Tobacco Alkaloid Assessment in a DSS-Induced Colitis Mouse Model with a Fully Humanized Immune System.

Inflammatory bowel disease (IBD) refers to chronic intestinal immune-mediated diseases including two main disease manifestations: ulcerative colitis (UC) and Crohn's disease (CD). Epidemiological, clinical, and preclinical evidence has highlighted the potential anti-inflammatory properties of naturally occurring alkaloids. In the present study, we investigated the potential anti-inflammatory activities of the tobacco alkaloids nicotine and anatabine in a dextran sulfate sodium (DSS)-induced UC mouse model with a fully humanized immune system. Our results show that nicotine significantly reduced all acute colitis symptoms and improved colitis-specific endpoints, including histopathologically assessed colon inflammation, tissue damage, and mononuclear cell infiltration. The tobacco alkaloid anatabine showed similar effectiveness trends, although they were generally weaker or not significant. Gene expression analysis in the context of biological network models of IBD further pinpointed a possible mechanism by which nicotine attenuated DSS-induced colitis in humanized mice. The current study enables further investigation of possible molecular mechanisms by which tobacco alkaloids attenuate UC symptoms.

GSK3732394: a Multi-specific Inhibitor of HIV Entry.

Long-acting antiretrovirals could provide a useful alternative to daily oral therapy for HIV-1-infected individuals. Building on a bi-specific molecule with adnectins targeting CD4 and gp41, a potential long-acting biologic, GSK3732394, was developed with three independent and synergistic modes of HIV entry inhibition that potentially could be self-administered as a long-acting subcutaneous injection. Starting with the bi-specific inhibitor, an α-helical peptide inhibitor was optimized as a linked molecule to the anti-gp41 adnectin, with each separate inhibitor exhibiting at least single-digit nanomolar (or lower) potency and a broad spectrum. Combination of the two adnectins and peptide activities into a single molecule was shown to have synergistic advantages in potency, the resistance barrier, and the ability to inhibit HIV-1 infections at low levels of CD4 receptor occupancy, showing that GSK3732394 can work in trans on a CD4+ T cell. Addition of a human serum albumin molecule prolongs the half-life in a human CD4 transgenic mouse, suggesting that it may have potential as a long-acting agent. GSK3732394 was shown to be highly effective in a humanized mouse model of infection. GSK3732394 is currently in clinical trials.IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1-infected patients is through the development of long-acting biologic inhibitors. Building on a bi-specific inhibitor approach targeting CD4 and gp41, a tri-specific molecule was generated with three distinct antiviral activities. The linkage of these three biologic inhibitors creates synergy that offers a series of advantages to the molecule. The addition of human serum albumin to the tri-specific inhibitor could allow it to function as a long-acting self-administered treatment for patients with HIV infection. This molecule is currently in early clinical trials.

Biopsy Induced Arteriovenous Fistula and Venous Stenosis in a Renal Transplant.

Renal transplant vein stenosis is a rare cause of allograft dysfunction. Percutaneous stenting appears to be safe and effective treatment for this condition. A 56-year-old Caucasian female with end stage renal disease received a deceased donor renal transplant. After transplant, her serum creatinine improved to a nadir of 1.2 mg/dL. During the third posttransplant month, her serum creatinine increased to 2.2 mg/dL. Renal transplant biopsy showed BK nephropathy. Mycophenolate was discontinued. Over the next 2 months, her serum creatinine crept up to 6.2 mg/dL. BK viremia improved from 36464 copies/mL to 15398 copies/mL. A renal transplant ultrasound showed lower pole arteriovenous fistula and abnormal waveforms in the renal vein. Carbon dioxide (CO2) angiography demonstrated severe stenosis of the transplant renal vein. Successful coil occlusion of fistula was performed along with angioplasty and deployment of stent in the renal transplant vein. Serum creatinine improved to 1.5 mg/dL after.

Performance of the Cockcroft-Gault and modification of diet in renal disease equations in estimating GFR in ill hospitalized patients.

BACKGROUND: Estimating glomerular filtration rate (GFR) in severely ill inpatients is clinically important for therapeutic interventions and prognosis, but notoriously difficult to do accurately. The Modification of Diet in Renal Disease (MDRD) equation and Cockcroft-Gault (CG) formula are widely used to estimate renal function in sick hospitalized patients; however, neither method has been validated in this setting. METHODS: Iodine 125-iothalamate clearances (iGFR) performed in 107 sick inpatients with renal dysfunction were compared with estimated GFRs (eGFRs) from the 6- and 4-variable MDRD (MDRD eGFR) and CG (CG eGFR) equations. RESULTS: Mean serum creatinine (SCr) level was 3.5 +/- 2.0 mg/dL (309 +/- 177 micromol/L), and mean iGFR was 17.1 +/- 17.9 mL/min/1.73 m2 (0.29 +/- 0.30 mL/s/1.73 m2). Six-variable MDRD eGFR was 22.5 +/- 17.4 mL/min/1.73 m2 (0.38 +/- 0.29 mL/s/1.73 m2), 4-variable MDRD eGFR was 23.9 +/- 16.3 mL/min/1.73 m2 (0.40 +/- 0.27 mL/s/1.73 m2), and CG eGFR was 26.0 +/- 17.1 mL/min/1.73 m2 (0.43 +/- 0.29 mL/s/1.73 m2). Blood urea nitrogen (BUN)/SCr ratios greater than 20 were seen in 58% of patients. Overall, the CG and MDRD equations overestimated iGFR, with poor agreement. Overestimation of at least 25% of measured iGFR was seen in 63%, 67%, and 70% of all inpatients when using the 6-variable MDRD, 4-variable MDRD, and CG equations, respectively. Accuracy of eGFR within 50% of measured iGFR was 55% for the 6-variable MDRD equation, 49% for the 4-variable MDRD equation, and 40% for the CG formula. The performance of both methods deteriorated further in patients with a BUN/SCr ratio greater than 20. CONCLUSION: Estimation equations are performed poorly compared with iGFR and are not reliable measures of actual level of function in sick hospitalized patients, especially those with a high BUN/SCr ratio. Although use of the 6-variable MDRD equation provides a better estimation of GFR, it still is unsuitable for clinical application in this population.

