Ba proxies have been broadly used to reconstruct past oceanic export production. However, the precise mechanisms underlying barite precipitation in undersaturated seawater are not known. The link between bacterial production and particulate Ba in the ocean suggests that bacteria may play a role. Here we show that under experimental conditions marine bacterial biofilms, particularly extracellular polymeric substances (EPS), are capable of bioaccumulating Ba, providing adequate conditions for barite precipitation. An amorphous P-rich phase is formed at the initial stages of Ba bioaccumulation, which evolves into barite crystals. This supports that in high productivity regions where large amounts of organic matter are subjected to bacterial degradation, the abundant EPS would serve to bind the necessary Ba and form nucleation sites leading to barite precipitation. This also provides new insights into barite precipitation and opens an exciting field to explore the role of EPS in mineral precipitation in the ocean. Despite the broad use of barium as a proxy for past ocean export production, the underlying mechanisms of barite precipitation remain unknown. Here, the authors show, under experimental conditions, that barium bioaccumulation on bacterially produced biofilms is the crucial step for barite formation. Understanding past variations in the efficiency of the biological pump calls for the reconstruction of ocean export productivity, which fluctuates considerably over multiple time scales2. As there is a strong link between particulate organic carbon (POC) and particulate Ba fluxes in the ocean3, Ba proxies have been used to reconstruct carbon fluxes and past marine export production4. Nevertheless, a comprehensive understanding of nutrient cycling and export production in past oceans, including the significance of the biological pump and its efficiency, are hindered by our lack of full understanding of Ba cycling in the ocean and controls on barite (BaSO4) formation in the water column. It is well known that barite precipitates in undersaturated seawater as micron-sized crystals in association with sinking biogenic debris5, and its nutrient-like behavior and link to carbon remineralization at depth have been widely demonstrated6. However, the processes that lead to precipitation of barite in microenvironments within sinking particulate matter are not well constrained. It has been suggested that bacteria may play a role, since observations in the ocean water column support a close relation between bacterial activity and particulate non-lithogenic Ba in the water column9. It has also been demonstrated that under experimental conditions diverse marine bacteria from different habitats and of phylogenetically diverse species can promote barite precipitation11. Seawater Ba isotopic composition likewise points to a biological role in barite precipitation. This process preferentially incorporates the lighter isotopes of Ba with larger fractionation factors than for inorganic barite, and the residual waters remain isotopically heavier14.The biological pump plays a crucial role in ocean chemistry and the global carbon cycle16. In marine environments, bacterial involvement in barite formation has recently been reported for a cold seep where barite precipitated on filaments of sulfide-oxidizing bacteria. Laboratory experiments with isolates of these sulfide-oxidizing bacteria also show that under low sulfate conditions the sulfate generated by sulfide-oxidizing bacteria fosters rapid barite precipitation localized on the cell biomass17. In most previous experiments, bacteria provided the necessary sulfate for barite precipitation. However, this is not analogous to the process occurring in the open ocean, where sulfate concentrations in seawater are high; hence providing sulfate does not represent the role of bacteria in marine microenvironments. Given that ocean seawater is generally undersaturated with respect to barite due to low concentrations of Ba, the formation of barite depends upon a mechanism that serves to increase Ba concentrations at the sites of barite precipitation. Bioconcentration of Ba by phytoplankton has been reported through culture experiments and Ba isotope-spiking experiments18. Bearing in mind that the decay of phytoplankton is bacterially mediated, bacterial productivity would increase along with the availability of organic matter substrate and in turn with export productivity.Although the microbial role in precipitation of sulfates has been poorly investigated in comparison to the microbial involvement in carbonates formation, microbial barite precipitation has been described in hot springs environments, where bacteria play a role either in oxidizing sulfur compounds to generate sulfate or in accumulating Ba in microbial matsIn this context, we explore the potential of certain marine bacteria to bioaccumulate Ba in biofilms so as to shed light on the processes allowing marine bacteria in their natural habitat to promote barite precipitation. Although past experimental work and the close link between bacterial production and particulate Ba abundance in seawater suggest a link between bacterial activity and barite formation, the precise mechanisms for precipitation and the specific role of marine bacteria in the process have not been investigated. The capability of certain marine bacteria to provide sulfate in culture experiments promoting barite precipitation had served to demonstrate the microbial role in barite precipitation in low sulfate settings. However, this role is underplayed in the sulfate rich seawater. We show here that Ba bioaccumulation on bacterially produced biofilms is the crucial step for barite formation. We also show how barite forms under experimental conditions from an amorphous precursor that evolves to barite crystal, and speculate that a similar process occurs in the ocean.Idiomarina loihiensis MAH1, Marinobacter hydrocarbonoclasticus, and Planomicrobium okeanokoites . These strains were chosen because they belong to three phylogenetically different genera widely distributed in the marine environment. The first two are Gram-negative bacteria (γ−Proteobacteria) and the third is a Gram-positive bacterium (Firmicutes). Bacteria grown in a liquid medium and washed with NaCl solution were suspended in synthetic seawater without sulfate, allowing biofilm formation. Two concentrations of BaCl2 in the media (2 and 20 mM) were used.Three marine bacterial strains were selected for this study: Scanning electron microscopy (SEM) observations have demonstrated that Ba accumulated in the biofilms produced by all three marine bacteria in both media (2 and 20 mM Ba) after 24 h. Figures High-resolution transmission electron microscopy (HRTEM) analyses allowed the precise composition of the Ba precipitates to be determined; EDX maps and spectra show the elemental distributions of Ba, P, and S in the biofilms, and the presence of polyphosphate grains enriched in Ba in some cells Figs. , 3, 4. TBarite particles from sinking particulate matter collected using a multiple-unit large-volume in situ filtration system (MULVFS) from the ocean water column  were analyzed under SEM for comparison to the experimental precipitates. Similar to our experiments, in the natural samples, P was also localized with Ba, particularly in very tiny grains Fig. , while i4. The precipitation of barite in undersaturated seawater19 strongly suggests that biogeochemical processes rather than purely abiotic processes must be involved in such precipitation. Although no explicit mechanism has been identified for the nucleation and growth of barite crystal in the ocean water column, diverse lines of evidence suggest that bacteria play a major role. Dehairs et al.7 investigated particulate Ba content in the North Pacific and show that the vertical distribution of particulate non-lithogenic Ba in the water column (composed primarily from barite) closely follows trends in bacterial activity and oxygen consumption. In the Australian sector of the Southern Ocean, Jacquet et al.8 observed that mesopelagic Ba content is correlated with bacterial activity, with an increasing abundance of particulate Ba when bacterial activity is high. Similarly, Planchon et al.9 show that mesopelagic particulate Ba distribution reflects bacterial degradation of organic matter, and that this is related to oxygen consumption and bacterial carbon respiration in the Atlantic sector of the Southern Ocean. Experimental work also demonstrated that phytoplankton may play a crucial role in bioconcentrating Ba18, and since sinking organic matter serves as a substrate for heterotrophic bacteria, the link between bacterial abundance and export productivity may be a key factor for Ba bioaccumulation in microenvironments in the ocean water column. Indeed, in natural environments, Ba enrichment associated with microbial biomass has been described in diverse settings, including thermal springs20, biofilms in the Roman Catacombs21, bacterial EPS at fumaroles in Solfatara Crater, Italy22, and in filaments of sulfide-oxidizing bacteria in marine cold seep17. Likewise, culture experiments demonstrated the capability of bacteria to promote barite precipitation under laboratory conditions23. In such experiments, bacterial metabolism supplied the necessary sulfate for barite formation. Our experimental work also provided new insights into precipitation mechanisms, showing that phosphate groups are the main ligand for Ba binding and that phosphate is eventually substituted by sulfate to form barite. The binding of many elements to phosphate groups is a common biomineralization process leading to microbially induced mineral precipitation. For example, Rivadeneyra et al.24 studied aragonite precipitation by Chromohalobacter marismortui and demonstrated that, over time, the composition of bioliths changed from amorphous phases of Ca phosphate and carbonate to precipitates made essentially of aragonite and struvite. Furthermore, the ability of polyphosphates to chelate metal ions has been widely demonstrated. In fact, the intracellular chelation of heavy metals by polyphosphate may serve to reduce heavy metal toxicity and improve cellular tolerance to metals25. We surmised that, in seawater, phosphate groups on bacterial biofilms could play a major role in concentrating Ba, and act as nucleation sites for barite formation. Although phosphate and sulfate groups have different sizes (the phosphate group being larger), the mutual substitution of sulfate and phosphate groups is a common process in nature, as are solid solutions between phosphate and sulfate phases26. The adsorption of divalent cations on EPS and cell walls has been widely demonstrated27. Indeed, microbial mats and biofilms are commonly enriched in calcium and magnesium as compared to surrounding waters28. Trace metal incorporation by bacteria has been demonstrated for other metals as well (e.g. U29) specifically in the case of marine bacteria used in this study such as I. loihiensis.The importance of marine barite and Ba proxies for reconstructing past ocean export productivity has been widely demonstratedI. loihiensis, M. hydrocarbonoclasticus, and P. okeanokoites) can bioaccumulate Ba, which is essential for increasing Ba concentration and establishing nucleation sites in microenvironments in which barite precipitates. These results lead one to further hypothesize that barite precipitation in the ocean water column is mediated by Ba enrichment, initially bound to phosphate groups in biofilms and other organic substrates, and that eventually the phosphate is substituted by sulfate, which is readily available in seawater, to form crystalline