Synthesis of N-tetra-O-acetyl-β-d-glucopyranosyl-N′-(4′,6′-diarylpyrimidin-2′-yl)thioureas

Some 2-amino-4,6-diarylpyrimidines 2 have been prepared from substituted benzylideneacetophenones and guanidine hydrochloride in the presence of alkali by conventional heating in alcoholic medium and microwave heating in solvent-free conditions. N-(2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl)-N′-(4′,6′-diarylpyrimidin-2′-yl)thioureas 4 have been synthesized by reaction of per-O-acetylated glucopyranosyl isothiocyanate 1 and substituted 2-amino-4,6-diarylpyrimidines 2. Two different methods have been used, namely, refluxing in anhydrous dioxane and solvent-free microwave-assisted coupling. The second procedure afforded higher yields in much shorter reaction times. The compounds 2 and 4 were tested for their antibacterial and antifungal activities in vitro against Staphylococcus epidermidis, Enterobacter aerogenes and Candida albicans by disc diffusion method.     

Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism

Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.     

TPX2 Impacts Acetylation of Histone H4 at Lysine 16: Implications for DNA Damage Response

During interphase, the spindle assembly factor TPX2 is compartmentalized in the nucleus where its roles remain largely uncharacterized. Recently, we found that TPX2 regulates the levels of serine 139-phosphoryated H2AX (γ-H2AX) at chromosomal breaks induced by ionizing radiation. Here, we report that TPX2 readily associates with the chromatin in the absence of ionizing radiation. Overexpression of TPX2 alters the DAPI staining pattern of interphase cells and depletion of TPX2 constitutively decreases the levels of histone H4 acetylated at lysine16 (H4K16ac) during G1-phase. Upon ionizing irradiation, this constitutive TPX2 depletion-dependent decrease in H4K16ac levels correlates with increased levels of γ-H2AX. The inversely correlated levels of H4K16ac and γ-H2AX can also be modified by altering the levels of SIRT1, herein identified as a novel protein complex partner of TPX2. Furthermore, we find that TPX2 depletion also interferes with formation of 53BP1 ionizing radiation-induced foci, known to depend on γ-H2AX and the acetylation status of H4K16. In brief, our study is the first indication of a constitutive control of TPX2 on H4K16ac levels, with potential implications for DNA damage response.     

Conditional deletion of Shp2 tyrosine phosphatase in thymocytes suppresses both pre-TCR and TCR signals

It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4(+) T cells. Furthermore, mature Shp2(-/-) T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2(-/-) T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.     

Controlling Neglected Tropical Diseases (NTDs) in Haiti: Implementation Strategies and Evidence of Their Success

Lymphatic filariasis (LF) and soil-transmitted helminths (STH) have been targeted since 2000 in Haiti, with a strong mass drug administration (MDA) program led by the Ministry of Public Health and Population and its collaborating international partners. By 2012, Haiti’s neglected tropical disease (NTD) program had reached full national scale, and with such consistently good epidemiological coverage that it is now able to stop treatment for LF throughout almost all of the country. Essential to this success have been in the detail of how MDAs were implemented. These key programmatic elements included ensuring strong community awareness through an evidence-based, multi-channel communication and education campaign facilitated by voluntary drug distributors; strengthening community trust of the drug distributors by ensuring that respected community members were recruited and received appropriate training, supervision, identification, and motivation; enforcing a “directly observed treatment” strategy; providing easy access to treatment though numerous distribution posts and a strong drug supply chain; and ensuring quality data collection that was used to guide and inform MDA strategies. The evidence that these strategies were effective lies in both the high treatment coverage obtained– 100% geographical coverage reached in 2012, with almost all districts consistently achieving well above the epidemiological coverage targets of 65% for LF and 75% for STH—and the significant reduction in burden of infection– 45 communes having reached the target threshold for stopping treatment for LF. By taking advantage of sustained international financial and technical support, especially during the past eight years, Haiti’s very successful MDA campaign resulted in steady progress toward LF elimination and development of a strong foundation for ongoing STH control. These efforts, as described, have not only helped establish the global portfolio of “best practices” for NTD control but also are poised to help solve two of the most important future NTD challenges—how to maintain control of STH infections after the community-based LF “treatment platform” ceases and how to ensure appropriate morbidity management for patients currently suffering from lymphatic filarial disease.     

Automated tracking of wild hummingbird mass and energetics over multiple time scales using radio frequency identification (RFID) technology

We examined the feasibility of automating the collection of hummingbird mass data facilitated by low‐cost, low‐power radio frequency identification (RFID) technology. In a field study in southern Ontario, wild hummingbirds were captured, subcutaneously implanted with passive integrated transponder (PIT) tags, and released over a three‐year period. Tagged hummingbirds were detected at specially designed feeder stations outfitted with low‐cost, low‐power RFID readers coupled with a perch secured to a digital balance. When tagged birds visited the feeder, transponder detection initiated the recording of the perched hummingbird's mass at regular intervals continuing as long as the bird remained. This permitted a nearly continuous record of mass during each visit. Mass data collected from tagged hummingbirds showed consistent trends at multiple temporal scales: the individual feeder visit, single days, and even whole seasons. These results further confirm that RFID technology is safe for use in the smallest birds. The effective detection range is a function of RFID reader power, antenna, and tag size. Yet, we find that careful arrangement of feeders and detectors allows for reliable detection even when detection range is low. When coupled with additional technologies, such as a precision electronic balance, this approach can yield robust serial measures of physiological parameters such as mass, an indicator of energy balance over time.     

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.     

Temporal and Spatial Analysis of Clinical and Molecular Parameters in Dextran Sodium Sulfate Induced Colitis

Background

Inflammatory bowel diseases (IBD), including mainly ulcerative colitis (UC) and Crohn''s disease (CD), are inflammatory disorders of the gastrointestinal tract caused by an interplay of genetic and environmental factors. Murine colitis model induced by Dextran Sulfate Sodium (DSS) is an animal model of IBD that is commonly used to address the pathogenesis of IBD as well as to test efficacy of therapies. In this study we systematically analyzed clinical parameters, histological changes, intestinal barrier properties and cytokine profile during the colitic and recovery phase.

Methods

C57BL/6 mice were administered with 3.5% of DSS in drinking water for various times. Clinical and histological features were determined using standard criteria. Myeloperoxidase (MPO) activity, transepithelial permeability and proinflammatory mediators were determined in whole colon or proximal and distal parts of colon.

Results

As expected after administration of DSS, mice manifest loss of body weight, shortening of colon length and bloody feces. Histological manifestations included shortening and loss of crypts, infiltration of lymphocytes and neutrophil, symptoms attenuated after DSS withdrawal. The MPO value, as inflammation indicator, also increases significantly at all periods of DSS treatment, and even after DSS withdrawal, it still held at very high levels. Trans-mucosal permeability increased during DSS treatment, but recovered to almost control level after DSS withdrawal. The production of proinflammatory mediators by colonic mucosa were enhanced during DSS treatment, and then recovered to pre-treated level after DSS withdrawal. Finally, enhanced expression of proinflammatory mediators also revealed a different profile feature in proximal and distal parts of the colon.

Conclusion

Experimental colitis induced by DSS is a good animal model to study the mechanisms underlying the pathogenesis and intervention against IBD, especially UC.