Droplets generated from toilets during urination as a possible vehicle of carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: In the health care setting, infection control actions are fundamental for containing the dissemination of multidrug-resistant bacteria (MDR). Carbapenemase-producing Enterobacterales (CPE), especially Klebsiella pneumoniae (CR-KP), can spread among patients, although the dynamics of transmission are not fully known. Since CR-KP is present in wastewater and microorganisms are not completely removed from the toilet bowl by flushing, the risk of transmission in settings where toilets are shared should be addressed. We investigated whether urinating generates droplets that can be a vehicle for bacteria and explored the use of an innovative foam to control and eliminate this phenomenon. METHODS: To study droplet formation during urination, we set up an experiment in which different geometrical configurations of toilets could be reproduced and customized. To demonstrate that droplets can mobilize bacteria from the toilet bowl, a standard ceramic toilet was contaminated with a KPC-producing Klebsiella pneumoniae ST101 isolate. Then, we reproduced urination and attached culture dishes to the bottom of the toilet lid for bacterial colony recovery with and without foam. RESULTS: Rebound droplets invariably formed, irrespective of the geometrical configuration of the toilet. In microbiological experiments, we demonstrated that bacteria are always mobilized from the toilet bowl (mean value: 0.11 ± 0.05 CFU/cm2) and showed that a specific foam layer can completely suppress mobilization. CONCLUSIONS: Our study demonstrated that droplets generated from toilets during urination can be a hidden source of CR-KP transmission in settings where toilets are shared among colonized and noncolonized patients.

Effect of duration of exposure to diets differing in dietary cation-anion difference on Ca metabolism after a parathyroid hormone challenge in dairy cows.

Objectives of the experiment were to determine the length of exposure to an acidogenic diet that would elicit changes in acid-base balance, mineral digestion, and response to parathyroid hormone (PTH)-induced changes in blood Ca and vitamin D3 in prepartum dairy cows. Nonlactating parous Holstein cows (n = 20) at 242 d of gestation were blocked by lactation (1 or >1) and pretreatment dry matter (DM) intake and, within block, they were randomly assigned to a diet with a dietary cation-anion difference (DCAD) of +200 mEq/kg of DM (DCAD +200) or an acidogenic diet with -150 mEq/kg of DM (DCAD -150). Water and DM intake were measured and blood was sampled daily. Urine was sampled every 3 h for 36 h, and then daily. During PTH challenges on d 3, 8, and 13, cows received i.v. PTH 1-34 fragment at 0.05 µg/kg of body weight every 20 min for 9 h to mimic the pulsatile release of endogenous PTH. Blood was sampled at 0 h, and hourly thereafter until 10 h, and at 12, 18, 24, 36, and 48 h relative to each challenge. Acid-base measures and concentrations of ionized Ca (iCa) in whole blood, and total Ca, Mg, P, and vitamin D metabolites in plasma were evaluated. On d 2 and 7, Ca, Mg, and P balances were evaluated. Cows fed DCAD -150 had smaller blood pH (7.431 vs. 7.389) and HCO3- (27.4 vs. 22.8 mM) compared with DCAD +200, and metabolic acidosis in DCAD -150 was observed 24 h after dietary treatments started. Concentrations of iCa begin to increase 24 h after feeding the acidogenic diet, and it was greater in DCAD -150 compared with DCAD +200 by 3 d in the experiment (1.23 vs. 1.26 mM). During the PTH challenges, cows fed DCAD -150 had greater concentration of iCa and area under the curve for iCa than those fed DCAD +200 (48.2 vs. 50.7 mmol/L × hour), and there was no interaction between treatment and challenge day. Concentration of 1,25-dihydroxyvitamin D3 in plasma did not differ during the PTH challenge, but change in 1,25-dihydroxyvitamin D3 relative to h 0 of the challenge was smaller in cows fed DCAD -150 than cows fed DCAD +200 (44.1 vs. 32.9 pg/mL). Urinary loss of Ca was greater in cows fed DCAD -150 compared with DCAD +200 (1.8 vs. 10.8 g/d); however, because digestibility of Ca increased in cows fed DCAD -150 (19.7 vs. 36.6%), the amount of Ca retained did not differ between treatments. Diet-induced metabolic acidosis was observed by 24 h after dietary treatment started, resulting in increases in concentration of iCa in blood observed between 1 and 3 d. Collectively, present results indicate that tissue responsiveness to PTH and changes in blood concentrations of iCa and digestibility of Ca are elicited within 3 d of exposure to an acidogenic diet. The increased apparent digestibility of Ca compensated for the increased urinary loss of Ca resulting in similar Ca retention.

Effect of continued metabolic acidification into the first 3 days of lactation on blood calcium status in postpartum dairy cattle: A randomized controlled trial.

