Anti-SpCas9 IgY Polyclonal Antibodies Production for CRISPR Research Use.

The CRISPR/Cas adaptative immune system has been harnessed as an RNA-guided, programmable genome editing tool, allowing for diverse biotechnological applications. The implementation of the system relies on the ability to detect the Cas9 protein in biological samples. This task is facilitated by employing antibodies, which exhibit several advantageous features and applications in the context of tropical neglected diseases. This study reports a one-month immunization scheme with the Cas9 protein fromStreptococcus pyogenes to produce IgY polyclonal antibodies (anti-SpCas9), which can be rapidly isolated by combining yolk de-lipidation with protein salting out using pectin and ammonium sulfate, respectively. Immunodetection assays indicate that the antibodies are highly sensitive, specific, and useful for detecting the SpCas9 protein in promastigotes ofLeishmania braziliensisexpressing exogenous SpCas9. Thus, the simple method for producing anti-SpCas9 IgY antibodies will accelerate CRISPR/Cas-based studies in Leishmania spp. This approach serves as a valuable research tool in this parasite model and holds the potential for wide application in various other biological samples, promoting the implementation of the system. In fact, a bioinformatics approach based on the identification of antigenic determinants in the SpCas9 protein suggests the possibility of using the anti-SpCas9 IgY antibodies in applications such as Prime and Base editing.

Predicting no-show appointments in a pediatric hospital in Chile using machine learning.

The Chilean public health system serves 74% of the country's population, and 19% of medical appointments are missed on average because of no-shows. The national goal is 15%, which coincides with the average no-show rate reported in the private healthcare system. Our case study, Doctor Luis Calvo Mackenna Hospital, is a public high-complexity pediatric hospital and teaching center in Santiago, Chile. Historically, it has had high no-show rates, up to 29% in certain medical specialties. Using machine learning algorithms to predict no-shows of pediatric patients in terms of demographic, social, and historical variables. To propose and evaluate metrics to assess these models, accounting for the cost-effective impact of possible intervention strategies to reduce no-shows. We analyze the relationship between a no-show and demographic, social, and historical variables, between 2015 and 2018, through the following traditional machine learning algorithms: Random Forest, Logistic Regression, Support Vector Machines, AdaBoost and algorithms to alleviate the problem of class imbalance, such as RUS Boost, Balanced Random Forest, Balanced Bagging and Easy Ensemble. These class imbalances arise from the relatively low number of no-shows to the total number of appointments. Instead of the default thresholds used by each method, we computed alternative ones via the minimization of a weighted average of type I and II errors based on cost-effectiveness criteria. 20.4% of the 395,963 appointments considered presented no-shows, with ophthalmology showing the highest rate among specialties at 29.1%. Patients in the most deprived socioeconomic group according to their insurance type and commune of residence and those in their second infancy had the highest no-show rate. The history of non-attendance is strongly related to future no-shows. An 8-week experimental design measured a decrease in no-shows of 10.3 percentage points when using our reminder strategy compared to a control group. Among the variables analyzed, those related to patients' historical behavior, the reservation delay from the creation of the appointment, and variables that can be associated with the most disadvantaged socioeconomic group, are the most relevant to predict a no-show. Moreover, the introduction of new cost-effective metrics significantly impacts the validity of our prediction models. Using a prototype to call patients with the highest risk of no-shows resulted in a noticeable decrease in the overall no-show rate.

Phenol release from pNIPAM hydrogels: scaling molecular dynamics simulations with dynamical density functional theory.

We employed molecular dynamic simulations (MD) and the Bennett's acceptance ratio method to compute the free energy of transfer, ΔGtrans, of phenol, methane, and 5-fluorouracil (5-FU), between bulk water and water-pNIPAM mixtures of different polymer volume fractions, ϕp. For this purpose, we first calculate the solvation free energies in both media to obtain ΔGtrans. Phenol and 5-FU (a medication used to treat cancer) attach to the pNIPAM surface so that they show negative values of ΔGtrans irrespective of temperature (above or below the lower critical solution temperature of pNIPAM, Tc). Conversely, methane switches the ΔGtrans sign when considering temperatures below (positive) and above (negative) Tc. In all cases, and contrasting with some theoretical predictions, ΔGtrans maintains a linear behavior with the pNIPAM concentration up to large polymer densities. We have also employed MD to compute the diffusion coefficient, D, of phenol in water-pNIPAM mixtures as a function of ϕp in the diluted limit. Both ΔGtrans and D as a function of ϕp are required inputs to obtain the release halftime of hollow pNIPAM microgels through Dynamic Density Functional Theory (DDFT). Our scaling strategy captures the experimental value of 2200 s for 50 µm radius microgels with no cavity, for ϕp ≃ 0.83 at 315 K.

