Reactive oxygen species signalling is involved in alkamide-induced altered root development.

Alkamides are alpha unsaturated N-acylamides structurally related to N-acyl ethanolamides (NAEs) and N-acyl-L-homoserine lactones (AHLs). Studies have shown that alkamides induce prominent changes in root architecture, a significant metabolic readjustment, and transcriptional reprogramming. Some alkamide responses have been associated with redox signalling; however, this involvement and ROS sources have not been fully described. We utilized a genetic approach to address ROS signalling in alkamide-induced processes and found that in Arabidopsis, treatment with the alkamide affinin (50μM) increased the in-situ accumulation of H2O2 in lateral root emergence sites and reduced H2O2 accumulation in primary root meristems implying that altered root growth was dependent on endogenous H2O2. Results show that ROS sourced from PRX34, RBOHC and RBOHD were involved in promotion of lateral root emergence by alkamides. RBOHC was required for affinin-induced enhanced root hair expansion. Furthermore, affinin-induced changes in lateral root emergence, but not root hair length, were dependent on a change in extracellular pH. Finally, reverse genetic experiments suggest heterotrimeric G-proteins were involved in plant response to alkamides; nevertheless, further studies with additional higher order G-protein mutants will be required to resolve this question. These results support that alkamides recruit specific ROS signaling programs to mediate alterations in root architecture.

Anticipating future SARS-CoV-2 variants of concern through ab initio quantum mechanical modeling

Evolved SARS-CoV-2 variants are currently challenging the efficacy of first-generation vaccines, largely through the emergence of spike protein mutants. Among these variants, Delta is presently the most concerning. We employ an ab initio quantum mechanical model based on Density Functional Theory to characterize the spike protein Receptor Binding Domain (RBD) interaction with host cells and gain mechanistic insight into SARS-CoV-2 evolution. The approach is illustrated via a detailed investigation of the role of the E484K RBD mutation, a signature mutation of the Beta and Gamma variants. The simulation is employed to: predict the depleting effect of the E484K mutation on binding the RBD with select antibodies; identify residue E484 as a weak link in the original interaction with the human receptor hACE2; and describe SARS-CoV-2 Wuhan strand binding to the bat Rhinolophus macrotis ACE2 as more optimized than the human counterpart. Finally, we predict the hACE2 binding efficacy of a hypothetical E484K mutation added to the Delta variant RBD, identifying a potential future variant of concern. Results can be generalized to other mutations, and provide useful information to complement existing experimental datasets of the interaction between randomly generated libraries of hACE2 and viral spike mutants. We argue that ab initio modeling is at the point of being aptly employed to inform and predict events pertinent to viral and general evolution.

An extracellular matrix damage sensor signals through membrane-associated kinase DRL-1 to mediate cytoprotective responses in Caenorhabditis elegans

We and others previously identified circumferential bands of collagen named annular furrows as key components of a damage sensor in the cuticle of Caenorhabditis elegans that regulates cytoprotective genes. Mutation or loss of non-collagen secreted proteins OSM-7, OSM-8, and OSM-11 activate the same cytoprotective responses without obvious changes to the cuticle indicating that other extracellular proteins are involved. Here, we used RNAi screening to identify protein kinase DRL-1 as a key modulator of cytoprotective gene expression and stress resistance in furrow and extracellular OSM protein mutants. DRL-1 functions downstream from furrow disruption and is expressed in cells that induce cytoprotective genes. DRL-1 is not required for expression of cytoprotective genes under basal or oxidative stress conditions consistent with specificity to extracellular signals. DRL-1 was previously shown to regulate longevity via a ‘Dietary Restriction-Like’ state, but it functions downstream from furrow disruption by a distinct mechanism. The kinase domain of DRL-1 is related to mammalian MEKK3, and MEKK3 is recruited to a plasma membrane osmosensor complex by a scaffold protein. In C. elegans, DRL-1 contains an atypical hydrophobic C-terminus with predicted transmembrane domains and is constitutively expressed at or near the plasma membrane where it could function to receive extracellular damage signals for cells that mount cytoprotective responses.

Arterial and Venous Thrombosis Complicated in COVID-19: A Retrospective Single Center Analysis in Japan

