Faculty Bibliography

Solid lipid nanoparticles (SLNs) have emerged as an excellent substitute over polymeric nanoparticles and, when incorporated with chitosan which activates the macrophage to impart an immune response, produce excellent results to fight against deleterious diseases like leishmaniasis where its parasite diminishes the immunity of the host to induce resistance. Based upon this hypothesis, chitosan-coated SLNs were developed and loaded with amphotericin B (AmB) for immunoadjuvant chemotherapy of Leishmania infection. Both uncoated and chitosan-coated AmB-loaded SLNs (AmB-SLNs) were fabricated using solvent emulsification and evaporation method. The various processes and formulation parameters involved in AmB-SLN preparation were optimized with respect to particle size and stability of the particles. In vitro hemolytic test credited the formulations to be safe when injected in the veins. The cellular uptake analysis demonstrated that the chitosan-coated AmB-SLN was more efficiently internalized into the J774A.1 cells. The in vitro antileishmanial activity revealed their high potency against Leishmania-infected cells in which chitosan-coated AmB-SLNs were distinguishedly efficacious over commercial formulations (AmBisome and Fungizone). An in vitro cytokine estimation study revealed that chitosan-coated AmB-SLNs activated the macrophages to impart a specific immune response through enhanced production of TNF-alpha and IL-12 with respect to normal control. Furthermore, cytotoxic studies in macrophages and acute toxicity studies in mice evidenced the better safety profile of developed formulation in comparison to marketed formulations. This study indicates that the AmB-SLNs are a safe and efficacious drug delivery system which promises strong competence in antileishmanial chemotherapy and immunotherapy.

The miR-143/145 cluster regulates VSMC specific gene expression, thus controlling differentiation, plasticity and contractile function, and promoting the VSMC phenotypic switch from a contractile/non-proliferative to a migrating/proliferative state. More recently increased miR-145 expression was observed in human carotid atherosclerotic plaques from symptomatic patients. The goal of this study was to investigate the contribution of miR-143/145 during atherogenesis by generating mice lacking miR-143/145 on an Ldlr-deficient background. Ldlr-/- and Ldlr-/--miR-143/145-/- (DKO) were fed a Western diet (WD) for 16 weeks. At the end of the treatment, the lipid profile and the atherosclerotic lesions were assessed in both groups of mice. Absence of miR-143/145 significantly reduced atherosclerotic plaque size and macrophage infiltration. Plasma total cholesterol levels were lower in DKO and FLPC analysis showed decreased cholesterol content in VLDL and LDL fractions. Interestingly miR-143/145 deficiency per se resulted in increased hepatic and vascular ABCA1 expression. We further confirmed the direct regulation of miR-145 on ABCA1 expression by qRT-PCR, Western blotting and 3'UTR-luciferase reporter assays. In summary, miR-143/145 deficiency significantly reduces atherosclerosis in mice. Therapeutic inhibition of miR-145 might be useful for treating atherosclerotic vascular disease.

Intranasal administration of neuropeptide Y (NPY) is a promising treatment strategy to reduce traumatic stress-induced neuropsychiatric symptoms of posttraumatic stress disorder (PTSD). We evaluated the potential of intranasal NPY to prevent dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis, a core neuroendocrine feature of PTSD. Rats were exposed to single prolonged stress (SPS), a PTSD animal model, and infused intranasally with vehicle or NPY immediately after SPS stressors. After 7 days undisturbed, hypothalamus and hippocampus, 2 structures regulating the HPA axis activity, were examined for changes in glucocorticoid receptor (GR) and CRH expression. Plasma ACTH and corticosterone, and hypothalamic CRH mRNA, were significantly higher in the vehicle but not NPY-treated group, compared with unstressed controls. Although total GR levels were not altered in hypothalamus, a significant decrease of GR phosphorylated on Ser232 and increased FK506-binding protein 5 mRNA were observed with the vehicle but not in animals infused with intranasal NPY. In contrast, in the ventral hippocampus, only vehicle-treated animals demonstrated elevated GR protein expression and increased GR phosphorylation on Ser232, specifically in the nuclear fraction. Additionally, SPS-induced increase of CRH mRNA in the ventral hippocampus was accompanied by apparent decrease of CRH peptide particularly in the CA3 subfield, both prevented by NPY. The results show that early intervention with intranasal NPY can prevent traumatic stress-triggered dysregulation of the HPA axis likely by restoring HPA axis proper negative feedback inhibition via GR. Thus, intranasal NPY has a potential as a noninvasive therapy to prevent negative effects of traumatic stress.

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria and Moraxella have evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogen Neisseria meningitidis is reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogen Moraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin.

Three strains capable of rapidly degrading TBBPA by co-metabolism and utilizing formate as the carbon source, named as J-F-01, J-F-02, and J-F-03, respectively, were isolated from enrichment cultures, which have been treated with 0.5mg/L TBBPA for 240 d. Based on morphology and 16S rRNA gene sequence analysis, both J-F-01 and J-F-02 were determined to Pseudomonas sp., while J-F-03 was identified as Streptococcus sp. A shorter half-life (6.1d) of TBBPA was observed in pure culture of J-F-03 when compared with J-F-01 (22.5d) and J-F-02 (13.6d). Surprisingly, the degradation of TBBPA was significantly enhanced by the mixed culture of J-F-02 and J-F-03. The optimal degradation conditions for the mixed cultures were determined. Under the optimal conditions, TBBPA (0.5mg/L) was completely metabolized by the mixed culture within ten days. Moreover, bromide and the metabolisms were detected, and a possible metabolic pathway was deduced from the detection of metabolite production patterns.

With increasing numbers of people with Alzheimer's and other dementias across the globe, many countries have developed national plans to deal with the resulting challenges. In the United States, the National Alzheimer's Project Act, signed into law in 2011, required the creation of such a plan with annual updates thereafter. Pursuant to this, the US Department of Health and Human Services (HHS) released the National Plan to Address Alzheimer's Disease in 2012, including an ambitious research goal of preventing and effectively treating Alzheimer's disease by 2025. To guide investments, activities, and the measurement of progress toward achieving this 2025 goal, in its first annual plan update (2013) HHS also incorporated into the plan a set of short, medium and long-term milestones. HHS further committed to updating these milestones on an ongoing basis to account for progress and setbacks, and emerging opportunities and obstacles. To assist HHS as it updates these milestones, the Alzheimer's Association convened a National Plan Milestone Workgroup consisting of scientific experts representing all areas of Alzheimer's and dementia research. The workgroup evaluated each milestone and made recommendations to ensure that they collectively constitute an adequate work plan for reaching the goal of preventing and effectively treating Alzheimer's by 2025. This report presents these Workgroup recommendations.

I entered the field of cell biology during its formative phase and contributed technical advances in electron microscopy that gave new insights into the physiological roles of subcellular compartments. I concentrated for many years on the structure and function of the rough endoplasmic reticulum where I aimed at explaining mechanistically how the functional specialization of ribosomes bound to the endoplasmic reticulum membranes is achieved. This work eventually led to the formulation of the signal hypothesis with Gunter Blobel in 1971. Subsequently, my laboratory contributed to the birth of the field of protein traffic with the demonstration that membrane-bound ribosomes in the ER are also responsible for the synthesis of membrane and luminal proteins destined to other subcellular compartments, pointing to the existence of sorting signals in the newly synthesized polypeptides and corresponding discriminating trafficking mechanisms. I have derived great satisfaction from the fact that some of the work I will present, including the introduction with M. Cerejido of the MDCK cell line to study the development and properties of polarized epithelia and the discovery with Rodriguez-Boulan of the polarized budding of enveloped viruses, has helped many laboratories to continue to explore the fascinating mechanisms that contribute to generate the complex organization of eukaryotic cells

Polarization of early embryos along cell contact patterns-referred to in this paper as radial polarization-provides a foundation for the initial cell fate decisions and morphogenetic movements of embryogenesis. Although polarity can be established through distinct upstream mechanisms in Caenorhabditis elegans, Xenopus laevis, and mouse embryos, in each species, it results in the restriction of PAR polarity proteins to contact-free surfaces of blastomeres. In turn, PAR proteins influence cell fates by affecting signaling pathways, such as Hippo and Wnt, and regulate morphogenetic movements by directing cytoskeletal asymmetries.