The 800 m transient data provides slight improvements in precipitation estimates for the state, particularly at high elevations dominated by snow pack. For future projections, a broader suite of GCM and scenarios could be developed, while awaiting the next iteration of projections from the IPCC. To improve the snow‐driven module in BCM, calibration of the snow accumulation and snowmelt calculations will be done to match measured snow‐water equivalent at over 300 snow sensors and snow courses, and maps of persistent glaciers will also be included. The use of the 800 m PRISM climate data also contributes to a marked improvement in snow pack in some locations. SSURGO soils maps are far more detailed and accurate than STATSGO datasets and reflect topographic controls on soil properties. Therefore SSURGO soils maps are being developed statewide for model application. Geologic maps and local calibrations are being refined in those locations where geologic types are not well represented by the calibration basins,round plastic plant pot such as the volcanics of the Modoc Plateau and the upper Klamath River basin. Herpes viruses target the cell nucleus because of their need for the cellular DNA reproduction machinery.
The nuclear entry of viral DNA is followed by the nuclear accumulation of viral proteins and replication of viral DNA, leading to the formation of viral replication compartments . Electron microscopy and confocal microscopy studies have illustrated how profoundly these viruses transform the nuclear structure so as to optimise their multiplication. During cellular entry the nucleocapsid of herpes simplex virus type-1 undergoes partial disassembly at the nuclear pore complex , followed by viral genome delivery via the pore. When the infection proceeds, the VRCs emerge as distinct nuclear foci, which subsequently undergo fusion and expansion into a large, globular VRC. The infected cell expresses and multiples only a limited number of input HSV-1 genomes. Each small VRC, initially located at the nuclear periphery, originates from a single viral genome. These small VRCs undergo actin-mediated directed movement, resulting in their fusion and eventual enlargement into the entire nucleus. The expansion of the VRCs is accompanied by an increase in the nuclear volume and relocation of the host chromatin into the nuclear periphery. It is in these VRCs that viral nucleocapsid assembly occurs. Soon after assembly, each nucleocapsid penetrates the host chromatin layer and nuclear lamina to the inner nuclear envelope, the site of its egress. Despite their many achievements with biological applications, light- and electron-based imaging techniques suffer from fundamental limitations in 3D imaging of the subcellular architecture of the entire cell. Transmission electron microscopy and TEM tomography are limited by fixation-induced distortions of cellular features, damage caused by the electron beam and the limited range of angular sampling. Fluorescence microscopy can determine the positions of specific molecules, but only of those selected based on existing information, includingthe sizes of the labels and the density of the labelling. Furthermore, immunofluorescence imaging is prone to artefacts by fixation or permeabilization. We employed a strategy integrating 3D soft X-ray tomography imaging with cryogenic fluorescence microscopy , confocal and electron microscopy, and advanced data analysis in order to study in more detail the HSV-1-induced changes in nuclear architecture, molecular organization of the host chromatin and, in particular, the formation of channels across the chromatin layer. Moreover, since X-rays can penetrate 15 μm-thick biological material, SXT allows for quantitative assessment of the entire cell. For SXT image acquisition, the cells were placed in thin-walled capillaries of diameter up to 15 μm. B cells were used because of their small size and HSV-1 susceptibility. In fact, SXT permits cell imaging in a near-native state i.e. intact, unsliced, unstained, and fully hydrated. X-ray absorption depends on the concentration of organic material in each voxel. Therefore, SXT not only detects multiple cellular structures but can also provide quantitative assessment of their composition and structure. Contrast in soft X-ray microscopy is generated by differential attenuation of X-rays by the bio-molecules in the specimen and is not muted by weakly absorbing water. Attenuation of X-rays by the specimen follows the Beer-Lambert law and is therefore both a linear and a quantitative measure of the thickness and the chemical species present at each point in the cell. To gain insight into the spatial localization of the host chromatin and of the viral and cellular proteins and nucleocapsids, we complemented 3D SXT with confocal and electron microscopy imaging techniques.To follow the progress of infection, we analysed the expression of viral immediate-early, early and late genes by cytometry and real-time RT-PCR and of the virus yields by plaque assay. We detected the presence of viral proteins, as well as the expression of viral lytic genes of all three phases of the replication cycle and substantial production of the progeny virus from the B cells. The highest yield of virus was obtained at 24h p.i. . This suggests that not only could HSV-1 enter and infect B cells but also that its replication cycle was completed. Based on these findings, we decided to use a multiplicity of infection of 5 and the time point 24h p.i. in our subsequent experiments.To further analyse the nuclear architecture, we used SXT on hydrated cells in their near-native state. The image contrast of SXT is based on the absorption of X-rays by mainly carbon and nitrogen. This allows measurement of the linear absorption coefficients of cellular structures reflecting their concentration of cellular biomolecules. Owing to its high density of bio-molecules, the heterochromatin region of the nucleus has a er LAC than the less densely packed nucleoplasm, as is evident from computer-generated SXT ortho-slices through nuclei . The use of an HSV-1 strain expressing EYFP-ICP4 allowed the detection of infected cells with enlarged VRC by CFM used in SXT studies. When SXT ortho-slices were aligned with CFM images of the same cell, EYFP-ICP4 was found to be localized in distinct nuclear foci or in a few enlarged foci in the heterochromatin-depleted nuclear regions .The distinct LAC values of SXT were used to automatically segment the nuclear structures for 3D visualization of the spatial information in HSV-1 infected cells. Surface-rendered 3D tomographic reconstructions of the nuclear