Background: Thrombosis is a characteristic complication in coronavirus disease 2019 (COVID-19). Since coagulopathy has been observed over the entire clinical course, thrombosis might be a clue to understanding the specific pathology in COVID-19. Currently, there is limited epidemiological data of COVID-19-associated thrombosis in the Japanese population and none regarding variant strains of SARS-CoV-2. Here, we elucidate the risk factors and the pattern of thrombosis in COVID-19 patients.Methods: The patients consecutively admitted to Tokyo Medical and Dental University Hospital with COVID-19 were retrospectively analyzed. SARS-CoV-2 variants of concern/interest (VOC/VOI) carrying the spike protein mutants E484K, N501Y, or L452R were identified by PCR-based analysis. All thrombotic events were diagnosed by clinical symptoms, ultrasonography, and/or radiological tests.Results: Among the 516 patients, 32 patients experienced 42 thromboembolic events. Advanced age, severe respiratory conditions, and several abnormal laboratory markers were associated with the development of thrombosis. While thrombotic events occurred in 13% of the patients with a severe respiratory condition, those events still occurred in 2.5% of the patients who did not require oxygen therapy. Elevated D-dimer and ferritin levels on admission were independent risk factors of thrombosis (adjusted odds ratio 9.39 and 3.11, 95% confidence interval 2.08–42.3, and 1.06–9.17, respectively). Of the thrombotic events, 22 were venous, whereas 20 were arterial. While patients with thrombosis received anticoagulation and antiinflammatory therapies with a higher proportion, the mortality rate, organ dysfunctions, and bleeding complications in these patients were higher than those without thrombosis. The incidence of thrombosis in COVID-19 became less frequent over time, such as during the replacement of the earlier strains of SARS-CoV-2 by VOC/VOI and during increased use of anticoagulatory therapeutics.Conclusion: This study elucidated that elevated D-dimer and ferritin levels are useful biomarkers of thrombosis in COVID-19 patients. The comparable incidence of arterial thrombosis with venous thrombosis and the development of thrombosis in less severe patients required further considerations for the management of Japanese patients with COVID-19. Further studies would be required to identify high-risk populations and establish appropriate interventions for thrombotic complications in COVID-19.

DnaK response to expression of protein mutants is dependent on translation rate and stability

Chaperones play a central part in the quality control system in cells by clearing misfolded and aggregated proteins. The chaperone DnaK acts as a sensor for molecular stress by recognising short hydrophobic stretches of misfolded proteins. As the level of unfolded protein is a function of protein stability, we hypothesised that the level of DnaK response upon overexpression of recombinant proteins would be correlated to stability. Using a set of mutants of the lambda-repressor with varying thermal stabilities and a fluorescent reporter system, the effect of stability on DnaK response and protein abundance was investigated. Our results demonstrate that the initial DnaK response is largely dependent on protein synthesis rate but as the recombinantly expressed protein accumulates and homeostasis is approached the response correlates strongly with stability. Furthermore, we observe a large degree of cell-cell variation in protein abundance and DnaK response in more stable proteins.

DnaK response to expression of protein mutants is dependent on translation rate and stability

Chaperones play a central part in the quality control system in cells by clearing misfolded and aggregated proteins. The chaperone DnaK acts as a sensor for molecular stress by recognising short hydrophobic stretches of misfolded proteins. As the level of unfolded protein is a function of protein stability, we hypothesised that the level of DnaK response upon overexpression of recombinant proteins would be correlated to stability. Using a set of mutants of the λ-repressor with varying thermal stabilities and a fluorescent reporter system, the effect of stability on DnaK response and protein abundance was investigated. Our results demonstrate that the initial DnaK response is largely dependent on protein synthesis rate but as the recombinantly expressed protein accumulates and homeostasis is approached the response correlates strongly with stability. Furthermore, we observe a large degree of cell-cell variation in protein abundance and DnaK response in more stable proteins.

High individual heterogeneity of neutralizing activities against the original 4 strain and 9 different variants of SARS-CoV-2

Background. Since the beginning of the COVID-19 pandemic, several SARS-CoV-2 variants have sequentially emerged. In France, most cases were due to spike D641G-harbouring viruses that descended initially from the Wuhan strain, then by variant of B.1.160 lineage we called Marseille-4 since the summer of 2020, which was followed by the alpha (UK) and beta (South African) variants in early 2021, then delta (Indian) now.Methods and Findings. We determined the neutralizing antibody (nAb) titres in sera from convalescent individuals previously infected by these 4 major local variants and from vaccine recipients to the original Wuhan strain and 9 variants, including two recent circulating delta (Indian) isolates. The results show high inter-individual heterogeneity in nAbs, especially according to the variant tested. Unexpectedly, the major variations among nAbs are based on the genotype responsible for the infection. Patients previously infected with the beta and B.1.160 variants had the lowest nAb titres. We show that this heterogeneity is well explained by spike protein mutants modelling using in silico approaches. The highest titres were observed in patients vaccinated with the Pfizer/BioNTech COVID-19 vaccine, even against the delta variant.Conclusions. Immunity acquired naturally after infection is highly dependent on the infecting variant and unexpectedly mRNA-based vaccine efficacy is shown to be often better than natural immunity in eliciting neutralizing antibodies.

Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71–Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.

Stabilizing proteins through saturation suppressor mutagenesis.

While there have been recent, transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding, however reliable screens for mutants that improve protein stability do not exist, especially for proteins that are well folded and relatively stable. We demonstrate that incorporation of a single, specific destabilizing, (parent inactive) mutation into each member of a single-site saturation mutagenesis library followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. When coupled to FACS sorting of a yeast surface display library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations, multiple stabilizing mutations could be identified after a single round of sorting. Multiple libraries with different parent inactive mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Individual stabilizing mutations could be combined to result in a multimutant with increase in thermal melting temperature of about 20 degrees Celsius and enhanced tolerance to high temperature exposure. The method employs small library sizes and can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants.