Functional genomics of the yeast DNA-damage response

High-throughput approaches are beginning to have an impact on many areas of yeast biology. Two recent studies, using different experimental platforms, provide insight into new pathways involved in the response of yeast to DNA damage.     

The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9

The Cf-9 gene encodes an extracytoplasmic leucine-rich repeat protein that confers resistance in tomato to races of the fungus Cladosporium fulvum that express the corresponding avirulence gene Avr 9. We investigated whether the genomic Cf-9 gene functions in potato and tobacco. Transgenic tobacco and potato plants carrying Cf-9 exhibit a rapid hypersensitive cell death response (HR) to Avr 9 peptide injection. Cf 9 tobacco plants were reciprocally crossed to Avr 9-producing tobacco. A developmentally regulated seedling lethal phenotype occurred in F1 progeny when Cf9 was used as the male parent and Avr 9 as the female parent. However, when Cf9 was inherited in the maternal tissue and a heterozygous Avr 9 plant was used as the pollen donor, a much earlier reaction was caused, leading to no germination of any F1 seed. Detailed analysis of the Avr 9-induced responses in Cf 9 tobacco leaves revealed that (1) most mesophyll cells died within 3 hr (compared with 12 to 16 hr in tomato); (2) the macroscopic HR was visible at an Avr 9 titer five times lower than that which caused visible symptoms in tomato; (3) the HR invariably extended into noninjected panels of the tobacco leaf; (4) no HR occurred in leaves of young tobacco plants; (5) in older plants, the HR was dramatically enhanced by sequential Avr 9 challenges; and (6) coexpression of a salicylate hydroxylase transgene (nahG) from Pseudomonas putida reduced the severity of the macroscopic leaf HR and also restored germination to Cf 9 x 35S:Avr 9 F1 seedlings. Simultaneous introduction of Cf-9 homologs (Hcr 9-9 genes A and B or D) along with the native Cf-9 gene did not alter the responses that were specifically induced by Avr 9. Various ways to use the Cf-9-Avr 9 gene combination to engineer broad-spectrum disease resistance in several solanaceous species are discussed.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) decreases left ventricular function. An echocardiographic study in cancer patients

To study the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the heart, echocardiographic assessments of left ventricular (LV) end-diastolic and end-systolic (ES) diameters (D), ejection fraction (EF) and cardiac output (CO) were done in six male patients (28-70 years of age) with advanced sarcoma (Group 1), prior to (day -1-0), during (day 7-9) and after (day 20-21) a first course of i.v. doxorubicin (day 0) without GM-CSF and a second course (3 weeks after the first one) with GM-CSF 250 microg/m(2)subcutaneously and daily from day 1-11. A similar study was done in ten female patients with advanced breast cancer (31-58 years of age, Group 2) for a first course of doxorubicin+cyclophosphamide with GM-CSF (same schedule as in Group 1). As compared to the mean of values prior to and after the course with GM-CSF in Group 1 and 2, the LVESD during GM-CSF administration transiently increased by median 6% (range -19 to 30%, P<0.05) vs -9% (-21 to 6%, not significant) in the first course without GM-CSF in Group 1 (P<0.05 between courses). The CO and EF tended to decrease during GM-CSF. GM-CSF thus causes a small and transient decrease of LV contractility.     

Hip Arthrodesis with the Anterolateral Plate: An Innovating Technique for an Orphaned Procedure

Background

In developing countries hip osteoarthritis constitutes a major public health issue as it is highly prevalent in all age ranges of population, including the young. It often remains untreated because of the low accessibility of total hip prostheses. Hip arthrodesis still represents a major treatment option, but, for several reasons which are discussed in this paper, is nowadays infrequently performed. By means of reporting the results of a new simple technique, using a self-devised plate, the relevancy of hip arthrodesis in this particular environment is emphasized.

Methods and Findings

Our series included 35 patients with painful hip osteoarthritis who underwent a hip fusion with the anterolateral arthrodesis plate. Two of them had a concurrent femoral osteotomy for correction of a vicious position of the limb and another patient had a femoral diaphysis osteotomy and placement of a Wagner elongating device in order to proceed with a limb lengthening by callotasis. The follow-up period averaged 16,9 months (9 to 34). All hips, except two, achieved solid fusion between 6 and 15 months after surgery. One failure of fusion was in the oldest patient, who presented a loosening of plate and screws due to an advanced degree of osteoporosis; the other was in a young patient who admitted having walked on his leg too soon. Patient satisfaction was high. We concluded that this technique is reliable and effective.

Conclusions

The results of this study should convince the hesitant surgeon and patient to consider hip arthrodesis an acceptable treatment option for disabling hip arthritis, compared to no treatment at all.     

Effects of Localized Hydrophilic Mannitol and Hydrophobic Nelfinavir Administration Targeted to Olfactory Epithelium on Brain Distribution

Many nasally applied compounds gain access to the brain and the central nervous system (CNS) with varying degree. Direct nose-to-brain access is believed to be achieved through nervous connections which travel from the CNS across the cribriform plate into the olfactory region of the nasal cavity. However, current delivery strategies are not targeted to preferentially deposit drugs to the olfactory at cribriform. Therefore, we have developed a pressurized olfactory delivery (POD) device which consistently and non-invasively deposited a majority of drug to the olfactory region of the nasal cavity in rats. Using both a hydrophobic drug, mannitol (log P = -3.1), and a hydrophobic drug, nelfinavir (log P = 6.0), and POD device, we compared brain and blood levels after nasal deposition primarily on the olfactory region with POD or nose drops which deposited primarily on the respiratory region in rats. POD administration of mannitol in rats provided a 3.6-fold (p < 0.05) increase in cortex-to-blood ratio, compared to respiratory epithelium deposition with nose drop. Administration of nelfinavir provided a 13.6-fold (p < 0.05) advantage in cortex-to-blood ratio with POD administration, compared to nose drops. These results suggest that increasing the fraction of drug deposited on the olfactory region of the nasal cavity will result in increased direct nose-to-brain transport.     

Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.