Selective retention of recombinant plasmids coding for human insulin

Plasmids may be lost from Escherichia coli K-12 hosts that are cultured without selection for plasmid retention. This is particularly true for chimeric plasmids that incorporate genes for human insulin into vectors derived from pBR322. The cIts857 gene of bacteriophage lambda was inserted into the bla gene of the human-insulin-coding plasmids, pIA7 delta 4 delta 1, pIB7 delta 4 delta 1 and pHI7 delta 4 delta 1, generating the new plasmids pPR17, pPR18 and pPR19, respectively, which produced the thermosensitive lambda repressor. The cI gene was downstream from the pM and pbla promoters, so that it may have been expressed from either or both promoters. Separate E. coli K-12 RV308 host strains containing the new recombinants were lysogenized with the repressor-defective bacteriophage lambda cI90. Loss of the plasmid from the lysogens causes concomitant loss of the lambda repressor and cell death, because the prophage is induced to enter the lytic growth cycle. The system effectively forces retention of the plasmid in all viable cells in the culture.     

Therapeutic development by repurposing drugs targeting SARS-CoV-2 spike protein interactions by simulation studies

The human-to-human transmitted respiratory illness in COVID-19 affected by the pathogenic Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), which appeared in the last of December 2019 in Wuhan, China, and rapidly spread in many countries. Thereon, based on the urgent need for therapeutic molecules, we conducted in silico based docking and simulation molecular interaction studies on repurposing drugs, targeting SARS-CoV-2 spike protein. Further, the best binding energy of doxorubicin interacting with virus spike protein (PDB: 6VYB) was observed to be −6.38 kcal/mol and it was followed by exemestane and gatifloxacin. The molecular simulation dynamics analysis of doxorubicin, Reference Mean Square Deviation (RMSD), Root Mean Square fluctuation (RMSF), Radius of Gyration (Rg), and formation of hydrogen bonds plot interpretation suggested, a significant deviation and fluctuation of Doxorubicin-Spike RBD complex during the whole simulation period. The Rg analysis has stated that the Doxorubicin-Spike RBD complex was stable during 15,000–35,000 ps MDS. The results have suggested that doxorubicin could inhibit the virus spike protein and prevent the access of the SARS-CoV-2 to the host cell. Thus, in-vitro/in-vivo research on these drugs could be advantageous to evaluate significant molecules that control the COVID-19 disease.     

Photosynthetic variation and photosynthetic nitrogen use efficiency in <Emphasis Type="Italic">Brassica</Emphasis> species with different genetic constitution of ribulose-1,5-bisphosphate carboxylase

Net photosynthetic rate (P _N) was high in genotypes with ‘C’ genome both in the nucleus and cytoplasm. This may be attributed to the co-ordinated manner of acting of both genome sources. Leaf mass per area (LMA) and chlorophyll content increased with leaf nitrogen (N) content but did not show any correlation with P _N. The factors which affected P _N had the same effect on photosynthetic nitrogen use efficiency (pNUE). Thus, differential allocation of N to the various components influences plant pNUE which is not significantly affected by genome constitution.     

Detecting low fragmented sites surrounding European protected areas – Implications for expansion of the Natura 2000 network

EU member states have set an ambitious goal of establishing additional protected areas (PAs) preserving 30 % of terrestrial land by 2030, specifying that additions should be of high ecological quality. A targeted selection of existing PA expansions into surroundings marginally fragmented by human infrastructure, may be an efficacious strategy to secure high ecological quality by maximizing PA area, accommodating species movement, and boosting climate change resilience. We used high-resolution data on effective mesh density, a metric measuring landscape fragmentation, in the vicinity of Natura 2000 PAs (N2k) to assess their potential for expansion. Our results show that contrary to most of Central Europe, mountainous and remote territories exhibit the lowest degree of fragmentation in N2k surroundings. Fragmentation in N2k surroundings is highly correlated with national population density, while economic wealth, measured by GDP per capita, plays a minor role. To address the long-standing dilemma of where scarce economic resources in nature conservation do the most-good, we conducted a country-level comparison between fragmentation in N2k surroundings and national expenditures on nature conservation relative to N2k area. Our results show a vast incongruity in resource availability for nature conservation among EU countries. Eastern European states, especially Romania, host underfunded N2k PAs while holding the highest potential for expanding N2k PAs into low fragmented lands. If protecting low fragmented lands is accepted as an efficacious strategy to meet EU biodiversity targets our results could be used to formulate pragmatic conservation decisions, while also ensuring high ecological quality of PA additions under climate change.     

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.     

Latitudinal effects on treehopper species richness (Hornoptera: Membracidae)

Abstract. 1. Membracid species richness declines with increasing latitude in the Western hemisphere but begins to increase again in temperate regions. In northern latitudes this transition occurs in the highlands of Mexico and is the result of the emergence of a new tribe and greater host specialization.
2. The relationship between number of species per genus and latitude is parabolic.
3. We suggest the low number of species per genus in tropical regions may be due to the lack of host specialization and reduced coordination of life history with host phenology.     

Comparison of Cox Model Methods in A Low-dimensional Setting with Few Events

Prognostic models based on survival data frequently make use of the Cox proportional hazards model. Developing reliable Cox models with few events relative to the number of predictors can be challenging, even in low-dimensional datasets, with a much larger number of observations than variables. In such a setting we examined the performance of methods used to estimate a Cox model, including (i) full model using all available predictors and estimated by standard tech-niques, (ii) backward elimination (BE), (iii) ridge regression, (iv) least absolute shrinkage and selec-tion operator (lasso), and (v) elastic net. Based on a prospective cohort of patients with manifest coronary artery disease (CAD), we performed a simulation study to compare the predictive accu-racy, calibration, and discrimination of these approaches. Candidate predictors for incident cardio-vascular events we used included clinical variables, biomarkers, and a selection of genetic variants associated with CAD. The penalized methods, i.e., ridge, lasso, and elastic net, showed a compara-ble performance, in terms of predictive accuracy, calibration, and discrimination, and outperformed BE and the full model. Excessive shrinkage was observed in some cases for the penalized methods, mostly on the simulation scenarios having the lowest ratio of a number of events to the number of variables. We conclude that in similar settings, these three penalized methods can be used interchangeably. The full model and backward elimination are not recommended in rare event scenarios.