Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene

A 180,388-bp contig encompassing the human retinoblastoma gene was sequenced in its entirety. Partial analysis of the sequence revealed (1) a high (A + T)/(G + C) ratio and a high density of Line-1 (L1) repeat sequences, suggesting that the locus maps to G-bands 13q14.12 or 13q14.2; (2) Alu repeats that are asymmetrically oriented over a region extending 87 kb; (3) an overabundance of non-Alu-associated poly(A) tracts 10 bp or larger oriented in the antisense rather than the sense direction (36 vs 6); (4) an Alu sequence nested within an L1 repeat, indicating that the expansion of L1 repeats predates at least some of the Alu expansions; (5) at least three newly discovered microsatellite polymorphisms, one of which was subsequently found to be identical to a polymorphism in a microsatellite-based linkage map of the human genome published by another group; and (6) the basis of previously discovered intragenic RFLPs. This sequence should enhance studies of this locus and of the organization of the human genome.     

Diversity,equity, and inclusion in the melanoma research community

The inaugural Diversity and Inclusion in Science Session was held during the 2021 Society for Melanoma Research (SMR) congress. The goal of the session was to discuss diversity, equity, and inclusion in the melanoma research community and strategies to promote the advancement of underrepresented melanoma researchers. An international survey was conducted to assess the diversity, equity, and inclusion (DEI) climate among researchers and clinicians within the Society for Melanoma Research (SMR). The findings suggest there are feelings and experiences of inequity, bias, and harassment within the melanoma community that correlate with one's gender, ethnic/racial group, and/or geographic location. Notably, significant reports of inequity in opportunity, discrimination, and sexual harassment demonstrate there is much work remaining to ensure all scientists in our community experience an academic workplace culture built on mutual respect, fair access, inclusion, and equitable opportunity.     

Particle size influences decay rates of environmental DNA in aquatic systems

Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on targets. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish-derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium-sized particles (1–10 μm) decayed more slowly than other size classes (i.e., <1 and > 10 μm), and thus made up an increasing proportion of eDNA particles over time. We also observed distinct eDNA particle size distribution (PSD) between our Common carp and Rainbow trout samples, suggesting that target-specific assays are required to determine starting eDNA PSDs. Additionally, we found evidence that different sizes of eDNA particles do not decay independently, with particle size conversion replenishing smaller particles over time. Nonetheless, a parsimonious mathematical model where particle sizes decay independently best explained the data. Given these results, we suggest a framework to discern target distance and abundance with eDNA data by applying sequential filtration, which theoretically has both metabarcoding and single-target applications.     

Small-scale variation in growing season length affects size structure of scarlet monkeyflower

Growing season length can control plant size over altitudinal and biogeographic scales, but its effect at the scale of meters is largely unexplored. Within the riparian zone of a northern California river, scarlet monkeyflower, Mimulus cardinalis , grows significantly larger at sites high in the channel as compared to sites low in the channel, and even larger where tributaries meet the main stem of the river. We explored the hypothesis that markedly different growing season length controls this size variation. Due to the very gradual retreat of the water level following winter flooding, emergence time is three months longer for plants growing at tributary confluences than for plants growing at low elevations in the channel. Consistent with the growing season length hypothesis, we found no difference in transplant growth between river and tributary confluence sites in an experiment where we equalized growing season length at these locations. Moreover, a second experiment showed that individuals planted earlier in the year gain a distinct size advantage over those planted later, even though growing conditions are less ideal. These results suggest that emergence time may be a key determinant of plant size structure along rivers, an important result considering forecasted variation in water flows with climate change.     

Area, shape and isolation of tropical forest fragments: effects on tree species diversity and implications for conservation

Aim To quantify the influences of forest area, shape and isolation on tree species diversity in Ghana and to compare their significance with the influences of climate (average annual rainfall) and disturbance (fire burn, logging, agriculture). Location The forest zone of southern Ghana, West Africa (between 5 and 8° N). Methods For twenty‐two forest fragments (1) bivariate regression analyses of tree species diversity (number and composition) were employed with forest spatial geometry, climate and disturbance variables. (2) Multivariate regression analyses of tree species number and all seven environmental variables were used to determine the variability in tree species number that could be accounted for by these environmental variables. Results Forest area, shape and isolation accounted for sharply decreasing proportions of variability in tree species diversity. Large forest fragments contained the greatest numbers of tree species and the highest proportions of rare tree species; irregular fragments had high proportions of regenerating, light‐demanding pioneers and mature, animal‐dispersed species and isolated fragments were floristically similar to less isolated fragments. Fire burn and average annual rainfall accounted for small, but nevertheless significant, proportions of variability in tree species diversity. Logging and agriculture were non‐significant variables. Main conclusions (1) Forest area is the most important consideration when planning tropical forest reserves. (2) Management of disturbance should take priority over management of forest shape if higher levels of tree diversity and species quality are to be maintained. (3) If new reserves are to be designated, they should be located within different climatic zones in order to capture a large fraction of the regional biota. (4) Biogeographers have an important role to play in formulating and testing hypotheses at a broad spatial scale and ultimately, informing conservation management within the tropical biome.