barite. While bacteria are known to play a role in providing sulfate for barite precipitation in certain natural environments such as sulfidic springs20, in the ocean water column, where sulfate is abundant, the bacterial role would consist primarily of providing biofilms that bind Ba as the first step for barite precipitation. The bacteria would thus play an indirect role in the precipitation process, providing binding sites on EPS, cell walls, or in polyphosphate granules within the cells where Ba can bind to phosphate, followed by substitution of phosphate with sulfate to form barite. The observation of amorphous phosphate precursor associated with Ba in natural sinking particulate matter collected using a MULVFS reinforces the suggestion that in natural ocean water the bacterial biofilms would provide phosphate groups as binding sites for barite formation.Our experimental work represents a step forward in this field and demonstrates that common marine bacteria  to functional groups of marine EPS  has also been demonstrated33 implying that a similar process may be prevalent for diverse authigenic minerals. Notably, the role of the EPS in the formation of other minerals (carbonates) has also been demonstrated even in the absence of living cells34.The bioaccumulation of Ba in the analyzed bacterial EPS would also suggest that barite formation is not associated with a specific organism as EPS is produced by an ample range of aquatic microorganisms, particularly phytoplankton and bacteria. This finding therefore opens a novel field surrounding the role of transparent exopolymer particles (TEPs) in the binding of Ba. TEPs play a major role in marine biogeochemical cycles and have received considerable attention since they were first described in the ocean36. Additional evidence supporting biological mediation includes the distribution of Ba isotopes in seawater and particulate barites14.Crystallization of barite in the presence of organic compounds is also consistent with the typical rounded and elliptical barite morphologies found in seawater and marine sediments38 and serve to bind the necessary Ba and form nucleation sites, leading to barite precipitation. This finding is consistent with the observed relationship between export productivity, bacterial activity, and barite formation in the ocean. Although additional research is needed to precisely describe the role of EPS and other biofilms in barite crystal nucleation and growth in the water column, our experimental work reveals a mechanism by which Ba bioaccumulation on microbial biofilms, and specifically in EPS, through phosphate binding groups would serve as the nucleation foci leading to barite formation in the ocean water column.Based on all the above observations, we suggest that in regions where productivity is high and large amounts of organic matter are exported to depth and undergo bacterial degradation, EPS are commonly produced by the abundant phytoplankton and bacterial cells2, 8.8; peptone, 5; Na2SO3, 3.24; CaCl2, 1.8; yeast extract, 1; KCl, 0.55; NaHCO3, 0.16; ferric citrate, 0.1; KBr, 0.08; SrCl2, 0.03; H3BO3, 0.02; Na2HPO4, 8; Na2SiO3, 4; NaF, 2.4; NH4NO3, 1.6. The medium was used in liquid and solid forms; to prepare the solid medium, 2% purified agar-agar  was added. For the Ba bioaccumulation experiments, sulfate-free synthetic seawater (SSW) was used to avoid inorganic barite precipitation. The composition of the SSW was (in g/l): NaCl, 19.45; MgCl2, 5.9; CaCl2, 1.8; KCl, 0.55; NaHCO3, 0.16; KBr, 0.08; SrCl2, 0.03; H3BO3, 0.02; ferric citrate, 0.1; Na2HPO4, 0.008; Na2SiO3, 0.004; NaF, 0.002; NH4NO3, 0.001; and pH 7.6. This SSW was used with two concentrations of BaCl2: 2 mM (SSW1) and 20 mM (SSW2), both being higher than those found naturally in seawater. These concentrations served to expedite the process and allow for easy detection of the Ba bioaccumulation under the experimental conditions.Bacterial strains were cultured and maintained in a marine broth (MB) medium , with the following composition (in g/l): NaCl, 19.45; MgClBacterial cells were grown and maintained in MB medium. Bacterial growth was monitored in the liquid MB by measuring the optical density at 620 nm. Ba accumulation tests were performed using bacterial biofilms produced by each of the selected strains. Cultures destined to biofilm formation were grown in liquid MB medium (100 ml) in 250 ml sterile Erlenmeyer flasks, incubated at 28 °C, and shaken continuously at a speed of 180 r.p.m. Cells were grown to late exponential phase and aliquots of 10 ml of the bacterial cultures were centrifuged at 10,000 r.p.m. for 10 min at 4 °C. The collected cells were rinsed three times with NaCl solution (3.5% w/v) to remove ingredients from the medium, in particular sulfates. The cells were re-suspended in sterile test tubes containing 10 ml of either SSW1 or SSW2. To promote biofilm formation, pieces of glass coverslips sterilized by autoclave (for 20 min at 120 °C) were aseptically placed in these previously prepared liquid-bacterial suspensions (one per each test tube). A minimum of three replicates per run were performed. The test tubes were incubated for 24, 48, and 96 h at 28 °C under shaking at 180 r.p.m.SEM and HRTEM were used to analyze the Ba bioaccumulated on bacterial biofilms. For this purpose, the glass coverslips were aseptically collected from the test tubes and immediately fixed in 1 ml of 2.5% glutaraldehyde solution (prepared in 0.2 M cacodylate buffer with 0.4 M sucrose and 0.1% NaCl to reach an osmolarity of 1220 mOsm similar to that of MB and SSW media). Samples were kept at 4 °C until their preparation for electron microscopy analyses. The fixed glass coverslips were dehydrated using a series of ethanol rinses , exposed to critical-point drying  and coated with carbon prior to observations. The biofilms were characterized using SEM (AURIGA FIB-FESEM Carl Zeiss SMT and Zeiss SUPRA40VP), equipped with EDX detector system . Biofilms were also analyzed by means of HRTEM; for these analyses, the coverslip surfaces with biofilms were scraped with a scalpel and then dispersed in ethanol by sonication for ~3 min. Samples were then collected and deposited on carbon-film-coated copper grids for observation. Data were collected using a FEI TITAN G2 60-300 microscope with a high brightness electron gun (X-FEG) operated at 300 kV and equipped with a Cs image corrector (CEOS). For analytical electron microscopy (AEM), a SUPER-X silicon-drift windowless EDX detector was used. Digital X-ray maps were collected on selected areas of the samples and mapped for Ba, S, and P. SAED patterns were also acquired for identification of crystalline phases. Regarding the analyses of barite particles from the ocean water column, representative pieces of the filters were coated with carbon for SEM observation.The datasets generated during the current study are available from the corresponding author.Supplementary InformationPeer Review File


Orally Disintegrating Tablets (ODT) have the advantages of both solid dosage form specially the stability and ease of handling and liquid dosage forms including ease of swallowing and pre-gastric absorption. We focused on taste masking and formulation of ranitidine ODT which disintegrates rapidly in the mouth within 60 sec using super-disintegrants, special polymers, water soluble and even insoluble excipients, sweeteners and essence. Various formulations were designed and made in four series. The amount of ranitidine in each formulation was 150 mg, and the final weight of tablets was around 500 mg. Prepared formulations were evaluated in terms of several physicochemical tests including powder/granule flowability, appearance, thickness, uniformity of weight, hardness, friability and disintegration time. Several taste masking techniques were investigated in each series of formulation, in order to cover the bitter taste of wranitidine. These included the addition of sweetener, granulation, solid dispersion with soluble and insoluble agents and complex formation with cellulose derivatives. The best formulation(s) in each group was/were chosen for taste evaluations with the help of 10 volunteers. Finally, formulation F14 was selected as the ultimate formulation, based on its better taste and shorter disintegration time (around 5 seconds). Formulation F14 contained Na CMC, avicel, Na starch glycolate, xylitol, saccharin, Na benzoate and menthol. The chosen formulation successfully passed the complementary evaluations such as assay of active ingredient and dissolution time. Na CMC was found to be acceptable in terms of decreasing disintegration time and enhanced taste masking potential and can be used in further ODT formulations.  Since pharmaceutical scientists have discovered advanced information about the physicochemical and biochemical parameters relevant to their profession, drug delivery systems have been increasingly promoted over the past decades . Recently, patient-friendly dosage forms are growing higher interests among pharmaceutical companies and medical society members in addition to patients. Most of therapeutic agents considered for systemic drug delivery have a tendency to be administered via oral route, owing to their numerous advantages, particularly greater patient compliance due to their ease of administration. Among these dosage forms are the orodispersible or orally disintegrating tablets (ODT) , 3. Orange Book an ODT as ″A solid dosage form containing medicinal substances, which disintegrates rapidly, usually within a matter of seconds, when placed upon the tongue”  has a definition in the  tongue” . The Eurallowing . Over thCertain studies concluded increased bioavailability and proved rapid absorption of drugs through pre-gastric absorption from mouth, pharynx & esophagus, as saliva passes down, therefore rapid onset of action and increased bioavailability will be expected . MoreoveThe target communities in ODT consumption are children, the elderly, hospitalized patients, bodily and mental cripples, those with mastication and deglutition problems, patients with resistant chronic nausea, patients under chemotherapy, psychotic patients who hide their tablets beneath their tongue and those people or travelers who have no access to water. ODTs have also been recently used in animals -11.Whereas ODTs are going to disintegrate in the buccal cavity and in direct contact with tongue’s taste buds , pleasan2-receptor antagonist that inhibits stomach acid production. It is commonly used in the treatment of peptic ulcer and gastro-esophageal reflux disease. Certain preparations of ranitidine are available over the counter (OTC) in various countries. It is on the World Health Organization›s List of Essential Medicines, the most important medications needed in a basic health system  and the percentage of drug release (90% in 15 min) was also higher than the other superdisintegrants investigated . Xylitol , sodium saccharine (food grade: FAD 8), sodium starch glycolate (SSG) (primojel), sodium benzoate, poly ethylene glycol (PEG 4000) and menthol all were purchased from Merck (Germany). Croscarmellose was obtained from FMC Biopolymer (Ireland), Na CMC grade 30000, HPMC and Poly vinyl pyrrolidone 10 (PVP 10) were all obtained from sigma (USA).Preliminary studies on ranitidine powderFirst, physicochemical properties of ranitidine powder were investigated such as powder purity, organoleptic properties  and flowability (by measuring the Carr’s index and Hussner’s ratio). Moreover, compressibility and disintegration time of the compressed drug powder was evaluated.Formulation of ranitidine orally disintegrating tabletsDue to ranitidine’s bitter taste, its’ ODT formulation