The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.The alphaherpesviruses herpes simplex virus (HSV) types 1 (HSV-1) and 2, varicella-zoster virus (VZV), and pseudorabies virus (PRV) replicate primarily in epithelial tissues before spreading into neurons where they establish latency. These viruses are extraordinarily adept at spreading rapidly from cell to cell in both these tissues. Given that alphaherpesviruses establish lifelong infections, cell-to-cell spread appears to be especially important in order for these viruses to outrun the immune system, especially after reactivation in hosts that have fully primed immunity. Consistent with this, HSV, PRV, and VZV all remain largely cell associated; large numbers of progeny virions accumulate on cell surfaces, especially at cell junctions in polarized epithelial cells (23). Moreover, virus mutants with specific defects in cell-to-cell spread that do not affect entry at apical surfaces are extremely attenuated in vivo (reviewed in references 15 and 21). It has been proposed elsewhere that alphaherpesviruses have evolved specialized mechanisms to allow specific sorting of virions to epithelial and neuronal cell junctions and the efficient transfer across junctions to promote infection of adjacent cells (14,21).HSV, PRV, and VZV all express a glycoprotein heterodimer, gE/gI, that promotes cell-to-cell spread (19,20,25,49,50). In the case of HSV and PRV, gE/gI was originally thought to be nonessential based on observations that gE Ϫ or gI Ϫ mutants enter cultured laboratory cells and replicate normally in the cells. However, PRV and HSV gE Ϫ or gI Ϫ mutants are severely compromised in epithelial and neuronal tissues (1,6,7,12,13,33,37,43,44,46...

Use of a TG/DTA/Raman system enabled real time collection of Raman spectra during the dehydration of Risedronate, the active pharmaceutical ingredient in the osteoporosis drug Actonel. Raman spectra collected during the dehydration of this material show a crystal lattice adjustment below the boiling point of water facilitating interpretation of the TG/DTA thermal profile. Raman spectra also revealed recrystallization processes subsequent to loss of lattice water that were not evident in the thermal analysis profile. Spectral evidence showing the formation and disappearance of a second hydrate form are observed during the dehydration of this material. In addition, spectral evidence indicating the presence of an anhydrate form of this material are observed prior to thermal induced degradation of this material.

Modification of a commercially available TG/DTA instrument enabled development of a working TG/DTA/Raman system. The TG/DTA was modified by replacing a portion of the furnace wall above the sample and reference holders with a double layer insulated quartz window. The quartz window provided ready access for Raman spectroscopy, enabling simultaneous collection of TGA, DTA, and Raman data during thermal analytical experiments. Insertion of the quartz glass window did not impact TGA or DTA performance as demonstrated by the comparison of baseline drift and temperature calibration for modified and unmodified furnaces.

TGA-DTA plays a central role in the strategy outlined for early evaluation of the solid state forms available to pharmaceutical new chemical entities. At this early stage of development, compound and time are typically at a premium, so a successful strategy requires making the best possible use of the materials and time available. In addition, because of time and compound limitations, the goal of a solid state investigation is focused upon early stage objectives rather than development of a complete understanding of all available solid state forms. The TGA-DTA is well suited to addressing these needs.

TGA-DTA plays a central role in the strategy outlined for early evaluation of the solid state forms available to pharmaceutical new chemical entities. Understanding of the solid state forms becomes more difficult when individual samples present as mixed forms, especially when it is not immediately recognized that the samples represent a mixture. In this study, TGA-DTA, in combination with light microscopy and powder X-ray diffraction, provided immediate evidence that samples represented mixed solid state forms. The initial assessment was made using as little as 5 mg of sample. Hygroscopicity challenges provided further proof for mixed forms. To make a definite assignment of the solid state forms present, isolation of pure phases of the suspected individual forms was necessary. Success of this testing strategy is illustrated using an example of mixed salt stoichiometry and mixed hydration states. A hierarchy is suggested for efficient isolation efforts when a complex mixture of solid state samples is present.