Although incidence of clinical hypocalcemia in postpartum dairy cows is low in US dairies, subclinical hypocalcemia after calving is common and has been associated with metabolic and infectious disease. It is widespread farm practice to feed a diet rich in anions to prepartum dairy cattle to support calcium homeostasis. However, this diet is typically discontinued at parturition, when calcium needs are still high. The objective of this trial was to determine the effects of extending metabolic acidification into the first 3 d of lactation in multiparous Holstein cows with the use of magnesium chloride (MgCl2) hexahydrate drenches on blood ionized calcium concentrations. Adult Holstein cows at a commercial dairy in their second or higher lactation, with a urine pH of 6.8 or less on the day of calving, were randomly assigned to either treatment or control groups, resulting in 13 cows in the treatment group and 14 cows in the control group. Treatment cows received 480 g of oral MgCl2 hexahydrate once daily for 3 d for continued acidification starting on the day of calving, whereas cows in the control group received no treatment. Urine pH was measured daily for 5 d, starting on the day of calving (0 DIM), to assess acidification status; blood was collected on day of calving (0 DIM), 2 DIM, and 4 DIM and analyzed for ionized calcium concentrations. Differences in blood ionized calcium and urine pH over time were compared using longitudinal data analysis. Urine pH was lower in treatment cows compared with control cows at 1, 2, and 3 DIM. Blood ionized calcium concentrations were different from baseline, taken at enrollment (0 DIM) and at 2 and 4 DIM in both treatment and control cows. However, no difference was detectable between treatment and control cows at 2 or 4 DIM with respect to blood ionized calcium concentrations. Oral supplementation with MgCl2 hexahydrate resulted in the desired acidification of urine pH in the treatment group, similar to feeding of an anionic close-up diet. Continued acidification of dairy cows until 2 DIM did not result in clinically meaningful higher blood calcium concentrations compared with controls, and further research is needed, to identify physiological reasons for this finding.

A simple POM clusters for in vivo NIR-II photoacoustic imaging-guided NIR-II photothermal therapy.

Photoacoustic (PA) imaging in the second near-infrared (NIR-II) window exhibits enhanced deep-tissue imaging capability. Likely, cancer therapy in the NIR-II window could provide deeper penetration depth and higher exposure to laser over NIR-I. However, the traditional application of excitation light is still in the NIR-I window. In view of the excellent imaging and therapeutic capabilities of NIR-II window, we have demonstrated a simple polyoxometalate (POM) clusters (molecular formula: (Na)n(PMo12O40) or (NH4+)n(PMo12O40)), which integrates NIR-II photoacoustic imaging and NIR-II photothermal therapy into an "all-in-one" theranostic nanoplatform, and could be used for PA imaging-guided photothermal therapy in the NIR-II window. In vivo experiments demonstrate that the POM clusters with good water solubility and biocompatibility were effective to kill tumor without recurrence and metastasis under 1064 nm laser illumination.

Effects of anionic supplement source in prepartum negative dietary cation-anion difference diets on serum calcium, feed intake, and lactational performance of multiparous dairy cows.

Incidence of subclinical hypocalcemia in early postpartum dairy cows continues to be an animal welfare concern and an economic burden for producers. Feeding prepartum negative dietary cation-anion difference (DCAD) diets produces metabolic acidosis, which supports mobilization of bone calcium and reduces the incidence of hypocalcemia. Achieving a sufficient degree of metabolic acidosis without reducing dry matter intake (DMI) can be difficult. This study compared the ability of MegAnion (MA; Origination O2D Inc., Maplewood, MN), a new DCAD supplement designed to be more palatable than typical anionic salt sources, and another palatable commercial DCAD product, SoyChlor (SC; Landus Cooperative, Ralston, IA), to reduce urine pH (a surrogate for metabolic acidosis) without reducing prepartum DMI. A secondary objective was to assess the effect of these anionic supplements on postpartum serum calcium concentrations and DMI. Prepartum multiparous Holstein (HO) and crossbred (XX) cows were blocked by breed and expected calving date and randomly assigned within breed to total mixed rations (TMR) with MA or SC and DCAD values of -215 mEq/kg of DM. Cows (n = 56; 15 MA-HO, 12 SC-HO, 15 MA-XX, 14 SC-XX) consumed the treatment TMR for at least 19 d and completed the 28 d in milk (DIM) phase of the study. Urine and blood samples were collected weekly and at 1, 2, and 3 DIM. Data were analyzed as a randomized block design by repeated measures with week or DIM as the repeated effect. Prepartum urine pH decreased from 8.15 ± 0.27 before treatment to 6.12 ± 0.14 during treatment, was not affected by anionic supplement, and increased immediately after calving when all cows consumed the same early-lactation TMR. Prepartum serum calcium concentrations were not affected (2.34 vs. 2.33 ± 0.02 mmol/L) by treatment, whereas nonesterified fatty acids were lower (86 vs. 120 ± 10 mmol/L) and insulin was greater (215 vs. 174 ± 10 pmol/L) in cows fed MA than in cows fed SC. These differences are supported by the numerically greater prepartum DMI (1.2 kg/d) and energy balance (1.8 Mcal/d) of cows fed MA. However, pre- and postpartum DMI and other production variables, including body weight, body condition score, milk yield, and energy balance, were not affected by treatment. This lack of difference indicates that MA provides another effective source of anionic salts for diets designed to reduce urine pH and induce metabolic acidosis in prepartum dairy cows.

Effect of subclinical and clinical hypocalcemia and dietary cation-anion difference on rumination activity in periparturient dairy cows.

Rumination involves a complex series of muscle contractions that bring a bolus of ingesta to the mouth for further mastication before it is swallowed again. Healthy cows ruminate 8 to 9 h/d. Hypocalcemia is known to disrupt nerve and muscle function. Our hypothesis was that hypocalcemia in periparturient cows would reduce rumination activity. Twenty-six Holstein cows entering their third lactation or greater were fed a control diet [dietary cation-anion difference (DCAD) = +196 mEq/kg of dry matter (DM)] or a low DCAD diet supplemented with anions (DCAD = -9 mEq/kg of DM) prepartum. Periparturient plasma Ca concentration and rumination rate were determined. Four of 12 control cows developed clinical milk fever, necessitating intravenous Ca therapy. Rumination rate decreased in all cows around the time of calving. Rumination rate on the first and second day of lactation was highly correlated with the cow's plasma Ca concentration on the first day of lactation. In one of our statistical models, a normocalcemic cow was defined as a cow whose plasma Ca concentration remained above 2.00 mM. Cows were retrospectively classified as normocalcemic, subclinically hypocalcemic, or clinically hypocalcemic (milk fever). Only 4 cows were considered normocalcemic, and all had been fed the low DCAD diet. Normocalcemic cows spent more time ruminating on the first day after calving than subclinically hypocalcemic cows or cows with milk fever. Cows with milk fever had a lower rumination rate than normocalcemic cows through d 3 of lactation. Rumination activity in cows with milk fever was almost nondetectable in the hours before and after intravenous Ca treatment for an extended period, despite the return of muscle function that allowed the cows to stand and eructate following treatment. Other statistical models using different definitions of normocalcemia gave qualitatively similar results. Diet had a great effect on plasma Ca concentration and rumination rate. Even when cows with clinical milk fever were removed from the control cow data set, cows on the low DCAD diet had significantly greater plasma Ca concentrations in the first 36 h after calving and a higher rumination rate on d 1 of lactation (248 ± 26 min) than control cows (158 ± 32 min).

Effect of a commercial anionic dietary supplement on urinary pH and concentrations of electrolytes and pH in blood of horses.

Aims: To compare urine urinary pH, blood pH and concentration of electrolytes in blood of healthy horses fed an anionic salt supplement to achieve diets with a dietary cation-anion difference (DCAD) of -40 or 0 mEq/kg DM, with horses a fed a diet with a DCAD of 85 mEq/kg DM.Methods: Eight healthy horses received each of three diets in a randomised crossover design. Diets consisted of grass hay and concentrate feed, with a varying amount of an anionic supplement to achieve a DCAD of 85 (control), 0 or -40 mEq/kg DM. They were fed for 14 days each with a washout period of 7 days between. Urine pH was measured daily and blood samples were collected on Days 0, 7 and 14 of each study period for the measurement of pH and concentration of electrolytes.Results: Four horses voluntarily consumed the anionic supplement with their feed, but four horses required oral supplement administration via dose syringe. During the study period mean urine pH was lower in horses fed diets with a DCAD of 0 (6.91; SD 0.04) and -40 (6.83; SD 0.04) mEq/kg DM compared to the control diet (7.30; SD 0.04). Compared with horses fed the control diet, mean urine pH was lower in horses fed the 0 and -40 mEq/kg DM diets on Days 1-12 and 14 (p < 0.05) of the study period. On Day 13 it was only lower in horses fed the -40 mEq/kg DM diet (p < 0.01). Urine pH was similar for horses fed the 0 and -40 mEq/kg DM diets (p = 0.151). The DCAD of the diet had no effect on blood pH, ionised Ca or anion gap. Mean concentrations of bicarbonate in blood were affected by diet (p = 0.049); they were lower when horses were fed the 0 mEq/kg diet relative to the control diet on Day 14.Conclusions and clinical relevance: The anionic supplement reduced urine pH in horses fed diets with a DCAD of 0 or -40 mEq/kg DM compared with 85 mEq/kg DM. However as urinary pH did not fall below pH 6.5, the pH below which calcium carbonate uroliths do not form, this reduction in urine pH is unlikely to be clinically significant. The supplement was variably palatable and showed minimal promise as an effective urinary acidifier at the doses administered in this study.

Meta-analysis of the effects of prepartum dietary cation-anion difference on performance and health of dairy cows.

The objectives were to use meta-analytic methods to determine the effects of changes in dietary cation-anion difference (DCAD) prepartum on productive performance and health of dairy cows. The literature was systematically reviewed, searching randomized experiments with transition cows that manipulated the prepartum DCAD or experiments with acidogenic diets in which dietary Ca, P, or Mg was manipulated. Forty-two experiments, including 134 treatment means and 1,803 cows, were included in the meta-analysis. Of those, 5 experiments with 15 treatment means reported responses for 151 nulliparous cows. Data collected included the mineral composition of prepartum diets, parity group prepartum, breed, days on treatment, and means and respective measure of variance for urine pH, dry matter intake (DMI), body weight, body condition, productive performance, concentrations of minerals and metabolites in blood, and incidence of diseases. Mixed effects meta-analyses were conducted weighting by the inverse of standard error of the means squared to account for the precision of each experiment. Models include the effects of DCAD, parity group prepartum, interaction between DCAD and parity group, and other covariates that showed significance in univariable analysis. Final models were selected based on parsimony and model fit. Reducing the prepartum DCAD reduced intake prepartum but improved intake postpartum in both parity groups. Interactions between DCAD and parity group occurred for yields of milk, fat-corrected milk (FCM), fat, and protein because reducing the DCAD improved those responses in parous cows; however, reducing the DCAD either had no effect on yields of milk and protein or reduced the yield of FCM and fat in nulliparous cows. The resulting equations from the statistical models predicted that reducing the DCAD from +200 to -100 mEq/kg would increase blood total Ca on the day of calving from 1.86 to 2.04 ± 0.05 mM, DMI postpartum 1.0 kg/d, and milk yield 1.7 kg/d in parous cows. The increased concentrations of blood total Ca at calving and postpartum explained the marked reduction in risk of milk fever in parous cows with a reduction in DCAD. As the DCAD decreased, the risk of retained placenta and metritis also decreased, resulting in fewer disease events per cow in both nulliparous and parous cows. Dietary concentrations of Ca, P, or Mg prepartum had no effect on DMI or yields of milk and FCM; however, increasing dietary Ca within the study range of 0.16 to 1.98% of dry matter tended to increase the risk of milk fever in parous cows regardless of DCAD fed. Collectively, results support the recommendation of prepartum acidogenic diets to result in a negative DCAD to parous cows with improvements in lactation performance and reduced risk of diseases; however, the range of DCAD fed did not allow for detection of an optimum value for postpartum performance. On the other hand, despite improvements in blood concentrations of Ca and reduction in uterine diseases with a reduction in DCAD fed to nulliparous cows, productive performance was either depressed or unaffected and the limited number of experiments did not provide sufficient evidence for a recommended DCAD for this group of cows.

Effects of prepartum dietary cation-anion difference intake on production and health of dairy cows: A meta-analysis.

Prepartum diets influence cow performance for weeks to months postpartum. This observation leads to questions about milk yield and physiological and health responses to diets with negative dietary cation-anion difference (DCAD). Further, responses to increased intake of a diet with lower DCAD (Eq/d) have not been explored using meta-analysis. Our objectives were to explore the effects of prepartum DCAD intake on metabolism and production and health as well as the potential for differences in intake of other macrominerals to influence responses to differences in DCAD intake using classical meta-analytical methods. Not all treated groups were fed a diet with negative DCAD, and the effect studied is that of reducing the DCAD. We hypothesized that reducing DCAD intake would improve Ca metabolism and postpartum performance. We used a maximum of 58 comparisons from 31 experiments and a total of 1,571 cows. Intakes of DCAD were 2.28 Eq/d and -0.64 Eq/d for the control, higher DCAD and treated, lower DCAD groups, respectively. Diets with lower DCAD reduced urine pH [standardized mean difference (SMD) = 1.90 and weighted mean difference (WMD) -1.23 pH]. Intake of lower DCAD decreased prepartum DMI (SMD = 0.23; WMD = 0.29 kg/d), increased postpartum DMI (SMD = 0.40; WMD = 0.63 kg/d), and increased milk yield (SMD = 0.172). However, we found an interaction with parity; diets with lower DCAD increased milk yield in parous cows (SMD = 0.29; WMD = 1.1 kg/d) but resulted in numerically lower milk yield in nulliparous cows (SMD = -0.20; WMD = 1.28 kg/d) compared with controls. The FCM yield increased with treatment (SMD = 0.12; WMD = 0.56 kg/d); however, yield of treated cows tended to be greater in parous cows but smaller for nulliparous cows compared with controls. Milk fat percentage, milk fat yield, and milk protein percentages were not affected by treatment, although milk protein yield tended to increase in cows fed the lower DCAD diet (SMD = 0.21; WMD = 0.02 kg/d). Treatment increased blood Ca (SMD = 0.53; WMD = 0.13 mM) and P (SMD = 0.40; WMD = 0.13 mM) on the day of calving and Ca postpartum (SMD = 0.36; WMD = 0.06 mM). Treated cows had smaller concentration of blood BHB before calving than controls (SMD = -0.39; WMD = -0.04 mM). Reducing DCAD in cows resulted in decreased risks of clinical hypocalcemia (risk ratio = 0.60) and retained placenta (risk ratio = 0.59), and reduced the odds of metritis (odds ratio = 0.46) and overall disease (OR = 0.61). We observed no effect on risk of abomasal displacement or mastitis and no effect of differences between treated and control cows in Ca intake (g/d) on the outcomes evaluated. A positive role for increased Mg intake between groups for increased milk fat yield and in reducing the risk of retained placenta was identified. Diets with lower DCAD improved performance of parous dairy cows, and our findings suggest a need for more studies on the effects of a lower DCAD on nulliparous transition cows.

Comparison of 0.46% calcium diets with and without added anions with a 0.7% calcium anionic diet as a means to reduce periparturient hypocalcemia.

Most studies demonstrating that diets with low dietary cation-anion difference (DCAD) reduce hypocalcemia in cows add enough anions to the diet to reduce urine pH below 7.0. One objective of these experiments was to determine whether there is any benefit to periparturient plasma Ca concentration if diet anion addition results in a lesser degree of acidification of the cow and urine pH does not go below 7.0. Another method for reducing hypocalcemia involves feeding a prepartal diet that is Ca deficient. This places the cow in negative Ca balance before calving, stimulating parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D secretion before calving and thus promoting Ca homeostasis at calving. As practiced in the field, low-Ca diets are often about 0.5% Ca. Our second objective was to determine whether a 0.46% Ca diet would be sufficiently low in Ca to stimulate PTH secretion before calving. A meta-analysis of the literature suggests that a 0.5% Ca, low-DCAD diet will reduce hypocalcemia better than a 0.7% Ca diet. A third objective was to compare periparturient plasma Ca in cows fed 0.46 or 0.72% Ca diets with similar DCAD. In experiment 1, anions (primarily chloride) or anions plus Ca were added to a 1.4% K basal diet to create the following diets: 0.46% Ca and +167 mEq/kg of DCAD, 0.46% Ca and -13 mEq/kg of DCAD, and 0.72% Ca and -17 mEq/kg of DCAD. In experiment 2, the same amounts of anion were added to a 2.05% K basal diet to create the following diets: 0.46% Ca and +327 mEq/kg of DCAD, 0.46% Ca and +146 mEq/kg of DCAD, and 0.72% Ca and +140 mEq/kg of DCAD. In experiment 1, cows fed the diet with 0.46% Ca and +167 mEq/kg of DCAD had significantly lower plasma Ca concentration after calving than cows fed the 0.46 or 0.72% Ca diets with anions. Periparturient plasma Ca concentrations did not differ in cows fed the low-DCAD diets with 0.46 or 0.72% Ca. Urine pH was reduced from 8.27 in the diet with 0.46% Ca and +167 mEq/kg of DCAD to 7.07 and 7.41 in the 0.46 and 0.72% Ca anion diets, respectively. Precalving plasma PTH and 1,25-dihydroxyvitamin D concentrations were similar in cows fed the 0.46% Ca diets and the 0.72% Ca diets, suggesting that the 0.46% Ca diets were not low enough in Ca to place the cow in negative Ca balance before calving. In experiment 2, adding the anion supplements to a 2.05% K diet did not reduce urine pH below 8.0. Periparturient plasma Ca concentrations did not differ in cows in any group in experiment 2. Precalving diets that are 0.46% Ca fed ad libitum are too high in Ca to stimulate Ca homeostasis before calving. Adding anions to a diet can benefit periparturient cow plasma Ca concentration, but only if it alters acid-base status enough to reduce urine pH below 7.5.

Drug Clearance from Cerebrospinal Fluid Mediated by Organic Anion Transporters 1 (Slc22a6) and 3 (Slc22a8) at Arachnoid Membrane of Rats.

Although arachnoid mater epithelial cells form the blood-arachnoid barrier (BAB), acting as a blood-CSF interface, it has been generally considered that the BAB is impermeable to water-soluble substances and plays a largely passive role. Here, we aimed to clarify the function of transporters at the BAB in regulating CSF clearance of water-soluble organic anion drugs based on quantitative targeted absolute proteomics (QTAP) and in vivo analyses. Protein expression levels of 61 molecules, including 19 ATP-binding-cassette (ABC) transporters and 32 solute-carrier (SLC) transporters, were measured in plasma membrane fraction of rat leptomeninges using QTAP. Thirty-three proteins were detected; others were under the quantification limits. Expression levels of multidrug resistance protein 1 (Mdr1a/P-gp/Abcb1a) and breast cancer resistance protein (Bcrp/Abcg2) were 16.6 and 3.27 fmol/µg protein (51.9- and 9.82-fold greater than in choroid plexus, respectively). Among those organic anion transporters detected only at leptomeninges, not choroid plexus, organic anion transporter 1 (oat1/Slc22a6) showed the greatest expression (2.73 fmol/µg protein). On the other hand, the protein expression level of oat3 at leptomeninges was 6.65 fmol/µg protein, and the difference from choroid plexus was within two-fold. To investigate oat1's role, we injected para-aminohippuric acid (PAH) with or without oat1 inhibitors into cisterna magna (to minimize the contribution of choroid plexus function) of rats. A bulk flow marker, FITC-inulin, was not taken up from CSF up to 15 min, whereas uptake clearance of PAH was 26.5 µL/min. PAH uptake was completely blocked by 3 mM cephalothin (inhibits both oat1 and oat3), while 17% of PAH uptake was inhibited by 0.2 mM cephalothin (selectively inhibits oat3). These results indicate that oat1 and oat3 at the BAB provide a distinct clearance pathway of organic anion drugs from CSF independently of choroid plexus.

Effect of negative dietary cation-anion differences on carcass characteristics and beef tenderness of Japanese Black steers.

Lowering dietary cation-anion differences (DCAD) can enhance responsiveness to Ca-homeostatic hormones and increase Ca availability, which might have potential to activate a Ca-dependent protease, calpain, and to enhance postmortem myofibrillar proteolysis. In this study, we investigated the effects of DCAD manipulation on calpain activity and beef tenderness in Japanese Black cattle which are characterized by their high marbling. Thirty-six Japanese Black steers were allotted to one of two treatments: (i) control (CON; DCAD +6.09 mEq/100 g of dry matter (DM)) or (ii) negative DCAD (NEGD; DCAD -8.27 mEq/100 g DM) for 70 days before slaughter. Lowering DCAD decreased DM and energy intake (P < 0.01) even though it did not negatively affect the growth performance or carcass characteristics. In NEGD, urine pH was decreased by acidification caused by the negative DCAD (P < 0.01). Calpain activities tended to be improved in NEGD (P = 0.09), but Warner-Bratzler shear force values were not affected by treatment. Although calpain activities tended to improve, lowering DCAD to -8.27 for 70 days before slaughter was insufficient to enhance beef tenderness in Japanese Black steers.

Dietary cation and anion difference: Effects on milk production and body fluid distribution in lactating dairy goats under tropical conditions.

This study aimed to determine the effect of dietary cation and anion difference (DCAD) on milk production and body fluid distribution in lactating dairy goats. Ten dairy goats were selected and divided into two groups, five animals each. Animals received either control DCAD (control, 22.81 mEq/100 g dry matter (DM)) or high DCAD (DCAD, 39.08 mEq/100 g DM). The results indicated that rectal temperature (Tr), respiration rate, milk yield and compositions did not differ between groups. But the percentage change of Tr from the DCAD group was lower than the control group between 09.00 and 13.00 hours. DM intake tended to increase in the DCAD group. Dairy goats in the DCAD group drank more water, but urinary excretion and plasma antidiuretic hormone concentration remained unchanged. Apparent water balance was higher from the DCAD group over 24 h. There was no effect of DCAD on plasma and blood volumes, but tended to increase in extracellular fluid and thereby increased total body water. The present results indicate that animals supplemented with high DCAD increase their total body water and apparent water balance. These results have contributed to the process of adaptation for evaporative cooling and would be useful in slowing down the elevation in Tr.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Lactation performance and energy metabolism.

The objectives of this experiment were to evaluate the effects of feeding diets with 2 dietary cation-anion difference (DCAD) levels and supplemented with either cholecalciferol (CH) or calcidiol (CA) during late gestation on lactation performance and energetic metabolism in dairy cows. The hypothesis was that combining a prepartum acidogenic diet with calcidiol supplementation would benefit peripartum Ca metabolism and, thus, improve energy metabolism and lactation performance compared with cows fed an alkalogenic diet or cholecalciferol. Holstein cows at 252 d of gestation were blocked by parity (28 nulliparous and 51 parous cows) and milk yield within parous cows, and randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial, with 2 levels of DCAD (positive, +130, and negative, -130 mEq/kg) and 2 sources of vitamin D, CH or CA, fed at 3 mg per 11 kg of diet dry matter (DM). The resulting treatment combinations were positive DCAD with CH (PCH), positive DCAD with CA (PCA), negative DCAD with CH (NCH), or negative DCAD with CA (NCA), which were fed for the last 21 d of gestation. After calving, cows were fed the same lactation diet. Body weight and body condition were evaluated prepartum and for the first 49 d postpartum. Blood was sampled thrice weekly prepartum, and on d 0, 1, 2, 3, and every 3 d thereafter until 30 d postpartum for quantification of hormones and metabolites. Lactation performance was evaluated for the first 49 d postpartum. Feeding a diet with negative DCAD reduced DM intake in parous cows by 2.1 kg/d, but no effect was observed in nulliparous cows. The negative DCAD reduced concentrations of glucose (positive = 4.05 vs. negative = 3.95 mM), insulin (positive = 0.57 vs. negative = 0.45 ng/mL), and insulin-like growth factor-1 (positive = 110 vs. negative = 95 ng/mL) prepartum. Treatments did not affect DM intake postpartum, but CA-supplemented cows tended to produce more colostrum (PCH = 5.86, PCA = 7.68 NCH = 6.21, NCA = 7.96 ± 1.06 kg) and produced more fat-corrected milk (PCH = 37.0, PCA = 40.1 NCH = 37.5, NCA = 41.9 ± 1.8 kg) and milk components compared with CH-supplemented cows. Feeding the negative DCAD numerically increased yield of fat-corrected milk by 1.0 kg/d in both nulliparous and 1.4 kg/d in parous cows. Minor differences were observed in postpartum concentrations of hormones and metabolites linked to energy metabolism among treatments. Results from this experiment indicate that replacing CH with CA supplemented at 3 mg/d during the prepartum period improved postpartum lactation performance in dairy cows.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Vitamin D, mineral, and bone metabolism.

Pregnant Holstein cows, 28 nulliparous and 51 parous, were blocked by parity and milk yield and randomly allocated to receive diets that differed in dietary cation-anion difference (DCAD), +130 or -130 mEq/kg, and supplemented with either calcidiol or cholecalciferol at 3 mg/11 kg of dry matter from 255 d of gestation until parturition. Blood was sampled thrice weekly prepartum, and on d 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 postpartum to evaluate effects of the diets on vitamin D, mineral and bone metabolism, and acid-base status. Blood pH and concentrations of minerals, vitamin D metabolites, and bone-related hormones were determined, as were mineral concentrations and losses in urine and colostrum. Supplementing with calcidiol increased plasma concentrations of 25-hydroxyvitamin D3, 3-epi 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D3, and 24,25-dihydroxyvitamin D3 compared with supplementing with cholecalciferol. Cows fed the diet with negative DCAD had lesser concentrations of vitamin D metabolites before and after calving than cows fed the diet with positive DCAD, except for 25-hydroxyvitamin D2. Feeding the diet with negative DCAD induced a compensated metabolic acidosis that attenuated the decline in blood ionized Ca (iCa) and serum total Ca (tCa) around calving, particularly in parous cows, whereas cows fed the diet with positive DCAD and supplemented with calcidiol had the greatest 1,25-dihydroxyvitamin D3 concentrations and the lowest iCa and tCa concentrations on d 1 and 2 postpartum. The acidogenic diet or calcidiol markedly increased urinary losses of tCa and tMg, and feeding calcidiol tended to increase colostrum yield and increased losses of tCa and tMg in colostrum. Cows fed the diet with negative DCAD had increased concentrations of serotonin and C-terminal telopeptide of type 1 collagen prepartum compared with cows fed the diet with positive DCAD. Concentrations of undercarboxylated and carboxylated osteocalcin and those of adiponectin did not differ with treatment. These results provide evidence that dietary manipulations can induce metabolic adaptations that improve mineral homeostasis with the onset of lactation that might explain some of the improvements observed in health and production when cows are fed diets with negative DCAD or supplemented with calcidiol.

Effects of prepartum dietary cation-anion difference and source of vitamin D in dairy cows: Health and reproductive responses.

The objectives of the experiment were to evaluate the effects of feeding diets with distinct dietary cation-anion difference (DCAD) levels and supplemented with 2 sources of vitamin D during the prepartum transition period on postpartum health and reproduction in dairy cows. The hypotheses were that feeding acidogenic diets prepartum would reduce the risk of hypocalcemia and other diseases, and the benefits of a negative DCAD treatment on health would be potentiated by supplementing calcidiol compared with cholecalciferol. Cows at 252 d of gestation were blocked by parity (28 nulliparous and 52 parous cows) and milk yield within parous cows, and randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial, with 2 levels of DCAD, positive (+130 mEq/kg) or negative (-130 mEq/kg), and 2 sources of vitamin D, cholecalciferol or calcidiol, fed at 3 mg for each 11 kg of diet dry matter. The resulting treatment combinations were positive DCAD with cholecalciferol (PCH), positive DCAD with calcidiol (PCA), negative DCAD with cholecalciferol (NCH), and negative DCAD with calcidiol (NCA), which were fed from 252 d of gestation to calving. After calving, cows were fed the same lactation diet supplemented with cholecalciferol at 0.70 mg for every 20 kg of dry matter. Blood was sampled 7 d before parturition, and at 2 and 7 d postpartum to evaluate cell counts and measures of neutrophil function. Postpartum clinical and subclinical diseases and reproductive responses were evaluated. Feeding a diet with negative DCAD eliminated clinical hypocalcemia (23.1 vs. 0%) and drastically reduced the incidence and daily risk of subclinical hypocalcemia, and these effects were observed in the first 48 to 72 h after calving. The diet with negative DCAD tended to improve the intensity of oxidative burst activity of neutrophils in all cows prepartum and increased the intensity of phagocytosis in parous cows prepartum and the proportion of neutrophils with killing activity in parous cows postpartum (58.5 vs. 67.6%). Feeding calcidiol improved the proportion of neutrophils with oxidative burst activity (60.0 vs. 68.7%), reduced the incidences of retained placenta (30.8 vs. 2.5%) and metritis (46.2 vs. 23.1%), and reduced the proportion of cows with multiple diseases in early lactation. Combining the negative DCAD diet with calcidiol reduced morbidity by at least 60% compared with any of the other treatments. Cows with morbidity had lower blood ionized Ca and serum total Ca concentrations than healthy cows. Treatments did not affect the daily risk of hyperketonemia in the first 30 d of lactation. Despite the changes in cow health, manipulating the prepartum DCAD did not influence reproduction, but feeding calcidiol tended to increase the rate of pregnancy by 55%, which reduced the median days open by 19. In conclusion, feeding prepartum cows with a diet containing a negative DCAD combined with 3 mg of calcidiol benefited health in early lactation.

A New Noncanonical Anionic Peptide That Translocates a Cellular Blood-Brain Barrier Model.

The capacity to transport therapeutic molecules across the blood-brain barrier (BBB) represents a breakthrough in the development of tools for the treatment of many central nervous system (CNS)-associated diseases. The BBB, while being protective against infectious agents, hinders the brain uptake of many drugs. Hence, finding safe shuttles able to overcome the BBB is of utmost importance. Herein, we identify a new BBB-translocating peptide with unique properties. For years it was thought that cationic sequences were mandatory for a cell-penetrating peptide (CPP) to achieve cellular internalization. Despite being anionic at physiological pH, PepNeg (sequence (SGTQEEY) is an efficient BBB translocator that is able to carry a large cargo (27 kDa), while maintaining BBB integrity. In addition, PepNeg is able to use two distinct methods of translocation, energy-dependent and -independent, suggesting that direct penetration might occur when low concentrations of peptide are presented to cells. The discovery of this new anionic trans-BBB peptide allows the development of new delivery systems to the CNS and contributes to the need to rethink the role of electrostatic attraction in BBB-translocation.

Effects of prepartum dietary cation-anion difference on aspects of peripartum mineral and energy metabolism and performance of multiparous Holstein cows.

