The global disease burden due to the HIV/AIDS epidemic continues to rise as efforts to prevent or treat the virus are met with more and more challenges. The advent of potent antiretroviral treatment options that appear to reduce viral loads to undetectable levels in infected patients, are thwarted by HIV’s ability to establish latency and a long term persistent virus reservoir. A preventative approach that can establish persistent immunity against HIV-1 is urgently needed. This study seeks to examine the in vitro effect of a candidate vaccine targeted against highly conserved amino acid sequences, known as Protease Cleavage Sites (PCS), in HIV-1. Preliminary testing of the vaccine consisting of 20mer PCS overlapping peptides was conducted in Cynomolgus macaques and revealed that the highest frequency of mutation in two of the twelve sites, were located in the PCS2 and PCS12 regions. Sequencing of these mutants isolated from the peripheral blood of vaccinated monkeys facilitated the creation, by molecular cloning, of a library viral clones harboring the same mutations. These viral clones were then characterized in vitro by determining their replicative abilities as compared to wild type virus. While more work is required to determine the mechanisms by which these mutations impair viral fitness, the results thus far, are informative towards the continued development of this candidate vaccine. / 2017-06-16T00:00:00Z

Angiogenesis is the formation of new blood vessels from preexisting vessels, which involves endothelial cell migration, differentiation and proliferation. Angiogenesis is mediated by a variety of cell surface receptors including growth factor receptors, cell adhesion molecules and others with pro- and anti-angiogenic functions. The primary goal of this study was to determine the expression of KIAA1024 in endothelial cells and elucidate its functional importance in endothelial cells. Our study, for the first time, demonstrated that KIAA1024 is expressed in human endothelial and various cancer cell lines. We further demonstrated that ectopic expression of KIAA1024 in porcine aortic endothelial (PAE) cells inhibits cell migration in both Boyden chamber and cell scratch assays. Additionally, overexpression of KIAA1024 in PAE cells significantly altered actin stress fiber formation and reduced capillary tube formation. Interestingly, unlike its inhibitory effect on capillary tube formation, overexpression of KIAA1024 in PAE cells increased cell migration, cell survival and inhibited phosphorylation of pro-apoptosis p38MAPK. Taken together, the data presented in this study identifies KIAA1024 as a novel cell surface receptor expressed in endothelial cells and regulates angiogenic properties of endothelial cells.

OBJECTIVE: Clinical gene therapy is increasingly becoming a favorable method for the targeting and treatment of various human diseases. One such gene-transfer method is the use of adeno-associated viruses (AAVs) as a tool for tissue-specific delivery of genes. AAV vectors are attractive vector candidates due to their low pathogenicity, biological safety, and ability to infect both dividing and quiescent cells for sustained gene expression. We sought to develop a protocol for the production, propagation, purification, and characterization of various AAV serotypes to determine their tropism and to observe their integrative and expressive abilities using different reporter genes in vitro and in vivo. METHODS: We used subcloning techniques to introduce our genes of interest into an AAV expression plasmid and recombined with two other AAV vectors required for packaging, cell-specific targeting, and integration capabilities. Serotypes AAV1/8/9 and 5 which are cross-packaged with the prototype AAV2 were used to individually target cardiac cells/skeletal muscle and neuronal cells respectively in eC57BL/6J mice. Viral particles were propagated in vitro and purified using an iodixanol gradient. Purified AAV vectors were introduced into cells in culture and injected into live mice via tail vein injection or using robotic stereotaxic surgery directly into brain opposite saline controls to determine expression capabilities in vitro and in vivo, respectively. Live mice were imaged using the IVIS bioluminescent imaging system to measure luciferase luminescent readout. Separately, sections were acquired from the brains of the mice injected with serotype AAV2/5 and imaged using live cell imaging fluorescence microscopy to demonstrate integrative ability of the vectors into neurons based on GFP reporter signal. RESULTS: A protocol for the generation of neuron-specific AAVs was developed for the successful integration of the reporter gene GFP. The reporter gene was cloned into the AAV expression cassette. Efficient transfection methods were determined along with optimal culture conditions for the propagation of the viral particles in vitro. Promoter-driven reporter gene expression was observed in serotype-targeted cell types in vitro and in vivo. Expression was limited to neuronal cells based on AAV serotype specificity and SYN1 promoter. Integration and expression of the luciferase gene was not observed in the IVIS system. CONCLUSION: Here we demonstrate expression of a GFP reporter in specific neuronal cell types which indicates successful integration and expression abilities of AAV vectors. This specific tissue-targeting technique using the AAV vector highlights the potential for the further development of these vectors as a promising gene transfer system.

White adipose tissue (WAT) fibrosis is now recognized to contribute to obesity-associated metabolic dysfunction. In obesity, WAT expands through both adipocyte hypertrophy and hyperplasia and regulators that control these remodeling events are only partially understood. However these pathways are likely interconnected with those that regulate fibrosis, the excessive pericellular extracellular matrix (ECM) deposition that occurs in WAT in obesity. Thus it is postulated that WAT fibrosis may limit WAT expansion and further contribute to metabolic dysfunction. Several studies have indicated that WAT progenitors reside within the vascular niche and in other organs these cells respond to numerous proinflammatory and profibrotic mediators. Previous studies have identified that the secreted ECM protein, aortic carboxypeptidase-like protein (ACLP), is expressed in vascular smooth muscle cells and enhances lung myofibroblast differentiation. It is also recognized that in vitro, ACLP expression decreases with adipogenic differentiation however its role in this process is unknown. The goal of this research was to test the hypothesis that ACLP stimulates the differentiation of WAT progenitors into myofibroblasts and represses adipogenesis. Using in vitro adipogenesis assays, ACLP expression was found to be rapidly diminished with adipogenesis in mouse and human adipose progenitors and was not expressed by differentiated adipocytes. In gain of function studies, recombinant ACLP repressed adipogenesis and promoted myofibroblast differentiation in both mouse and human adipose progenitors. In loss of function studies utilizing chemical inhibitors, ACLP signaling in adipose progenitors was dependent on transforming growth factor β receptor activity. Analysis of fibrotic WAT revealed ACLP protein expression increased in the stromal-vascular fraction (SVF) and co-localized with pericellular ECM deposition. Analysis of mRNA expression in fibrotic WAT sub-populations revealed that immune cell depleted SVF was the primary source of ACLP. Additional studies investigated the cellular origin of WAT myofibroblasts, which used transgenic mice with fluorescent reporters for α smooth muscle actin and collagen I. Analysis of SVF demonstrated that multiple stromal-vascular cell types are capable of differentiating into both adipocytes and myofibroblasts. Taken together, these studies identified ACLP as a stromal derived mediator of adipose tissue progenitor differentiation and the accumulation of ACLP may limit adipocyte expansion in fibrotic WAT.

abstract: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is widely accepted as the world's most abundant enzyme and represents the primary entry point for inorganic carbon into the biosphere. Rubisco's slow carboxylation rate of ribulose-1,5-bisphosphate (RuBP) and its susceptibility to inhibition has led some to term it the "bottle neck" of photosynthesis. In order to ensure that Rubisco remains uninhibited, plants require the catalytic chaperone Rubisco activase. Activase is a member of the AAA+ superfamily, ATPases associated with various cellular activities, and uses ATP hydrolysis as the driving force behind a conformational movement that returns activity to inhibited Rubisco active sites. A high resolution activase structure will be an essential tool for examining Rubisco/activase interactions as well as understanding the activase self-association phenomenon. Rubisco activase has long eluded crystallization, likely due to its infamous self-association (polydispersity). Therefore, a limited proteolysis approach was taken to identify soluble activase subdomains as potential crystallization targets. This process involves using proteolytic enzymes to cleave a protein into a few pieces and has previously proven successful in identifying crystallizable protein fragments. Limited proteolysis, utilizing two different proteolytic enzymes (alpha-chymotrypsin and trypsin), identified two tobacco activase products. The fragments that were identified appear to represent most of what is considered to be the AAA+ C-terminal all alpha-domain and some of the AAA+ N-terminal alpha beta alpha-domain. Identified fragments were cloned using the pET151/dTOPO. The project then moved towards cloning and recombinant protein expression in E. coli. NtAbeta(248-383) and NtAbeta(253-354) were successfully cloned, expressed, purified, and characterized through various biophysical techniques. A thermofluor assay of NtAbeta(248-383) revealed a melting temperature of about 30°C, indicating lower thermal stability compared with full-length activase at 43°C. Size exclusion chromatography suggested that NtAbeta(248-383) is monomeric. Circular dichroism was used to identify the secondary structure; a plurality of alpha-helices. NtAbeta(248-383) and NtAbeta(253-354) were subjected to crystallization trials. / Dissertation/Thesis / M.S. Biochemistry 2012

Mykoplasmat ovat pieniä, soluseinättömiä bakteereja, jotka elävät loisina ihmis-, eläin- tai kasvisolujen sisällä tai niiden pinnalla. Monet mykoplasmat ovat patogeenisiä ja pääsääntöisesti myös elin- ja kudosspesifisiä. Niiden genomi on erittäin pieni ja aineenvaihdunta yksinkertaista (Razin et al. 1998). Pisaratartuntana leviävä Mycoplasma pneumoniae aiheuttaa ihmisillä enimmäkseen hengitystieinfektioita, jotka ovat usein hyvin pitkäkestoisia. Infektioiden spesifinen diagnosointi on vaikeaa ja mykoplasmojen soluseinän puutteen vuoksi monet perinteiset mikrobilääkkeet, kuten beetalaktaamiantibiootit ovat niitä vastaan tehottomia (Razin et al. 1998, Kashyap & Sarkar 2010). Mycoplasma pneumoniae on pääsääntöisesti solunulkoinen patogeeni, ja sen selviytyminen on isäntäsolusta riippuvaista. Organismilla on erityinen kiinnittymisorganelli, jonka avulla pisaratartuntana hengitysteihin kulkeutuneet bakteerisolut kykenevät kiinnittymään epiteelisolujen värekarvoihin ja liikkumaan niitä pitkin solun pintaan ja infektoimaan solun. Onnistuneen adheesion seurauksena aktivoituu useita eri mekanismeja, jotka osaltaan selittävät infektion aiheuttamia oireita (Razin et al. 1998, Shimizu 2016).

Influenssa A- ja B-virukset ovat negatiivisäikeisiä RNA-viruksia, joiden genomi on segmentoitunut kahdeksaan osaan. Influenssa A-virus voidaan luokitella alatyyppeihin kalvoglykoproteiinien: hemagglutiniinin (HA) ja neuraminidaasin (NA) avulla. Influenssavirukset aiheuttavat kausittaisia epidemioita sekä ajoittain odottamattomia pandemioita. Kausi-influenssaepidemiat aiheutuvat kahdesta influenssa A-viruksen alatyypistä (H1N1 ja H3N2) sekä kahdesta influenssa B-viruksen linjasta (Yamagata ja Victoria). Influenssaviruksilta voi suojautua rokotteen avulla, jonka tehokkuus vaihtelee 60–90 % välillä. Tällä hetkellä käytössä olevia kausi-influenssarokotteita ovat trivalenttinen influenssarokote (TIV), LAIV-rokote sekä syksyllä 2018 Suomessa käyttöön otettava nelivalenttinen influenssarokote (QIV). Kehitteillä olevat universaalit influenssarokotteet pyrkivät antamaan mahdollisimman laajan ja kestävän suojan influenssaviruksia vastaan. Nämä universaalit influenssarokotteet ovat keskittyneet aktivoimaan spesifisten vasta-aineiden tuotantoa mm. HA:a, NA:a sekä M2-ionikanavia vastaan. Lisäksi kehitteillä on T-soluvastetta aktivoiva universaali influenssarokote.

