Size matters: lower body weight pigs have a different response to immune challenge and amino acids supplementation above the estimated requirement compared to heavy pigs.

The immune response varies between pigs, as not all pigs have the same response to a stressor. This variation may exist between individuals due to body weight (BW) or body composition, which may impact the capacity for coping with an immune challenge (IC). Tryptophan (Trp), threonine (Thr), and methionine (Met) requirements might also play a considerable part in supporting immune system activation while reducing variation between pigs; however, the latter has yet to be reported. This exploratory study investigated the effect of initial BW (light vs. heavy-weight) and supplementation of Trp, Thr, and Met above National Research Council (NRC) requirements on feeding behavior and the coping capacity of growing pigs under an IC. Eighty gilts were categorized into 2 groups according to BW: light-weight (LW, 22.5 kg) and heavy-weight pigs (HW, 28.5 kg). Both BW groups were group-housed for a 28-d trial in a good or poor sanitary condition (SC). Pigs within a poor SC were orally inoculated with 2 × 109 colony units of Salmonella Typhimurium, and fresh manure from a pig farm was spread on the floor. Pigs within good SC were not inoculated, nor was manure spread. Two diets were provided within each SC: control (CN) or supplemented (AA+) with Trp, Thr, and Met at 120% of NRC recommended levels. A principal component analysis was performed in R, and a feeding behavior index was calculated in SAS. Results showed that LW and HW pigs were clustered separately on day 0, where LW pigs had a positive correlation with body lipid percentage (r = 0.83), and HW pigs had a positive correlation with body protein percentage (r = 0.75). After the IC, the cluster configuration changed, with diets influencing LW more than HW pigs within poor SC. On day 14, LW fed AA + diet in poor SC was clustered separately from LW pigs fed CN diet, whereas LW fed AA + and CN diets in good SC were clustered together. For feeding behavior, in both analyzed periods (period 1: days 7 to 14; period 2: days 21 to 28), LW had lower total feed intake and shorter meals than HW pigs (P < 0.10), independent of the SC. Furthermore, LW pigs fed AA + diet had a more regular feed intake pattern than those fed CN diet, while a more irregular pattern was observed for HW pigs fed AA + diet than CN diet at period 2. These findings suggest that supplementing Trp, Thr, and Met above requirements may be a nutritional strategy for LW pigs under IC by improving feed intake regularity and reducing the probability of being susceptible to IC.

An immune challenge impacts pig welfare and may decrease growth and protein deposition. These may happen due to the different nutrient requirements of immune-challenged pigs compared to non-challenged. Dietary supplementation of tryptophan, threonine, and methionine has been proven to be a strategy to mitigate performance losses by supporting immune system functioning, maintaining gut barrier integrity, and reducing oxidative status. However, individuals within a population with similar age and genetics have distinct responses to dietary strategies due to different coping abilities to an immune challenge, which may be due to body weight (BW)/body composition and feeding behavior patterns. In this context, this study investigated the effect of BW (light-weight vs. heavy-weight) and tryptophan, threonine, and methionine supplementation on feeding behavior and the coping capacity of growing pigs under an immune challenge. Heavy-weight pigs had greater feed intake regularity and coping abilities over time when compared to light-weight pigs. However, increasing the amino acid level in the diet improved feed intake regularity in light-weight pigs. The amino acid supplementation may be a potential precision nutrition strategy for light-weight pigs by improving feed intake regularity over time, reducing susceptibility to an immune challenge.

Normal vision and development in mice with low functional expression of Kir7.1 in heterozygosis for a blindness-producing mutation inactivating the channel.

