Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication

Replication of flock house virus (FHV) RNA1 and production of subgenomic RNA3 in the yeast Saccharomyces cerevisiae provide a useful tool for the dissection of FHV molecular biology and host-encoded functions involved in RNA replication. The replication template activity of RNA1 can be separated from its coding potential by supplying the RNA1-encoded replication factor protein A in trans. We constructed a trans-replication system in yeast to examine cis-acting elements in RNA1 that control RNA3 production, as well as RNA1 and RNA2 replication. Two cis elements controlling RNA3 production were found. A proximal subgenomic control element was located just upstream of the RNA3 start site (nucleotides [nt] 2282 to 2777). A short distal element also controlling RNA3 production (distal subgenomic control element) was identified 1.5 kb upstream, at nt 1229 to 1239. Base pairing between these distal and proximal elements was shown to be essential for RNA3 production by covariation analysis and in vivo selection of RNA3-expressing replicons from plasmid libraries containing random sequences in the distal element. Two distinct RNA1 replication elements (RE) were mapped within the 3' quarter of RNA1: the intRE (nt 2322 to 2501) and the 3'RE (nt 2735 to 3011). The 3'RE significantly overlaps the RNA3 region in RNA1, and this information was applied to produce improved RNA3-based vectors for foreign-gene expression. In addition, replication of an RNA2 derivative was dependent on RNA1 templates capable of forming the long-distance interaction that controls RNA3 production.     

Quality control in the yeast secretory pathway: a misfolded PMA1 H+-ATPase reveals two checkpoints

The yeast plasma-membrane H(+)-ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway and has recently emerged as an excellent system for identifying quality control mechanisms along the pathway. In the present study, we have tracked the biogenesis of Pma1-G381A, a misfolded mutant form of the H(+)-ATPase. Although this mutant ATPase is arrested transiently in the peripheral endoplasmic reticulum, it does not become a substrate for endoplasmic reticulum-associated degradation nor does it appear to stimulate an unfolded protein response. Instead, Pma1-G381A accumulates in Kar2p-containing vesicular-tubular clusters that resemble those previously described in mammalian cells. Like their mammalian counterparts, the yeast vesicular-tubular clusters may correspond to specific exit ports from the endoplasmic reticulum, since Pma1-G381A eventually escapes from them (still in a misfolded, trypsin-sensitive form) to reach the plasma membrane. By comparison with wild-type ATPase, Pma1-G381A spends a short half-life at the plasma membrane before being removed and sent to the vacuole for degradation in a process that requires both End4p and Pep4p. Finally, in a separate set of experiments, Pma1-G381A was found to impose its phenotype on co-expressed wild-type ATPase, transiently retarding the wild-type protein in the ER and later stimulating its degradation in the vacuole. Both effects serve to lower the steady-state amount of wild-type ATPase in the plasma membrane and, thus, can explain the co-dominant genetic behavior of the G381A mutation. Taken together, the results of this study establish Pma1-G381A as a useful new probe for the yeast secretory system.     

Coherent patterns of abundance and size of a tropical snapper in dynamic estuary systems

Although tropical estuaries are important juvenile habitats for the snapper, Lutjanus russelli, the extent of this utilisation, and relationships to physical variation are unknown. We used fish trapsto investigate spatial and temporal patterns of distribution, abundance and size of L. russelli within and between three tropical estuaries (Cattle, Barramundi and Alligator Creeks) over a two year period. L. russelli showed no indication of spatial segregation by size along the lengths of the estauries. The probability of capturing L. russelli was similar throughout Barramundi Creek and the seaward parts of Cattle Creek. However, the probability of capture fell markedly in upstream regions of Cattle Creek. These areas where characterised by depressed salinity levels due to seasonal flooding. High probabilities of capture of L. russelli during the wet and post-wet seasons in the whole of Barramundi Creek correlated with the time of smallest modal sizes. Thisreflected the entry of large numbers of small recruits into the trappable population. Temporal patterns in mean length were consistent for both years. The major feature of the size data was a clear pattern of seasonal change in the mean fork length of L. russelli, with largest mean sizes occurring in the pre-wet and the smallest mean sizes in the wet and post-wet seasons. This pattern probably reflected the interaction of four processes; the entry of new recruits into the trappable population, growth and mortality during residence in the estuarine nursery habitats, and migration of large fish away from the estuaries. The three creeks studied provided L. russelli with a harsh and extremely variable environment. Over the three creeks, salinities ranged from extremely hyposaline (0) to very hypersaline (55.5) and varied up to 39 within a location. However, despite the substantial physical differences and the spatial separation of the three estuaries, the patterns of temporal change were clear and consistent. Overall, the annual pattern of change insize seemed to relate more to biological processes of recruitment, mortality and migration than to responses to physical variability.     