The objectives of this study were to determine the effect of decreasing dietary cation-anion difference [DCAD; (Na+ + K+) - (Cl- + S2-)] of the prepartum diet on aspects of mineral metabolism, energy metabolism, and performance of peripartum dairy cows. Multiparous Holstein cows (n = 89) were enrolled between 38 and 31 d before expected parturition and randomized to treatments in a completely randomized design (restricted to balance for previous 305-d mature equivalent milk production, parity, and body condition score) at 24 d before expected parturition. Treatments consisted of a low-K ration without anion supplementation [CON; n = 30, DCAD = +18.3 mEq/100 g of dry matter (DM)]; partial anion supplementation to a low-K ration (MED; n = 30, DCAD = +5.9 mEq/100 g of DM); and anion supplementation to a low-K ration to reach a targeted average urine pH between 5.5 and 6.0 (LOW; n = 29, DCAD = -7.4 mEq/100 g of DM). Cows were fed a common postpartum diet and data collected through 63 d in milk. Urine pH (CON = 8.22, MED = 7.89, and LOW = 5.96) was affected quadratically by decreasing prepartum DCAD. A linear relationship between urine pH and urine Ca:creatinine ratio was observed (r = -0.81). Plasma Ca concentrations in the postpartum period (d 0 to 14; CON = 2.16, MED = 2.19, and LOW = 2.27 mmol/L) were increased linearly with decreasing prepartum DCAD. A treatment by parity (second vs. third and greater) interaction for postpartum plasma Ca concentration suggested that older cows had the greatest response to the low DCAD diet and older cows fed LOW had decreased prevalence of hypocalcemia after calving. A quadratic effect of decreasing DCAD on prepartum DMI was observed (CON = 13.6, MED = 14.0, and LOW = 13.2 kg/d). Milk production in the first 3 wk postpartum was increased linearly with decreasing DCAD (CON = 40.8, MED = 42.4, and LOW = 43.9 kg/d) and DMI in this period also tended to linearly increase (CON = 20.2, MED = 20.9, and LOW = 21.3 kg/d). Overall, effects on intake and milk yield analyzed over wk 1 to 9 postpartum were not significant. This study demonstrates that feeding lower DCAD diets prepartum improves plasma Ca status in the immediate postpartum period and results in increased DMI and milk production in the 3 wk after parturition. Compared with no anion supplementation or lower levels of anion supplementation, greater improvements were observed with the lower DCAD feeding strategy, in which an average urine pH of 5.5 to 6.0 was targeted.

The effect of cation source and dietary cation-anion difference on rumen ion concentrations in lactating dairy cows.

Many studies have focused on the influence of dietary cation-anion difference (DCAD) on animal performance but few have examined the effect of DCAD on the rumen ionic environment. The objective of this study was to examine the effects of DCAD, cation source (Na vs. K), and anion source (Cl vs. bicarbonate or carbonate) on rumen environment and fermentation. The study used 5 rumen-fistulated dairy cows and 5 dietary treatments that were applied using a 5×5 Latin square design with 2-wk experimental periods. Treatments consisted of (1) the basal total mixed ration (TMR); (2) the basal TMR plus 340mEq/kg of Na (dry matter basis) using NaCl; (3) the basal TMR plus 340mEq/kg of K using KCl; (4) the basal TMR plus 340mEq/kg of Na using NaHCO3; and (5) the basal TMR plus 340mEq/kg of K using K2CO3. On the last day of each experimental period, rumen samples were collected and pooled from 5 different locations at 0, 1.5, 3, 4.5, 6, 9, and 12h postfeeding for measurement of rumen pH and concentrations of strong ions and volatile fatty acids (VFA). Dietary supplementation of individual strong ions increased the corresponding rumen ion concentration. Rumen Na was decreased by 24mEq/L when K was substituted for Na in the diet, but added dietary Na had no effect on rumen K. Rumen Cl was increased by 10mEq/L in diets supplemented with Cl. Cation source had no effect on rumen pH or total VFA concentration. Increased DCAD increased rumen pH by 0.10 pH units and increased rumen acetate by 4mEq/L but did not increase total VFA. This study demonstrated that rumen ion concentrations can be manipulated by dietary ion concentrations. If production and feed efficiency responses to DCAD and ionophores in the diet are affected by rumen Na and K concentrations, then manipulating dietary Na and K could be used either to enhance or diminish those responses.

Hypoglycemic activity and stability enhancement of human insulin-tat mixture loaded in elastic anionic niosomes.

This study aimed to investigate the synergistic effect of trans-activator of transcription (Tat) and niosomes for the improvement of hypoglycemic activity of orally delivered human insulin. The elastic anionic niosomes composing of Tween 61/cholesterol/dicetyl phosphate/sodium cholate at 1:1:0.05:0.02 molar ratio loaded with insulin-Tat mixture (1:3 molar ratio) was prepared. Deformability of the elastic anionic niosomes decreased after loaded with the mixture of 1.35 times. For the in vitro release, the insulin (T10 = 4 h) loaded in the elastic anionic niosomes indicated the slower release rate than insulin in the mixture (T10 = 3 h) loaded in niosomes. At room temperature (30 ± 2 °C), the mixture loaded in elastic anionic niosomes was more chemical stable than the free mixture of 1.3, 1.4 and 1.7 times after stored for 4, 8 and 12 weeks, respectively. Oral administration in the alloxan-induced diabetic mice of the mixture loaded in elastic anionic niosomes with the insulin doses at 25, 50 and 100 IU/kg body weight indicated significant hypoglycemic activity with the percentage fasting blood glucose reduction of 1.95, 2.10 and 2.10 folds of the subcutaneous insulin injection at 12 h, respectively. This study has demonstrated the synergistic benefits of Tat and elastic anionic niosomes for improving the hypoglycemic activity of the orally delivered human insulin as well as the stability enhancement of human insulin when stored at high temperature. The results from this study can be further developed as an effective oral insulin delivery.