Mexico City during and after the September 19, 2017 earthquake: Assessment of seismic resilience and ongoing recovery process.

Mexico City was severely affected by the September 19, 2017 Puebla-Morelos earthquake (M w = 7.1). City authorities confirmed that more than 12,000 structures for all uses were damaged as a consequence of this earthquake. In this paper, the focus of attention is devoted to trying to quantify in a simple manner how resilient the built environment in Mexico City was during the September 19, 2017 earthquake. Global statistics compiled for the severity of damage observed during this seismic event from detailed information gathered from well-documented and detailed damage surveys were used for this purpose. Also, an update is provided on how the seismic reconstruction and recovery processes of the built environment in Mexico City have been after this earthquake. This adaptive resilience has been assessed from reliable information and statistics of the ongoing reconstruction process of the affected built environment in Mexico City. The implementation of structural health monitoring programs for typical, representative buildings within the city would be germane to detect and correct potential structural deficiencies on time before the next strong earthquake may strike, then helping to improve the seismic resilience of the built environment.

Yield degradation due to laser drive asymmetry in D3He backlit proton radiography experiments at OMEGA.

Mono-energetic proton radiography is a vital diagnostic for numerous high-energy-density-physics, inertial-confinement-fusion, and laboratory-astrophysics experiments at OMEGA. With a large number of campaigns executing hundreds of shots, general trends in D3He backlighter performance are statistically observed. Each experimental configuration uses a different number of beams and drive symmetry, causing the backlighter to perform differently. Here, we analyze the impact of these variables on the overall performance of the D3He backlighter for proton-radiography studies. This study finds that increasing laser drive asymmetry can degrade the performance of the D3He backlighter. The results of this study can be used to help experimental designs that use proton radiography.

A genome-wide association study identifies novel gene associations with facial skin wrinkling and mole count in Latin Americans.

BACKGROUND: Genome-wide association studies (GWASs) have identified genes influencing skin ageing and mole count in Europeans, but little is known about the relevance of these (or other genes) in non-Europeans. OBJECTIVES: To conduct a GWAS for facial skin ageing and mole count in adults < 40 years old, of mixed European, Native American and African ancestry, recruited in Latin America. METHODS: Skin ageing and mole count scores were obtained from facial photographs of over 6000 individuals. After quality control checks, three wrinkling traits and mole count were retained for genetic analyses. DNA samples were genotyped with Illumina's HumanOmniExpress chip. Association testing was performed on around 8 703 729 single-nucleotide polymorphisms (SNPs) across the autosomal genome. RESULTS: Genome-wide significant association was observed at four genome regions: two were associated with wrinkling (in 1p13·3 and 21q21·2), one with mole count (in 1q32·3) and one with both wrinkling and mole count (in 5p13·2). Associated SNPs in 5p13·2 and in 1p13·3 are intronic within SLC45A2 and VAV3, respectively, while SNPs in 1q32·3 are near the SLC30A1 gene, and those in 21q21·2 occur in a gene desert. Analyses of SNPs in IRF4 and MC1R are consistent with a role of these genes in skin ageing. CONCLUSIONS: We replicate the association of wrinkling with variants in SLC45A2, IRF4 and MC1R reported in Europeans. We identify VAV3 and SLC30A1 as two novel candidate genes impacting on wrinkling and mole count, respectively. We provide the first evidence that SLC45A2 influences mole count, in addition to variants in this gene affecting melanoma risk in Europeans.

Trimethylamine N-oxide levels are associated with NASH in obese subjects with type 2 diabetes.

AIMS: Trimethylamine N-oxide (TMAO), choline and betaine serum levels have been associated with metabolic diseases including type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). These associations could be mediated by insulin resistance. However, the relationships among these metabolites, insulin resistance and NAFLD have not been thoroughly investigated. Moreover, it has recently been suggested that TMAO could play a role in NAFLD by altering bile acid metabolism. We examined the association between circulating TMAO, choline and betaine levels and NAFLD in obese subjects. METHODS: Serum TMAO, choline, betaine and bile acid levels were measured in 357 Mexican obese patients with different grades of NAFLD as determined by liver histology. Associations of NAFLD with TMAO, choline and betaine levels were tested. Moreover, association of TMAO levels with non-alcoholic steatohepatitis (NASH) was tested separately in patients with and without T2D. RESULTS: TMAO and choline levels were significantly associated with NAFLD histologic features and NASH risk. While increased serum TMAO levels were significantly associated with NASH in patients with T2D, in non-T2D subjects this association lost significance after adjusting for sex, BMI and HOMA2-IR. Moreover, circulating secondary bile acids were associated both with increased TMAO levels and NASH. CONCLUSIONS: In obese patients, circulating TMAO levels were associated with NASH mainly in the presence of T2D. Functional studies are required to evaluate the role of insulin resistance and T2D in this association, both highly prevalent in NASH patients.

A coil-to-globule transition capable coarse-grained model for poly(N-isopropylacrylamide).

We present a model for mesoscopic molecular dynamics simulations of poly(N-isopropyl-acrylamide) (pNIPAM). The model uses a coarse-grained scheme based on the explicit-solvent Martini force field. The mapping of the polymer accounts for three beads per monomer. Similarly to the Martini water bead, the amide moieties of the polymer include an electric dipole. The model is tested by building polymer chains of different sizes and proved to accurately capture the thermal response of pNIPAM without including any temperature-dependent parameters. The critical temperature of the model is observed at (302.1 ± 1.1) K for a 35-mer and it keeps invariant when increasing the chain length. We deployed a series of replica-exchange molecular dynamics simulations that evidence the oligomer reaches thermodynamic equilibrium irrespective of the starting configuration. Finally, the model is applied to a membrane structure of pNIPAM where a good agreement with previous atomistic simulations is observed.

Energetic metabolism, milk production, and inflammatory response of transition dairy cows fed rumen-protected glucose.

Objectives were to evaluate the effects of rumen-protected glucose (RPG) supplementation on milk production, post-absorptive metabolism, and inflammatory biomarkers in transition dairy cows. Fifty-two multiparous cows were blocked by previous 305-d mature-equivalent milk (305ME) yield and randomly assigned to 1 of 2 iso-energetic and iso-nitrogenous treatments: (1) control diet (CON; n = 26) or (2) a diet containing RPG (pre-fresh 5.3% of dry matter and 6.0% of dry matter postpartum; n = 26). Cows received their respective dietary treatments from d -21 to 28 relative to calving, and dry matter intake was calculated daily during the same period. Weekly body weight, milk composition, and fecal pH were recorded until 28 d in milk (DIM), and milk yield was recorded through 105 DIM. Blood samples were collected on d -7, 3, 7, 14, and 28 relative to calving. Data were analyzed using repeated measures in the MIXED procedure (SAS Institute Inc., Cary, NC) with previous 305ME as a covariate. Fecal pH was similar between treatments and decreased (0.6 units) postpartum. Dry matter intake pre- and postpartum were unaffected by treatment, as was milk yield during the first 28 or 105 DIM. Milk fat, protein, and lactose concentration were similar for both treatments. Blood urea nitrogen and plasma glucose concentrations were unaffected by treatment; however, results showed increased concentration of circulating insulin (27%), lower nonesterified fatty acids (28%), and lower postpartum ß-hydroxybutyrate (24%) in RPG-fed cows. Overall, circulating lipopolysaccharide-binding protein and haptoglobin did not differ by treatment, but at 7 DIM, RPG-fed cows had decreased lipopolysaccharide-binding protein and haptoglobin concentrations (31 and 27%, respectively) compared with controls. Supplemental RPG improved some biomarkers of post-absorptive energetics and inflammation during the periparturient period, changes primarily characterized by increased insulin and decreased nonesterified fatty acids concentrations, with a concomitant reduction in acute phase proteins without changing milk production and composition.

Validating a heat stress model: The effects of an electric heat blanket and nutritional plane on lactating dairy cows.

