Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors.

Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. On the basis of their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N and T368A mutations enhanced serum resistance, and additionally K66T, T368A and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N+T368A or K66T+S162T+T230N+T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy

Nanoanalysis of a sub-nanometre reaction layer in a metal inserted high-k gate stack

Reactions at the interfaces can occur in metal inserted high-k gate stacks and are likely to evolve during device processing. Such reactions may affect the electrical properties of the stack and hence these could change during processing. The key interfaces are often not atomically flat and characterising the reaction layers on the near atomic scale required is a challenge. Aberration corrected scanning transmission electron microscopy (STEM) and spectrum imaging (SI) using electron energy loss spectroscopy (EELS) is used to characterise an HfN or Hf(O,N) reaction layer, ∼0.25 nm wide, between HfO<sub>2</sub> and TiN. This demonstrates the very significant advances in high spatial resolution characterisation made in recent years

A procedure for implanting a spinal chamber for longitudinal in vivo imaging of the mouse spinal cord.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed

Diverse Inflammatory Response After Cerebral Microbleeds Includes Coordinated Microglial Migration and Proliferation.

BACKGROUND AND PURPOSE:Cerebral microbleeds are linked to cognitive decline, but it remains unclear how they impair neuronal function. Infarction is not typically observed near microbleeds, suggesting more subtle mechanisms, such as inflammation, may play a role. Because of their small size and largely asymptomatic nature, real-time detection and study of spontaneous cerebral microbleeds in humans and animal models are difficult. METHODS:We used in vivo 2-photon microscopy through a chronic cranial window in adult mice to follow the inflammatory response after a cortical microhemorrhage of ≈100 µm diameter, induced by rupturing a targeted cortical arteriole with a laser. RESULTS:The inflammatory response included the invasion of blood-borne leukocytes, the migration and proliferation of brain-resident microglia, and the activation of astrocytes. Nearly all inflammatory cells responding to the microhemorrhage were brain-resident microglia, but a small number of CX3CR1+ and CCR2+ macrophages, ultimately originating from the invasion of blood-borne monocytes, were also found near the lesion. We found a coordinated pattern of microglia migration and proliferation, where microglia within 200 µm of the microhemorrhage migrated toward the lesion over hours to days. In contrast, microglia proliferation was not observed until ≈40 hours after the lesion and occurred primarily in a shell-shaped region where the migration of microglia decreased their local density. These data suggest that local microglia density changes may trigger proliferation. Astrocytes activated in a similar region as microglia but delayed by a few days. By 2 weeks, this inflammatory response had largely resolved. CONCLUSIONS:Although microhemorrhages are small in size, the brain responds to a single bleed with an inflammatory response that involves brain-resident and blood-derived cells, persists for weeks, and may impact the adjacent brain microenvironment

New Hampshire: the story of a state.

Thesis (M.Ed.)--Boston Universit

A constitutional study of a dual phase steel containing 12% chromium

Bibliography: pages 79-86.This thesis involved a study of the phase transfonnations in a chromium containing corrosion resistant dual phase steel, designated 3CR12. The objectives included the detennination of time-temperature-transformation (TTT) diagrams for the transformations between austenite and ferrite and an investigation into the factors controlling these reactions. The austenite decomposition reaction for a high nickel alloy, 3CR12Ni, and the effect of varying titanium concentrations on the equilibrium phases present in 3CR12, were also examined. Dilatometry was used to determine the transformation temperatures between austenite and ferrite and the Ms temperatures for the alloys investigated. The kinetics of the reactions were investigated by optical microscopy using two different etching techniques while the volume fractions of the various constituents were determined by a point counting method. Transmission electron microscopy was used to study the carbide morphologies and the nucleation and growth modes of the phases during the transformations. The distribution of the alloy elements were determined by microhardness measurements, an electronprobe microanalysis and a Kevex spectrometer attached to a scanning electron microscope. The 3CR12 alloy used in this study did not become fully austenitic above the Ae₃; it lies in the nose of the gamma loop of the Fe-Cr phase diagram. Two temperature regimes were identified on the decomposition of austenite. At 750°c the existing ferrite grains grew into the austenite matrix, while at 650° C and 700°C new ferrite was sympathetically nucleated i.e. it was heterogeneously nucleated on existing ferrite/austenite grain boundaries. Two types of carbide morphologies were formed. These were random precipitation within the ferrite grains and interphase precipitation. The TTI diagram showed conventional "C" curve kinetics. The austenitisation reaction occurred by a para-equilibrium mechanism. The rate controlling process was the structural change from ferrite to austenite; the reaction was not long range diffusion controlled. The speed of the reaction increased continuously with increasjng transformation temperature. No growth of ferrite occurred on isothermal transformation of 3CR12Ni at temperatures below the Ae₁. Increasing the bulk titanium content increased the Ms, Ae₁ and Ae₃ temperatures of 3CR12 due to the removal of carbon from, and the addition of titanium to, solution

The Relationship Between Degree of Sobriety in Male Alcoholics and Coping Styles Used by their Wives

The present study was designed primarily to study the relationship between modes of coping used by wives of alcoholics and the outcome of their husbands\u27 drinking behavior. Previous research had provided some understanding of the wife\u27s personality, but little controlled research had studied the relationship between the wife\u27s behavior and her husband\u27s drinking. Orford and his associates (Orford and Guthrie, 1968, Orford, et aj_., 1975) provided the initial stimulus for this study by demonstrating that relatively consistent modes of coping could be identified. However, the Orford, et a]_. study found a minimal relationship between modes of coping and the eventual outcome of the husband\u27s drinking. Based on other studies (e.g., Smith, 1969, Rae, 1972, Wright, 1975), which indicated that there is a relationship between the husband\u27s outcome and certain aspects of the wife\u27s behavior, the present study hypothesized specific weaknesses in the Orford, et aj_. (1975) study which precluded finding a relationship. Using previous research (e.g., Jackson, 1954, Kogan and Jackson, 1961, 1963, Lemert, 1960, Bailey, 1967) additional (predictor) variables (stage of the wife\u27s reactive pattern, the wife\u27s perception of her husband, whether there was a drinking problem before marriage, and the wife\u27s educational level) were hypothesized to interact with modes of coping. Hypotheses were formulated concerning the relationship of drinking outcome and the modes of coping and other predictor variables. A questionnaire containing subscales for each of the variables hypothesized to be relevant was administered to wives of alcoholics. In addition, four measures of sobriety were administered to both the wives and their husbands. A general regression design was used to analyze the relationship between drinking outcome and the predictor variables. Two groups of wives (HR and HNR), identified on the basis of whether the husband completed his questionnaire, were found to differ significantly on all of the measures of sobriety. Subsequent analyses were performed separately on the two groups. A factor analysis of the modes of coping questionnaire revealed nine identifiable factors, six of which corresponded closely with the factors in the Orford, et al_. (1975) study. Thus, specific, relatively stable modes of coping were identified. The data provided no support for Jackson\u27s (1954) stage theory. Possible reasons for this failure were discussed. The results provided support for the general hypothesis that the styles of coping used by wives of alcoholics in response to their husbands\u27 drinking are related to the drinking outcome. In both groups (HR and HNR) certain behaviors were more highly correlated with eventual sobriety than other behaviors. In both groups the crucial variable seemed to be the safety of the atmosphere between the husband and wife. Those modes of coping which communicated the wife\u27s feelings of distress and frustration to the drinking husband in a way which was safe for him were the most highly related to his attainment of sobriety. There were a number of interactions between modes of coping and the wife\u27s perception of her husband. Again threat seemed to be the important variable. Responses which might have threatened an insecure husband, and thus showed a low correlation with eventual sobriety, might have been significantly correlated with sobriety when the husband was seen to be relatively secure and well adjusted. Limitations of the present study were discussed and future research, particularly regarding the cause-effect relationship between the wife\u27s behavior and the husband\u27s drinking, was suggested

New Hampshire: the story of a state.

Thesis (M.Ed.)--Boston Universit