Fluorophotometric Assessment of Tear Volume and Turnover Rate in Healthy Dogs and Cats

Purpose: The study establishes normative data of tear volume (TV) and tear turnover rate (TTR) in healthy dogs and cats, 2 species commonly used for translational research in ophthalmology. Methods: Thirty-six dogs and 24 cats were enrolled, encompassing a variety of breeds with diverse skull conformations (brachycephalic, mesocephalic, and dolichocephalic). Two microliters of 10% fluorescein were instilled onto the upper bulbar conjunctiva of both eyes, followed by tear collection with 2-μL capillary tubes at 0, 2, 4, 6, 10, 15, and 20 min. Fluorescein concentrations were measured with a computerized scanning ocular fluorophotometer. The TV and TTR were estimated based upon nonlinear mixed-effects analysis of fluorescein decay curves. Results: In dogs, median (interquartile range) TV, basal TTR (bTTR), and reflex TTR (rTTR) were 65.3 μL (42.3–87.9), 12.2%/min (3.7–22.1), and 50.0%/min (25.9–172.3), respectively. In cats, median (interquartile range) TV, bTTR, and rTTR were 32.1 μL (29.5–39.9), 10.9%/min (3.0–23.7), and 50.0%/min (28.4–89.4), respectively. Body weight (r = 0.44) and age (r = 0.30) were positively correlated (P ≤ 0.019) with TV in dogs. Age was negatively correlated (P ≤ 0.018) with TTR in dogs (r = −0.33) and cats (r = −0.24). However, TV and TTR were not associated with skull conformation in either species. Conclusions: Dogs have greater TV than cats but similar basal and rTTR. Tear parameters were impacted by body weight and age, but not by skull conformation. In both clinical and research settings, successive lacrimal tests should be spaced by ≥10 min to provide sufficient time for the tear film to replenish, as bTTR is ∼11%/min–12%/min in both species

Orchestrating Tuple-based Languages

The World Wide Web can be thought of as a global computing architecture supporting the deployment of distributed networked applications. Currently, such applications can be programmed by resorting mainly to two distinct paradigms: one devised for orchestrating distributed services, and the other designed for coordinating distributed (possibly mobile) agents. In this paper, the issue of designing a pro- gramming language aiming at reconciling orchestration and coordination is investigated. Taking as starting point the orchestration calculus Orc and the tuple-based coordination language Klaim, a new formalism is introduced combining concepts and primitives of the original calculi. To demonstrate feasibility and effectiveness of the proposed approach, a prototype implementation of the new formalism is described and it is then used to tackle a case study dealing with a simplified but realistic electronic marketplace, where a number of on-line stores allow client applications to access information about their goods and to place orders

Melengestrol Acetate at Greater Doses Than Typically Used for Estrous Synchrony in Bovine Females Does Not Mimic Endogenous Progesterone in Regulation of Secretion of Luteinizing Hormone and 17β-Estradiol

Our working hypothesis was that doses of melengestrol acetate (MGA) greater than those typically administered in estrous synchrony regimens would regulate secretion of LH and 17β-estradiol (E2) as endogenous progesterone (P4) does during the midluteal phase of the estrous cycle. We also hypothesized that endogenous P4 from the CL would interact with MGA to further decrease the frequency of LH pulses and E2. Cows on Day 5 of their estrous cycle (Day 0 = estrus) were randomly assigned to an untreated control group (CONT, n = 5) or to one of six MGA treatment groups (n = 5 per group): 1) MGA administered orally each day via a gelatin capsule at a dose of 0.5 mg MGA/cow with the CL present (0.5CL); 2) 0.5 mg MGA/cow daily in the absence of CL (0.5NO); 3) 1.0 mg MGA with CL present (1.OCL); 4) 1.0 mg MGA without CL (1.ONO); 5) 1.5 mg MGA with CL present (1.5CL); 6) 1.5 mg without CL (1.5NO). MGA was administered for 10 days (Day 5 = initiation of treatment). To regress CL, cows assigned to groups without CL received injections of prostaglandin F2α (PGF, 0; 25 mg) on Days 6 and 7 of their estrous cycle. All cows were administered PGF2α. at the end of the 10-day treatment period. During the treatment period, daily blood samples were collected to determine concentrations of E2. Serial blood samples were collected at 15-min intervals for 24 h on Days 8, 11, and 14 to determine pattern of LH secretion. Frequency of LH pulses on Days 8, 11, and 14 was greater (p \u3c 0.05) in cows without CL (0.5NO, 1.ONO, and 1.5NO) than in cows with CL (0.5CL, 1.OCL, 1.5CL, and CONT). Mean concentrations of LH were greater (p \u3c 0.05) in cows from the 0.5NO group on Days 8 and 11 and were greater (p \u3c 0.05) in cows from the 0.5NO, 1.ONO, and 1.5NO groups on Day 14 as compared to cows with CL. Overall mean concentrations of LH across Days 8, 11, and 14 were greatest (p \u3c 0.05) in cows from the 0.5NO group and were also greater (p \u3c 0.05) in cows from the 0.5NO, 1.ONO, and 1.5NO groups as compared to cows in the 0.5CL, 1.OCL, 1.5CL, and CONT groups. Mean concentrations of E2 during the treatment period were greater (p \u3c 0.05) in cows from the 0.5NO group than in cows from either the 1.ONO or the 1.5NO group; these values were also greater (p \u3c 0.05) in cows of the 0.5NO, 1.ONO, and 1.5NO groups as compared to cows of the 1.OCL and CONT groups. Therefore, we reject our working hypothesis because doses of MGA greater than those typically used in estrous synchrony protocols did not suppress LH and E2 to the same extent that endogenous P4 does. In addition, MGA treatment when CL were present did not result in a further suppression of LH pulse frequency or of E2 as compared to the values in control cows with functional CL

Comparison of baricitinib, upadacitinib, and tofacitinib mediated regulation of cytokine signaling in human leukocyte subpopulations

BACKGROUND: The in vitro pharmacology of baricitinib, upadacitinib, and tofacitinib was evaluated to understand differences among these JAK inhibitors (JAKis) at the cellular level. METHODS: Peripheral blood mononuclear cells from healthy donors were incubated with different JAKis, levels of phosphorylated signal transducer and activator of transcription (pSTAT) were measured following cytokine stimulation, and half maximum inhibitory concentration (IC50) values were calculated in phenotypically gated leukocyte subpopulations. Therapeutic dose relevance of the in vitro analysis was assessed using calculated mean concentration-time profiles over 24 h obtained from JAKi-treated subjects. Time above IC50 and average daily percent inhibition of pSTAT formation were calculated for each JAKi, cytokine, and cell type. RESULTS: Distinct JAKis displayed different in vitro pharmacologic profiles. For example, tofacitinib and upadacitinib were the most potent inhibitors of the JAK1/3-dependent cytokines tested (interleukin [IL]-2, IL-4, IL-15, and IL-21) with lower IC50 values and increased time above IC50 translating to a greater overall inhibition of STAT signaling during the dosing interval. All JAKis tested inhibited JAK1/2-dependent cytokines (e.g., IL-6 and interferon [IFN]-γ), the JAK1/tyrosine kinase 2 (TYK2)-dependent cytokines IL-10 and IFN-α, the JAK2/2-dependent cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the JAK2/TYK2-dependent cytokine granulocyte colony-stimulating factor (G-CSF), but often to significantly differing degrees. CONCLUSIONS: Different JAKis modulated distinct cytokine pathways to varying degrees, and no agent potently or continuously inhibited an individual cytokine signaling pathway throughout the dosing interval. Notably, baricitinib inhibited JAK1/3 signaling to a lesser extent than upadacitinib and tofacitinib, while upadacitinib, baricitinib, and tofacitinib inhibited the signaling of JAK2/2-dependent cytokines, including GM-CSF and IL-3, as well as the signaling of the JAK2/TYK2-dependent cytokine G-CSF

Conformational Plasticity of proNGF

Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central and peripheral nervous system. The unprocessed precursor of NGF, proNGF, seems to be endowed with biological functions distinct from those of the mature protein, such as chaperone-like activities and apoptotic and/or neurotrophic properties. We have previously suggested, based on Small Angle X-ray Scattering data, that recombinant murine proNGF has features typical of an intrinsically unfolded protein. Using complementary biophysical techniques, we show here new evidence that clarifies and widens this hypothesis through a detailed comparison of the structural properties of NGF and proNGF. Our data provide direct information about the dynamic properties of the pro-peptide and indicate that proNGF assumes in solution a compact globular conformation. The N-terminal pro-peptide extension influences the chemical environment of the mature protein and protects the protein from proteolytic digestion. Accordingly, we observe that unfolding of proNGF involves a two-steps mechanism. The distinct structural properties of proNGF as compared to NGF agree with and rationalise a different functional role of the precursor

Single Cycle Structure-Based Humanization of an Anti-Nerve Growth Factor Therapeutic Antibody

Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates

Phase I Hydroxylated Metabolites of the K2 Synthetic Cannabinoid JWH-018 Retain In Vitro and In Vivo Cannabinoid 1 Receptor Affinity and Activity

K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ(9)-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2's high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).JWH-018, five potential monohydroxylated metabolites (M1-M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [(3)H]CP-55,940, and then for CB1R intrinsic efficacy using an [(35)S]GTPγS binding assay. JWH-018 and M1-M5 bound CB1Rs with high affinity, exhibiting K(i) values that were lower than or equivalent to Δ(9)-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ(9)-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ(9)-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ(9)-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways

Intranasal “painless” Human Nerve Growth Factors Slows Amyloid Neurodegeneration and Prevents Memory Deficits in App X PS1 Mice

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that “painless” hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of “painless” hNGF variants as a new generation of therapeutics for neurodegenerative diseases