Evolutionary relationships of the Tas2r receptor gene families in mouse and human.

The early molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCRs) encoded by the Tas2r family of taste receptor genes. The identification of the complete TAS2R receptor family repertoire in mouse and a comparative study of the Tas2r gene families in mouse and human might help to better understand bitter taste perception. We have identified, cloned, and characterized 13 new mouse Tas2r sequences, 9 of which encode putative functional bitter taste receptors. The encoded proteins are between 293 and 333 amino acids long and share between 18% and 54% sequence identity with other mouse TAS2R proteins. Including the 13 sequences identified, the mouse Tas2r family contains approximately 30% more genes and 60% fewer pseudogenes than the human TAS2R family. Sequence and phylogenetic analyses of the proteins encoded by all mouse and human Tas2r genes indicate that TAS2R proteins present a lower degree of sequence conservation in mouse than in human and suggest a classification in five groups that may reflect a specialization in their functional activity to detect bitter compounds. Tas2r genes are organized in clusters in both mouse and human genomes, and an analysis of these clusters and phylogenetic analyses indicates that the five TAS2R protein groups were present prior to the divergence of the primate and rodent lineages. However, differences in subsequent evolutionary processes, including local duplications, interchromosomal duplications, divergence, and deletions, gave rise to species-specific sequences and shaped the diversity of the current TAS2R receptor families during mouse and human evolution.

Functional expression of a mammalian olfactory receptor in Caenorhabditis elegans.

The olfactory system in both vertebrates and invertebrates can recognize and distinguish thousands of chemical signals. Olfactory receptors are responsible for the early molecular events in the detection of volatile compounds and the perception of smell. Recently, candidate olfactory receptor genes have been identified in several organisms, but their characterization is far from been completed due to the difficulty to functionally express them in heterologous systems. To circumvent such difficulty, we expressed a mammalian olfactory gene, rat I7, in the nematode. We generated transgenic worms expressing I7 in AWA or AWB chemosensory neurons and performed behavioural assays using different concentrations of the rat I7 receptor agonist octanal. Pure octanal was repellent for wild-type worms whereas a 1:10 dilution was attractant. Expression of I7 in AWB neurons counteracted the volatile attraction to diluted octanal observed in control wild-type worms. Furthermore, expression of I7 in AWA neurons counteracted the volatile avoidance to pure octanal observed in wild-type worms. These results indicate that it is possible to functionally express mammalian olfactory receptors in providing a research tool to efficiently search for specific olfactory receptor ligands and to extend our understanding of the molecular basis of olfaction.

Neurosecretion competence. A comprehensive gene expression program identified in PC12 cells.

The phenotype of neurosecretory cells is characterized by clear vesicles and dense granules, both discharged by regulated exocytosis. However, these organelles are lacking completely in a few neurosecretion-incompetent clones of the pheochromocytoma PC12 line, in which other specific features are maintained (incompetent clones). In view of the heterogeneity of PC12 cells, a differential characterization of the incompetent phenotype based on the comparison of a single incompetent and a single wild-type clone would have been inconclusive. Therefore, we have compared two pairs of PC12 clones, studying in parallel the transcript levels of 4,200 genes and 19,000 express sequence tags (ESTs) by high density oligonucleotide arrays. After accurate data processing for quality control and filtration, a total of 755 transcripts, corresponding to 448 genes and 307 ESTs, was found consistently changed, with 46% up-regulated and 54% down-regulated in incompetent versus wild-type clones. Many but not all neurosecretion genes were profoundly down-regulated in incompetent cells. Expression of endocytosis genes was normal, whereas that of many nuclear and transcription factors, including some previously shown to play key roles in neurogenesis, was profoundly changed. Additional differences appeared in genes involved in signaling and metabolism. Taken together these results demonstrate for the first time that expression of neurosecretory vesicles and granules is part of a complex gene expression program that includes many other features that so far have not been recognized.

How We Smell: The Molecular and Cellular Bases of Olfaction.

Three models for the perception of odor molecules are suggested for the first time by experimental data. These studies illustrate how the nose may smell. Moreover, they suggest additional role(s) for odor receptors within and outside the olfactory system.