Guardians' Perceptions of Dogs' Welfare and Behaviors Related to Visiting the Veterinary Clinic

A large survey of Italian dog guardians (n = 906) was conducted to assess dog behavior and welfare at the veterinary clinic and to investigate how guardians and veterinarians affect them. This study confirmed that the veterinary clinic is a source of stress for most dogs, who showed impaired welfare in all phases: in the waiting room, entering the examination room, on the examination table, and when approached by the vet. This study also characterizes some factors related to the guardians' and veterinarians' behavior that affect the dogs' behavior and welfare during the veterinary examination. If dogs had not been examined by a vet since puppyhood, if they did not accept treatments by their guardians, and if they were scolded when refusing a treatment, the risk for having problems with dog welfare and behavior at the veterinary clinic increased. The attention paid by the vet to the dog was found to be positively related with a good response of the dog to the vet. Prevention seems to be the key for the protection of dog welfare related to veterinary care.     

Preliminary analysis of reproductive success in a large mammal with alternative mating tactics,the Northern chamois,Rupicapra rupicapra

In polygynous mating systems, reproductive skew depends on the ability of males to monopolize females, which in turn may promote the development of contrasting traits in the two sexes. Although dominant individuals normally enjoy a higher reproductive success (RS) than subordinates, the use of genetic markers has shown that behavioural observations of male mating success may not provide reliable clues of RS. We report the preliminary results of the first DNA‐based paternity analysis on the Northern chamois (Rupicapra rupicapra), a scarcely dimorphic mountain ungulate described as highly polygynous, in relation to mating tactic and age. Because of sampling difficulties, the success in parentage assignment was low, and the interpretation of results requires caution. Territorial males had a greater RS than nonterritorial ones but they were unable to monopolize mating events. Age had a weak effect on paternity outcome but only males ≥ 6 years showed siring success. Although future studies are needed to assess the opportunity for sexual selection in male chamois, the concurrence of limited sexual size dimorphism, compensatory growth, unbiased sex‐specific survival, RS of alternative mating tactics and, possibly, long breeding tenure, may hint at the adoption of a conservative mating strategy in this species.     

Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement

We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.     

Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS

The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.     

New platinum-oxicam complexes as anti-cancer drugs. Synthesis, characterization, release studies from smart hydrogels, evaluation of reactivity with selected proteins and cytotoxic activity in vitro

The reaction of aqueous cis-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ with Na⁺HMEL⁻ (H₂MEL, meloxicam, 4-hydroxy-2-methyl-N-(5-methyl-1,3-thiazol-2-yl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide), and Na⁺HISO⁻ (H₂ISO, isoxicam, 4-hydroxy-2-methyl-N-(5-methylisoxazol-3-yl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide) at pH 7 produced micro-crystalline cis-[Pt(NH₃)₂(N^1′-HMEL)₂], 5 and cis-[Pt(NH₃)₂(N^1′-HISO)₂], 6. The X-ray diffraction structure of 5 shows two HMEL⁻ anions donating through the thiazole nitrogen atoms and adopting a head-to-tail (HT) conformation. The ¹H NMR spectrum for 5 from DMSO-d₆ shows inertness of the complex up to at least 24 h. Delivery studies for 5 and 6 from vinyl hydrogel based on l-phenylalanine (pH 6.5, 25 °C) show that concentrations of complexes ranging between 2.5 and 5 μM can be reached after a day. Compounds 5 and 6 show strong anti-proliferative effects on CH1 cells (ovarian carcinoma, human) in vitro, IC₅₀ values being 0.60 and 0.37 μM, respectively (0.16 μM for reference, cis-diamminodichloridoplatinum(II), cisplatin). ESI-MS measurements clearly documented that both 5 and 6 form adducts with the three model proteins ubiquitin (UBI), cytochrome c (CYT C) and superoxide dismutase (SOD), the HISO⁻ complex being significantly more effective than the HMEL⁻ one. Density functional methods help in finding rationale for the easiest dissociation of Pt-H₂ISO/HISO bonds when compared to the Pt-N^1′-H₂MEL/N^1′-HMEL linkages.     

Immune Regulatory Neural Stem/Precursor Cells Protect from Central Nervous System Autoimmunity by Restraining Dendritic Cell Function

Background

The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinico-pathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs.

Methodology/Principal Findings

To achieve the exclusive targeting of the peripheral immune system, SJL mice with PLP-induced EAE were injected subcutaneously with NPCs and the treatment commenced prior to disease onset. NPC-injected EAE mice showed significant clinical improvement, as compared to controls. Exogenous NPCs lacking the expression of major neural antigens were reliably (and for long-term) found at the level of draining lymph nodes, while establishing sophisticated anatomical interactions with lymph node cells. Importantly, injected NPCs were never found in organs other than lymph nodes, including the brain and the spinal cord. Draining lymph nodes from transplanted mice showed focal up-regulation of major developmental stem cell regulators, such as BMP-4, Noggin and Sonic hedgehog. In lymph nodes, injected NPCs hampered the activation of myeloid dendritic cells (DCs) and steadily restrained the expansion of antigen-specific encephalitogenic T cells. Both ex vivo and in vitro experiments identified a novel highly NPC-specific–BMP-4-dependent–mechanism hindering the DC maturation.

Conclusion/Significance

The study described herein, identifies the first member of the TGF β/BMP family of stem cell regulators as a novel tolerogenic factor released by NPCs. Full exploitation of this pathway as an efficient tool for vaccination therapy in autoimmune inflammatory conditions is underway.     

ENDOPHYTIC BACTERIA FROM SEEDS OF NICOTIANA TABACUM CAN REDUCE CADMIUM PHYTOTOXICITY

Although endophytic bacteria seem to have a close association with their host plant, little is known about the influence of seed endophytic bacteria on initial plant development and on their interactions with plants under conditions of metal toxicity. In order to further elucidate this close relationship, we isolated endophytic bacteria from surface sterilized Nicotiana tabacum seeds that were collected from plants cultivated on a cadmium-(Cd) and zinc-enriched soil. Many of the isolated strains showed Cd tolerance. Sterilely grown tobacco plants were inoculated with either the endogenous microbial consortium, composed of cultivable and noncultivable strains; single strains; or defined consortia of the most representative cultivable strains. Subsequently, the effects of inoculation of endophytic bacteria on plant development and on metal and nutrient uptake were explored under conditions with and without exposure to Cd. In general, seed endophytes were found to have a positive effect on plant growth, as was illustrated by an increase in biomass production under conditions without Cd. In several cases, inoculation with endophytes resulted in improved biomass production under conditions of Cd stress, as well as in a higher plant Cd concentration and total plant Cd content compared to noninoculated plants. These results demonstrate the beneficial effects of seed endophytes on metal toxicity and accumulation, and suggest practical applications using inoculated seeds as a vector for plant beneficial bacteria.     

Synthesis and antiviral evaluation of thieno[3,4-d]pyrimidine C-nucleoside analogues of 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-adenosine and -inosine

Several thieno[3,4-d]pyrimidine derivatives, including four hitherto unknown 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-C-nucleoside analogues of adenosine and inosine have been synthesized. When evaluated in cell culture experiments against human immunodeficiency virus, none of the tested compounds exhibited any significant antiviral effect, while two of them showed some cytotoxicity.     

Effects of Ambient Temperature on Sleep and Cardiovascular Regulation in Mice: The Role of Hypocretin/Orexin Neurons

The central neural pathways underlying the physiological coordination between thermoregulation and the controls of the wake-sleep behavior and cardiovascular function remain insufficiently understood. Growing evidence supports the involvement of hypocretin (orexin) peptides in behavioral, cardiovascular, and thermoregulatory functions. We investigated whether the effects of ambient temperature on wake-sleep behavior and cardiovascular control depend on the hypothalamic neurons that release hypocretin peptides. Orexin-ataxin3 transgenic mice with genetic ablation of hypocretin neurons (n = 11) and wild-type controls (n = 12) were instrumented with electrodes for sleep scoring and a telemetric blood pressure transducer. Simultaneous sleep and blood pressure recordings were performed on freely-behaving mice at ambient temperatures ranging between mild cold (20°C) and the thermoneutral zone (30°C). In both mouse groups, the time spent awake and blood pressure were higher at 20°C than at 30°C. The cold-related increase in blood pressure was significantly smaller in rapid-eye-movement sleep (REMS) than either in non-rapid-eye-movement sleep (NREMS) or wakefulness. Blood pressure was higher in wakefulness than either in NREMS or REMS at both ambient temperatures. This effect was significantly blunted in orexin-ataxin3 mice irrespective of ambient temperature and particularly during REMS. These data demonstrate that hypocretin neurons are not a necessary part of the central pathways that coordinate thermoregulation with wake-sleep behavior and cardiovascular control. Data also support the hypothesis that hypocretin neurons modulate changes in blood pressure between wakefulness and the sleep states. These concepts may have clinical implications in patients with narcolepsy with cataplexy, who lack hypocretin neurons.