Numerical analysis of reflectance spectra

(1) Samples of yeast which varied over an arbitrary 0–100% scale with respect, to cytochrome concentration were prepared. (2) The reflectance spectrum of each sample was scanned. (3) These reflectance spectra were analyzed numerically on a digital computer to determine the bandwidths and intensities as a function of cytochrome concentration. Band shapes using the profiles of Cauchy, Gauss, and Giovanlelli were used. The correlations may be used to estimate cytochrome concentrations with a standard deviation of about 3%.     

Salmonella: Now you see it,now you don't

Diseases caused by Salmonella species are characterized by bacterial invasion of host cells. Salmonella invasion requires a genetic locus (inv) with homology to bacterial systems involved in specific protein export and organelle assembly. Until recently, the actual Salmonella invasion factors exported or assembled by the inv system remained unidentified. It now appears that Salmonella produces novel appendages upon contact with host cells. These appendages are transient, appearing and disappearing rapidly from the bacterial surface. Appendages are altered in strains unable to invade due to mutations within the inv/spa locus. Therefore, a role for the invasion locus has been identified, providing another example of bacterial pathogens responding to signals provided by the host cell surface.     

The effects of coumarin on the frequency of deletions in a duplication strain of Aspergillus nidulans.

Summary Strains of A. nidulans with a chromosome segment in duplicate show instability resulting from deletions in either of the duplicate segments. In Dp (I, II) strains, with the terminal segment of IR attached terminally to IIR, spontaneous deletions occur most frequently, though not exclusively, from the translocated segment. Coumarin, at concentrations which did not affect viability or growth rate, enhanced the instability of Dp (I, II) strains by selectively increasing only the deletion class of highest spontaneous frequency. This selective action is interpreted tentatively as due to inhibition of the repair of a particular class of DNA lesion occurring spontaneously in the attachment region of Dp (I, II) strains.     

Rapid Microscopy and Use of Vital Dyes: Potential to Determine Viability of Cryptococcus neoformans in the Clinical Laboratory

Background

Cryptococcus neoformans is the commonest cause of fungal meningitis, with a substantial mortality despite appropriate therapy. Quantitative culture of cryptococci in cerebrospinal fluid (CSF) during antifungal therapy is of prognostic value and has therapeutic implications, but is slow and not practicable in many resource-poor countries.

Methods

We piloted two rapid techniques for quantifying viable cryptococci using mixtures of live and heat-killed cryptococci cultured in vitro: (i) quantitative microscopy with exclusion staining using trypan blue dye, and (ii) flow cytometry, using the fluorescent dye 2′-7′-Bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM). Results were compared with standard quantitative cryptococcal cultures. Quantitative microscopy was also performed on cerebrospinal fluid (CSF) samples.

Results

Both microscopy and flow cytometry distinguished between viable and non-viable cryptococci. Cell counting (on log scale) by microscopy and by quantitative culture were significantly linearly associated (p<0.0001) and Bland-Altman analysis showed a high level of agreement. Proportions of viable cells (on logit scale), as detected by flow cytometry were significantly linearly associated with proportions detected by microscopy (p<0.0001) and Bland-Altman analysis showed a high level of agreement.

Conclusions

Direct microscopic examination of trypan blue-stained cryptococci and flow-cytometric assessment of BCECF-AM-stained cryptococci were in good agreement with quantitative cultures. These are promising strategies for rapid determination of the viability of cryptococci, and should be investigated in clinical practice.     

Selective Allosteric Inhibition of MMP9 Is Efficacious in Preclinical Models of Ulcerative Colitis and Colorectal Cancer

Expression of matrix metalloproteinase 9 (MMP9) is elevated in a variety of inflammatory and oncology indications, including ulcerative colitis and colorectal cancer. MMP9 is a downstream effector and an upstream mediator of pathways involved in growth and inflammation, and has long been viewed as a promising therapeutic target. However, previous efforts to target matrix metalloproteinases (MMPs), including MMP9, have utilized broad-spectrum or semi-selective inhibitors. While some of these drugs showed signs of efficacy in patients, all MMP-targeted inhibitors have been hampered by dose-limiting toxicity or insufficient clinical benefit, likely due to their lack of specificity. Here, we show that selective inhibition of MMP9 did not induce musculoskeletal syndrome (a characteristic toxicity of pan-MMP inhibitors) in a rat model, but did reduce disease severity in a dextran sodium sulfate-induced mouse model of ulcerative colitis. We also found that MMP9 inhibition decreased tumor growth and metastases incidence in a surgical orthotopic xenograft model of colorectal carcinoma, and that inhibition of either tumor- or stroma-derived MMP9 was sufficient to reduce primary tumor growth. Collectively, these data suggest that selective MMP9 inhibition is a promising therapeutic strategy for treatment of inflammatory and oncology indications in which MMP9 is upregulated and is associated with disease pathology, such as ulcerative colitis and colorectal cancer. In addition, we report the development of a potent and highly selective allosteric MMP9 inhibitor, the humanized monoclonal antibody GS-5745, which can be used to evaluate the therapeutic potential of MMP9 inhibition in patients.