K+ channel Kir7.1 expressed at the apical membrane of the retinal pigment epithelium (RPE) plays an essential role in retinal function. An isoleucine-to-threonine mutation at position 120 of the protein is responsible for blindness-causing vitreo-retinal dystrophy. We have studied the molecular mechanism of action of Kir7.1-I120T in vitro by heterologous expression and in vivo in CRISPR-generated knockin mice. Full-size Kir7.1-I120T reaches the plasma membrane but lacks any activity. Analysis of Kir7.1 and the I120T mutant in mixed transfection experiments, and that of tandem tetrameric constructs made by combining wild type (WT) and mutant protomers, leads us to conclude that they do not form heterotetramers in vitro. Homozygous I120T/I120T mice show cleft palate and tracheomalacia and do not survive beyond P0, whereas heterozygous WT/I120T develop normally. Membrane conductance of RPE cells isolated from WT/WT and heterozygous WT/I120T mice is dominated by Kir7.1 current. Using Rb+ as a charge carrier, we demonstrate that the Kir7.1 current of WT/I120T RPE cells corresponds to approximately 50% of that in cells from WT/WT animals, in direct proportion to WT gene dosage. This suggests a lack of compensatory effects or interference from the mutated allele product, an interpretation consistent with results obtained using WT/- hemizygous mouse. Electroretinography and behavioral tests also show normal vision in WT/I120T animals. The hypomorphic ion channel phenotype of heterozygous Kir7.1-I120T mutants is therefore compatible with normal development and retinal function. The lack of detrimental effect of this degree of functional deficit might explain the recessive nature of Kir7.1 mutations causing human eye disease.NEW & NOTEWORTHY Human retinal pigment epithelium K+ channel Kir7.1 is affected by generally recessive mutations leading to blindness. We investigate one such mutation, isoleucine-to-threonine at position 120, both in vitro and in vivo in knockin mice. The mutated channel is inactive and in heterozygosis gives a hypomorphic phenotype with normal retinal function. Mutant channels do not interfere with wild-type Kir7.1 channels which are expressed concomitantly without hindrance, providing an explanation for the recessive nature of the disease.

Muscle growth affects the metabolome of the pectoralis major muscle in red-winged tinamou (Rhynchotus rufescens).

The aim of the present study was to identify and quantify the metabolites (metabolome analysis) of the pectoralis major muscle in male red-winged tinamou (Rhynchotus rufescens) selected for growth traits. A selection index was developed for females [body weight (BW), chest circumference (CC), and thigh circumference (TC)] and males [BW, CC, TC, semen volume, and sperm concentration] in order to divide the animals into 2 experimental groups: selection group with a higher index (TinamouS) and commercial group with a lower index (TinamouC). Twenty male offspring of the 2 groups (TinamouS, n = 10; TinamouC, n = 10) were confined for 350 d. The birds were slaughtered and pectoralis major muscle samples were collected, subjected to polar and apolar metabolites extractions and analyzed by proton nuclear magnetic resonance (1H NMR) spectroscopy. Analysis of the polar metabolomic profile identified 65 metabolites; 29 of them were differentially expressed between the experimental groups (P < 0.05). The TinamouS groups exhibited significantly higher concentrations (P < 0.05) of 25 metabolites, including anserine, aspartate, betaine, carnosine, creatine, glutamate, threonine, 3-methylhistidine, NAD+, pyruvate, and taurine. Significantly higher concentrations of cysteine, beta-alanine, lactose, and choline were observed in the TinamouC group (P < 0.05). The metabolites identified in the muscle provided information about the main metabolic pathways (higher impact value and P < 0.05), for example, phenylalanine, tyrosine and tryptophan biosynthesis; alanine, aspartate and glutamate metabolism; D-glutamine and D-glutamate metabolism; ß-alanine metabolism; glycine, serine and threonine metabolism; taurine and hypotaurine metabolism; histidine metabolism; phenylalanine metabolism. The NMR spectra of apolar fraction showed 8 classes of chemical compounds. The metabolome analysis shows that the selection index resulted in the upregulation of polyunsaturated fatty acids, unsaturated fatty acids, phosphocholines, phosphoethanolamines, triacylglycerols, and glycerophospholipids. The present study suggests that, despite few generations, the selection based on muscle growth traits promoted changes in metabolite concentrations in red-winged tinamou.

The Existing Drug Nifuroxazide as an Antischistosomal Agent: In Vitro, In Vivo, and In Silico Studies of Macromolecular Targets.

Schistosomiasis is a parasitic disease that afflicts approximately 250 million people worldwide. There is an urgent demand for new antiparasitic agents because praziquantel, the only drug available for the treatment of schistosomiasis, is not universally effective and may derail current progress toward the WHO goal of eliminating this disease as a public health problem by 2030. Nifuroxazide (NFZ), an oral nitrofuran antibiotic, has recently been explored to be repurposed for parasitic diseases. Here, in vitro, in vivo, and in silico studies were conducted to evaluate the activity of NFZ on Schistosoma mansoni. The in vitro study showed significant antiparasitic activity, with 50% effective concentration (EC50) and 90% effective concentration (EC90) values of 8.2 to 10.8 and 13.7 to 19.3 µM, respectively. NFZ also affected worm pairing and egg production and induced severe damage to the tegument of schistosomes. In vivo, a single oral dose of NFZ (400 mg/kg of body weight) to mice harboring either prepatent or patent S. mansoni infection significantly reduced the total worm burden (~40%). In patent infection, NFZ achieved a high reduction in the number of eggs (~80%), but the drug caused a low reduction in the egg burden of animals with prepatent infection. Finally, results from in silico target fishing methods predicted that serine/threonine kinases could be one of the potential targets for NFZ in S. mansoni. Overall, the present study revealed that NFZ possesses antischistosomal properties, mainly in terms of egg burden reduction in animals with patent S. mansoni infection. IMPORTANCE The increasing recognition of the burden imposed by helminthiasis, associated with the limited therapeutic arsenal, has led to initiatives and strategies to research and develop new drugs for the treatment of schistosomiasis. One of these strategies is drug repurposing, which considers low-risk compounds with potentially reduced costs and shorter time for development. In this study, nifuroxazide (NFZ) was evaluated for its anti-Schistosoma mansoni potential through in vitro, in vivo, and in silico studies. In vitro, NFZ affected worm pairing and egg production and induced severe damage to the tegument of schistosomes. In vivo, a single oral dose of NFZ (400 mg/kg) to mice harboring either prepatent or patent S. mansoni infection significantly reduced the total worm burden and egg production. In silico investigations have identified serine/threonine kinases as a molecular target for NFZ. Collectively, these results implied that NFZ might be a potential therapeutic candidate for the treatment of schistosomiasis.

Increased dietary Trp, Thr, and Met supplementation improves growth performance and protein deposition of salmonella-challenged growing pigs under poor housing conditions.

Highly intensified rearing conditions and precarious sanitary management predispose pigs to immune system activation, altered amino acid (AA) metabolism, and decreased growth performance. Thus, the main objective of this study was to evaluate the effects of increased dietary tryptophan (Trp), threonine (Thr), and methionine + cysteine (Met + Cys) supplementation on performance, body composition, metabolism, and immune responses of group-housed growing pigs under challenging sanitary conditions. A hundred and twenty pigs (25.4 ± 3.7 kg) were randomly assigned to a 2 × 2 factorial arrangement, consisting of two sanitary conditions (SC, good [GOOD] or salmonella-challenge and poor housing condition [Salmonella Typhimurium (ST) + POOR]) and two diets, control (CN) or supplemented with AA (Trp, Thr, and Met + Cys:Lys ratios 20% higher than those of the CN diet [AA>+]). Pigs were followed during the growing phase (25-50 kg) and the trial lasted 28 d. The ST + POOR SC pigs were challenged with Salmonella Typhimurium and raised in a poor housing condition. The ST + POOR SC increased rectal temperature, fecal score, serum haptoglobin, and urea concentration (P < 0.05) and decreased serum albumin concentration (P < 0.05) compared with GOOD SC. Body weight, average daily feed intake, average daily gain (ADG), feed efficiency (G:F), and protein deposition (PD) were greater in GOOD SC than in ST + POOR SC (P < 0.01). However, pigs housed in ST + POOR SC fed with AA+ diet had lower body temperature (P < 0.05), increased ADG (P < 0.05) and nitrogen efficiency (P < 0.05), and a tendency for improved PD and G:F (P < 0.10) compared with CN diet fed pigs. Regardless of the SC, pigs fed AA+ diet had lower serum albumin (P < 0.05) and tended to decrease serum urea levels (P < 0.10) compared with CN diet. The results of this study suggest that the ratio of Trp, Thr, and Met + Cys to Lys for pigs are modified by sanitary conditions. Furthermore, supplementation of diets with a blend of Trp, Thr, and Met + Cys improves performance, especially under salmonella-challenge and poor housing conditions. Dietary tryptophan, threonine, and methionine supplementation can modulate immune status and influence resilience to sanitary challenges.

Immune system activation alters pigs' physiology and metabolism, increasing maintenance requirements and reducing voluntary feed intake and weight gain. Dietary functional amino acid supplementation (tryptophan, threonine, and methionine) is a strategy to support the immune system activation for immune components production, maintenance of the gut barrier integrity, and reduction of the oxidative status. Additionally, amino acid supplementation may mitigate growth performance losses. In this context, this study was conducted to investigate the effect of diets with or without tryptophan, threonine, and methionine supplementation on the performance and immune system activation of growing pigs under a sanitary challenge. The amino acid supplementation mitigated the immune system activation of challenged growing pigs and improved growth performance when compared to pigs fed diets with no supplementation. The functional amino acid supplementation may be an efficient nutritional strategy to optimize health and growth performance of immune-challenged pigs.

