compared to controls, 40 (±8) min. In addition, only two out of seven mice "knocked down" when the antidote was administered compared to the controls given toxicant only (100% knockdown).

Immobilization of novel inorganic nano-complexes onto MWCNT nanomaterials as a novel adsorbent and anti-inflammatory therapy in an induced model of rheumatoid arthritis.

Novel supported inorganic metal nano-complexes of Ag(I) and Co(II) derived from 4-amino-N-(4-methylpyrimidin-2-yl) benzene sulfonamide (SulMer) were synthesized using olive leaf extract as a reducing agent with grinding and microwave methods. The prepared samples were denoted as Comp1-6. The surface morphologies of the synthesized nanomaterials were analyzed using C, H, N, S analysis, Fourier-transform infrared spectroscopy, UV- visible spectroscopy, proton and carbon nuclear magnetic resonance, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, and x-ray powder diffraction (XRD) analysis. The data revealed that all the synthesized complexes exhibited a 1:1 metal-to-ligand ratio with a coordination number of 4 or 6. The mean particle size of the nanomaterial samples was 25-35 nm. The XRD patterns indicated a crystalline nature for the complexes. The supported inorganic metal nano-complexes displayed good activity in the adsorptive removal of Direct Red 81 (DR-81) from aqueous solutions. In addition, the effect of the supported metal nano-complexes on the immune system was studied as well as how these anti-inflammatory compounds could be used to treat many autoimmune diseases, most notably rheumatoid arthritis. An experimental model for arthritis can be induced using complete Freund's adjuvant. It was shown that the supported complex offers several advantages such stability, eco-friendliness, simple experimental conditions, short reaction times, and easy work- up.

Endogenous synthesis of milk cis-9,trans-11 conjugated linoleic acid in dairy ewes: Quantification using 13C-labeled vaccenic acid and comparison with estimates based on cobalt administration.

Isotopic tracers are used to directly quantify the effect of mammary Δ9-desaturation on milk fatty acid (FA) composition, but very few studies have applied this method to measuring the endogenous synthesis of rumenic acid (RA; cis-9,trans-11 conjugated linoleic acid) in cows and goats, and no publications exist in ewes. In sheep, knowledge about the contribution of stearoyl-coenzyme A desaturase (SCD) to milk FA secretion is derived mostly from indirect estimates based on inhibition of the enzyme by oral administration of cobalt, a cost-effective method that has not been validated to date. To fill both gaps, we conducted an isotopic tracer assay in sheep to quantify the proportion of endogenous RA in milk for the first time in this species. We then compared the results with estimates derived from a Co administration assay performed on the same animals. First, 5 lactating ewes received an intravenous injection of 200 mg of [1-13C]trans-11 18:1 (vaccenic acid, VA), the precursor for RA production by SCD activity. At -24, -15, 0, 9, 24, 33, 48, 57, 72, 81, and 96 h post-injection, we recorded milk yield and collected milk samples to examine fat concentration and FA profile. We conducted compound-specific isotope analysis of VA and RA by gas chromatography-combustion isotope ratio mass spectrometry. Afterward, in the Co administration assay, ewes received a daily dose of 7 mg of Co/kg of body weight for 5 d. We analyzed milk samples for composition before and on the last days of cobalt dosing. On average, 17% of the injected amount of [1-13C]VA was transferred to milk within 96 h post-injection, and up to 29% of the VA taken up by the mammary gland was desaturated to milk RA. Under our conditions, the mean proportion of this conjugated linoleic acid isomer deriving from Δ9-desaturation represented 82 to 90% of the amount secreted in milk. However, the proportion estimated in the Co assay with calculations based on individual FA concentrations was lower (on average, 46%). When we calculated the same estimates based on changes in Δ9-desaturation ratios after Co dosing, the higher values of endogenous RA (75%) did not differ from the results of the isotopic tracer assay. Nevertheless, correlation analysis between the results obtained through [1-13C]VA or Co administration revealed no significant relationship, which would prevent acceptance of the latter as a reliable alternative to isotopic labeling to examine mammary Δ9-desaturation in dairy ewes.

Ameliorative effects of melatonin against nano and ionic cobalt induced genotoxicity in two in vivo Drosophila assays.

The aim of this study is to evaluate the ameliorative effect of melatonin (MEL) against induced genotoxicity by cobalt (II) chloride (CoCl2) and cobalt nanoparticles (CoNPs) (50 nm). Genotoxicity of CoCl2 and CoNPs were investigated using single cell gel electrophoresis (COMET) in Drosophila melanogaster hemocytes, which are blood cells of the Drosophila, and the somatic mutation and recombination test (SMART) was used to investigate mutant effects on the Drosophila wings. Three concentrations (0.1, 1, and 10 mM) of CoNPs and CoCl2 were applied to demonstrate their genotoxic potential. Both CoNPs and CoCl2 have mutagenic potential for the three concentrations tested in the COMET assay; however, only the 10 mM concentration of the ionic form and two high concentrations (1 and 10 mM) of CoNPs induced genotoxicity in the Drosophila SMART assay. Three different concentrations of MEL (0.1, 0.5 and 2.5 mM) were used against cobalt at highest concentration (10 mM) of both CoCl2 and CoNPs in both the SMART and COMET assays. MEL ameliorated the genotoxicity induced by CoCl2 and CoNPs in vivo Drosophila COMET and SMART assays.

Ruminal metagenomic analyses of goat data reveals potential functional microbiota by supplementation with essential oil-cobalt complexes.

BACKGROUND: Essential Oils (EO) are complex mixtures of plant secondary metabolites that have been proposed as promising feed additives for mitigating methane and ammonia emissions. We have previously demonstrated that Essential Oil-Cobalt (EOC) supplementation resulted in increased average daily gain and improved phenotypes (cashmere fiber traits, carcass weight, and meat quality) when cashmere goats received supplementation at approximately 2 mg/kg of body weight. However, the ruminal microbiological effects of EO remain poorly understood with regard to the extent to which ruminal populations can adapt to EO presence as feed ingredients. The effects of varying levels of EO require additional study. RESULTS: In this study, we conducted metagenomic analyses using ruminal fluid samples from three groups (addition of 0, 52, and 91 mg) to evaluate the influence of dietary EOC supplementation on goat rumen bacterial community dynamics. EOC addition resulted in changes of ruminal fermentation types and the EOC dose strongly impacted the stability of ruminal microbiota. The Bacteroides sp. and Succinivibrio sp. type bacterial community was positively associated with improved volatile fatty acid production when the diet was supplemented with EOC. CONCLUSIONS: A clear pattern was found that reflected rapid fermentative improvement in the rumen, subsequent to butyrate metabolism and EOC based feed additives may affect rumen microbes to further improve feed conversion. This observation indicates that EOC can be safely used to enhance animal productivity and to reduce ammonia and waste gas emissions, thus positively impacting the environment.

Electrospinning synthesis and assessment of physicochemical properties and biocompatibility of cobalt nitrate fibers for wound healing applications.

The aim of this study was to develop polyurethane (PU) wound dressing incorporated with cobalt nitrate using electrospinning technique. The morphology analysis revealed that the developed composites exhibited reduced fiber and pore diameter than the pristine PU. The electrospun membranes exhibited average porosity in the range of 67% - 71%. Energy-dispersive X-ray spectra (EDS) showed the presence of cobalt in the PU matrix. The interaction of cobalt nitrate with PU matrix was evident in Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The contact angle results indicated the improved wettability of the prepared PU/cobalt nitrate composites (82° ± 2) than the pure PU (100° ± 1). The incorporation of cobalt nitrate into the PU matrix enhanced the surface roughness and mechanical strength as evident in the atomic force microscopy (AFM) and tensile test analysis. The blood compatibility assays revealed the anticoagulant nature of the prepared composites by displaying prolonged blood clotting time than the PU control. Further, the developed composite exhibited less toxicity nature as revealed in the hemolysis and cytotoxicity studies. It was observed that the PU wound dressing added with cobalt nitrate fibers exhibited enhanced physicochemical, better blood compatibility parameters and enhanced fibroblast proliferation rates which may serve as a potential candidate for wound dressings.

Production responses and cost-benefit of long-acting pre-lambing anthelmintic treatment of yearling ewes in two commercial flocks in New Zealand.

AIMS: To investigate the production responses and cost-benefit of administering a controlled-release anthelmintic capsule (CRC) to pregnant yearling ewes prior to lambing. METHODS: Yearling ewes from two commercial sheep flocks (A, n=489; B, n=248) in the North Island of New Zealand were enrolled in the study. Prior to lambing, CRC containing albendazole and abamectin were administered to half the ewes while the other half remained untreated. Ewe liveweights and body condition scores were measured prior to lambing, at weaning and, for Flock B, prior to subsequent mating. Lambs were matched to dams shortly after birth and the weight and number of lamb weaned per ewe were determined. A cost-benefit analysis was undertaken for Flock B considering the increased weight of lamb weaned per ewe, and the weight of ewes at the next mating and the benefit in terms of lambs born. RESULTS: The mean weight at weaning of treated ewes was greater for treated than untreated ewes by 2.76 (95% CI 0.64-4.88) kg in Flock A (p<0.001) and 2.35 (95% CI -0.41-5.12) kg in Flock B (p=0.003); the weight of lamb weaned per ewe was greater for treated than untreated ewes by 1.43 (95% CI -0.71 to -3.49) kg in Flock A (p=0.041) and 3.97 (95% CI 1.59-6.37) kg in Flock B (p<0.001), and ewe liveweight prior to subsequent mating was greater for treated than untreated ewes in Flock B by 4.60 (95% CI 3.6-5.6) kg (p<0.001). There was no difference in the percentage of lambs reared to weaning between treated and untreated ewes in either flock (p>0.8). The overall cost-benefit of treatment for Flock B was NZ$9.44 per treated ewe. CONCLUSIONS AND CLINICAL RELEVANCE: Pre-lambing CRC administration to yearling ewes resulted in increased ewe weaning weights and weight of lamb weaned in both the flocks studied. There was an economic benefit in the one flock where this was assessed.