The efficacy of an electric heat blanket (EHB) has previously been confirmed as an alternative method to evaluate heat stress (HS). However, a pair-feeding design has not been used with the EHB model. Therefore, study objectives were to determine the contribution of the nutritional plane to altered metabolism and productivity during EHB-induced HS. Multiparous Holstein cows (n = 18; 140 ± 10 d in milk) were subjected to 2 experimental periods (P); during P1 (4 d), cows were in thermoneutral conditions with ad libitum feed intake. During P2 (4 d), cows were assigned to 1 of 2 treatments: (1) thermoneutral conditions and pair-fed (PF; n = 8) or (2) EHB-induced HS with ad libitum feed intake (n = 10). Overall, the EHB increased rectal temperature, vaginal temperature, skin temperature, and respiration rate (1.4°C, 1.3°C, 0.8°C, and 42 breaths/min, respectively) relative to PF cows. The EHB reduced dry matter intake (DMI; 47%) and, by design, PF cows had a similar pattern and extent of decreased DMI. Milk yield decreased in EHB and PF cows by 27.3% (12.1 kg) and 13.4% (5.4 kg), respectively, indicating that reduced DMI accounted for only ∼50% of decreased milk synthesis. Milk fat content tended to increase (19%) in the EHB group, whereas in the PF cows it remained similar relative to P1. During P2, milk protein and lactose contents tended to decrease or decreased (1.3 and 2.2%, respectively) in both EHB and PF groups. Milk urea nitrogen remained unchanged in PF controls but increased (34.2%) in EHB cows relative to P1. The EHB decreased blood partial pressure of CO2, total CO2, HCO3, and base excess levels (17, 16, 17, and 81%, respectively) compared with those in PF cows. During P2, the EHB and PF cows had similar decreases (4%) in plasma glucose content, but no differences in circulating insulin were detected. However, a group by day interaction was detected for plasma nonesterified fatty acids; levels progressively increased in PF controls but remained unaltered in the EHB cows. Blood urea nitrogen increased in the EHB cows (61%) compared with the PF controls. In summary, utilizing the EHB model indicated that reduced nutrient intake explains only about 50% of the decrease in milk yield during HS, and the postabsorptive changes in nutrient partitioning are similar to those obtained in climate-controlled chamber studies. Consequently, the EHB is a reasonable and economically feasible model to study environmental physiology of dairy cows.

Effects of dietary zinc source on the metabolic and immunological response to lipopolysaccharide in lactating Holstein dairy cows.

The objectives of this study were to evaluate the effects of replacing 40 mg/kg of Zn from Zn sulfate (control; CON) with Zn AA complex (AvZn) on metabolism and immunological responses following an intravenous lipopolysaccharide (LPS) challenge in lactating cows. Cows were randomly assigned to 1 of 4 treatments: (1) pair-fed (PF) control (PF-CON; 5 mL of saline; n = 5), (2) PF AvZn (PF-AvZn; 5 mL of saline; n = 5), (3) LPS euglycemic clamp control (LPS-CON; 0.375 µg of LPS/kg of BW; n = 5), and (4) LPS euglycemic clamp AvZn (LPS-AvZn; 0.375 µg of LPS/kg of BW; n = 5). Cows were enrolled in 3 experimental periods (P). During period 1 (3 d), cows received their respective dietary treatments and baseline data were obtained. During period 2 (P2; 2 d), a 12-h LPS euglycemic clamp was conducted or cows were PF to their respective dietary counterparts. During period 3 (P3; 3 d), cows received their dietary treatment and consumed feed ad libitum. Mild hyperthermia (1°C) was observed in LPS cows at 3 h postbolus. Throughout P2, the rectal temperature of LPS-AvZn cows was decreased (0.3°C) relative to LPS-CON cows. Administrating LPS decreased dry matter intake (47%) during P2, and by experimental design the pattern was similar in PF cohorts. During P3, dry matter intake from LPS cows remained decreased (15%) relative to PF cows. Milk yield from LPS cows decreased (54%) during P2 relative to PF cows, but it was similar during P3. During P2, somatic cell count increased 3-fold in LPS cows relative to PF controls. Dietary AvZn tended to decrease somatic cell count (70%) during P3 relative to LPS-CON cows. Insulin increased 7-fold in LPS cows at 12 h postbolus and remained increased (4-fold) for the duration of P2. Circulating glucagon from LPS cows increased (65%) during P2, and supplementing AvZn blunted the increase (30% relative to LPS-CON). During P2, circulating cortisol increased 7-fold post-LPS infusion relative to PF cows, and supplementing AvZn decreased cortisol (58%) from 6 to 48 h postbolus relative to LPS-CON cows. Administrating LPS increased circulating LPS-binding protein and serum amyloid A (3- and 9-fold, respectively) relative to PF cows. Compared with LPS-CON, LPS-AvZn cows had increased circulating serum amyloid A (38%) 24 h postbolus. The 12-h total glucose deficit was 36 and 1,606 g for the PF and LPS treatments, respectively, but was not influenced by Zn source. In summary, replacing a portion of the Zn sulfate with Zn AA complex appeared to reduce the inflammatory response but had no effect on the glucose deficit.

Control over the transverse structure and long-distance fiber propagation of light at the single-photon level.

Quantum entanglement is arguably the cornerstone which differentiates the quantum realm from its classical counterpart. While entanglement can reside in any photonic degree of freedom, polarization permits perhaps the most straightforward manipulation due to the widespread availability of standard optical elements such as waveplates and polarizers. As a step towards a fuller exploitation of entanglement in other degrees of freedom, in this work we demonstrate control over the transverse spatial structure of light at the single-photon level. In particular we integrate in our setup all the technologies required for: (i) fibre-based photon pair generation, (ii) deterministic and broadband single-photon spatial conversion relying on a passive optical device, and (iii) single-photon transmission, while retaining transverse structure, over 400 m of few-mode fibre. In our experiment, we employ a mode selective photonic lantern multiplexer with the help of which we can convert the transverse profile of a single photon from the fundamental mode into any of the supported higher-order modes. We also achieve conversion to an incoherent or coherent addition of two user-selected higher order modes by addressing different combinations of inputs in the photonic lantern multiplexer. The coherent nature of the addition, and extraction of usable orbital angular momentum at the single-photon level, is further demonstrated by far-field diffraction through a triangular aperture. Our work could enable studies of photonic entanglement in the transverse modes of a fibre and could constitute a key resource quantum for key distribution with an alphabet of scalable dimension.

P-NIPAM in water-acetone mixtures: experiments and simulations.

The lower critical solution temperature (LCST) of poly-N-isopropylacrylamide (p-NIPAM) diminishes when a small volume of acetone is added to the aqueous polymer solution, and then increases for further additions, producing a minimum at a certain acetone concentration. Here this behavior is observed through the variation of the hydrodynamic radius RH of p-NIPAM microgels with temperature, measured by dynamic light scattering (DLS), when adding increasing amounts of acetone in the molar fraction range of 0.00 to 0.25. This size trend of microgels with temperature is well captured by all-atom molecular dynamic simulations, which are implemented for a single 30-mer, at similar solvent and temperature conditions. Both DLS measurements and simulations indicate that the shrunken state continuously augments its size with increasing acetone content. This, in turn, leads to a minimum of the globule-to-coil transition temperature, which should correspond to the minimum of the LCST. Furthermore, density profiles, as obtained by considering a membrane arrangement of oligomers, reveal a preferential interaction of the polymer with acetone to the detriment of water. We observe how the membrane loses water content as the temperature is increased while keeping a similar amount of acetone in its interior. This competition between water and acetone for the polymer surface plays a major role in the enthalpy driven dependence of the critical temperature with acetone concentration.

Effects of continuous and increasing lipopolysaccharide infusion on basal and stimulated metabolism in lactating Holstein cows.

Experimental objectives of this study were to characterize the systemic and intracellular metabolic response to continuous lipopolysaccharide (LPS) infusion in mid-lactation Holstein cows (169 ± 20 d in milk; 681 ± 16 kg of body weight). Following 3 d of acclimation, cows were enrolled in 2 experimental periods (P). During P1 (3 d), cows were fed ad libitum and baseline data were collected. In P2 (8 d), cows were assigned to 1 of 2 treatments: (1) saline-infused and pair-fed (CON-PF; i.v. sterile saline at 40 mL/h; n = 5) or (2) LPS-infused and fed ad libitum (LPS-AL; Escherichia coli O55:B5 at 0.017, 0.020, 0.026, 0.036, 0.055, 0.088, 0.148, and 0.148 µg/kg of body weight per hour for d 1 through 8, respectively; n = 6). During P2, CON-PF cows were pair-fed to LPS-AL cows to eliminate confounding effects of dissimilar nutrient intake. Blood samples were collected on d 1 and 2 of P1 and d 1, 3, 5, and 7 of P2. Following the P2 d 7 a.m. milking, adipose tissue, skeletal muscle, and liver biopsies were collected for reverse transcription quantitative PCR and Western blot analysis. To assess whole-body nutrient trafficking, an i.v. glucose tolerance test (GTT) was performed following the a.m. milking on P2 d 8; 4 h after the GTT, cows received an epinephrine challenge. During P2, there were no treatment differences in circulating glucose. Relative to P1, CON-PF cows had or tended to have decreased plasma ß-hydroxybutyrate and insulin (29 and 47%, respectively) during P2, whereas neither variable changed in LPS-AL cows, leading to an overall increase in ß-hydroxybutyrate and insulin (41 and 140%, respectively) relative to CON-PF cows. Circulating nonesterified fatty acids were increased from d 1 to 3 and subsequently decreased from d 3 to 7 in cows from both treatments. Blood urea nitrogen gradually decreased in CON-PF cows and increased in LPS-AL cows from d 1 to 5 of P2, resulting in an overall 25% increase in LPS-AL versus CON-PF cows. In response to the GTT, the glucose and insulin area under the curve were increased 33 and 56%, respectively, in LPS-AL compared with CON-PF cows; changes reflective of whole-body insulin resistance. However, protein abundance of insulin signaling markers within muscle, liver, and adipose tissue were similar between treatments. There were no observable treatment differences in the glucose or nonesterified fatty acids response to the epinephrine challenge. No treatment differences were observed in hepatic mRNA abundance of key gluconeogenic or lipid export enzymes. In conclusion, chronic LPS exposure altered multiple parameters of basal and stimulated metabolism, but did not appear to affect the molecular machinery evaluated herein.