Threonine-to-lysine ratio in laying hens: physiological parameters and organ weight

The objective of this study was to evaluate the effect of threonine:digestible lysine ratio in the diet on the physiological variablesand weight of organs of light laying hens. Two hundred and ten 47 week-old Dekalb White laying henswere distributed in a completely randomized design, with five levels of threonine (0.507; 0.552; 0.597; 0.642 and 0.677%) and seven replicates of six birds each. The experimental period was 10 weeks, totaling 62 days and more eight days for the animals to adapt. The physiological parameters of cloacal temperature (CT), respiratory rate (RR) and average surface temperature (AST) were recorded weekly (7:00 am, 10:00 am, 1:00 pm, 4:00 pm, and 7:00 pm); after solid and water fasting,the birds were slaughtered to assess the absolute weight of the organs. The time of day influenced (p < 0.05) the physiological parameters RR and AST, and CT showed a significant effect (p < 0.05) of increasing levels of digestible threonine. The total weight of the pancreas, proventriculus and lung showed a significant effect (p < 0.05) of the increase in the levels of digestible threonine. The respiratory rate is affected by the levels of threoninein the diet. The 0.687% level promoted hypertrophy of the pancreas, proventriculus and lung, promoting more significant activity of these organs.(AU)

Sexual dimorphism in the molecular mechanisms of insulin resistance during a critical developmental window in Wistar rats.

BACKGROUND: Insulin resistance (IR) is a condition in which the response of organs to insulin is impaired. IR is an early marker of metabolic dysfunction. However, IR also appears in physiological contexts during critical developmental windows. The molecular mechanisms of physiological IR are largely unknown in both sexes. Sexual dimorphism in insulin sensitivity is observed since early stages of development. We propose that during periods of accelerated growth, such as around weaning, at postnatal day 20 (p20) in rats, the kinase S6K1 is overactivated and induces impairment of insulin signaling in its target organs. This work aimed to characterize IR at p20, determine its underlying mechanisms, and identify whether sexual dimorphism in physiological IR occurs during this stage. METHODS: We determined systemic insulin sensitivity through insulin tolerance tests, glucose tolerance tests, and blood glucose and insulin levels under fasting and fed conditions at p20 and adult male and female Wistar rats. Furthermore, we quantified levels of S6K1 phosphorylated at threonine 389 (T389) (active form) and its target IRS1 phosphorylated at serine 1101 (S1101) (inhibited form). In addition, we assessed insulin signal transduction by measuring levels of Akt phosphorylated at serine 473 (S473) (active form) in white adipose tissue and skeletal muscle through western blot. Finally, we determined the presence and function of GLUT4 in the plasma membrane by measuring the glucose uptake of adipocytes. Results were compared using two-way ANOVA (With age and sex as factors) and one-way ANOVA with post hoc Tukey's tests or t-student test in each corresponding case. Statistical significance was considered for P values < 0.05. RESULTS: We found that both male and female p20 rats have elevated levels of glucose and insulin, low systemic insulin sensitivity, and glucose intolerance. We identified sex- and tissue-related differences in the activation of insulin signaling proteins in p20 rats compared to adult rats. CONCLUSIONS: Male and female p20 rats present physiological insulin resistance with differences in the protein activation of insulin signaling. This suggests that S6K1 overactivation and the resulting IRS1 inhibition by phosphorylation at S1101 may modulate to insulin sensitivity in a sex- and tissue-specific manner. Video Abstract.

Insulin regulates the synthesis of carbohydrates, lipids and proteins differently between males, and females. One of its primary functions is maintaining adequate blood glucose levels favoring glucose entry in muscle and adipose tissue after food consumption. Insulin resistance (IR) is a condition in which the response of organs to insulin is impaired. IR is frequently associated with metabolic dysfunction such as inflammation, obesity, or type 2 diabetes. However, physiological IR develops in healthy individuals during periods of rapid growth, pregnancy, or aging by mechanisms not fully understood. We studied the postnatal development, specifically around weaning at postnatal day 20 (p20) of Wistar rats. In previous works, we identified insulin resistance during this period in male rats. This work aimed to characterize IR at p20, determine its underlying mechanisms, and identify whether sexual dimorphism in physiological IR occurs during this stage. We found that p20 rats of both sexes have elevated blood glucose and insulin levels, low systemic insulin sensitivity, and glucose intolerance. We identified differences in insulin-regulated protein activation (S6K1, IRS1, Akt, and GLUT4) between sexes in different tissues and adipose tissue depots. Studying these mechanisms and their differences between males and females is essential to understanding insulin actions and their relationship with the possible development of metabolic diseases in both sexes.

The protein phosphatase 2A catalytic subunit StPP2Ac2b enhances susceptibility to Phytophthora infestans and senescence in potato.

The serine/threonine protein phosphatases type 2A (PP2A) are involved in several physiological responses in plants, playing important roles in developmental programs, stress responses and hormone signaling. Six PP2A catalytic subunits (StPP2Ac) were identified in cultivated potato. Transgenic potato plants constitutively overexpressing the catalytic subunit StPP2Ac2b (StPP2Ac2b-OE) were developed to determine its physiological roles. The response of StPP2Ac2b-OE plants to the oomycete Phytophthora infestans, the causal agent of late blight, was evaluated. We found that overexpression of StPP2Ac2b enhances susceptibility to the pathogen. Further bioinformatics, biochemical, and molecular analyses revealed that StPP2Ac2b positively regulates developmental and pathogen-induced senescence, and that P. infestans infection promotes senescence, most likely through induction of StPP2Ac2b expression.

Lung Tumor Cells with Different Tn Antigen Expression Present Distinctive Immunomodulatory Properties.

Lung cancer is the first leading cause of cancer-related deaths in the world. Aberrant glycosylation in lung tumors leads to the expression of tumor-associated carbohydrate structures, such as the Tn antigen, consisting of N-acetyl-galactosamine (GalNAc) linked to a serine or threonine residue in proteins (α-GalNAc-O-Ser/Thr). The Tn antigen can be recognized by the Macrophage Galactose/GalNAc lectin (MGL), which mediates various immune regulatory and tolerogenic functions, mainly by reprogramming the maturation of function of dendritic cells (DCs). In this work, we generated two different Tn-expressing variants from the Lewis-type lung murine cancer cell line LL/2, which showed different alterations in the O-glycosylation pathways that influenced the interaction with mouse MGL2 and the immunomodulatory properties of DCs. Thus, the identification of the biological programs triggered by Tn+ cancer cells might contribute to an improved understanding of the molecular mechanisms elicited by MGL-dependent immune regulatory circuits.

A mutation in THREONINE SYNTHASE 1 uncouples proliferation and transition domains of the root apical meristem: experimental evidence and in silico proposed mechanism.

A continuum from stem to transit-amplifying to a differentiated cell state is a common theme in multicellular organisms. In the plant root apical meristem (RAM), transit-amplifying cells are organized into two domains: cells from the proliferation domain (PD) are displaced to the transition domain (TD), suggesting that both domains are necessarily coupled. Here, we show that in the Arabidopsis thaliana mto2-2 mutant, in which threonine (Thr) synthesis is affected, the RAM lacks the PD. Through a combination of cell length profile analysis, mathematical modeling and molecular markers, we establish that the PD and TD can be uncoupled. Remarkably, although the RAM of mto2-2 is represented solely by the TD, the known factors of RAM maintenance and auxin signaling are expressed in the mutant. Mathematical modeling predicts that the stem cell niche depends on Thr metabolism and that, when disturbed, the normal continuum of cell states becomes aborted.

Prediction of semen analysis parameter improvement after varicocoelectomy using 1 H NMR-based metabonomics assays.

BACKGROUND: Varicocoele is the most common correctable cause of male infertility; however, predicting varicocoelectomy outcomes is difficult. "Omics" techniques have been increasingly used to develop new diagnostic and prognostics tools for several male infertility causes, and could be applied to study varicocoele. OBJECTIVES: The objective is to create metabolomics models capable of segregating men who improved semen analysis (SA) parameters or achieved natural pregnancy after microsurgical varicocoelectomy (MV) from those who did not, using hydrogen-1 nuclear magnetic resonance (1 H NMR) spectra of seminal plasma of pre-operative samples. MATERIAL AND METHODS: We recruited 29 infertile men with palpable varicocoele. 1 H NMR spectra of seminal plasma were obtained from pre-operative samples and used to create metabonomics models. Improvement was defined as an increase in the total motile progressive sperm count (TMC) of the post-operative SA when compared to the baseline, and pregnancy was assessed for 24 months after MV. RESULTS: Using linear discriminant analysis (LDA), we created a model that discriminated the men who improved SA from those who did not with accuracy of 93.1%. Another model segregated men who achieved natural pregnancy from men who did not. We identified seven metabolites that were important for group segregation: caprylate, isoleucine, N-acetyltyrosine, carnitine, N-acetylcarnitine, creatine, and threonine. DISCUSSION: We described the use of metabonomics model to predict with high accuracy the outcomes of MV in infertile men with varicocoele. The most important metabolites for group segregation are involved in energy metabolism and oxidative stress response, highlighting the pivotal role of these mechanisms in the pathophysiology of varicocoele. CONCLUSIONS: 1 H NMR spectroscopy of seminal plasma can be used in conjunction with multivariate statistical tools to create metabonomics models useful to segregate men with varicocoele based on the reproductive outcomes of MV. These models may help counseling infertile men with varicocoele regarding their prognosis after surgery.

Specificity and Reactivity of Mycobacterium tuberculosis Serine/Threonine Kinases PknG and PknB.

Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis, has 11 eukaryotic-like serine/threonine protein kinases, which play essential roles in cell growth, signal transduction, and pathogenesis. Protein kinase G (PknG) regulates the carbon and nitrogen metabolism by phosphorylation of the glycogen accumulation regulator (GarA) protein at Thr21. Protein kinase B (PknB) is involved in cell wall synthesis and cell shape, as well as phosphorylates GarA but at Thr22. While PknG seems to be constitutively activated and recognition of GarA requires phosphorylation in its unstructured tail, PknB activation is triggered by phosphorylation of its activation loop, which allows binding of the forkhead-associated domain of GarA. In the present work, we used molecular dynamics and quantum-mechanics/molecular mechanics simulations of the catalytically competent complex and kinase activity assays to understand PknG/PknB specificity and reactivity toward GarA. Two hydrophobic residues in GarA, Val24 and Phe25, seem essential for PknG binding and allow specificity for Thr21 phosphorylation. On the other hand, phosphorylated residues in PknB bind Arg26 in GarA and regulate its specificity for Thr22. We also provide a detailed analysis of the free energy profile for the phospho-transfer reaction and show why PknG has a constitutively active conformation not requiring priming phosphorylation in contrast to PknB. Our results provide new insights into these two key enzymes relevant for Mtb and the mechanisms of serine/threonine phosphorylation in bacteria.

In ovo threonine supplementation affects ileal gene expression of nutrient transporters in broilers inoculated post-hatch with Salmonella Enteritidis.

The effect of in ovo threonine (Thr) supplementation on the ileal expression of glucose, peptide and amino acid transporters was assessed in Salmonella Enteritidis-challenged broiler chicks. At 17.5 days of incubation, fertile eggs were supplemented in the amniotic fluid with sterile saline or 3.5% threonine. Hatchlings were individually weighed, and Salmonella Enteritidis negative status was confirmed. At 2 days of age, half of the birds of each group were inoculated with sterile nutrient broth or Salmonella Enteritidis inoculum. Relative expression of sodium-dependent glucose transporter 1 (SGLT1), glucose transporter 2 (GLUT2), di- and tri-peptide transporter 1 (PepT1) and alanine, serine, cysteine, threonine transporter (ASCT1) was assessed at hatch, 2 and 9 days of age, i.e., before inoculation and 7 days post-inoculation (dpi). At 9 days of age (7dpi), threonine increased SGLT1 and GLUT2 expression, whereas GLUT2 expression decreased in Salmonella-challenged birds. There was a significant interaction between threonine and Salmonella for PepT1 and ASCT1. Threonine increased PepT1 expression only in non-challenged birds. In addition, in ovo supplementation increased expression of ASCT1 regardless of post-hatch inoculation; Salmonella inoculation resulted in decreased expression of ASCT1 only in supplemented birds. The results suggest that while intra-amniotic threonine administration in broiler embryos increases the expression of genes related to the absorption of monosaccharides and amino acids, Salmonella challenge may negatively affect the expression of protein related transporters in the ileum of broilers.

Cadmium-induced hypertension is associated with renal myosin light chain phosphatase inhibition via increased T697 phosphorylation and p44 mitogen-activated protein kinase levels.

Dietary intake of the heavy metal cadmium (Cd2+) is implicated in hypertension, but potassium supplementation reportedly mitigates hypertension. This study aims to elucidate the hypertensive mechanism of Cd2+. Vascular reactivity and protein expression were assessed in Cd2+-exposed rats for 8 weeks to determine the calcium-handling effect of Cd2+ and the possible signaling pathways and mechanisms involved. Cd2+ induced hypertension in vivo by significantly (p < 0.001) elevating systolic blood pressure (160 ± 2 and 155 ± 1 vs 120 ± 1 mm Hg), diastolic blood pressure (119 ± 2 and 110 ± 1 vs 81 ± 1 mm Hg), and mean arterial pressure (133 ± 2 and 125 ± 1 vs 94 ± 1 mm Hg) (SBP, DBP, and MAP, respectively), while potassium supplementation protected against elevation of these parameters. The mechanism involved augmentation of the phosphorylation of renal myosin light chain phosphatase targeting subunit 1 (MYPT1) at threonine 697 (T697) (2.58 ± 0.36 vs 1 ± 0) and the expression of p44 mitogen-activated protein kinase (MAPK) (1.78 ± 0.20 vs 1 ± 0). While acetylcholine (ACh)-induced relaxation was unaffected, 5 mg/kg b.w. Cd2+ significantly (p < 0.001) attenuated phenylephrine (Phe)-induced contraction of the aorta, and 2.5 mg/kg b.w. Cd2+ significantly (p < 0.05) augmented sodium nitroprusside (SNP)-induced relaxation of the aorta. These results support the vital role of the kidney in regulating blood pressure changes after Cd2+ exposure, which may be a key drug target for hypertension management. Given the differential response to Cd2+, it is apparent that its hypertensive effects could be mediated by myosin light chain phosphatase (MLCP) inhibition via phosphorylation of renal MYPT1-T697 and p44 MAPK. Further investigation of small arteries and the Rho-kinase/MYPT1 interaction is recommended.

Threonine incorporation in tissues of growing broiler chickens using L-[15 N] threonine.

Most amino acid requirement trials appear for whole-body responses, but there is little information concerning amino acid incorporation in individual tissues, which may vary according to the age. L-[15 N] threonine was used to evaluate its incorporation rate and distribution among broiler tissues in different ages. Seventy-two male broiler chickens were distributed into three different phases: starter (4 to 9 days old), grower (18 to 23 days old) and finisher phase (32 to 37 days old). L-[15 N] threonine was added on balanced diets, and birds were fed for five days in each phase. Enriched samples of breast muscle, feathers, liver, jejunum and plasma were collected at 0, 6, 12, 24, 48, 72, 96 and 120 hr after fed birds with the tracer in each phase. In the tissues were analysed dry matter, nitrogen and stable nitrogen. The 15 N isotope abundance according to the time was fitted into exponential or linear equations using a same intercept. The ratio of the steepness or slope coefficients was determined to compare the L-[15 N] threonine incorporation according to the age. In addition, L-[15 N] threonine mass balances were performed to assess the L-[15 N] threonine distribution among the evaluated tissues. Except for feathers, the L-[15 N] threonine incorporation rate decreased with ageing. Taking into account the L-[15 N] threonine distribution in the tissues, only in the jejunum was not observed an increase as the broiler grew. The L-[15 N] incorporation varied in each tissue and according to the age of the broiler chickens. These outcomes could be useful to comprehend changes in amino acid requirements tissue-specific according to age.

A Novel Mutation in the FSH Receptor (I423T) Affecting Receptor Activation and Leading to Primary Ovarian Failure.

CONTEXT: Follicle-stimulating hormone (FSH) plays an essential role in gonadal function. Loss-of-function mutations in the follicle-stimulating hormone receptor (FSHR) are an infrequent cause of primary ovarian failure. OBJECTIVE: To analyze the molecular physiopathogenesis of a novel mutation in the FSHR identified in a woman with primary ovarian failure, employing in vitro and in silico approaches, and to compare the features of this dysfunctional receptor with those shown by the trafficking-defective D408Y FSHR mutant. METHODS: Sanger sequencing of the FSHR cDNA was applied to identify the novel mutation. FSH-stimulated cyclic adenosine monophosphate (cAMP) production, ERK1/2 phosphorylation, and desensitization were tested in HEK293 cells. Receptor expression was analyzed by immunoblotting, receptor-binding assays, and flow cytometry. Molecular dynamics simulations were performed to determine the in silico behavior of the mutant FSHRs. RESULTS: A novel missense mutation (I423T) in the second transmembrane domain of the FSHR was identified in a woman with normal pubertal development but primary amenorrhea. The I423T mutation slightly impaired plasma membrane expression of the mature form of the receptor and severely impacted on cAMP/protein kinase A signaling but much less on ß-arrestin-dependent ERK1/2 phosphorylation. Meanwhile, the D408Y mutation severely affected membrane expression, with most of the FSH receptor located intracellularly, and both signal readouts tested. Molecular dynamics simulations revealed important functional disruptions in both mutant FSHRs, mainly the loss of interhelical connectivity in the D408Y FSHR. CONCLUSIONS: Concurrently, these data indicate that conformational differences during the inactive and active states account for the distinct expression levels, differential signaling, and phenotypic expression of the I423T and D408Y mutant FSHRs.