requires challenging taste masking strategies. In this study we were looking for simple industrially operative strategies. First, a basic formulation was designed and prepared for ranitidine ODT, containing avicel as the filler, sodium starch glycolate as the super-disintegrant and sodium benzoate as the water soluble lubricant. The direct compression method, alongside the granulation technique in some formulations, was applied for tablet preparation and four series of taste masked formulations were prepared.In series A formulations, sweeteners and essence were added to formulation. Based on previous investigations, saccharin and xylitol are among the sweeteners which are widely used for masking the bitter taste, at a ratio of 1:1 or 1:2, mainly due to their non-glycogenic characteristic which is ideal for use in pediatric and diabetic patients . Series In series B formulations, drug powder was granulated with PEG 4000, using the solid dispersion method. In here the water soluble polymer PEG 4000 was melted using the bain-marie technique and mixed with equal amount of drug powder and was then cooled down to a semi solid state. Next, it was passed through a 30 mesh sieve and formed into granules in order to lessen the drug powder contact with taste buds. It is also hypothesized that PEG can temporarily and partially cover the drug particles to lessen the bitter taste sensation. Granules were then used in different formulations, alongside the sweeteners and menthol, as presented in In Series C formulations, drug powder was granulated with white wax, using solid dispersion method. In the next step granulation with white wax was performed with the same method of PEG. Due to its lower water solubility, it’s supposed that drug release will decrease in oral cavity and bitter taste will be sensed less. Four formulations were designed alongside with sweeteners and menthol as shown in In series D formulations, ranitidine was complexed with the cellulose-based polymer sodium carboxymethyl cellulose (NaCMC), in order to coat drug particles with the polymer. NaCMC was hydrated with water to form a gel with a viscosity of about 70 cps. Next, the drug powder was added to the gel and the mixture was dried in a vacuum oven at 50 °C for two hours. The dried gel was triturated and passed through a 30 mesh sieve. Different ratios of polymer to drug were prepared, also containing sweetener and menthol. Formulations prepared have been shown in Physicochemical evaluations of ranitidine ODT formulationsDifferent ranitidine ODT formulations prepared, underwent physicochemical tests mentioned below:(I) Power/granule flowability: ꞌs index [(C = 100(1-ρB/ρT)],where ρB is the freely settled bulk density of the powder, and ρT is the tapped bulk density of the powder) and Hausnerꞌs ratio (H = ρT/ ρB).flowability was measured in terms of calculating the Carr(II) Tablet appearance: tablets should have a flat and smooth surface, with no chipping, cracking or any other defect. (III)Thickness:a digital vernier caliper was employed for measuring tablet thicknessin mm.(IV) Uniformity of weight: this was determined by weighing 20 randomly selected tablets. (V)Hardness:Hardness of tablets was determined using a Monsanto hardness tester, and expressed in kp.(VI)Friability:Friability of the tablets was examined, using aRoche friabilator and was expressed in %.(VII) Disintegration time:an ODT formulation should disintegrate or dissolve in water quickly. Hence, for conducting the disintegration time test, 6 tablets from each formulation were chosen randomly and each individually dropped into a basket connected to the wall of a beaker containing 10 mL pH 7.2 phosphate buffer . Then, the beaker was placed on a shaker with a speed of 30 rpm, for imitation of light flow of saliva. The duration of time required for disintegration of each tablet was recorded. This test was performed at room temperature. Taste masking and complementary tests on the selected formulationsFormulations which showed optimum physicochemical properties, were found to be acceptable for further studies. They underwent additional studies, as follows: (I)Consumer’sacceptance:Ten healthy and non-smoking volunteers; 5 females and 5 males, aged between 20 to 29 years; took part in this study and expressed their opinion on the appropriateness of the taste of selected formulations. This was based on a scale from 1 to 5, with 1 representing the worst formulation and 5 the best formulation. It should be mentioned that the test formulations were coded in order to prevent any bias in the volunteers. Following the comparison of the taste of formulations, the most suitable formulation was considered as the ultimate formulation. This formulation was studied in terms of supplementary physicochemical tests.(II) Assay of active ingredient:HPLC was used for this test. The column was C18 and the mobile phase consisted of aqueous ammonium acetate: methanol (85:15 v/v) with a flow rate of 1.5 mL/min. A UV detector was employed for detection of ranitidine in the wavelength of 322nm. 20 tablets were powdered randomly, and 500 mg  was weighted to be assayed. The powder was dissolved in mobile phase at a concentration of 0.112 mg/mL, same as the standard solution, and passed through a 0.22 µM filter and then injected into the HPLC injection port . Finally(III) Dissolution time test: dissolution test was performed on 10 tablets, using the paddle system in distilled water medium, at a speed of 50 rpm for 45 min based on the USP guideline . A UV deStatistical analysisFor the purpose of comparison between two different test samples, independent sample t-test was used and for more than two samples, one way ANOVA was utilized. In cases in which significant differences existed in the ANOVA test, Tukey post-hoc test was used to specify those samples having significant differences with each other. In qualitative samples (taste studies), Friedman and Wilcoxon paired statistical test was used. The Friedman test shows the existence of an overally significant difference between formulations and the Wilcoxon paired test compares formulations with each other pair-wise, in order to specify samples with significant differences. In all the above mentioned tests, a p-value ≤0.05 was considered as significant.The results obtained from the studies conducted on ranitidine powder and the various ranitidine ODT formulations prepared, have been presented as follows:Preliminary studies on ranitidine powderAt first physicochemical characteristics of ranitidine powderwas examined, including the organoleptic characteristics, powder flowability, compressibility and disintegration time.ꞌs index and Hausnerꞌs ratio, showed poor powder flow which needs the addition of appropriate lubricants for modification. Results showed that ranitidine is a yellowish powder with undesirable bitter taste and sulfur-like odor, complying with the specifications. The results obtained for flowability, based on the CarrThe maximum amount of powder compressibility, following the preparation of compressed powder discs was 3.12 ± 0.46 KP (n = 10), showing an acceptable degree of powder compressibility for the direct compression method. Disintegration time of compacted powder discs was over 10 min indicating the need for the inclusion of effective disintegrants in formulation.Ranitidine ODT formulationsVarious formulations were prepared using different ingredients in four series (A-D), and physicochemical properties of each series including flowability, appearance, thickness, uniformity of weight, hardness, friability and disintegration time were investigated. The results obtained have been shown in Flowability of all formulations was improved in comparison with the ranitidine powder, due to the addition of property modifying excipients such as sodium benzoate . In series A formulation the disintegration time was longer than 60 sec and also the addition of the sweeteners could not mask the bitter taste completely. Therefore, F1 formulation was omitted from further complementary evaluations. In series B formulations, only the F4 formulation had a disintegration time of less than 60 sec (21.83 ± 6.56 s). Formulation F4 was a basic formulation, merely formed by granulating the drug with PEG4000, and did not contain any sweetener or essence. Hence, formulations F4a and F4b were designed and made using xylitol, saccharin and menthol. Based on the results, formulation F4b had a desirable appearance and quicker disintegration time than formulation F4a, and was therefore chosen for further evaluations. In series C formulations in which white wax was utilized for taste masking, none of the formulations had an appropriate disintegration time, but formulation F7 had a desirable appearance and moderate flowability and hence was selected for complementary studies. In the last series of formulations (series D),in which NaCMC was incorporated for masking the bitter taste, the disintegration time was impressively decreased to nearly below 30 s  and the tablet appearance was desirable in all formulations. Formulations F14 and F15 were chosen due to their lower disintegration times for complementary evaluations. The selected formulations physicochemical test results are shown in Complementary studies on the selected formulationsFormulations F4b, F7, F14 and F15 were subjected to taste masking evaluation. Following the comparison of the scores given by ten volunteers to the tastes of the selected formulations, formulation F14 was chosen as the ultimate formulation . When examining the dissolution profile (time) of formulation F14, more than 85% of its’ drug content was released over a period of 45 minutes, which is within the acceptable range stated in the literature(27).2 antagonist and blocks the production of acid by acid-producing cells in the stomach.  It is commonly used in treatment of peptic ulcer disease and gastroesophageal reflux disease, which are prevalent nowadays in all ages especially elderlies. In this study we aimed to design a novel and simple ranitidine ODT formulation, using polymers, water-soluble excipients and super-disintegrants. These formulation strategies could be cost-beneficial and can be easily adopted in industrial scale in the pharmaceutical companies.Ranitidine is an oral drug that is widely used as an effective Het al. developed a new fast disintegrating tablet with ranitidine as the model drug (et al. designed a ranitidine fast disintegrating tablet, in which they used different super-disintegrant including sodium starch glycolate, pregelatinized starch and NaCMC (Generally speaking in ODT formulations, disintegration time and taste masking are the two most important issues which should be considered in their formulation design . In seridel drug . They alnd NaCMC . They suAs shown in Overall, based on the physicochemical tests conducted as well as the taste masking studies, formulation F14 seems to be the most suitable formulation for the preparation of ranitidine ODT.In this study we aimed to formulate a novel and simple ranitidine orally disintegrating tablet, capable of disintegrating rapidly within the buccal cavity. The ultimate ranitidine ODT formulation selected was formulation F14, which contained ranitidine and NaCMC as the intra-granular components and xylitol alongside saccharin and menthol, avicel, sodium starch glycolate and Na benzoate as the extra-granular components. This formulation complied well with all the physicochemical tests conducted, particularly disintegrating within 5 sec. In addition, this formulation also provided a desirable taste and after taste within the buccal cavity and hence could be considered as an appropriate ODT formulation for ranitidine.


