Mechanisms of Newcastle Disease Virus-Mediated Membrane Fusion: A Dissertation

For many paramyxoviruses, including Newcastle disease virus (NDV), syncytia formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (23, 73). Because a potential interaction in paramyxovirus infected cells has never been clearly demonstrated, such an interaction was explored in Chapter 2 using coimmunoprecipitation and crosslinking. Both HN and F proteins could be precipitated with heterologous antisera after a five minute radioactive pulse as well as after a two hour chase in non-radioactive media, but at low levels. Chemical crosslinking increased detection of complexes containing HN and F proteins at the cell surface. After crosslinking, intermediate as well as high molecular weight species containing both proteins were precipitated with monospecific antisera. Precipitation of proteins with anti-HN after crosslinking resulted in the detection of complexes which electrophoresed in the stacker region of the gel, from 160-300 kD, at 150 kD and at 74 kD. Precipitates obtained with anti-F after crosslinking contained species which migrated in the stacker region of the gel, between 160-300 kD, at 120 kD and at 66 kD. The 3-4 discrete complexes ranging in size from 160-300 kD contained both HN and F proteins when precipitated with either HN or F antisera. That crosslinking of complexes containing both HN and F proteins was not simply a function of overexpression of viral glycoproteins at the cell surface was addressed by demonstrating crosslinking at early time points post infection, when levels of viral surface glycoproteins are low. Use of cells infected with an avirulent strain of NDV showed that chemically crosslinked HN and F proteins were precipitated independent of cleavage of F0. Furthermore, under conditions that maximized HN protein binding to its receptor, there was no change in the percentages of HN and F0 proteins precipitated with heterologous antisera, but a decrease in F1protein precipitated was observed upon attachment. These data argue that the HN and F proteins interact in the RER. Upon attachment of the HN protein to its receptor, the HN protein undergoes a conformational change which causes a subsequent change in the associated F protein, releasing the hydrophobic fusion peptide into the target membrane and initiating fusion. Chapter 3 explores the stalk region of the NDV HN protein, which has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (7, 51, 57). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled-coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha-helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic a position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins

Caltech's Throop Hall

THROOP HALL first opened its doors to the public on February 5, 1910. Known then as Pasadena Hall, it was the first building on the new campus of Throop Polytechnic Institute

Drawing From Objects: Objective and Subjective Realities

Moving a drawing tool over a surface, paper, metal, has always been a very felicitous, satisfying, and sensuous experience for me. Causing an illusory shape, with an edge and a texture and suggested volume, to appear is still nothing short of amazing. But making a mark or network of marks, unrelated to objects seen and responded to, has never been sufficiently interesting and involving. I'm acutely aware of specific, tangible objects in the world, and aware as well of how they represent, or connote, or embody complex moments in my personal, emotional history. My perception of them is heightened and conditioned by experiences I've had in their vicinity. I tend to stare at objects in my environment for long periods of time, in a kind of brown study, until they seem to seethe with personal significance. The problem of "choosing" significant objects was indeed a problem in the past, but recently certain things have simply imposed themselves on or risen to the surface of my consciousness and demand to be drawn. Their shapes, details, textures, volumes, and intersections with other objects offer sufficient visual and emotional material for a lifetime of drawing activity. &nbsp;</p

The Biosimilars Act: The United States’ Entry into Regulating Biosimilars and its Implications, 12 J. Marshall Rev. Intell. Prop. L. 322 (2013)

The Patient Protection and Affordable Care Act is most well-known for creating a mandate requiring individuals to have health insurance. However, another provision of the Act, the Biologics Price Competition and Innovation Act, created a new process for companies to introduce biosimilars, products that are highly similar to licensed drugs in terms of purity, safety, and potency, but have minor differences in the inactive ingredients. This provision seeks to alleviate strain on companies introducing biosimilars by creating an abbreviated pathway for their approval by the Food and Drug Administration, similar to an Abbreviated New Drug Application under the Hatch-Waxman Act. This article provides a comprehensive overview of the Biologics Price Competition and Innovation Act and contrasts it with the Hatch-Waxman Act and European Law on Biosimilars. Strategies for patent claiming and resolving patent disputes are then discussed

Liquid chromatography high-resolution TOF analysis: investigation of MSE for broad-spectrum drug screening.