Feeding reduced-fat dried distillers grains with solubles to lactating Holstein dairy cows does not alter milk composition or cause late blowing in cheese.

Feeding dried distillers grains with solubles (DDGS) to lactating dairy cows has been implicated as a cause of late blowing defects in the production of Swiss-style cheeses. Our objectives were (1) to test the effect of feeding reduced-fat DDGS (RF-DDGS; ∼6% fat) to lactating dairy cows on the composition of milk and on the suitability of the milk for production of baby Swiss cheese and (2) to evaluate the effect of diet on cow lactation performance. Lactating Holstein dairy cows were fed both dietary treatments in a 2 × 2 crossover design. Cows were housed in a 48-cow freestall pen equipped with individual feeding gates to record feed intake. The control diet was a corn, corn silage, and alfalfa hay diet supplemented with mechanically expelled soybean meal. The experimental diet was the same base ration, but 20% (dry matter basis) RF-DDGS were included in place of the expelled soybean meal. The RF-DDGS diet was additionally supplemented with rumen-protected lysine; diets were formulated to be isoenergetic and isonitrogenous. Cows were allowed ad libitum access to feed and water, fed twice daily, and milked 3 times daily. For cheese production, milk was collected and pooled 6 times for each dietary treatment. There was no treatment effect on milk yield (35.66 and 35.39 kg/d), milk fat production (1.27 and 1.25 kg/d), milk fat percentage (3.65 and 3.61%), milk protein production (1.05 and 1.08 kg/d), lactose percentage (4.62 and 4.64%), milk total solids (12.19 and 12.28%), and somatic cell count (232.57 and 287.22 × 103 cells/mL) for control and RF-DDGS, respectively. However, dry matter intake was increased by treatment, which implied a reduction in feed efficiency. Milk protein percentage also increased (3.01 and 3.11%), whereas milk urea nitrogen decreased (14.18 and 12.99 mg/dL), indicating that protein utilization may be more efficient when cows are fed RF-DDGS. No differences in cheese were observed by a trained panel except cheese appearance; control cheese eyes were significantly, but not practically, larger than the RF-DDGS cheese. These results indicate that RF-DDGS can be effectively used in the rations of lactating Holstein cows with no deleterious effects on milk production and composition and metrics of the physiology of the cow (i.e., blood glucose and nonesterified fatty acids); however, feeding RF-DDGS increased dry matter intake, which decreased feed efficiency. Finally, feeding RF-DDGS did not negatively influence quality and suitability of milk for production of baby Swiss cheese.

CD38 protein deficiency induces autoimmune characteristics and its activation enhances IL-10 production by regulatory B cells.

CD38 is a transmembrane protein expressed in B lymphocytes, and is able to induce responses as proliferation, differentiation or apoptosis. Several reports propose that CD38 deficiency accelerates autoimmune processes in murine models of autoimmune diabetes, lymphoproliferation and rheumatoid arthritis. Other reports have shown elevated CD38 expression in B and T cells from patients with autoimmunity; however, the role of CD38 is still unclear in the development of autoimmunity. Recently, it has been characterized as CD1dhi CD5+ regulatory B cell subpopulation able to produce IL-10, and the loss of these cells exacerbates the autoimmunity in murine models. Here, we report that CD38-/- mice exhibited elevated titres of ANAS, anti-dsDNA autoantibodies from 12 months of age and were higher by 16 months of age and mice presented kidney damage. Interestingly, there is a reduction in the survival of CD38-/- mice compared to the WT. Furthermore, CD38 is highly expressed by CD1dhigh CD5+ regulatory B cells, and the agonistic anti-CD38 stimulus plus LPS was able to increase the percentage of this cell subset and its ability to induce IL-10 production. Together, these results suggest that CD38 could play a role in the control of autoimmune diseases through their expression on regulatory B cells.

Effect of chromium on bioenergetics and leukocyte dynamics following immunoactivation in lactating Holstein cows.

Activated immune cells are insulin sensitive and utilize copious amounts of glucose. Because chromium (Cr) increases insulin sensitivity and may be immunomodulatory, our objective was to evaluate the effect of supplemental Cr (KemTrace Cr propionate, 20 g/d; Kemin Industries Inc., Des Moines, IA) on immune system glucose utilization and immune system dynamics following an intravenous endotoxin challenge in lactating Holstein cows. Twenty cows (320 ± 18 d in milk) were randomly assigned to 1 of 4 treatments: (1) pair-fed (PF) control (PF-CON; 5 mL of saline; n = 5), (2) PF and Cr supplemented (PF-Cr; 5 mL of saline; n = 5), (3) lipopolysaccharide (LPS)-euglycemic clamp and control supplemented (LPS-CON; 0.375 µg/kg of body weight LPS; n = 5), and (4) LPS-euglycemic clamp and Cr supplemented (LPS-Cr; 0.375 µg/kg of body weight LPS; n = 5). The experiment was conducted serially in 3 periods (P). During P1 (3 d), cows received their respective dietary treatments and baseline values were obtained. At the initiation of P2 (2 d), either a 12-h LPS-euglycemic clamp was conducted or cows were PF to their respective dietary counterparts. During P3 (3 d), cows consumed feed ad libitum and continued to receive their respective dietary treatment. During P2, LPS administration decreased dry matter intake (DMI; 40%) similarly among diets, and by experimental design the pattern and magnitude of reduced DMI were similar in the PF cohorts. During P3, LPS-Cr cows tended to have decreased DMI (6%) relative to LPS-CON cows. Relative to controls, milk yield from LPS-challenged cows decreased (58%) during P2 and LPS-Cr cows produced less (16%) milk than LPS-CON cows. During P3, milk yield progressively increased similarly in LPS-administered cows, but overall milk yield remained decreased (24%) compared with PF controls. There were no dietary treatment differences in milk yield during P3. Circulating insulin increased 9- and 15-fold in LPS-administered cows at 6 and 12 h postbolus, respectively, compared with PF controls. Compared with LPS-CON cows, circulating insulin in LPS-Cr cows was decreased (48%) at 6 h postbolus. Relative to PF cows, circulating LPS binding protein and serum amyloid A from LPS-administered cows increased 2- and 5-fold, respectively. Compared with PF cows, blood neutrophil counts in LPS-infused cows initially decreased, then gradually increased 163%. Between 18 and 48 h postbolus, the number of neutrophils was increased (12%) in LPS-Cr versus LPS-CON cows. The 12-h total glucose deficit was 220 and 1,777 g for the PF and LPS treatments, respectively, but glucose utilization following immune activation was not influenced by Cr. In summary, supplemental Cr reduced the insulin response and increased circulating neutrophils following an LPS challenge but did not appear to alter the immune system's glucose requirement following acute and intense activation.

Composition of gut microbiota in obese and normal-weight Mexican school-age children and its association with metabolic traits.

BACKGROUND: Childhood obesity is a serious public health problem in Mexico. Adult gut microbiota composition has been linked to obesity, but few studies have addressed the role of gut microbiota in childhood obesity. OBJECTIVES: The aim of this study is to compare gut microbiota composition in obese and normal-weight children and to associate gut microbiota profiles with amino acid serum levels and obesity-related metabolic traits. METHODS: Microbial taxa relative abundance was determined by 16S rRNA sequencing in 67 normal-weight and 71 obese children aged 6-12 years. Serum amino acid levels were measured by mass spectrometry. Associations between microbiota composition, metabolic parameters and amino acid serum levels were tested. RESULTS: No significant differences in phyla abundances or Firmicutes/Bacteroidetes ratios were observed between normal-weight and obese children. However, Bacteroides eggerthii abundance was significantly higher in obese children and correlated positively with body fat percentage and negatively with insoluble fibre intake. Additionally, Bacteroides plebeius and unclassified Christensenellaceae abundances were significantly higher in normal-weight children. Abundance of both these species correlated negatively with phenylalanine serum levels, a metabolite also found to be associated with obesity in Mexican children. CONCLUSIONS: The study identified bacterial species associated with obesity, metabolic complications and amino acid serum levels in Mexican children.

Effect of partially replacing a barley-based concentrate with flaxseed-based products on the rumen bacterial population of lactating Holstein dairy cows.

AIMS: The effects of partial replacement of a barley-based concentrate with flaxseed-based products on the rumen bacterial population of lactating Holstein dairy cows were evaluated. METHODS AND RESULTS: Treatments fed were CONT, a normal diet that included barley silage, alfalfa hay and a barley-based concentrate that contained no flaxseed or faba beans; FLAX, inclusion of a nonextruded flaxseed-based product containing 55·0% flaxseed, 37·8% field peas and 6·9% alfalfa; EXT, similar to FLAX, but the product was extruded and EXTT, similar to FLAX, but product was extruded and field peas were replaced by high-tannin faba beans. The rumen bacterial population was evaluated by utilizing 16S rRNA gene sequencing. Most abundant phyla, families and genera were unaffected. However, some taxa were affected; for example, unsaturated fatty acid content was negatively correlated with Clostridiaceae, and tannin content was negatively correlated with BS11 and Paraprevotellaceae. CONCLUSIONS: Predominant rumen bacterial taxa were not affected, but the abundance of some taxa found in lower proportions shifted, possibly due to sensitivity to unsaturated fatty acids or tannins. SIGNIFICANCE AND IMPACT OF THE STUDY: Flaxseed-based products were effective for partially replacing barley-based concentrate in rations of lactating dairy cows. No negative effects of these products were observed on the abundance of predominant rumen bacterial taxa, with only minor shifts in less abundant bacteria.

Diseño y producción de diversas proteínas fusión de la nicotinamida/ nicotinato mononucleotido adenilil transferasa (NMNAT) de Plasmodium falciparum / Design and production of various fusion proteins of the nicotinamide/ nicotinate mononucleotide adenilil transferase (NMNAT) of Plasmodium falciparum / Desenho e produção de várias proteínas fusão da nicotinamida/nicotinato mononucleotido adenilil transferasa (NMNAT) do Plasmodium falciparum

Resumen Las proteínas recombinantes se han convertido en herramientas útiles en la investigación bioquímica. Sin embargo, durante su producción, aparecen cuerpos de inclusión (IB), debido, por un lado, a la alta expresión de proteína producida a partir de los vectores usados que poseen promotores de alta eficiencia y, por otro lado, a características propias de la proteína. Ahora bien, la nicotinamida/nicotinato mononucleótido adenililtransferasa (NMNAT) es una proteína central en la biosíntesis del NAD(H)+, molécula esencial en el metabolismo celular, y ha sido estudiada en parásitos protozoos. Para el estudio de la NMNAT de estos parásitos se ha recurrido a la expresión de su versión recombinante en E. coli, obteniéndose gran cantidad de proteína como IB. Con el fin de aumentar la solubilidad de la proteína, se clonó la secuencia codificante de la NMNAT de Plasmodium falciparum en diferentes vectores de expresión, se indujo la expresión de la proteína recombinante en E. coli BL21 (DE3) y se analizó la solubilidad. La proteína fusión con mayor solubilidad fue purificada y evaluada enzimáticamente. La adición de la etiqueta MBP (proteína de unión a maltosa) a la PfNMNAT incrementó su solubilidad y permitió obtener una proteína funcional con una alta pureza.

Abstract Recombinant proteins have become useful tools in biochemistry research. During their production, however, inclusion bodies (IB) appear, on the one hand, due to the high expression rate from the recombinant plasmids, which have high efficiency promoters, and, on the other hand, intrinsic characteristics of the expressed protein. Furhtermore, the nicotinamide/nicotinate mononucleotide adenilyl transferase (NMNAT) is a central protein in NAD(H)+biosynthesis, an essential cofactor in cell metabolism, and in protozoon parasite has been studied. To study the NMNAT protein of these parasites, their recombinant version in E. coli has been expressed, getting a great quantity of IB as a by-product. To increase the solubility of the protein, the coding sequence of the NMNAT enzyme of Plasmodium falciparum was cloned in different expression plasmids which were subsequently transformed into E. coli BL21 (DE3) expression strain. The solubility of the recombinant proteins was assessed and the one with the highest presence in the soluble fraction was subsequently purified and its enzyme activity was determined. The recombinant protein with a MBP (maltose-binding protein) tag showed an increased solubility and purity.

Resumo As proteínas recombinantes tem convertido em ferramentas úteis na investigação bioquímica. Um problema frequente durante a sua produção é a aparição de corpos de inclusão (IB), devida principalmente à elevada expressão da proteína produzida a partir dos vectores utilizados que têm promotores de elevada eficiência; bem como características das próprias proteínas. Além disso, a nicotinamida/nicotinato mononucleótido adenililtransferasa é uma proteína central na biossíntese do NAD(H)+, molécula essencial no metabolismo celular, e tem sido estudada em protozoários parasitos. Para o estudo da NMNAT destes parasitos, foi realizada a expressão da sua versão recombinante em E. coli, e foi obtido uma grande quantidade de proteína em forma de IB. Com a finalidade de aumentar a quantidade de proteína na fração solúvel, a sequência codificadora para NMNAT de Plasmodium falciparum foi clonada em diferentes vectores de expressão, foi induzida a expressão da proteína recombinante em E. coli BL21(DE3) e foi analisada a solubilidade. A proteína fusão com maior solubilidade foi purificada e avaliada enzimáticamente. A adição da etiqueta MBP (proteína de união a maltosa) à PfNMNAT incrementou a solubilidade da proteína e permitiu obter uma proteína funcional com una alta pureza. espécie de planta.