The regional exception to this trend was Europe, where suitability in temperate dryland portions will decline substantially. These results clarify how rising temperatures interact with other key drivers of moisture availability to determine the sustainability of rainfed agriculture and help policymakers, resource managers, and the agriculture industry anticipate shifts in areas suitable for rainfed cultivation.The distribution of rainfed agriculture, which accounts for approximately ¾ of global croplands, is expected to respond to climate change and human population growth and these responses may be especially pronounced in water limited areas. Because the environmental conditions that support rainfed agriculture are determined by climate, weather, and soil conditions that affect overall and transient water availability, predicting this response has proven difficult, especially in temperate regions that support much of the world’s agriculture. Here, we show that suitability to support rainfed agriculture in temperate dryland climates can be effectively represented by just two daily environmental variables: moist soils with warm conditions increase suitability while extreme high temperatures decrease suitability. 21 Approximately 1.5–1.6 billion hectares, or roughly 12–15% of the earth’s terrestrial land surface, are currently in cultivation or exist within a matrix of substantial cultivation, and in many regions cultivation is nearly ubiquitous3. Expansion of cultivation into newly suitable locations is an anticipated societal response to both changing climate4 and shifting geographic patterns of human populations5. Strategies for adapting our agricultural systems to climate change6 include enhanced irrigation9, although future water supplies for irrigation are uncertain, especially as societal water demand grows and changing climatic conditions alter both temperature and precipitation patterns10. Consequently, rainfed agriculture, which currently represents approximately 75% of global croplands11, is likely to remain a major component of the global food system. As cultivated lands shift4, the importance of rainfed agriculture will become especially pronounced for dryland areas where irrigation infrastructure and water supply are already limited. Quantifying the climatic and edaphic controls of rainfed cultivation in drylands is necessary to facilitate accurate forecasts of local, national, and global food supply12. In addition, understanding the potential future distribution of agricultural land use is important to forecast the impact of human activities on a range of ecological processes and services13. Cultivation is a transformative land-use practice that influences ecosystem water balance14, carbon dynamics15, and nutrient cycling16, and modifies landscape attributes such as biodiversity, habitat connectivity, and potential migration pathways17.Meeting the nutritional demands of a burgeoning human population in coming decades will require shifts in the distribution of global cultivation20 that are not well estimated by mean climate conditions alone23. Climatic variables that influence the distribution of rainfed agriculture include the degree of overlap between warm and wet seasons24, growing degree days, aridity, and winter temperatures25. Studies of crop yields have indicated that high temperature extremes, which lead to high vapor pressure deficits, can decrease rainfed crop yields in a variety of crops and regions32, especially during sensitive periods of crop development33. In addition, the adverse impacts on crop yields appear to be mediated by transient deficits of soil water availability, rather than directly by temperature21. Despite this evidence for interactive effects of temperature extremes and transient soil moisture on crop yields, the roles that these variables play in dictating overall areas suitable for agriculture have not been explored. Understanding these effects is particularly important considering expectations for increasing temperature stress34 throughout the 21st century.The suitability of drylands for rainfed agriculture is dictated by interactions among temperature, precipitation, and soil texture36. By contrast, climate change impacts on agricultural suitability are uncertain for temperate regions37 that support a majority of global agricultural land38 and tend to have higher yields than tropical areas due in large part to higher soil fertility39. Globally, the temperate zone accounts for roughly 1/6 of cultivation and represents approximately 31% and 17%, of cultivated areas for wheat and maize, respectively40. Understanding the controls over agricultural suitability and the potential impact of climate change on the distribution of suitable areas is especially challenging in temperate drylands because suitability in areas with high annual temperature amplitude is driven by complex interactions among seasonal and soil-depth patterns of both soil moisture and temperature. Plant growth and overall agricultural suitability in these areas is restricted to the mostly frost-free, warm season, which may not coincide with the period of highest soil moisture availability.Climate change is expected to decrease agricultural suitability at the global scale, although many studies have suggested that the most negative outcomes are likely to occur in tropical and sub-tropical systems5, we also included a static metric of access to markets in our candidate variables. Second, we quantified how potential climate change-driven shifts in both weather extremes and transient soil moisture conditions may alter the abundance and extent of areas in temperate drylands that can support rainfed agriculture.Here, we examined climate and soil texture controls of rainfed agriculture suitability in temperate drylands. Our goal was to assess how climate change, and particularly changing weather extremes, may impact the distribution and abundance of areas suitable to support rainfed agriculture in temperate dryland regions. First, we examined current climate, weather, and edaphic controls of rainfed agriculture suitability in temperate drylands, and we identified the metrics of daily soil moisture availability and weather extremes that were most strongly related to the current distribution of rainfed agriculture, from a list of potential explanatory variables used by previous studies , and MKT is market access.We examined how remotely-sensed measures of current rainfed agriculture in temperate drylands Fig.  related Our best model contained just two variables representing soil moisture and climate, suggesting that soil and climatic controls over agricultural suitability can be effectively represented by the interactive effects of the long-term mean annual frequency of wet degree days in the top soil layers and the long-term mean annual number of days with maximum temperature greater than 34 °C. Rainfed agriculture was positively related to wet degree days and market access, and negatively related to extreme hot days Fig. . The pos2 suitable for rainfed agriculture across all temperate dryland regions, close to the 2.51 million km2 of realized rainfed agriculture estimated by remote sensing2. Our estimates . We estimate approximately 2.32 million kmPredictions based on wet degree days, extreme temperatures and market access  would result in a 41% increase in global temperate dryland area suitable to support rainfed agriculture , in West Asia by 30% (0.35 million km2), in North America by 43% (0.26 million km2), in South America by 35% (0.02 million km2), and decrease in Europe by 31% (0.014 million km2). The directions of these predictions for change in suitability were generally robust to uncertainty in both statistical model structure and future climate represented by variation among two emissions scenarios and 16 climate models  suggests potential increases for most regions. Compared to current modeled suitable area, our best model predicted that climate change represented by the median GCM under RCP8.5 at the end of the 21els Fig. , althougels Fig. . The top30, its role in restricting the distribution of areas suitable for rainfed agriculture has not been previously demonstrated. Second, extreme heat interacts with the dominant control exerted by transient soil moisture availability in these dryland regions such that rainfed agriculture can be restricted by any combination of dry conditions or extreme heat. These interacting influences are especially important in the context of climate change, because a growing potential for extreme heat events, more frequent ecological drought periods, and enhanced aridity in drylands are among the most reliable aspects of climate projections41 . As temperatures continue to rise in coming decades, this limitation will have potentially important negative consequences for future global food supply43.We found that the number of days with maximum temperature greater than 34Consistent with our hypothesis about the positive influence of proximity to population centers, our model selection indicated a positive relationship with market access. Although market access patterns may shift in the future, we did not attempt to anticipate those changes and, for the purpose of this analysis, assumed that the geographic patterns of market access will not change in the future. The inclusion of market access in our best model suggests that it provides useful information that can complement climate and ecohydrological metrics to enhance future studies of agricultural suitability.23. For example, recent climatic warming trends have been related to decreased crop yield in many temperate regions, including soy and maize in North America and Asia, and wheat in Asia, and increased yield in other areas, including wheat in North America and rice in the Northern parts of Asia6. While most of these yield patterns were consistently linked to temperature trends, growing season precipitation trends, which are less consistent than temperature trends, showed weak relationships with crop yield at global scales29 but are likely much more important in water-limited arid and semiarid climates.For temperate dryland regions, the importance of both wet degree days and high temperature extremes, and the interaction between them, suggests a simple framework for interpreting both historical relationships between climate and agriculture, as well as for developing future projections of climate change impacts that explicitly incorporate edaphic influences over moisture availability44. In particular, the northern portions of East Asia, West Asia and North America and the southern portion of South America all display increases in suitability for rainfed agriculture. Some portions of these areas of increasing suitability are already heavily cultivated, while others have relatively low current prevalence of cultivated agriculture. This increased suitability is driven primarily by increases in temperature that when combined with slight precipitation increases, result in higher wet degree days in the top soil layers , and migration of cultivated land use practices into areas previously considered too cold4. Likewise, areas of declining suitability for rainfed agriculture, notably Europe and the southern portion of the U.S. Great Plains, may also experience substantial changes in ecosystem structure and potentially enhanced erosion and dust production as a result of crop failures. While land use decisions are influenced by many factors other than environmental suitability, changes in suitability may have dramatic impacts on ecosystems in temperate dryland regions16 and the non-agricultural ecosystem services that they currently provide.Cultivated agriculture is a transformative, high-impact land use practice that fundamentally alters biodiversity and ecosystem structure and function. Broad geographic changes in cultivation intensity, including both large parcels of previously cropped lands left fallow on the trailing edge as well as conversion of land to cropping due to increased suitability, would impact ecological processes and ecosystem structure at a range of spatial scales. The widespread areas of increasing suitability for rainfed agriculture in temperate drylands that we identified, combined with growing global demand for food and fiberMAT) > 0 °C, b) ≥ 4 but < 8 months have mean monthly temperatures ≥ 10 °C, c) Trewartha climate group category is D46, and d) aridity  is considered arid or semiarid (0.05 ≤ AI < 0.5). We included areas meeting these temperate dryland criteria under either current conditions or future conditions for any of the scenarios we examined and our comparisons of current and future rainfed agricultural suitability utilize a consistent area (described below) which is larger than current temperate drylands. We applied a geographic raster with 0.3125-degree square cells so that exactly one cell center of the NCEP/CFSR T382 Gaussian grid47 fell in each of our cells (resolution of about 0.312° × ~0.312°). Our raster contained 1152 × 576 cells and had its origin at 90 °S and 179.84375 °W. From these criteria, we identified 20,020 cells for running simulations. Our results are based on 5 geographic regions from the UN geoscheme48: ‘South America’ (<15 °N & >25 °W); ‘Eastern Asia’ including the eastern portion of Southern Asia  and the eastern portion of Eastern Europe ; ‘Western and Central Asia’ including the western portion of Southern Asia  and western portion of Eastern Europe (<87 °E); ‘Western Mediterranean basin’ (W of the Dinaric and Pindus Mountains) including Europe and Northern Africa, but excluding Eastern Europe (>0 °N & (<25 °W & <14 °E); ‘North America’ (>25 °N & >50 °W).We defined temperate drylands as areas where a) mean annual temperature  daily maximum and minimum temperature (2 m above ground) and 6-hourly precipitation from NCEP/CFSR data47. We examined two RCPs, RCP4.5 and RCP8.5, to represent emissions uncertainty, and 16 GCMs  from the “Downscaled CMIP3 and CMIP5 Climate and Hydrology Projections” archive at http://gdo-dcp.ucllnl.org/downscaled_cmip_projections/ . We combined historical daily data (NCEP/NFSR) with monthly GCM predictions of historical and future conditions with a hybrid-delta downscaling approach to obtain future daily forcing54.As described in Schlaepfer et al.55 and Schlaepfer et al.49 and the algorithms for estimating vegetation from climate were applied under both current climate and future climate for each time period and climate model examined. Soil texture data were derived from the ISRIC-WISE global soil dataset at 5 arc-min spatial resolution and at 20-cm depth intervals. Soil depth was estimated from ISRIC-WISE unless the soil was deeper that 1 m, in which case depth was estimated as 95% of the maximum root depth with 50-cm depth intervals56 and soil texture was assumed to be the same as the deepest ISRIC-WISE layer. We extracted elevation information from the GAEZ 2008 30-arcsec global elevation dataset at http://webarchive.iiasa.ac.at/Research/LUC/External-World-soil-database/HTML/global-terrain-slope-download.html?sb=7 and calculated area-weighted median and elevation range for each cell.Seasonal patterns of vegetation structure and function were estimated from climate variables as described in Bradford et al.2 who synthesized four previous remote-sensing studies of global croplands and developed two products relating to global cropland distribution: crop dominance and major crops, both which included estimates of rainfed vs. irrigated cultivation  Within each simulation grid cell  the prevalence of dryland agriculture was calculated from the remote-sensing derived agriculture estimates (1 km2 resolution) generated by Teluguntla et al.2. We averaged both products, with rainfed major crop abundance  calculated as the sum of the rainfed major crop categories  multiplied by 0.7 to approximate the actual subpixel abundance of agriculture, based on57, and rainfed crop dominance  estimated as class 3 (“croplands rainfed”), class 4 “croplands, rainfed minor fragments”, and class 5 (“rainfed very minor fragments”) multiplied by 0.7, 0.25, and 0.1, respectively (reflecting within-pixel abundance57). Our assumptions about within-pixel abundances  influences the regional and global estimates of land area currently cultivated. However, we explored a range of sub-pixel abundance values and found that statistical model performance and estimates of percent change in area suitable to support rainfed agriculture as a result of climate change were not substantially influenced by sub-pixel abundance values. Consequently, we focus on proportional change in suitable area by region, and present estimates of actual land area in supplementary materials. The abundance of rainfed agriculture can be influenced not only by the climatic and soil moisture conditions that support viable crop yields, but also by the economic demand for food and proximity to population centers5. We estimated access to markets for each cell from a dataset developed as part of the Global Land Project, accessed from  on October 25, 2015. These market access data were highly right skewed, so we applied a natural log transformation adding a constant of 0.0001.The proportion of each cell that is currently cultivated in dryland agriculture was estimated from the results of Teluguntla 60 and specific simulations for this analysis are described in Schlaepfer et al.49. SOILWAT has been applied and found to be reliable in dryland ecosystems including temperate grasslands61 and temperate shrub-dominated ecosystems60. Inputs to SOILWAT include daily weather conditions (mean daily maximum and minimum temperature and daily precipitation), mean monthly climate conditions , latitude, elevation, vegetation  and soil properties (texture of each soil layer). SOILWAT estimates processes for each functional plant group including interception by vegetation and litter, evaporation of intercepted water, transpiration and hydraulic redistribution from each soil layer. SOILWAT estimates hydrological processes including partitioning of precipitation into snowfall and rain, snow accumulation, melt and loss, infiltration into the soil profile, percolation for each soil layer, bare soil evaporation and deep drainage60. We executed these simulations on Yellowstone at the National Center for Atmospheric Research-Wyoming Supercomputing Center and the Advanced Research Computing Center’s Mount Moran/Bighorn facilities at the University of Wyoming.We utilized SOILWAT, a daily time step, multiple soil layer, process-based, simulation model of ecosystem water balanceWe considered a set of seven variables as candidates for main effects that represent specific hypotheses about environmental and economic controls over the distribution of rainfed agriculture , the predicted proportion of land used for agricultural, y,  and an overdispersion term, y was modeled as a function of covariates, X, and estimated coefficients, β, via a logit link:We modeled the number of square kilometers of dryland rainfed agriculture, a priori; then, we created a set of global models using combinations of the selected covariates and interactions; finally, we ranked the global models using AIC. First, we defined three major regions  for model fitting. We conducted three separate model selection iterations with each major region as an out-of-sample dataset and performed forward stepwise model selection where coefficients and overdispersion parameter were estimated via maximum likelihood using the remaining two major regions as in-sample data and the performance of a model was based on the negative log-likelihood (NLL) of the out-of-sample data given the estimated β and 62 (version × 64 3.2.3), which are included in the stats62 and emdbook63 packages respectively. We used AIC to rank the 10 global models; the ranking was confirmed by RMSE (based on in-sample data) (SI Appendix, Table nd, 8th and 15th GCM rank to represent variability among GCMs. To quantify the relative influence of statistical uncertainty vs. climate uncertainty, we partitioned variance in predictions of future land area suitable to support rainfed agriculture into variance attributable to statistical models, climate scenarios and residual variance (SI Appendix, Table We first selected covariates from among the seven candidates to include in our final models by a regional model selection process defined Supplementary Material


Vulpes vulpes display great variability in home range size. Yet, there has been little consensus regarding the reasons why home range sizes of red foxes vary so extensively. In this study, we examine possible causes of variation in red fox home range sizes using data from 52 GPS collared red foxes from four study areas representing a gradient of landscape productivity and human landscape alteration in Norway and Sweden. Using 90% Local Convex Hull home range estimates, we examined how red fox home range size varied in relation to latitude, elevation, vegetation zone, proportion of agricultural land and human settlement within a home range, and sex and age. We found considerable variation in red fox home range sizes, ranging between 0.95 km2 to 44 km2 (LoCoH 90%) and 2.4 km2 to 358 km2 (MCP 100%). Elevation, proportion of agricultural land and sex accounted for 50% of the variation in home range size found amongst foxes, with elevation having the strongest effect. Red foxes residing in more productive landscapes (those in more southern vegetation zones), had home ranges approximately four times smaller than the home ranges of foxes in the northern boreal vegetation zone. Our results indicate that home range size was influenced by a productivity gradient at both the landscape (latitude) and the local (elevation) scale. The influence of the proportion of agriculture land on home range size of foxes illustrates how human landscape alteration can affect the space use and distribution of red foxes. Further, the variation in home range size found in this study demonstrates the plasticity of red foxes to respond to changing human landscape alteration as well as changes in landscape productivity, which may be contributing to red fox population increases and northern range expansions.Home range size is a fundamental concept for understanding animal dispersion and ecological needs, and it is one of the most commonly reported ecological attributes of free-ranging mammals. Previous studies indicate that red foxes  The location and size of the area that an animal uses to secure resources and mates (i.e. its’ home range ) is fundHome range size can vary greatly across taxa, populations and individuals –7, and iHowever, there has been much debate as to the relative importance of these factors in shaping home range size, and often, such mechanisms are examined separately despite their synergistic effects on home range size , 20. ThuVulpes vulpes is a species that demonstrates great flexibility in distribution, foraging behaviour and social structure [The red fox tructure –26. Red tructure , 28. Simtructure , 29 and tructure .Previous studies indicate that red foxes display high variability in home range size . YeAdditionally, much of our previous knowledge regarding red fox spatial ecology has relied on VHF technology, with the choice of home range estimation technique and sampling scheme further influencing reported home range sizes , 36. AdvThe objective of this study was to examine possible causes of variation in home range sizes of red foxes using data from 52 GPS collared foxes from four study areas representing a gradient of environmental productivity and human landscape alteration. Specifically, we examined variation in home range size in relation to extrinsic factors  and intrinsic factors (sex and age) of red foxes.We predicted variation in home range sizes of red foxes along both a landscape (latitude) and localized (elevation) gradient, with home ranges being smallest in the south and increasing in size to the north and towards higher elevations, as increasing latitude and elevation have been shown to constrain environmental productivity and increase seasonality which alters resource availability , 33, 37.Picea abies) and Scots pine (Pinus sylvestris) dominate the forests in all areas, but birch (Betula pubescens and B. verrucosa) and other deciduous tree species are present increasingly to the south.We conducted this study within four different areas in Sweden and Norway representing a gradient of decreasing landscape productivity and human land use from Kolmården, Sweden, in the south to Hedmark County, Norway, in the north  lies at an average altitude of 24 meters above sea level (a.s.l.), and the southern portion of the study area is coastal. This area is a mosaic of productive agriculture areas, boreonemoral forests and human settlements, thus representing a productive and more anthropogenically modified landscape. Daily mean temperatures range from 20°C in summer to –5°C in winter. Snow covers the ground irregularly from December to March.Grimsö (59°40'N-15°25'E) and Hedemora (60°16′N-15°59′E) are both located in south-central Sweden and consist of a transitional border zone between boreonemoral forests in the south and boreal forests in the north. Grimsö is a 140km2 wildlife research area dominated by mixed coniferous forest (74%) and bogs (18%) with farmland comprising approximately 3% [ately 3% , 44. HedHedmark County, Norway (61°53′N 12°2′E) is a transitional border zone between northern boreal forest of low productivity and alpine tundra. The study area lies in the eastern part of Hedmark county, Norway, which extends from the Swedish border in the east to the Glomma River in the west. Most of the area lies 600m-800m a.s.l. Less than 1% of the area is cultivated or residential land, one third is productive forest, and the remainder consists of tundra, mountains, lakes, and rivers. Daily mean temperatures range from 10°C in summer to –25°C in winter, and the ground is generally snow covered from November to May.Between 2012 and 2016, we captured and equipped 80 red foxes with GPS radio collars . Animal capture and handling protocols differed in Norway and Sweden, however all capture and handling procedures were approved by and followed the ethical guidelines required by the Swedish Animal Ethics Committee  and the Norwegian Experimental Animal Ethics Committee . In addition, permits to capture wild animals were provided by the Norwegian Directorate for Nature Management and the Swedish Environmental Protection Board NV-03459-11). All foxes were initially captured using baited wooden box traps. Foxes captured in Sweden were either immobilized using a mixture of 2 mg/kg ketamine and 0.08mg/kg medetomedine, where the medetomedine was later reversed with 0.4mg/kg atipamizole, or with 10 mg/kg tiletamine-zolazepam, for which there is no reversal [9-11. AllWe determined the minimum monitoring duration of red foxes needed for home ranges to reach a stable asymptote based on area-observation curves . This waWe derived home ranges using two different non parametric methods: MCP, for comparison with previous red fox studies, and Local Convex Hull, a nonparametric kernel method using a fixed number of nearest neighboring points (LoCoH-k) , 50. We We obtained land cover and elevation from digitized topographic maps of Sweden  and Norway (Norwegian Mapping Authority). Using ArcGIS 10.1 , we convC) [C value as the best model though we considered models within two AICC units to be of similar quality [We compared differences in mean home range sizes between all reciprocal levels of MCP and LoCoH-k estimates, and tested for statistical significance using a paired (Student’s) one-way t-test. For analysis of variation in home range size, we selected the more conservative 90% LoCoH home ranges estimates,   using thC) . We sele quality . Model at-test, 90% t51 = 3.13, p = 0.003, 95% t51 = 2.96, p = 0.005, 100% t51 = 3.35, p = 0.002). The GPS data emphasized the occurrence of excursions and exploratory movement patterns, which resulted in outlying positions greatly increasing home range sizes, depending on the estimator used. LoCoH-k estimates decrease substantially when outlying fixes were removed, compared to MCP estimates, resulting in overall more conservative home range size estimates. Specifically, using 90% of the core relocations resulted in the average MCP home range size almost triple the size compared to LoCoH-k estimates  and 2.4 km2 to 358 km2 (MCP 100%). Home ranges averaged 7.1 km2 ± 1.3 SE (90% LoCoH-k) or 52 km2 ± 10 SE (100% MCP) which varied depending on estimator and level  while only one home range in the northern study area was smaller than 10 km2 .Red fox home ranges in this study showed considerable individual variation in size as well, ranging between 0.95 kmnd level . In genend level . Only thThe red fox home ranges in this study contained on average 23% agricultural land  and 3% human settlement , which varied along a landscape gradient . Notably2 = 0.50) and 52% of cumulative model weight  was three times larger than estimates from comparable studies of red foxes, in similar boreal and tundra landscapes [The red fox home range sizes in our study were much larger than those previously reported for red foxes , 30. Remndscapes –62. Our Furthermore, the GPS collars used in this study are not limited to our ability to follow and relocate animals. They therefore have the ability to increase our knowledge of movements and behaviors across landscapes and far distances. Our use of GPS technology not only highlights larger sized home ranges than previously known, but also the exploratory movement patterns of red foxes, indicating that excursions may be more common among red foxes than previously thought. Several foxes within our study showed a pattern of utilizing multiple, separate core areas within large home ranges. Further, six foxes, not included in this study, used two distinct home ranges and regularly traveled between them. Meia and Weber  cautioneRed fox home ranges at higher elevations and in the northern boreal vegetation zone were approximately four times larger than those of foxes at lower elevations and in the two southern vegetation zones, indicating that home range size was influenced by a productivity gradient at both the landscape (latitude) and the local (elevation) scales. Larger home ranges at higher latitudes and elevations have also been found for wolves in Scandinavia , and thiElevation showed the strongest effect on home range size. This is possibly because the changes in environmental productivity along a latitude gradient were not as evident as the environmental variation  experienced at a local scale with increasing elevation. Further, increasing seasonality has been found to decrease population density . It may At lower elevations, where the amount of available agricultural land increased, red foxes with a higher proportion of agriculture land maintained smaller home ranges. Fragmented agricultural landscapes often allow for higher prey densities compared to northern areas dominated by boreal forest  and incrRed foxes did not demonstrate clear intersexual differences in home range size. However, the inclusion of sex in the final model indicates intersexual differences within elevation gradients and proportion of agricultural land in home ranges. We do not know breeding status of female foxes, thus it is possible the impact of sex may have been related to seasonal differences related to breeding status  or an arThe overall flexibility of the red fox in its space use, social structure and resource utilization makes disentangling the sources of intraspecific variation in home range size complex. Further, both population density  and terrS1 TableVulpes vulpes) included in this study. All foxes were resident individuals that were monitored ≥ 3 months, with the exception of two females monitored for 84 and 87 days (fox number 16 and 41). The LoCoH-k 90% estimates highlighted in grey represent the home range sizes used in data analysis and modeling.Summary of home range sizes of all red foxes ((DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Introduction: Spinal Epidural abscess (SEA) is an uncommon pathology that needs an urgent intervention to decompress the pressure on the spinal epidural sac, cord, and roots. The authors report a rare case of a young adult with lumbar spinal epidural tuberculous abscess occupying the spinal canal from L2–L5 vertebrae with extesion to the posterior paraspinal muscles and presenting with bilateral progressive lower limb weakness.  Case report: A 42 years old male teacher presented with a 15-day history of progressive difficulty to walking and bilateral lower limb weakness associated with fever, malaise and later on urinary incontinence. A magnetic resonance imaging (MRI) scan revealed a paraspinal intermuscular abscess and an abscess occupying the spinal canal compressing the dural sac from L2–L4/5, without any signs of vertebral involvement. Surgery was done by a posterior midline incision. Pus was evacuated from multiple pockets through the paraspinal muscle layers. Laminectomy for L3/4, and hemilaminectomy for L2/3, and L4/5 were performed. Pus and bone specimens were negative for acid-fast bacilli. However, both histopathological studies and Polymerase Chain Reaction (PCR) testing confirmed the presence of tuberculosis (TB). The patient received TB antibiotics, and a follow-up MRI scan at 2 months showed complete evacuation of the abscess. However, signs of L5 spondylitis were evident. No further surgery was needed as there was no vertebral collapse or neural compression and the patient's clinical condition was improving. He had normal right lower limb power and sensation and grade 4+ motor power of the left lower limb. Bowels and bladder function was normal.Conclusion: Isolated tuberculous spinal epidural abscess is a rare disease and should be treated urgently with evacuation and decompression. Signs of spondylitis or spondylodiscitis may appear later and therefore long follow up is recommended in tuberculous cases presenting with an isolated epidural abscess. A 42 years old male teacher referred to our hospital complaining of the 15-day history of progressive back pain radiating to both thighs. He subsequently developed weakness affecting both lower limbs, but mainly on the left side, with urinary incontinence and constipation. It was associated with low-grade fever, loss of appetite and ended with the inability to walk.On examination, he looked pale and uncomfortable. He had a pulse rate of 95 beats per minute, blood pressure 110/70, temperature 38.3 °C, and a respiratory rate of 19 cycles per minute.Inability to walk, severe tenderness in the back around the midline and paraspinal region, and severe limitation of spinal motion.    Sensation:  paresthesia below the inguinal ligaments in both lower limbs.grade 2 knee extension;grade 1 knee flexion;grade 1 ankle dorsiflexion;grade 1 ankle plantar flexion.Left side:grade 3 knee extension and flexion;grade 2 ankle plantar flexion and dorsiflexion.Right side:diminished knee and ankle jerk;equivocal Babiniski sign.urinary incontinence;constipation.9 with lymphocytosis (60%), essential sedimentation rate 95 mm/hour, C-reactive protein 90, random blood sugar 114 g/dL, blood urea 78 mg/dL, and serum creatinine 1.3.Hemoglobin 11.1 g/dL, white blood cell count 11 × 10Plain X-ray of the lumbosacral spine in both Antero-posterior and lateral view revealed no abnormality. MRI of lumbosacral spine in both T1, T2, and STIR showed a localized collection of pus in the canal compressing the dural sac at L3, L4 and L5 levels with a small collection of pus at L2–L3 level. Also, there were multiple pockets of pus in the paraspinal muscles –3.  After discussing the treatment options with the patients, we decided to proceed with surgery. Using a posterior midline approach, in a prone position and under general anesthesia, the paraspinal muscles were dissected. We noticed multiple pockets of pus imbedded in the paraspinal muscles, mainly on the left side, which were evacuated. Then a laminectomy of L3–L4 was done with hemilaminectomy at L2–L3 and L4–L5. Pus was draining from these levels through the spinal canal. Samples were taken from pus, laminae bone, and soft tissue and sent for histopathological and microbiological studies.Thorough irrigation and debridement were done, a drain was applied, and the wound was closed in layers.The patient was kept on broad-spectrum antibiotics for 14 days pending microbiological laboratory results.Microbiological results showed no growth of bacteria, Acid Fast Bacilli was negative, but Polymerase chain reaction (PCR) was positive.Histopathological study: sections showing calcified mature bony trabeculae and degenerative cartilaginous tissues component in between marked hematopoietic depletion, dense chronically inflamed fibrous replacement including heavy mixed chronic inflammatory cells which include foamy histiocytic and mature lymphocytic cells infiltrate. Multiple ill-defined granulomas were seen with obvious extra bony inflammatory extension to the surrounding soft tissue and skeletal, muscular tissues. The final impression was a chronic specific tuberculous granulomatous spinal cold abscess.Anti TB antibiotics were started after the above results became available.Postoperatively, gradual neurological recovery occurred in both lower limbs. At one month follow up, the right lower limb had full motor recovery (grade 5), while the left lower limb was weaker on knee flexion and ankle dorsiflexion (grade 4). The patient was still incontinent to urine and constipated.At two months after surgery, the left lower limb showed further neurological improvement (grade 4+), and though the patient reported some improvement of his sphincter control, he was still complaining of nocturnal incontinence and constipation. A follow-up MRI at two months of index surgery showed complete abscess resolution with good decompression of the cauda equina region . SurprisFive months later, he recovered both lower limbs motor strength and full control of his sphincters.Vertebral TB accounts for less than 1% of all TB infections in the body and more than 50% of musculoskeletal infections. It is considered the most serious type of skeletal TB, with possible neurological symptoms due to compression of the neural structures. It may also lead to deformity and significant vertebral structure destruction and instability .Cases of the SEA due to TB infection have been reported in developing countries. The clinical presentation of the tubercular SEA may be more insidious, leading to failure of early diagnosis treatment .Initial presentation of the tubercular SEA may include back pain, low-grade fever, and severe neurological deficit. The classical triad of fever, back pain and neurological deficits present in only 2% of the tubercular SEA . MRI is There are only a few reported cases of the tubercular SEA. Most of these infections are secondary to pyogenic infection –8.Treatment options depend on many factors like the site and severity of compression, and clinical presentation. Medical treatment can be offered in certain cases of the tubercular SEA. Surgery is indicated when there is compression of neurological structures, failure of resolution, doubtful diagnosis, and other reasons like mechanical instability. The options for surgical treatment may vary from decompression laminectomy, multilevel laminotomy or laminectomy with or without fixation; all this can be determined by the adequacy of decompression intraoperatively .Infection of the spine may lead to wide range of problems including the neurological deficit, mechanical instability, and deformity . Very feInvolvement of vertebral body of the lumbar spine in T1W follow up MRI with low signal intensity could be due to direct spread from the abscess or a reactivation of a dormant focus. The clinical presentation in our patient suggested compression in the cauda equine region with the highest level of involvement at L2.Tubercular SEA is amenable to both non-surgical and surgical management . In our This case report highlights the importance of early diagnosis and early treatment of tuberculous SEA. Isolated tuberculous epidural abscess is rare and can precede the development of tuberculous spondylitis, which mandates long follow up.The authors declare that they have no conflicts of interest in relation to this article.


Despite long hours of sunlight in Qatar and other regions of the Middle East, vitamin D deficiency has been rising. In parallel, the prevalence of metabolic syndrome has also been increasing in Qatar. Vitamin D levels have been associated with metabolic syndrome but the data are inconsistent and no studies have addressed these inter-relationships in a Middle Eastern population where the prevalence of these conditions is high. The objective is to investigate the prevalence of vitamin D deficiency and its association with metabolic syndrome and its components in the Qatar Biobank population.−1 serum vitamin D levels) adjusting for age, sex, ethnicity, season of blood collection, physical activity and education. Odds ratios and 95% confidence intervals were calculated for all analyses.A cross-sectional study of 1205 participants (702 women and 503 men) from the Qatar Biobank, comprising Qataris and non-Qataris between the ages of 18 and 80 years, was used to perform multivariate linear regression analyses to examine the association between metabolic syndrome and prevalence of vitamin D deficiency (defined as <20 ng ml−1) with more men being deficient (68.6%) than women (61.3%). Serum vitamin D was 8% lower in individuals with metabolic syndrome  compared to individuals without metabolic syndrome. Waist circumference and HDL as well as high triglyceride levels were also significantly positively associated with vitamin D deficiency. No association was found between the other components of metabolic syndrome or diabetes and the presence of vitamin D deficiency.Approximately 64% of participants were vitamin D deficient (<20 ng mlVitamin D deficiency is prevalent in this Qatari population. Presence of metabolic syndrome was associated with presence of vitamin D deficiency. Future prospective studies need to be conducted to investigate the potential for causality. For example, exposure to 0.5 minimal erythemal dose is equivalent to supplementing ~3000 IU of vitamin D3.1 One of the main physiological functions of vitamin D is to maintain calcium and phosphorous levels in the body to sustain various metabolic functions, including bone metabolism.3 The most abundant circulating biomarker of vitamin D status is 25-hydroxyvitamin D (25(OH)D), which also has a longer half-life (25 days) compared to the active metabolite; 1,25-dihydroxyvitamin D (7 h).4 However, the threshold used to define vitamin D deficiency often varies according to the population and outcome of interest.7 The most common definition for optimal 25(OH)D levels is the concentration at which it supresses the parathyroid hormone to its minimum, however, using this definition results in a wide range of minimal optimal values from 12 ng ml−1 to 40 ng ml−1.7Vitamin D deficiency is highly prevalent worldwide and is associated with many adverse health outcomes.12 The association between vitamin D levels and a range of disease outcomes could be explained by intermediate disease risk factors such as the metabolic syndrome (MetS); a constellation of risk factors, including increased obesity, hypertriglyceridemia, hypertension and diabetes. Some studies have suggested an inverse associations between serum vitamin D and MetS,16 while others have not confirmed this observation.18 It is also unclear which components of the MetS might drive this association with vitamin D with some studies, suggesting obesity and others glucose haemostasis.20A number of studies have shown that vitamin D levels are inversely associated with the risk of a diverse set of diseases, including several cancers, diabetes and cardiovascular diseases.26 For example, a Kuwaiti and an Emirati study reported that ~98 and 83% (respectively) of the participants had serum 25(OH)D less than 20 ng ml−1.25 Studies thus far conducted in Qatar on vitamin D were specific to a certain population . At the same time, the prevalence of MetS was measured to be ~26.5%, according to the International Diabetes Federation (IDF) criteria, amongst Qataris aged 20 and over.27 Similarly, there was a high prevalence of MetS, according to both the IDF and Adult Treatment Panel (ATPIII) criteria, and its components in neighbouring regions.31 Given the high prevalence of these two conditions in the region and the limited epidemiological evidence in this population so far, we aimed to investigate the prevalence of vitamin D deficiency, as well as the association between MetS and its components with vitamin D, in the well-characterised Qatar Biobank.Despite the long hours of sunlight in Qatar and surrounding regions, vitamin D deficiency has been shown to be highly prevalent in this region.32 Questionnaires on health, lifestyle, and diet were completed. Anthropometric measurements and body composition were also obtained. Biological samples, including blood, a urine sample and a saliva sample, were collected from each participant after the questionnaires were filled out.The data for this study was obtained from the Qatar Biobank (QBB), which consisted of 1205 participants; with 702 females and 503 males, at the time of investigation. Participants who were either Qatari or non-Qataris but who were long-term residents of Qatar (that have lived there for over 15 years) were included in the cohort. Recruitment for these participants occurred mostly by personal recommendations of friends and family between December 2012 and February 2014. Participation was voluntary and registration was conducted via online and telephone bookings. A maximum of 10 participants were seen per day. All participants gave informed consent. Detailed methods for recruitment and study design have been reported elsewhere.A total of 66 clinical biomarkers  were routinely measured for