<p> Short interfering RNA (siRNA) causes sequence specific gene silencing of mRNA and has been shown to be a very promising therapeutic agent for a wide range of diseases. We have developed a hybrid collagen/cell penetrating peptide (CHP), that contains a triple helical domain (POG)_n that provides stability, and a cell penetrating domain (R_n) which contains positively charged arginine residues allowing for internalization into cells. We determined that the CHPs form highly crystalline nanoparticles with siRNA with molar ratios of 1:18 and 1:9 depending on the number of arginines in the CPP domain. CHPs are able to effectively deliver and release siRNA into 3T3 Swiss mouse fibroblast cells with higher efficiency and gene silencing ability than that of Lipofectamine. The data suggest that our strategy for development of the CHP-siRNA complex can provide an avenue for effective delivery of siRNA.</p><p>

<p> Ion mobility spectrometry &ndash; mass spectrometry (IMS-MS) based techniques have significantly advanced the study of both the structure and stability of biomolecules. Solution phase, biologically relevant peptide/protein conformations have been analyzed utilizing electrospray ionization (ESI) coupled to IMS-MS. The work presented here focuses on the development and application of IMS-MS for the study of the structure and stability of peptides and proteins. Structures originating in solution can be related to the biological activity of the system. Specifically, we examine the influence of both proline isomerization and metal binding on the conformational preferences and stabilities of a series of peptides and proteins. </p><p> The first study uses IMS-MS to examine the conformation specific binding of Zn²⁺ to oxytocin (OT). When bound to Zn, OT has an increased affinity for its receptor. We observe two conformations which are the result of the <i>cis-trans</i> orientation of the Cys⁶-Pro⁷ bond. We find that Zn²⁺ binds preferentially to the trans configuration demonstrating that <i>cis-trans</i> isomerization regulates the binding of Zn²⁺ to OT, therefore linking the orientation of the Cys⁶-Pro⁷ bond to the biological activity of the molecule. In another series of experiments, we look at the role of penultimate proline on the stability of bradykinin along with a library of penultimate proline containing peptides. For BK, we find that the Pro²-Pro³ bond, which is enzymatically resistant, is cleaved with 100% specificity when incubated at high temperatures. With IMS-MS, we are able to monitor the mass spectral and conformational changes as a function of time, and find that <i>cis-trans</i> isomerization of a single bond regulates the rate of dissociation, and hence the stability of the system. Finally, we look at the thermal stability of the insulin-zinc hexamer complex. Utilizing temperature-controlled nano-ESI IMS-MS, we capture the dissociation of the insulin hexamer and the unfolding of insulin dimer and monomer, occurring simultaneously with increasing solution temperature. We show the ability of IMS-MS to observe the conformation specific melting temperatures of a protein complex, demonstrating a fast and sensitive assay for stability characterization.</p><p>

<p> This research is aimed at understanding the quantum mechanical concept of the tunneling in hydride-transfer reactions in solution. The reaction is a redox reaction involving the hydride transfer from 2-propanol to a series of substituted carbocationic oxidizing agents, 9-arylxanthylium ions (<i> G</i>-PhXn⁺), in acetonitrile. Explanations in the contemporary tunneling models utilize the concept of the longer donor-acceptor distance (DAD) at tunneling ready state (TRS) in protium (H)-tunneling as compared to deuterium (D)-tunneling. Our group&rsquo;s hypothesis is that different DADs in H- vs. D-tunneling TRSs will lead to TRS conformations of different steric environment and possibly different electronic structures. This opens a new research direction that studies how primary (1&deg;) H isotope substitution with D affects the kinetic isotope effect (KIE) at the in-place secondary 2&deg; H/D positions (2&deg; KIE) as well as the Hammett correlations. This dependence provides information about the structure of a TRS. Our group has previously conducted investigations on the structural effect on the 1&deg; isotope dependence of 2&deg; KIEs in hydride transfer reactions to differentiate the isotopically different TRS conformations. It has been revealed that the 1&deg; isotope dependence of the 2&deg; KIEs is increased by the steric effects supporting the assumption of different DADs in H- <i>versus </i> D-tunneling, which leads to the different conformations of TRS. My approach in this research is focused on the study of 1&deg; isotope effect on the Hammett correlations at the <i>G</i>-PhXn⁺ to attempt to differentiate the isotopically different TRS electronic structures. The degree of electronic interactions between the neighboring groups from reactants is shown to vary from H- to D-tunneling reactions, suggesting different electronic structures of two isotopic TRSs. The evidence for this arises from the non-zero isotopic slope difference <b>(&rho;H - &rho;D &ne; 0)</b> for the corresponding Hammett correlations.</p><p>