Divergent Effects of Chaperone Overexpression and Ethanol Supplementation on Inclusion Body Formation in RecombinantEscherichia coli

The proper folding of aggregation-prone recombinant proteins inEscherichia colican be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps). We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S′-β-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30°C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S′-β-galactosidase 1.5- to 2-fold. When cultures overexpressing thednaKJoperon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S′-β-galactosidase aggregation was almost entirely abolished. Combining DnaK–DnaJ overexpression and growth of the cells at temperatures lower than 30°C did not result in a comparable increase in activity. Although the individual effects of ethanol supplementation anddnaKJoverproduction were more limited when the culture volume was raised, a synergistic improvement in preS2-S′-β-galactosidase activity was observed when the two approaches were used in concert. In contrast, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S′-β-galactosidase. Our results show that ethanol can exert complex and divergent effects on inclusion body formation and that the beneficial effect of the solvent on recombinant protein folding cannot simply be explained by an increase in the intracellular concentration of molecular chaperones.     

A selective sweep driven by pyrimethamine treatment in southeast asian malaria parasites

Malaria parasites (Plasmodium falciparum) provide an excellent system in which to study the genomic effects of strong selection in a recombining eukaryote because the rapid spread of resistance to multiple drugs during the last the past 50 years has been well documented, the full genome sequence and a microsatellite map are now available, and haplotype data can be easily generated. We examined microsatellite variation around the dihydrofolate reductase (dhfr) gene on chromosome 4 of P. falciparum. Point mutations in dhfr are known to be responsible for resistance to the antimalarial drug pyrimethamine, and resistance to this drug has spread rapidly in Southeast (SE) Asia after its introduction in 1970s. We genotyped 33 microsatellite markers distributed across chromosome 4 in 61 parasites from a location on the Thailand/Myanmar border. We observed minimal microsatellite length variation in a 12-kb (0.7-cM) region flanking the dhfr gene and diminished variation for approximately 100 kb (6 cM), indicative of a single origin of resistant alleles. Furthermore, we found the same or similar microsatellite haplotypes flanked resistant dhfr alleles sampled from 11 parasite populations in five SE Asian countries indicating recent invasion of a single lineage of resistant dhfr alleles in locations 2000 km apart. Three features of these data are of especially interest. (1). Pyrimethamine resistance is generally assumed to have evolved multiple times because the genetic basis is simple and resistance can be selected easily in the laboratory. Yet our data clearly indicate a single origin of resistant dhfr alleles sampled over a large region of SE Asia. (2). The wide valley ( approximately 6 cM) of reduced variation around dhfr provides "proof-of-principle" that genome-wide association may be an effective way to locate genes under strong recent selection. (3). The width of the selective valley is consistent with predictions based on independent measures of recombination, mutation, and selection intensity, suggesting that we have reasonable estimates of these parameters. We conclude that scanning the malaria parasite genome for evidence of recent selection may prove an extremely effective way to locate genes underlying recently evolved traits such as drug resistance, as well as providing an opportunity to study the dynamics of selective events that have occurred recently or are currently in progress.     

Smooth muscle of the dorsal aorta shares a common clonal origin with skeletal muscle of the myotome

We show that cells of the dorsal aorta, an early blood vessel, and of the myotome, the first skeletal muscle to form within the somite, derive from a common progenitor in the mouse embryo. This conclusion is based on a retrospective clonal analysis, using a nlaacZ reporter targeted to the alpha-cardiac actin gene. A rare intragenic recombination event results in a functional nlacZ sequence, giving rise to clones of beta-galactosidase-positive cells. Periendothelial and vascular smooth muscle cells of the dorsal aorta are the main cell types labelled, demonstrating that these are clonally related to the paraxial mesoderm-derived cells of skeletal muscle. Rare endothelial cells are also seen in some clones. In younger clones, arising from a recent recombination event, myotomal labelling is predominantly in the hypaxial somite, adjacent to labelled smooth muscle cells in the aorta. Analysis of Pax3(GFP/+) embryos shows that these cells are Pax3 negative but GFP positive, with fluorescent cells in the intervening region between the aorta and the somite. This is consistent with the direct migration of smooth muscle precursor cells that had expressed Pax3. These results are discussed in terms of the paraxial mesoderm contribution to the aorta and of the mesoangioblast stem cells that derive from it.     

In vitro cardiac performance in triploid brown trout at two acclimation temperatures

The maximum values for heart rate ( f _H), stroke volume ( V _H), cardiac output ( Q ) and myocardial power output, measured in vitro with a perfused heart preparation, as well as the isometric force-frequency relationship for atrial and ventricular muscle strips, in triploid brown trout Salmo trutta were all comparable with established information for diploid rainbow trout Oncorhynchus mykiss . Therefore, it was concluded that triploidy is not associated with a major deficiency in maximum cardiac performance. However, a heightened sensitivity to ryanodine was discovered, which indicated an enhanced role for the sarcoplasmic reticulum in excitation-contraction coupling in these triploid fish. It is suspected that the enhanced role of the ryanodine receptor may be a cellular compensation related to larger cardiac myocytes. It was also clearly established that there was a plateau in maximum cardiac performance between 14 and 18° C and this plateau might be a contributing factor to the reduced factorial aerobic scope and increased fish mortality observed at 18° C.     

Altered expression of flowering class B and class C genes in the Appendix tobacco mutant

 In the tobacco (Nicotiana tabacum) Appendix mutant, anthers are tipped by a miniature style and stigma. The outgrowth appears on the anther when it is already differentiating and follows the developmental timing of the central carpel. The Appendix mutation thus represents a late homeotic transformation suggesting that the APPENDIX (APX) gene either could be a misregulated organ identity gene or could be involved in regulating the expression of such genes. RFLP analysis with two class B (TM6 and NTGLO) and a class C (NAG) probes revealed that the Appendix phenotype is not caused by a mutation in one of these genes. However, in situ hybridization showed important changes in the expression of NTGLO and NAG in the mutant when compared with wild-type tobacco. Surprisingly, although no phenotypic alteration other than the style and stigma outgrowth is observed in the Appendix mutant, changes in class B and class C gene expession were not restricted to the anther tip cells from which the outgrowth originates. As expected, NAG was expressed in the Appendix outgrowth but it was also overexpressed in the normal third and fourth whorl organs at the time the outgrowth, as well as the central styles and stigmas, differentiated. Overexpression of a class C gene is probably responsible for the Appendix phenotype. In normal and mutant flowers, NTGLO was expressed in the second, third and fourth whorls up to the time of carpel fusion. Expression of this class B gene then ceased in the fourth whorl organs but was reactivated at later stages only in the styles and stigmas as well as in the outgrowths of the mutant. It thus seems that the function of the APX gene is either to regulate the late expression of organ identity genes or to control cell proliferation in such a way that, in the mutant, some cells are in a state where they respond in an unusual way to developmental signals. Received: 17 October 1997 / Revision accepted: 24 March 1998     

DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain

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Highlights? DCC and netrin-1 are enriched at synapses in the adult mouse forebrain ? DCC is enriched in the PSD and regulates dendritic spine morphology ? LTP induction and memory formation require DCC expression by neurons ? DCC activation of Src is required for NMDAR-dependent LTP in adult CNS     

Growth Monitoring: A Survey of Current Practices of Primary Care Paediatricians in Europe

Objective

We aimed to study current practices in growth monitoring by European primary care paediatricians and to explore their perceived needs in this field.

Methods

We developed a cross-sectional, anonymous on-line survey and contacted primary care paediatricians listed in national directories in the 18 European countries with a confederation of primary care paediatricians. Paediatricians participated in the survey between April and September 2011.

Results

Of the 1,198 paediatricians from 11 European countries (response rate 13%) who participated, 29% used the 2006 World Health Organization Multicentre Growth Reference Study growth charts, 69% used national growth charts; 61% used software to draw growth charts and 79% did not use a formal algorithm to detect abnormal growth on growth charts. Among the 21% of paediatricians who used algorithms, many used non-algorithmic simple thresholds for height and weight and none used the algorithms published in the international literature. In all, 69% of paediatricians declared that a validated algorithm to monitor growth would be useful in daily practice. We found important between-country variations.

Conclusion

The varied growth-monitoring practices declared by primary care paediatricians reveals the need for standardization and evidence-based algorithms to define abnormal growth and the development of software that would use such algorithms.     

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.     

Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells

Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A–Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A–Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A–Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A–Br in HT1080 cells are discussed.