BackgroundHigh-resolution mass spectrometry (HRMS) has the potential to supplement other drug screening platforms used in toxicology laboratories. HRMS offers high analytical specificity, which can be further enhanced by incorporating a fragment ion for each analyte. The ability to obtain precursor ions and fragment ions using elevated collision energies (MS(E)) can help improve the specificity of HRMS methods.MethodsWe developed a broad-spectrum screening method on an ultraperformance liquid chromatography TOF mass spectrometer (UPLC-TOF-MS) using the MS(E) mode. A diverse set of patient samples were subjected to a simple dilute, hydrolyze, and shoot protocol and analyzed in a blind manner. Data were processed with 3 sets of criteria with increasing stringency, and the results were compared with the reference laboratory results.ResultsA combination of retention time match (±0.2 min), a protonated analyte, and fragment ion mass accuracy of ±5 ppm produced zero false-positive results. Using these criteria, we confirmed 92% (253/275) of true positives. The positive confirmation rate increased to 98% (270/275) when the requirement for a fragment ion was dropped, but also produced 53 false positives. A total of 136 additional positive drug findings not identified by the reference methods were identified with the UPLC-TOF-MS.ConclusionsMS(E) provides a unique way to incorporate fragment ion information without the need of precursor ion selection. A primary limitation of requiring a fragment ion for positive identification was that certain drug classes required high-energy collisions, which formed many fragment ions of low abundance that were not readily detected

A PDA-based dietary self-monitoring intervention to reduce sodium intake in an in-center hemodialysis patient

Objective: The purpose of the BalanceWise-hemodialysis study is to determine the efficacy of a dietary intervention to reduce dietary sodium intake in patients receiving maintenance, in-center hemodialysis (HD). Personal digital assistant (PDA)-based dietary self-monitoring is paired with behavioral counseling. The purpose of this report is to present a case study of one participant's progression through the intervention. Methods: The PDA was individually programmed with the nutritional requirements of the participant. With 25 minutes of personalized instruction, the participant was able to enter his meals into the PDA using BalanceLog® software. Nutritional counseling was provided based on dietary sodium intake reports generated by BalanceLog®. Results: At initiation of the study the participant required 4 HD treatments per week. The participant entered 342 meals over 16 weeks (≥3 meals per day). BalanceLog® revealed that the participant consumed restaurant/fast food on a regular basis, and consumed significant amounts of corned beef as well as canned foods high in sodium. The study dietitian worked with the participant and his wife to identify food alternatives lower in sodium. Baseline sodium consumption was 4,692 mg, and decreased at a rate of 192 mg/week on average. After 11 weeks of intervention, interdialytic weight gains were reduced sufficiently to permit the participant to reduce HD treatments from 4 to 3 per week. Because of a low serum albumin at baseline (2.9 g/dL) the study dietitian encouraged the participant to increase his intake of high quality protein. Serum albumin level at 16 weeks was unchanged (2.9 g/dL). Because of intense pruritis and a high baseline serum phosphorus (6.5 mg/dL) BalanceLog® electronic logs were reviewed to identify sources of dietary phosphorus and counsel the participant regarding food alternatives. At 16 weeks the participant's serum phosphorus fell to 5.5 mg/dL. Conclusions: Self-monitoring rates were excellent. In a HD patient who was willing to self-monitor his dietary intake, BalanceLog® allowed the dietitian to target problematic foods and provide counseling that appeared to be effective in reducing sodium intake, reducing interdialytic weight gain, and alleviating hyperphosphatemia and hyperkalemia. Additional research is needed to evaluate the efficacy of the intervention. © 2008 Sevick et al, publisher and licensee Dove Medical Press Ltd

The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: interactions at ferrous heme and cysteine thiols

It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols