Effects of the GingivalStat approach on early gingival margin remodeling following esthetic clinical crown lengthening: A case series.

OBJECTIVES: The aim of this case series is to present the potential applications of the GingivalStat approach, that is, the use of temporary gingival stabilizers, to favor early gingival margin remodeling and prevent the occurrence of gingival rebound following esthetic clinical crown lengthening. CLINICAL CONSIDERATIONS: Four patients requiring clinical crown lengthening were treated for esthetical and functional reasons. The surgical approach included: (a) gingival margin recontouring; (b) full-thickness flap elevation; (c) osteotomy (to achieve an adequate dimension between the alveolar bone crest and the CEJ) and osteoplasty (to reduce the bone thickness and improve the buccal bone anatomic profile, where indicated); (d) temporary gingival stabilizer placement using a block-out resin or a composite (the GingivalStat approach); and (e) flap repositioning, adaptation, and suture. One- to five-year follow-ups, reported in the different case scenarios, show evidence of clinically stable gingival margins around the treated teeth. CONCLUSIONS: Within the limits of this case series, it can be concluded that the GingivalStat approach appears as a further maneuver to cope with clinical crown lengthening procedures at esthetic sites. GingivalStat seems to favor gingival margin contour remodeling during the early phase of healing as well as prevent the occurrence of gingival rebound. CLINICAL SIGNIFICANCE: GingivalStat approach may guide gingival margin remodeling and prevent gingival rebound after wound healing of sites submitted to esthetic clinical crown lengthening.

Errors and complications in clinical periodontal practice due to methodologic bias and bad interpretation of the evidence.

Different types of errors and complications may arise during and after the execution of periodontal or implant-related procedures. Some of the most relevant, although also controversial, and less commented, causative agents of errors and complications are methodological biases and bad interpretation of the evidence. Proper assessment of the literature requires of solid clinical knowledge combined with a systematic approach built on the recognition of common methodological biases and the avoidance of interpretive errors to critically retrieve, dissect, and judiciously apply available information for the promotion of periodontal and peri-implant health. This review addresses common types of methodological bias and interpretive errors that can alter the reader's perceptions on the real effect and potential ramifications of the reported outcomes of a given therapeutic approach due to bad interpretation of the available evidence: (1) types of methodological biases; (2) spin and interpretive bias; (3) interpretation pitfalls when assessing the evidence (4) choice of relevant endpoints to answer the question(s) of interest; and (5) balance between statistical significance and clinical relevance.

Periodontal phenotype modification of complexes periodontal-orthodontic case scenarios: A clinical review on the applications of allogenous dermal matrix as an alternative to subepithelial connective tissue graft.

OBJECTIVES: The aim of this review is to address the potential applications of allogenous dermal matrix (ADM), as an alternative to subepithelial connective tissue graft (SCTG), in promoting periodontal phenotype modification (PPM) of challenging periodontal-orthodontic clinical scenarios. OVERVIEW: The rationale behind the need of changing thin to thick gingival tissues is associated to the superior and more stable treatment outcomes promoted by PPM therapy. PPM, via soft tissue grafting, leads to clinical and histological changes of the pre-established original genetic conditions of the gingiva. Although SCTG-based procedures are recognized as the "gold standard" for the treatment of sites requiring root coverage and gingival augmentation, ADM has been recognized as the most suitable alternative to SCTG, particularly in clinical scenarios where the use of autogenous grafts is not possible. Thus, ADM is considered an optimal option for the treatment of patients with a history (or in need) of orthodontic tooth movement, due its two-fold potential indication: (1) the promotion of periodontal soft tissue phenotype modification; and (2) its use, as a barrier membrane, in hard tissues augmentation procedures. CONCLUSIONS: ADM is a viable option for soft tissue augmentation, as well as for treatment approaches involving buccal bone gain. CLINICAL SIGNIFICANCE: Periodontal phenotype modification therapy, when applied in challenging periodontal-orthodontic clinical scenarios, promotes root coverage and prevents the onset and development clinical attachment loss.