all participants. Haematology and blood biochemistry were analysed by the laboratories at Hamad Medical Corporation (HMC).2 and 25(OH)D3 were measured. In this study, vitamin D deficiency was defined by the US Endocrine Society (USES) guidelines as <20 ng ml−1 (50 nmol l−1) in circulation, while individuals with 21–29 ng ml−1 were considered insufficient, and individuals with >30 ng ml−1 were considered sufficient.33 To convert between ng ml−1 and nmol l-1 of 25(OH)D, multiply the concentration of 25(OH)D in ng ml−1 by 2.5.25(OH)D was analysed in one lab at HMC, which is College of American Pathologist (CAP) accredited, using a LIAISON 25 OH Vitamin D TOTAL Assay, where serum 25(OH)D−2). Waist and hip measurements were measured using a non-stretchable sprung measuring tape by Seca. The waist was identified as the smallest part of the trunk after folding their arms across their chest. If it was not possible to find a natural indent of the trunk, the circumference around the umbilicus was measured in centimetres. For the hip measurement, the Seca measuring tape was placed at the widest part of the hips and measured in centimetres. Waist-to-hip ratio was calculated as waist in centimetres divided by hip circumference also in centimetres.Weight, height, body mass index (BMI), and waist and hip circumference were all measured by a trained nurse. Participants wore gowns or light weight clothing, and shoes and socks were removed. Weight was measured using the TANITA BC-418 MA instrument; a multi-frequency segmental body composition analyser, or a digital floor scale Seca-876 for manual weight measurement when TANITA was contraindicated. BMI was calculated as weight in kilogram divided by height in metres squared (kg m−1),2 (2) reduced HDL cholesterol , (3) raised blood pressure (BP) (systolic BP ⩾130 mm Hg or diastolic BP ⩾85 mm Hg), (4) raised fasting plasma glucose (defined as HbA1c levels⩾5.7 mmol l−1).34 HbA1c was measured with turbidimetric inhibition immunoassay (TINIA). Cholesterol was measured using CHOD-PAP GEN2 STAND ID/MS, HDL was measured using the HDL–C plus third generation, LDL was measured using the LDL-C plus second generation and triglyceride was measured using GPO-PAP.Metabolic syndrome was defined according to the new IDF definition as being centrally obese  as well as at least two of the following four factors: (1) raised triglycerides  a third measurement was performed. The participant rested for 30 s between the measurements. If the measurements did not differ, the average of the first two measurements was taken. However, if the measurements did differ, the average of the second and third measurement was used. Diabetes was reported in two ways: (1) self-reported; where 15.4% reported having diabetes and (2) laboratory measurements of HbA1c levels (⩾6.5% was considered evidence for diabetes),χ2 test was performed, while a t-test was performed to test the differences between a categorical and a continuous variable after stratifying by sex. A geometric mean for serum 25(OH)D was calculated by performing a one-way Analysis of Covariance (ANCOVA) adjusting for sex, age and season of blood collection. Partial Spearman's correlation coefficient was used to measure the cross-sectional association between anthropometric measurements, circulating lipids, haemoglobin A1c (HbA1c), glucose, insulin, C-peptide, folate, vitamin D, calcium and SHBG.To test the difference in the distributions of categorical variables, a 34 Univariate (crude) and multivariable linear regression models were used to calculate the regression coefficients and associated 95% confidence intervals (CI). Multivariable model (Model 2) was adjusted for age (continuous), sex, ethnicity, education, physical activity and season. Participants with missing ethnicity values (n=242) were excluded from all analyses.Vitamin D was modelled as a continuous measurements. Metabolic syndrome and its components were categorised into having abnormal levels or normal levels based on the IDF definition.Approximately 58% of the study population were females; mean age was similar between males and females . There w−1) with slightly more men (69%) being vitamin D deficient compared to women (61%). Another 25% of the population had insufficient vitamin D levels. 33 The percentage of people reported taking vitamin D supplements was high with 49% of females and 25% of males  and serum 25(OH)D levels. There was a significant positive association between the presence of MetS and vitamin D deficiency after adjusting for Model 2  . Components of the MetS that were inversely associated with serum 25(OH)D levels, including high waist circumference  and elevated triglycerides . While increasing levels of HDL was positively associated with 25(OH)D levels . Elevated blood pressure showed weak inverse association with vitamin D; the association was stronger with the presence of vitamin D deficiency  . The association between vitamin D deficiency or 25(OH)D levels with presence of diabetes was not statistically significant.09–2.18) . Howevere: 0.03)  compared26 At the same time, the percentage of participants reporting supplementation for vitamin D, especially in women was very high (49%), higher than that observed in white populations,38 potentially showing a greater awareness of vitamin D deficiency in this population.In this unique study in Qatar, we showed that despite being a country with high levels of sun exposure, the prevalence of vitamin D deficiency is very high (64%),with the majority of the examined population showing signs of deficiency. This is consistent with previous observations which estimated the prevalence of vitamin deficiency (<20 ng/ml) to range between 72–87%.−1), with slightly more men being deficient compared to women. Previous evidence from studies in the Middle East region supports higher level of vitamin D in males compared to females. A Saudi Arabian study performed on 10 709 participants reported more females being severely vitamin D deficient (<10 ng ml−1) compared to males.23 A Bahraini and Lebanese study also reported lower mean serum vitamin D in females compared to males.39 One hypothesis for this difference could be explained by vitamin D supplementation, as women in the Qatar Biobank were more likely to take vitamin D supplements compared to men. This was not reported in both the Saudi and Bahraini studies due to exclusion of participants that consumed vitamin D supplements.23Approximately 64% of the participants in the Qatar Biobank were vitamin D deficient  study, reported significant inverse associations between continuous serum vitamin D and overall MetS, based on the IDF criteria.40 Studies examining the direction of the effect between vitamin D, cholesterol and lipids suggest that lipids levels and BMI may be a cause for decreased vitamin D and not vice versa.44 Other cross-sectional studies did not support these associations.46 However, this might have been due to the small sample sizes47 and small variation in the exposure .46 Studies on Middle Eastern population where the metabolism of vitamin D could be different due to different lifestyles and darker skin pigmentation, are very limited and our results support an inverse association between the two examined phenotypes.MetS was significantly associated with the risk of vitamin D deficiency in the Qatar BioBank. Several cross-sectional and few prospective studies have reported similar positive associations between vitamin D deficiency and the risk of metabolic syndrome.49 Vitamin D has also been shown to inhibit the release of cytokines from the immune cell, which is harmful to β cells.50Many mechanisms have been proposed to explain the association between vitamin D and future risk of MetS component. Since vitamin D is fat soluble and could be stored in adipose tissue, it can be sequestered in the subcutaneous fat in obese individuals, reducing the levels of circulating vitamin D in the blood leading to less release of vitamin D into the blood.40 the British Birth Cohort,47 Middle Eastern populations,51 and in other populations around the world.53 Conversely, several cross-sectional studies did not report a correlation between obesity and vitamin D deficiency.54 However, these are mainly small studies with limited power to observe associations.46 We also showed that high triglyceride levels were associated with lower levels of vitamin D. Studies also report significant inverse associations between vitamin D with high triglyceride levels.55 However, when stratified by sex, only the association in men remained significant in the Ling et al. study.52 On the contrary, Reis et al. reported a positive trend between vitamin D deficiency and high triglyceride levels only in women.46Obesity, measured as BMI, waist circumference, and waist-to-hip ratio were positively associated with vitamin D deficiency with stronger associations observed with waist circumference. Similar results were found in both prospective and cross-sectional studies, including the Aus-Diab study,32 Qatar Biobank, as of yet is not considered to have a fully representative sample, since the most common source of participant recruitment is through word of mouth to friends and families.32 However, the population of our study shows small differences with the Qatar Stepwise Survey (QSS), which is based on the WHO STEPS survey and consists of 2 496 participants. In the QSS, measurements of height, weight, and BMI were similar to this study. However, in Qatar Biobank, over 60% of the participants had a university degree compared to 35% of the QSS,56 suggesting a higher representation of more educated and higher socioeconomic status individuals in Qatar Biobank.This is the first study of vitamin D and metabolic syndrome performed in the Qatari population and long-term residents (⩾15 years living in Qatar) with high-quality data, which involves collection of extensive questionnaire information, clinical phenotyping and biological samples.Since this study is cross-sectional, the directionality of the relationship between adiposity, vitamin D, metabolic syndrome, and diabetes could not be elucidated. The non-significant results for the association between diabetes and vitamin D deficiency, may have been due to the relatively small sample size and low response rate for some questions regarding physical activity, smoking, supplements and so on. It could also have been due to other confounding factors such as specific medication use and dosage of supplements, which were not captured. Approximately 84% of participants were missing data on medication use for hypertension and 68% of participants were missing for medication on cholesterol usage, which may have caused some misclassification. Additionally, the low levels of circulating vitamin D found in this population may have been insufficient to observe any inverse relationships with metabolic syndrome components and diabetes.To conclude, the findings from this study support a positive association between the presence of metabolic syndrome and vitamin D deficiency. We also observed that MetS components, such as obesity and high triglyceride, were inversely associated with circulating serum vitamin D levels. Future prospective studies should elucidate the potential causal association between vitamin D and MetS using Mendelian randomisation approaches or through supplementation with Vitamin D. Moreover, mechanistic studies should concentrate on identifying pathophysiological pathways and molecular mechanisms linking vitamin D deficiency and metabolic syndrome.