Multivariate modeling strategies to predict nutritional requirements of essential amino acids in semiheavy second-cycle hens

An experiment with 23 diets was performed to evaluate the effect of digestible lysine (Lys), digestible methionine + cysteine (Met+Cys), and digestible threonine (Thr) on egg production of H&N Brown second-cycle laying hens (SCLH) for 20 weeks (92-111 weeks of age) in cages under environmental conditions. Body weight (BW), feed intake (FI), feed conversion ratio (FCR), egg weight (EW), number of hen-housed eggs, and livability were also evaluated during the experiment. Diets were formulated from a central composite design that combined five levels of Lys, Met+Cys, and Thr ranging from 727 to 1159, 662 to 1055, and 552 to 882 mg/kg, respectively. Egg production (EP) data were evaluated through three different modeling strategies: egg production models, multivariate polynomial models, and artificial neural networks (ANN). A cascade-forward neural network with logsigmoid transfer function was selected as the best model according to goodness-offit statistics in both identification and validation data. One of the best scenarios for EP of H&N Brown SCLH under specific outdoor conditions was established at Lys, Met+Cys, and Thr levels of 1138, 1031, and 717 mg/hen·day, respectively. The ANN model may be an appropriate tool to study and predict EP of H&N Brown SCLH based on the combination of three different levels of essential digestible amino acids. The strategies included in this work may contribute to improving poultry performance based on modeling techniques to study other production parameters in terms of different nutritional requirements and productive conditions.

Validation of the T86I mutation in the gyrA gene as a highly reliable real time PCR target to detect Fluoroquinolone-resistant Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking. METHODS: C. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene. RESULTS: We detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates. CONCLUSIONS: Detection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.

A linker of the proline-threonine repeating motif sequence is bimodal.

The linker of the endoglucanase from Xanthomonas campestris pv. campestris ((PT)12) has a specific sequence, a repeating proline-threonine motif. In order to understand its role, it has been compared to a regular sequence linker, in this work-the cellobiohydrolase 2 from Trichoderma reesei (CBH2). Elastic properties of the two linkers have been estimated by calculating free energy profile along the linker length from an enhanced sampling molecular dynamics simulation. The (PT)12 exhibits more pronounced elastic behaviour than CBH2. The PT repeating motif results in a two-mode energy profile which could be very useful in the enzyme motions along the substrate during hydrolytic catalysis.

Protein Phosphorylation in Serine Residues Correlates with Progression from Precancerous Lesions to Cervical Cancer in Mexican Patients.

Protein phosphorylation is a posttranslational modification that is essential for normal cellular processes; however, abnormal phosphorylation is one of the prime causes for alteration of many structural, functional, and regulatory proteins in disease conditions. In cancer, changes in the states of protein phosphorylation in tyrosine residues have been more studied than phosphorylation in threonine or serine residues, which also undergo alterations with greater predominance. In general, serine phosphorylation leads to the formation of multimolecular signaling complexes that regulate diverse biological processes, but in pathological conditions such as tumorigenesis, anomalous phosphorylation may result in the deregulation of some signaling pathways. Cervical cancer (CC), the main neoplasm associated with human papillomavirus (HPV) infection, is the fourth most frequent cancer worldwide. Persistent infection of the cervix with high-risk human papillomaviruses produces precancerous lesions starting with low-grade squamous intraepithelial lesions (LSIL), progressing to high-grade squamous intraepithelial lesions (HSIL) until CC is generated. Here, we compared the proteomic profile of phosphorylated proteins in serine residues from healthy, LSIL, HSIL, and CC samples. Our data show an increase in the number of phosphorylated proteins in serine residues as the grade of injury rises. These results provide a support for future studies focused on phosphorylated proteins and their possible correlation with the progression of cervical lesions.