Late-gestation maternal dietary methyl donor and cofactor supplementation in sheep partially reverses protection against allergic sensitization by IUGR.

Perinatal exposures are associated with altered risks of childhood allergy. Human studies and our previous work suggest that restricted growth in utero (IUGR) is protective against allergic disease. The mechanisms are not clearly defined, but reduced fetal abundance and altered metabolism of methyl donors are hypothesized as possible underlying mechanisms. Therefore, we examined whether late-gestation maternal dietary methyl donor and cofactor supplementation of the placentally restricted (PR) sheep pregnancy would reverse allergic protection in progeny. Allergic outcomes were compared between progeny from control pregnancies (CON; n = 49), from PR pregnancies without intervention (PR; n = 28), and from PR pregnancies where the dam was fed a methyl donor plus cofactor supplement from day 120 of pregnancy until delivery (PR + Methyl; n = 25). Both PR and PR + Methyl progeny were smaller than CON; supplementation did not alter birth size. PR was protective against cutaneous hypersensitivity responses to ovalbumin (OVA; P < 0.01 in singletons). Cutaneous hypersensitivity responses to OVA in PR + Methyl progeny were intermediate to and not different from the responses of CON and PR sheep. Cutaneous hypersensitivity responses to house dust mites did not differ between treatments. In singleton progeny, upper dermal mast cell density was greater in PR + Methyl than in PR or CON (each P < 0.05). The differences in the cutaneous allergic response were not explained by treatment effects on circulating immune cells or antibodies. Our results suggest that mechanisms underlying in utero programming of allergic susceptibility by IUGR and methyl donor availability may differ and imply that late-gestation methyl donor supplementation may increase allergy risk.

Revisiting cobalt chloride preconditioning to prevent hypobaric hypoxia-induced damage: identification of global proteomic alteration and key networks.

Several studies have supported the hypoxia mimetic roles and cytoprotective properties of cobalt chloride in vitro and in vivo. However, a clear understanding of biological process-based mechanism that integrates the available information remains unknown. This study was aimed to explore the potential mechanism of cobalt chloride deciphering its benefits and well-known physiological challenge caused by hypobaric hypoxia that reportedly affects nearly 24 % of the global population. In order to explore the mechanism of CoCl2, we used global proteomic and systems biology approach in rat model to provide a deeper insight into molecular mechanisms of preconditioning. Furthermore, key conclusions were drawn based on biological network analysis and their enrichment with ontological overlaps. The study was further strengthened by consistent identification of validation of proteins using immunoblotting. CoCl2-pretreated animals exposed to hypoxia showed two significant networks, one lipid metabolism and other cell cycle associated, with a total score of 23 and eight focus molecules. In this study, we delineated two primary routes: one, by direct modulation of reactive oxygen species metabolism and, second, by regulation of lipid metabolism which was not known until now. The previously known benefits of cobalt chloride during physiological challenge by hypobaric hypoxia are convincing and could be explained by some basic set of metabolic and molecular reorganization within the hypoxia model. Interestingly, we also observed some of the completely unknown roles of cobalt chloride such as regulation of lipid that could undulate the translational roles of cobalt chloride supplementation beyond hypoxia preconditioning.

Cobalt chloride-mediated protein kinase Cα (PKCα) phosphorylation induces hypoxia-inducible factor 1α (HIF1α) in the nucleus of gastric cancer cell.

Hypoxia promotes cancer progression, and metastasis. The major protein expressed in hypoxic solid cancer is hypoxia-inducible factor 1 (HIF1). We show that enhanced phosphorylation of a conventional protein kinase C isoform, PKCα, at threonine 638 (T(638)) by hypoxia-mimetic cobalt chloride induces HIF1α in nuclei of gastric epithelial cells (GECs). Moreover, phospho-T(638)-PKCα (P-PKCα) interacts with p300-HIF1α complex in the nuclei of hypoxic GECs and PKCα phosphorylation at T(638) enhances transcriptional activity of HIF1α. High P-PKCα expression in neoplastic gastric cancer biopsy samples as compared to nonneoplastic samples suggests that P-PKCα might act as an indicator of gastric cancer progression.

YPTB3816 of Yersinia pseudotuberculosis strain IP32953 is a virulence-related metallo-oligopeptidase.

BACKGROUND: Although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations. RESULTS: In this study we found that oligopeptidase OpdA of Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli. Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used. CONCLUSIONS: The results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-oligopeptidases in particular. This is the first study demonstrating that opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity.