Myosin V transports secretory vesicles via a Rab GTPase cascade and interaction with the exocyst complex.

Vesicle transport requires four steps: vesicle formation, movement, tethering, and fusion. In yeast, two Rab GTPases, Ypt31/32, are required for post-Golgi vesicle formation. A third Rab GTPase, Sec4, and the exocyst act in tethering and fusion of these vesicles.

Crystal structure of NAD+-dependent Peptoniphilus asaccharolyticus glutamate dehydrogenase reveals determinants of cofactor specificity.

Glutamate dehydrogenases (EC 1.4.1.

The activation cycle of Rab GTPase Ypt32 reveals structural determinants of effector recruitment and GDI binding.

Rab GTPases localize to distinct sub-cellular compartments and regulate vesicle trafficking in eukaryotic cells. Yeast Rabs Ypt31/32 and Sec4 have 68% homology and bind to common interactors, yet play distinct roles in the transport of exocytic vesicles. The structures of Ypt31/32 have not previously been reported in the uncomplexed state.

Simultaneous determination of timolol maleate, rosuvastatin calcium and diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV.

A novel HPLC-UV method was developed for the simultaneous determination of timolol (TM), rosuvastatin (RST), and diclofenac sodium (DS) in pharmaceuticals, human plasma and aqueous humor using naproxen sodium as internal standard (IS). The target compounds were analyzed on Hypersil BDS C(18) column (250 mm × 4.6 mm, 5 μm), applying 0.

Spectral-domain optical coherence tomography assessment of an epiretinal membrane with axoplasmic stasis from nerve fiber layer traction.

Purpose: To report a case of visible axoplasmic stasis on fundus examination correlating with spectral-domain optical coherence tomography findings of nerve fiber layer traction.

Methods: Case report.

Results: Spectral-domain optical coherence tomography images show nerve fiber layer traction and distortion at the sites where visible cotton wool spot-like changes were seen on examination.

Hepatitis C virus NS3/NS4A DNA vaccine induces multiepitope T cell responses in rhesus macaques mimicking human immune responses [corrected].

Numerous studies have suggested that an effective hepatitis C virus (HCV) vaccine must induce a strong T helper 1 (Th1) T cell response. While several therapeutic vaccine candidates have shown promise in clinical trials, response rates have been low suggesting that further optimization is important. However, such optimization has been hindered by a lack of a benchmark animal model in which to test vaccine-induced immune responses before clinical evaluation.

Structural insights into dissimilatory sulfite reductases: structure of desulforubidin from desulfomicrobium norvegicum.

Dissimilatory sulfite reductases (dSiRs) are crucial enzymes in bacterial sulfur-based energy metabolism, which are likely to have been present in some of the earliest life forms on Earth. Several classes of dSiRs have been proposed on the basis of different biochemical and spectroscopic properties, but it is not clear whether this corresponds to actual physiological or structural differences. Here, we describe the first structure of a dSiR from the desulforubidin class isolated from Desulfomicrobium norvegicum.

Poxvirus A46 protein binds to TIR domain-containing Mal/TIRAP via an α-helical sub-domain.

Poxviruses are large DNA viruses that replicate in the cytosol and express numerous proteins to subvert the host immunity. Vaccinia virus A46 is a 25kDa protein that antagonizes multiple components of the Toll-like/interleukin-1 receptor (TLR) pathway by targeting cytosolic adaptor proteins. A46 binds to MyD88, Mal/TIRAP, TRIF and TRAM and suppresses the activation of NF-κB and interferon regulatory factors.

Strontium doped lead zirconate titanate ceramics: study of calcination and sintering process to improve piezo effect.

Perovskite crystal structure is found in many ionic solids like CaTiO3, BaTiO3 and Lead Zirconate Titanates (PZT). In this structure off-center position of cations in oxygen octahedral causes polarization and produces direct and indirect piezoelectric responses in ceramic materials that are suitable for many ultrasonic applications. In the present study 9% Sr doped PZT ceramics were prepared and their dielectric and piezoelectric properties measured.

Long-term programming of antigen-specific immunity from gene expression signatures in the PBMC of rhesus macaques immunized with an SIV DNA vaccine.

While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances, including genetic optimization and in vivo electroporation (EP), have helped to dramatically boost their immunogenicity.

Immunogenicity of a novel engineered HIV-1 clade C synthetic consensus-based envelope DNA vaccine.

DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence.

Quantitative analysis of DNA methylation of imprinted genes in single human blastocysts by pyrosequencing.

We report the first quantitative assessment of DNA methylation for any gene in the human preimplantation embryo to reveal that imprints exist at KvDMR1, RB1, SNRPN, and GRB10 in the human blastocyst. For comparison, in two human embryonic stem cell lines, imprints were also observed at KvDMR1, SNRPN, GRB10, and other imprinted loci, whereas RB1 and MEG3 were hypermethylated.

Crystallization of an engineered RUN domain of Rab6-interacting protein 1/DENND5.