periphery of the infected cells revealed low-density gaps in the compact layer of marginalized host chromatin. Statistical analysis showed that 24.7± 1.2% of the surface area of the chromatin had a low-LAC value , suggesting the presence of low-density regions in the compact chromatin . In the non-infected cells the relative area of the region with a low LAC value was 7.5±1.2% . In order to further study the low-LAC regions, we analysed their ‘skeletonized’ versions. The skeletonized structure revealed that the low-LAC regions formed channels in the 0.5 μm-thick layer of host chromatin close to the nuclear envelope,25 liter round pot some of which penetrated the nuclear periphery across the layer of peripheral chromatin in both infected and non-infected cells . However, in the infected cells the total number of low-LAC breaks, 900± 300 , was significantly higher than in the non-infected cells . Note that the average volume of the nucleus , was significantly increased in the infected cells compared to the non-infected cells. Additionally, analysis of the density of these channels across the layer of marginalized heterochromatin as a function of distance from the nuclear envelope revealed that, in a 0.1 μm band close to the nuclear envelope, the area density of these channels was significantly higher in the infected than in the non-infected cells . Furthermore, analysis of their local thickness as a functionof distance from the nuclear envelope indicated that their diameter increased towards the nuclear centre, and that they finally merged with the nucleoplasm of the infected and non-infected cells. The smallest diameter of these channels at the nuclear periphery, at 0.1μm from the nuclear envelope in both cases, was at least 200nm . Their size was thus sufficient to allow the passage of at least one viral nucleocapsid at a time. It is known that heterochromatin-free gaps are associated with NPCs and involved in nucleo-cytoplasmic transport of non-infected cells. This prompted us to study whether the virus-induced channels are connected with NPCs. The number and distribution of low-DAPI gaps across the heterochromatin were compared with those of NPCs. Immunolabelling of cells with the Nup153 antibody revealed that NPCs frequently formed clusters in both infected and non-infected cells. Some of these clusters were significantly enlarged in the infected cells . The numbers of NPCs or NPC clusters in the infected cells were reduced compared to those in non-infected cells . Accordingly, in the infected cells the total number of low-LAC breaks was higher than the number of NPCs, whereas in the non-infected cells the number of breaks in the nuclear periphery was close to that of NPCs. Moreover, our studies showed that, in the infected cells, the low-DAPI regions were almost always located independently of NPCs, whereas in the non-infected cells, the low-DAPI regions were located adjacent to the NPCs . In summary, these results demonstrated that HSV-1 infection increases the number of virus-capsid-sized and NPC-independent gaps, which may facilitate the viral transport across the compacted layer of chromatin.Confocal microscopy was performed to detect the presence of nucleocapsid proteins in the low-density chromatin regions of the nuclear periphery. Viral capsid protein VP5 was frequently seen in low-DAPI regions in close proximity to the nuclear envelope . Next, we used transmission electron microscopy to examine the distribution of nucleocapsids with respect to the peripheral marginalized chromatin. The images showed that nucleocapsids were very often localized in thechromatin region next to the nuclear envelope. Consistently with the confocal data, these nucleocapsids were located in the low-density chromatin breaks and narrow virus-nucleocapsid-sized channels . The NPCs are mostly invisible in the infected cells, presumably because of virus induced structural changes of the NE. The distribution of chromatin and NPCs in the non-infected cells is shown in the Supplementary Fig. S5. This is in line with recent studies showing that herpesvirus infection increases the porosity of the nucleus, leading to an enhancement of nucleocapsid motility. Altogether, our findings showed that HSV-1 infection induces breakages penetrating the cellular chromatin barrier to permit nucleocapsid access to the nuclear envelope.Formation of a VRC as a result of HSV-1 lytic infection is followed by structural changes in the host chromatin. Profound reorganization of the nuclear chromatin by HSV-1 has been known to include chromatin marginalization to the nuclear periphery. A compact layer of host heterochromatin constitutes an accessibility barrier for the translocation of viral nucleocapsids toward the inner nuclear envelope across which they exit the nucleus. A previous indication of HSV-1 infection’s ability to disrupt marginalized chromatin, so as to allow access through it, has come from immunofluorescence studies showing fragmented distribution of histone H1, suggesting the presence of routes through chromatin5 . By combining SXT, CFM and confocal imaging techniques with advanced image analysis, we achieved a new insight into the 3D structure and distribution of such breakages.We observed that the low-density regions in the host chromatin formed gaps through it. In the absence of infection these openings were typically located near NPCs, in agreement with earlier results on the density distribution of the host chromatin. In the infected cells, the number of these low-density breakages was significantly increased, and they were most often located independent of NPCs. This independence was in agreement with the NPC-independent nuclear egress of the virus. The SXT analysis revealed channels wide enough to allow the passage of a 125-nm-wide HSV-1 nucleocapsid. However, the narrowest channels, with a diameter of 200 nm, allowed the passage of only one or a few nucleocapsids at a time. This leads to the presence of nucleocapsids in the discontinuous-density regions of the peripheral chromatin close to the nuclear envelope, which agrees well with the egress mechanism demonstrated previously. The exact nature of the molecular mechanisms involved in the intra-nuclear motility of the capsids is controversial. It has been suggested that nucleocapsids are transported in the nucleoplasm via an active process mediated by an intra-nuclear actin and myosin motor protein. However, a very recent study showed that their motility is based on passive diffusion36,46. In summary, we were able to create 3D reconstructions of intact and fully hydrated HSV-1 infected cell nuclei by using SXT.