Active alcohol consumption is associated with acute-on-chronic liver failure in Hispanic patients.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe clinical entity associated with elevated short-term mortality. We aimed to characterize patients with decompensated cirrhosis according to presence of ACLF, their association with active alcohol intake, and long-term survival in Latin America. METHODS: Retrospective cohort study of decompensated cirrhotic in three Chilean university centers (2017-2019). ACLF was diagnosed according EASL-CLIF criteria. We assessed survival using competing-risk and time-to-event analyses. We evaluated the time to death using accelerated failure time (AFT) models. RESULTS: We included 320 patients, median age of 65.3±11.7 years old, and 48.4% were women. 92 (28.7%) patients met ACLF criteria (ACLF-1: 29.3%, ACLF-2: 27.1%, and ACLF-3: 43.4%). The most common precipitants were infections (39.1%), and the leading organ failure was kidney (59.8%). Active alcohol consumption was frequent (27.7%), even in patients with a prior diagnosis of non-alcoholic fatty liver disease (NAFLD) (16.2%). Ninety-two (28.7%) patients had ACLF (ACLF-1: 8.4%, ACLF-2: 7.8%, and ACLF-3: 12.5%). ACLF patients had a higher MELD-Na score at admission (27 [22-31] versus 16 [12-21], p<0.0001), a higher frequency of alcohol-associated liver disease (36.7% versus 24.9%, p=0.039), and a more frequent active alcohol intake (37.2% versus 23.8%, p=0.019). In a multivariate model, ACLF was associated with higher mortality (subdistribution hazard ratio 1.735, 95%CI: 1.153-2.609; p<0.008). In the AFT models, the presence of ACLF during hospitalization correlated with a shorter time to death: ACLF-1 shortens the time to death by 4.7 times (time ratio [TR] 0.214, 95%CI: 0.075-0.615; p<0.004), ACLF-2 by 4.4 times (TR 0.224, 95%CI: 0.070-0.713; p<0.011), and ACLF-3 by 37 times (TR 0.027, 95%CI: 0.006-0.129; p<0.001). CONCLUSIONS: Patients with decompensated cirrhosis and ACLF exhibited a high frequency ofactive alcohol consumption. Patients with ACLF showed higher mortality and shorter time todeath than those without ACLF.

Plant responses to heterogeneous salinity: agronomic relevance and research priorities.

BACKGROUND: Soil salinity, in both natural and managed environments, is highly heterogeneous, and understanding how plants respond to this spatiotemporal heterogeneity is increasingly important for sustainable agriculture in the era of global climate change. While the vast majority of research on crop response to salinity utilizes homogeneous saline conditions, a much smaller, but important, effort has been made in the past decade to understand plant molecular and physiological responses to heterogeneous salinity mainly by using split-root studies. These studies have begun to unravel how plants compensate for water/nutrient deprivation and limit salt stress by optimizing root-foraging in the most favourable parts of the soil. SCOPE: This paper provides an overview of the patterns of salinity heterogeneity in rain-fed and irrigated systems. We then discuss results from split-root studies and the recent progress in understanding the physiological and molecular mechanisms regulating plant responses to heterogeneous root-zone salinity and nutrient conditions. We focus on mechanisms by which plants (salt/nutrient sensing, root-shoot signalling and water uptake) could optimize the use of less-saline patches within the root-zone, thereby enhancing growth under heterogeneous soil salinity conditions. Finally, we place these findings in the context of defining future research priorities, possible irrigation management and crop breeding opportunities to improve productivity from salt-affected lands.

Short-Term Effects of Kinesio Taping on Muscle Recruitment Order During a Vertical Jump: A Pilot Study.