Effectors of the Rab small GTPases are large multi-domain proteins which have proved difficult to express in soluble form in Escherichia coli. Generally, effectors are recruited to a distinct subcellular compartment by active (GTP-bound) Rabs, which are linked to membranes by one or two prenylated Cys residues at their C-termini. Following recruitment via their Rab-binding domain (RBD), effectors carry out various aspects of vesicle formation, transport, tethering and fusion through their other domains.

Infant formula alters surfactant protein A (SP-A) and SP-B expression in pulmonary epithelial cells.

Surfactant proteins A (SP-A) and SP-B are critical in the ability of pulmonary surfactant to reduce alveolar surface tension and provide innate immunity. Aspiration of infant milk formula can lead to lung dysfunction, but direct effects of aspirated formula on surfactant protein expression in pulmonary cells have not been described. The hypothesis that infant formula alters surfactant protein homeostasis was tested in vitro by assessing surfactant protein gene expression in cultured pulmonary epithelial cell lines expressing SP-A and SP-B that were transiently exposed (6 hr) to infant formula.

Effects of infant formula on cell homeostasis and cytokine levels in an in vivo and in vitro murine aspiration model.

Rationale: The role of infant formula aspiration in lung injury has not been studied extensively. We evaluated the effects of a single infant formula aspiration into the lungs of mice and the effect of infant formula exposure on cell lines representing murine alveolar macrophages and type II epithelial cells.

Objectives: To study the effects of exposure to infant formula on cell count histology and cytokine levels in an in vivo and in vitro model of aspiration.

Thoracic splenosis: know it--avoid unnecessary investigations, interventions, and thoracotomy.

Thoracic splenosis (TS) is autoimplantation of ectopic splenic tissue in the thoracic cavity that occurs following splenic injury. Most cases of TS are asymptomatic and are diagnosed during the course of an evaluation of incidentally discovered pulmonary lesions. Some cases may be difficult to diagnose, especially if features suggesting TS are not recognized.

Co-delivery of PSA and PSMA DNA vaccines with electroporation induces potent immune responses.

Prostate cancer (PCa) remains a significant public health problem. Current treatment modalities for PCa can be useful, but may be accompanied by deleterious side effects and often do not confer long-term control. Accordingly, additional modalities, such as immunotherapy, may represent an important approach for PCa treatment.

A DNA vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates.

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen.

A highly optimized DNA vaccine confers complete protective immunity against high-dose lethal lymphocytic choriomeningitis virus challenge.

Protection against infection is the hallmark of immunity and the basis of effective vaccination. For a variety of reasons there is a great demand to develop new, safer and more effective vaccine platforms. In this regard, while 'first-generation' DNA vaccines were poorly immunogenic, new genetic 'optimization' strategies and the application of in vivo electroporation (EP) have dramatically boosted their potency.

A novel prototype device for electroporation-enhanced DNA vaccine delivery simultaneously to both skin and muscle.

Electroporation (EP) of either muscle or skin has proven to be an efficient method for increasing DNA-based vaccine delivery and immunogenicity in small and large animals. Previous comparative studies in large animals suggest that intramuscular (i.m.

High antibody and cellular responses induced to HIV-1 clade C envelope following DNA vaccines delivered by electroporation.

Background: Clade C is the predominant HIV-1 strain infecting people in sub-Saharan Africa, India, and China and there is a critical need for a vaccine targeted to these areas. In this study we tested a DNA based vaccine that encodes the SIVgag, SIVpol and HIV-1 envelope clade C.

Methods: Rhesus macaques were immunized by electroporation with the DNA plasmid encoding optimized SIVgag, SIVpol and an HIV-1 env clade C with or without the adjuvant RANTES.

Electroporation of plasmid DNA to swine muscle.

For plasmid-mediated gene therapy applications, a major limitation to scale up from rodents to large animals is the low expression level of injected plasmid DNA. The electroporation technique, which results in the passage of foreign material through the cell membrane, is one method that has been shown to be effective at improving local plasmid uptake and consequently, expression levels. Previous studies have determined that optimized electroporation parameters (such as electric field intensity, number of pulses, lag time between plasmid injections and electroporations, and optimal plasmid formulation conditions) are dependent on the target muscle type and individual species.

Pharmacokinetics and dosage regimen of ciprofloxacin following single intramuscular administration in Teddy goats.

The objective of this study was to determine the pharmacokinetics and dosage regimen of ciprofloxacin in Teddy goats. Ciprofloxacin was administered intramuscularly at 5 mg/kg body weight in each of eight animals. Following drug administration, blood samples were collected at different time intervals and analyzed for ciprofloxacin using HPLC.

Multivalent smallpox DNA vaccine delivered by intradermal electroporation drives protective immunity in nonhuman primates against lethal monkeypox challenge.

The threat of a smallpox-based bioterrorist event or a human monkeypox outbreak has heightened the importance of new, safe vaccine approaches for these pathogens to complement older poxviral vaccine platforms. As poxviruses are large, complex viruses, they present technological challenges for simple recombinant vaccine development where a multicomponent mixtures of vaccine antigens are likely important in protection. We report that a synthetic, multivalent, highly concentrated, DNA vaccine delivered by a minimally invasive, novel skin electroporation microarray can drive polyvalent immunity in macaques, and offers protection from a highly pathogenic monkeypox challenge.