CONTEXT: Kinesio taping is commonly used in sports and rehabilitation settings with the aim of prevention and treatment of musculoskeletal injuries. However, limited evidence exists regarding the effects of 24 and 72 hours of kinesio taping on trunk and lower limb neuromuscular and kinetic performance during a vertical jump. OBJECTIVE: The purpose of this study was to analyze the short-term effects of kinesio taping on height and ground reaction force during a vertical jump, in addition to trunk and lower limb muscle latency and recruitment order. DESIGN: Single-group pretest-posttest. SETTING: University laboratory. PARTICIPANTS: Twelve male athletes from different sports (track and field, basketball, and soccer). INTERVENTIONS: They completed a single squat and countermovement jump at basal time (no kinesio taping), 24, and 72 hours of kinesio taping application on the gluteus maximus, biceps femoris, rectus femoris, gastrocnemius medialis, and longissimus. MAIN OUTCOME MEASURES: Muscle onset latencies were assessed by electromyography during a squat and countermovement jump, in addition to measurements of the jump height and normalized ground reaction force. RESULTS: The kinesio taping had no effect after 24 hours on either the countermovement or squat jump. However, at 72 hours, the kinesio taping increased the jump height (P = .02; d = 0.36) and normalized ground reaction force (P = .001; d = 0.45) during the countermovement jump. In addition, 72-hour kinesio taping reduced longissimus onset latency (P = .03; d = 1.34) and improved muscle recruitment order during a countermovement jump. CONCLUSIONS: These findings suggest that kinesio taping may improve neuromuscular and kinetic performance during a countermovement jump only after 72 hours of application on healthy and uninjured male athletes. However, no changes were observed on a squat jump. Future studies should incorporate a control group to verify kinesio taping's effects and its influence on injured athletes.

[Carriage of Staphylococcus aureus among food service workers]. / Portación de Staphylococcus aureus enterotoxigénico tipo A, en frotis nasofaríngeos en manipuladores de alimentos.

Background Staphylococcus aureus produces 11 serotypes of endotoxins that may cause food poisoning. Aim To determine the prevalence of type A enterotoxigenic Staphylococcus aureus carriage among food service workers in Chillan, Chile. Material and Methods Pharyngeal swabs were obtained from 100 food service workers and were cultured in Agar plates. After identifying the presence of Staphylococcus aureus, DNA was extracted to identify type A toxin by conventional PCR. Results Thirty eight percent of samples were colonized with Staphylococcus aureus. Among these, 26% were toxin A producers. Conclusions Half of the sampled workers carried Staphylococcus aureus and a quarter of these produced type A enterotoxin.

Evaluation of two novel 64Cu-labeled RGD peptide radiotracers for enhanced PET imaging of tumor integrin αvß3.

PURPOSE: Our goal was to demonstrate that suitably derivatized monomeric RGD peptide-based PET tracers, targeting integrin αvß3, may offer advantages in image contrast, time for imaging, and low uptake in nontarget tissues. METHODS: Two cyclic RGDfK derivatives, (PEG)2-c(RGDfK) and PEG4-SAA4-c(RGDfK), were constructed and conjugated to NOTA for (64)Cu labeling. Their integrin αvß3-binding properties were determined via a competitive cell binding assay. Mice bearing U87MG tumors were intravenously injected with each of the (64)Cu-labeled peptides, and PET scans were acquired during the first 30 min, and 2 and 4 h after injection. Blocking and ex vivo biodistribution studies were carried out to validate the PET data and confirm the specificity of the tracers. RESULTS: The IC50 values of NOTA-(PEG)2-c(RGDfK) and NOTA-PEG4-SAA4-c(RGDfK) were 444 ± 41 nM and 288 ± 66 nM, respectively. Dynamic PET data of (64)Cu-NOTA-(PEG)2-c(RGDfK) and (64)Cu-NOTA-PEG4-SAA4-c(RGDfK) showed similar circulation t 1/2 and peak tumor uptake of about 4 %ID/g for both tracers. Due to its marked hydrophilicity, (64)Cu-NOTA-PEG4-SAA4-c(RGDfK) provided faster clearance from tumor and normal tissues yet maintained excellent tumor-to-background ratios. Static PET scans at later time-points corroborated the enhanced excretion of the tracer, especially from abdominal organs. Ex vivo biodistribution and receptor blocking studies confirmed the accuracy of the PET data and the integrin αvß3-specificity of the peptides. CONCLUSION: Our two novel RGD-based radiotracers with optimized pharmacokinetic properties allowed fast, high-contrast PET imaging of tumor-associated integrin αvß3. These tracers may facilitate the imaging of abdominal malignancies, normally precluded by high background uptake.

FITC-conjugated cyclic RGD peptides as fluorescent probes for staining integrin αvß3/αvß5 in tumor tissues.

This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvß3/αvß5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvß3/αvß5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvß3/αvß5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1-0.3 g. Tumors were harvested for integrin αvß3/αvß5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvß3/αvß5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvß3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin ß3 antibody, their tumor localization and tumor cell binding are integrin αvß3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvß3/αvß5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin ß3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD2 (an integrin αvß3/αvß5-targeted radiotracer) and the relative integrin αvß3/αvß5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvß3/αvß5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvß3/αvß5 expression levels in tumor tissues.

(99m)Tc-Galacto-RGD2: a novel 99mTc-labeled cyclic RGD peptide dimer useful for tumor imaging.

This study sought to evaluate [(99m)Tc(HYNIC-Galacto-RGD2)(tricine)(TPPTS)] ((99m)Tc-Galacto-RGD2: HYNIC = 6-hydrazinonicotinyl; Galacto-RGD2 = Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(SAA-PEG2-(1,2,3-triazole)-1-yl-4-methylamide)]]2 (SAA = 7-amino-L-glycero-L-galacto-2,6-anhydro-7-deoxyheptanamide, and PEG2 = 3,6-dioxaoctanoic acid); and TPPTS = trisodium triphenylphosphine-3,3',3″-trisulfonate) as a new radiotracer for tumor imaging. Galacto-RGD2 was prepared via the copper(I)-catalyzed 1,3-dipolar azide-alkyne Huisgen cycloaddition. HYNIC-Galacto-RGD2 was prepared by reacting Galacto-RGD2 with sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (HYNIC-OSu) in the presence of diisopropylethylamine, and was evaluated for its integrin αvß3 binding affinity against (125)I-echistatin bound to U87MG glioma cells. The IC50 value for HYNIC-Galacto-RGD2 was determined to be 20 ± 2 nM. (99m)Tc-Galacto-RGD2 was prepared in high specific activity (∼ 185 GBq/µmol) and high radiochemical purity (>95%), and was evaluated in athymic nude mice bearing U87MG glioma xenografts for its tumor-targeting capability and biodistribution. The tumor uptake of (99m)Tc-Galacto-RGD2 was 10.30 ± 1.67, 8.37 ± 2.13, 6.86 ± 1.33, and 5.61 ± 1.52%ID/g at 5, 30, 60, and 120 min p.i., respectively, which was in agreement with high integrin αvß3 expression on glioma cells and neovasculature. Its lower uptake in intestines, lungs, and spleen suggests that (99m)Tc-Galacto-RGD2 has advantages over (99m)Tc-3P-RGD2 ([(99m)Tc(HYNIC-3P-RGD2)(tricine)(TPPTS)]: 3P-RGD2 = PEG4-E[PEG4-c(RGDfK)]2; PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) for imaging tumors in the chest and abdominal regions. U87MG tumors were readily detected by SPECT and the tumor uptake of (99m)Tc-Galacto-RGD2 was integrin αvß3-specific. (99m)Tc-Galacto-RGD2 also had very high metabolic stability. On the basis of results from this study, it was concluded that (99m)Tc-Galacto-RGD2 is an excellent radiotracer for imaging integrin αvß3-positive tumors and related metastases.

Food restriction, ghrelin, its antagonist and obestatin control expression of ghrelin and its receptor in chicken hypothalamus and ovary.

The purpose of the present study was to identify the role of age, nutritional state and some metabolic hormones in control of avian hypothalamic and ovarian ghrelin/ghrelin receptor system. We examined the effect of food restriction, administration of ghrelin 1-18, ghrelin antagonistic analogue (D-Lys-3)-GHRP-6, obestatin and combinations of them on the expression of ghrelin and ghrelin receptor (GHS-R1a) in hypothalamus and ovary of old (23months of age) and young (7months of age) chickens. Expression of mRNAs for ghrelin and GHS-R1a in both hypothalamus and largest ovarian follicle was measured by RT-PCR. It was observed that food restriction could promote the expression of ghrelin and GHS-R1a in hypothalamus and ovary of the old chickens, but in the young chickens it reduced expression of ghrelin and did not affect expression of GHS-R1a in the ovary. Administration of ghrelin 1-18 did not affect hypothalamic or ovarian ghrelin mRNA, but significantly increased the expression of GHS-R1a in hypothalamus, but not in ovary. (D-Lys-3)-GHRP-6, significantly stimulated accumulation of ghrelin, but not GHS-R1a mRNA in hypothalamus or ghrelin or GHS-R1a in the ovary. Ghrelin 1-18 and (D-Lys-3)-GHRP-6, when given together, were able either to prevent or to induce effect of these hormones. Obestatin administration increased expression of ghrelin gene in the hypothalamus, but not expression of hypothalamic GHS-R1a, ovarian ghrelin and GHS-R1a. Furthermore, obestatin was able to modify effect of both ghrelin and fasting on hypothalamic and ovarian mRNA for ghrelin GHS-R1a. Our results (1) confirm the existence of ghrelin and its functional receptors GHS-R1a in the chicken hypothalamus and ovary (2) confirm the age-dependent control of ovarian ghrelin by feeding, (3) demonstrate, that nutritional status can influence the expression of both ghrelin and GHS-R1a in hypothalamus and in the ovary (3) demonstrates for the first time, that ghrelin can promote generation of its functional receptor in the hypothalamus, but not in the ovary, (4) show that ghrelin1-18 and (D-Lys-3)-GHRP-6 could not only be antagonists in the action on chicken hypothalamus and ovaries, but also independent regulators and even agonists, and (5) provide first evidence for action of obestatin on hypothalamic ghrelin and on the response of hypothalamic and ovarian ghrelin/GHS-R1a system to food restriction. These data indicate the involvement of both hypothalamic and ovarian ghrelin/GHS-R1 systems in mediating the effects of nutritional status, ghrelin and obestatin on reproductive processes.

Use of pyrogallol red and pyranine as probes to evaluate antioxidant capacities towards hypochlorite.

Hypochlorite is a strong oxidant able to induce deleterious effects in biological systems. The goal of this work was to investigate the use of PGR and PYR as probes in assays aimed at evaluating antioxidant activities towards hypochorite and apply it to plant extracts employed in Chilean folk medicine. The consumption of PGR and PYR was evaluated from the decrease in the visible absorbance and fluorescence intensity, respectively. Total phenolic content was determined by the Folin Ciocalteau assay. PGR and PYR react with hypochlorite with different kinetics, being considerably faster the consumption of PGR. Different stoichiometric values were also determined: 0.7 molecules of PGR and 0.33 molecules of PYR were bleached per each molecule of added hypochlorite. Both probes were protected by antioxidants, but the rate of PGR bleaching was too fast to perform a kinetic analysis. For PYR, the protection took place without changes in its initial consumption rate, suggesting a competition between the dye and the antioxidant for hypochlorite. Plant extracts protected PYR giving a PYR-HOCl index that follows the order: Fuchsia magellanica ≈ Marrubium vulgare ≈ Tagetes minuta > Chenopodium ambrosoides ≈ Satureja montana > Thymus praecox. Based on both the kinetic data and the protection afforded by pure antioxidants, we selected PYR as the best probe. The proposed methodology allows evaluating an antioxidant capacity index of plant extracts related to the reactivity of the samples towards hypochlorite.

Osmotic Demyelination Syndrome in a Normonatremic Patient Under Treatment With Proton Pump Inhibitors.

A 66-year-old woman was admitted to the emergency department with diarrhea, nausea, and vomiting as well as low-grade fever. She was initially treated with ciprofloxacin and metronidazole with symptomatic improvement and was discharged. One week later, she returned to the emergency department for gait instability, dizziness, and vomiting and had a witnessed generalized tonic-clonic seizure in the hospital. During both admissions, the presence of ionic alterations such as severe hypomagnesemia, hypophosphatemia, and hypokalemia stood out, while sodium levels remained normal. Among her antecedents, she had a hiatal hernia and had been receiving treatment with omeprazole for years.

Probing the Binding Mechanism of Acylated Peptides to Human Serum Albumin.

Peptides represent an increasingly important class of pharmaceutical products. During the last decade or so, acylation with fatty acids has demonstrated considerable success in prolonging the circulating half-life of therapeutic peptides by exploiting the ability of fatty acids to reversibly bind to human serum albumin (HSA), thus significantly impacting their pharmacological profiles. Employing methyl-13C-labeled oleic acid or palmitic acid as probe molecules and exploiting HSA mutants designed to probe fatty acid binding, the signals in two-dimensional (2D) nuclear magnetic resonance (NMR) spectra corresponding to high-affinity fatty acid binding sites in HSA were assigned. Subsequently, using a set of selected acylated peptides, competitive displacement experiments by 2D NMR identified a primary fatty acid binding site in HSA utilized in acylated peptide binding. These results represent an important first step toward understanding the structural basis for acylated peptides binding to HSA.

Low 2-methoxyestradiol levels at the first trimester of pregnancy are associated with the development of pre-eclampsia.

OBJECTIVE: To determine whether maternal plasma levels of 2-methoxyestradiol (2-ME) are decreased early in pregnancies that subsequently develop pre-eclampsia (PE) and whether this difference could be attributed to the presence of Val158Met catechol-O-methyltransferase (COMT) polymorphism in the placenta. METHODS: Clinical characteristics and plasma samples were collected at 11 to 14 weeks prospectively in a cohort of patients. From them, 13 PE and 72 control pregnant women were chosen. Plasma soluble fms-like tyrosine kinase1 and placental growth factor levels were measured by electrochemiluminescence and 2-ME was measured by high-performance liquid chromatography with mass spectrometry/mass spectrometry detection. At delivery, placental tissue was collected and the Val158Met COMT polymorphism was determined by restriction fragment length polymorphism-PCR. RESULTS: At 11 to 14 weeks, patients who would develop PE have significantly lower plasma levels of 2-ME than controls [1.9 ± 2 standard error of the mean (SEM) vs 61.7 ± 27 pg/mL, P < 0.05]. The Val158Met polymorphism was more frequent in controls than in PE patients and the placental presence of COMT polymorphism was associated with a decreased risk of developing PE [PE: 23.1% vs control: 66.6%; χ(2) = 10.9, p = 0.0041]. CONCLUSIONS: Lower plasma concentrations of 2-ME during early pregnancy in patients who subsequently develop PE were found. Presence of placental Val158Met COMT polymorphism is associated with a decreased risk to develop PE, suggesting a protective role against PE.

Photoacoustic imaging reveals mechanisms of rapid-acting insulin formulations dynamics at the injection site.

OBJECTIVE: Ultra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog® and Lyumjev® formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. METHODS: We employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. RESULTS: Through experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. CONCLUSIONS: This study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and the formulation excipients, which could be useful to make faster and better controlled insulin formulations in the future.

Effect of inhibitor and activator of ghrelin receptor (GHS-R1a) on porcine ovarian granulosa cell functions.

It was previously shown, that ghrelin and its agonistic analogue, ghrelin 1-18, can be a stimulator of ovarian cell functions (promoter of proliferation, inhibitor of apoptosis and stimulator of hormones release). The aim of our studies was to compare the action of two ghrelin analogues - ghrelin 1-18, activator of ghrelin receptors (GHS-R1a), and (D-Lys3)-GHRP-6, its inhibitor, on porcine ovarian granulosa cell functions. Effects of (D-Lys3)-GHRP-6 added at doses of 0, 1, 10 or 100 ng/ml on the expression of markers of proliferation (PCNA, cyclin B1, MAPK/ERK1,2), apoptosis (bax, p53, caspase 3) and release of steroid hormones (progesterone, testosterone, estradiol) were examined. In addition, some effect of ghrelin 1-8 on some of these parameters (expression of MAPK/ERK1,2, bax, p53) were verified. It was shown, that (D-Lys3)-GHRP-6 promotes all markers of granulosa cell proliferation, inhibits all markers of apoptosis and stimulates the release of all three steroid hormones. Similar effects of (D-Lys3)-GHRP-6 (inhibitor of GHS-R1a) and ghrelin 1-18 (its stimulator) suggest that the examined effects of these substances on porcine ovaries are not mediated by GHS-R1a. Both chemical analogues could be potentially useful for stimulation of reproductive processes, at least in in vitro conditions.

The effects of time and dose of pregnant mare serum gonadotropin (PMSG) on reproductive efficiency in hair sheep ewes.

The aim of this study was to evaluate the effects of dose and application time of pregnant mare serum gonadotropin (PMSG) on reproductive performance of hair sheep ewes synchronized with fluorogesterone acetate (FGA) under tropical conditions of Northeastern Mexico. Ninety-nine hair ewes (63 Blackbelly and 36 Pelibuey) were treated with intravaginal sponges during 10 days. After insertion of FGA sponges, ewes were divided into four groups, and PMSG was injected intramuscularly at doses of 100, 200, and 400 IU. Relative to FGA sponge removal, PMSG was administrated at -48 h, -24 h, and at sponge removal. PMSG was not administered to the control group. Control ewes had similar (P > 0.05) lambing rate, fertility, and fecundity than those treated with 100 IU of PMSG, but lower (P < 0.05) percentages to these variables than those treated with 200 and 400 IU of PMSG. Time to estrus decreased linearly, and ovulation rate increased quadratically as PMSG dose increased (0 to 400 IU). Administration of PMSG before sponge removal increased (P < 0.01) response to estrus and decreased (P < 0.01) interval to estrus compared with control. Ovulation rate, lambing rate, fertility, and fecundity were not affected (P > 0.05) by administration time of PMSG. Both dose and time of PMSG application did not affect (P > 0.05) pregnancy rate, percentage of single and multiple lambing, and prolificacy. In conclusion, results show that the dose of 400 IU of PMSG administered before sponge withdrawal in an estrus synchronization protocol improved reproductive efficiency of hair sheep ewes.

Characterization of viral insulins reveals white adipose tissue-specific effects in mice.

OBJECTIVE: Members of the insulin/insulin-like growth factor (IGF) superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single-chain (sc), i.e., IGF-1-like, forms of the viral insulin/IGF-1-like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e., more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1) for the first time. METHODS: The dcVILPs were chemically synthesized. Using murine fibroblast cell lines overexpressing insulin receptor (IR-A or IR-B) or IGF1R, we first determined the binding affinity of dcVILPs to the receptors and characterized post-receptor signaling. Further, we used C57BL/6J mice to study the effect of dcVILPs on lowering blood glucose. We designed a 3-h dcVILP in vivo infusion experiment to determine the glucose uptake in different tissues. RESULTS: GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A and IR-B) and to the IGF1R, and for the latter, show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75 nmol/kg/min) stimulated a comparable glucose uptake in heart and skeletal muscle and brown adipose tissue, GIV dcVILP stimulated 2-fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin in WAT. CONCLUSIONS: Our results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogs that specifically target the tissues and provide new insights into their potential role in disease.

N-{N-[2-(3,5-Difluorophenyl)acetyl]-(S)-alanyl}-(S)-phenylglycine tert-butyl ester (DAPT): an inhibitor of γ-secretase, revealing fine electronic and hydrogen-bonding features.

The title compound, C(23)H(26)F(2)N(2)O(4), is a dipeptidic inhibitor of γ-secretase, one of the enzymes involved in Alzheimer's disease. The molecule adopts a compact conformation, without intramolecular hydrogen bonds. In the crystal structure, one of the amide N atoms forms the only intermolecular N-H...O hydrogen bond; the second amide N atom does not form hydrogen bonds. High-resolution synchrotron diffraction data permitted the unequivocal location and refinement without restraints of all H atoms, and the identification of the characteristic shift of the amide H atom engaged in the hydrogen bond from its ideal position, resulting in a more linear hydrogen bond. Significant residual densities for bonding electrons were revealed after the usual SHELXL refinement, and modeling of these features as additional interatomic scatterers (IAS) using the program PHENIX led to a significant decrease in the R factor from 0.0411 to 0.0325 and diminished the r.m.s. deviation level of noise in the final difference Fourier map from 0.063 to 0.037 e Å(-3).