High-level constitutive expression in Pichia pastoris and one-step purification of phospholipase D from cowpea (Vigna unguiculata L. Walp)

Phospholipase D (PLD) is one of the main enzymes involved in signal transduction, vesicle trafficking and membrane metabolism processes. Here we describe the heterologous high-yield expression in the yeast Pichia pastoris, one-step purification and characterization of catalytically active PLDalpha from cowpea (Vigna unguiculata L. Walp). Immunoblotting experiments showed that recombinant PLDalpha is recognized by a polyclonal antibody raised against native soybean PLDalpha. A single calcium-dependent octyl-Sepharose chromatography step was used to obtain a highly purified recombinant PLDalpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry data. From 1L of yeast culture medium, about 8 mg of pure recombinant PLDalpha was obtained and the specific activity measured on phosphatidylcholine was 27 micromol/min/mg. Contrary to what was observed previously with Vigna unguiculata PLDalpha expressed in insect cells, no proteolytic degradation of the N-terminal calcium-dependent C2 lipid binding domain was observed here. This functional recombinant PLDalpha should provide a valuable tool for performing detailed studies on the molecular characterization of enzymes as well as structural studies.     

Persistence of balsam fir and black spruce populations in the mixedwood and coniferous bioclimatic domain of eastern North America

The boreal ecocline (ca 49°N) between the southern mixedwood (dominated by balsam fir) and the northern coniferous bioclimatic domain (dominated by black spruce) may be explained by a northward decrease of balsam fir regeneration, explaining the gradual shift to black spruce dominance. 7,010 sample plots, with absence of major disturbances, were provided by the Quebec Ministry of Forest, Fauna, and Parks. The regeneration (sapling abundance) of balsam fir and black spruce were compared within and between the two bioclimatic domains, accounting for parental trees, main soil type (clay and till) and climate conditions, reflected by summer growing degree‐days above 5°C (GDD_5), total summer precipitation (May–August; PP_MA). Parental trees and soil type determined balsam fir and black spruce regeneration. Balsam fir and black spruce, respectively, showed higher regeneration in the mixedwood and the coniferous bioclimatic domains. Overall, higher regeneration was obtained on till for balsam fir, and on clay soils for black spruce. GDD_5 and PP_MA were beneficial for balsam fir regeneration on clay and till soils, respectively, while they were detrimental for black spruce regeneration. At a population level, balsam fir required at least 28% of parental tree basal area in the mixedwood, and 38% in the coniferous bioclimatic domains to maintain a regeneration at least equal to the mean regeneration of the whole study area. However, black spruce required 82% and 79% of parental trees basal area in the mixedwood and the coniferous domains, respectively. The northern limit of the mixedwood bioclimatic domain was attributed to a gradual decrease toward the north of balsam fir regeneration most likely due to cooler temperatures, shorter growing seasons, and decrease of the parental trees further north of this northern limit. However, balsam fir still persists above this northern limit, owing to a patchy occurrence of small parental trees populations, and good establishment substrates.     

Additional evaluations show that specific BWA‐aln settings still outperform BWA‐mem for ancient DNA data alignment

Xu et al. (2021) recently recommended a new parameterization of BWA‐mem as a superior alternative to the widely‐used BWA‐aln algorithm to map ancient DNA sequencing data. Here, we compare the BWA‐mem parameterization recommended by Xu et al. with the best‐performing alignment methods determined in the recent benchmarks of Oliva and colleagues (2021), demonstrating that BWA‐aln is still the gold‐standard for ancient DNA read alignment .     

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.     

Natural Killer Cells Induce Eosinophil Activation and Apoptosis

Eosinophils are potent inflammatory cells with numerous immune functions, including antigen presentation and exacerbation of inflammatory responses through their capacity to release a range of largely preformed cytokines and lipid mediators. Thus, timely regulation of eosinophil activation and apoptosis is crucial to develop beneficial immune response and to avoid tissue damage and induce resolution of inflammation. Natural Killer (NK) cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and eosinophils. Co-culture experiments revealed that human NK cells could trigger autologous eosinophil activation, as shown by up-regulation of CD69 and down-regulation of CD62L, as well as degranulation, evidenced by increased CD63 surface expression, secretion of eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). Moreover, NK cells significantly and dose dependently increased eosinophil apoptosis as shown by annexin V and propidium iodide (PI) staining. Direct contact was necessary for eosinophil degranulation and apoptosis. Increased expression of phosphorylated extracellular signal-regulated kinase (ERK) in cocultured eosinophils and inhibition of eosinophil CD63 expression by pharmacologic inhibitors suggest that MAPK and PI3K pathways are involved in NK cell-induced eosinophil degranulation. Finally, we showed that NK cells increased reactive oxygen species (ROS) expression by eosinophils in co-culture and that mitochondrial inhibitors (rotenone and antimycin) partially diminished NK cell-induced eosinophil apoptosis, suggesting the implication of mitochondrial ROS in NK cell-induced eosinophil apoptosis. Pan-caspase inhibitor (ZVAD-FMK) only slightly decreased eosinophil apoptosis in coculture. Altogether, our results suggest that NK cells regulate eosinophil functions by inducing their activation and their apoptosis.     

Interferon-alpha triggers B cell effector 1 (Be1) commitment

B-cells can contribute to the pathogenesis of autoimmune diseases not only through auto-antibody secretion but also via cytokine production. Therapeutic depletion of B-cells influences the functions and maintenance of various T-cell subsets. The mechanisms governing the functional heterogeneity of B-cell subsets as cytokine-producing cells are poorly understood. B-cells can differentiate into two functionally polarized effectors, one (B-effector-1-cells) producing a Th-1-like cytokine pattern and the other (Be2) producing a Th-2-like pattern. IL-12 and IFN-γ play a key role in Be1 polarization, but the initial trigger of Be1 commitment is unclear. Type-I-interferons are produced early in the immune response and prime several processes involved in innate and adaptive responses. Here, we report that IFN-α triggers a signaling cascade in resting human naive B-cells, involving STAT4 and T-bet, two key IFN-γ gene imprinting factors. IFN-α primed naive B-cells for IFN-γ production and increased IFN-γ gene responsiveness to IL-12. IFN-γ continues this polarization by re-inducing T-bet and up-regulating IL-12Rβ2 expression. IFN-α and IFN-γ therefore pave the way for the action of IL-12. These results point to a coordinated action of IFN-α, IFN-γ and IL-12 in Be1 polarization of naive B-cells, and may provide new insights into the mechanisms by which type-I-interferons favor autoimmunity.     

Improved survival of HIV-1-infected patients with progressive multifocal leukoencephalopathy receiving early 5-drug combination antiretroviral therapy

Background

Progressive multifocal leukoencephalopathy (PML), a rare devastating demyelinating disease caused by the polyomavirus JC (JCV), occurs in severely immunocompromised patients, most of whom have advanced-stage HIV infection. Despite combination antiretroviral therapy (cART), 50% of patients die within 6 months of PML onset. We conducted a multicenter, open-label pilot trial evaluating the survival benefit of a five-drug cART designed to accelerate HIV replication decay and JCV-specific immune recovery.

Methods and Findings

All the patients received an optimized cART with three or more drugs for 12 months, plus the fusion inhibitor enfuvirtide during the first 6 months. The main endpoint was the one-year survival rate. A total of 28 patients were enrolled. At entry, median CD4+ T-cell count was 53 per microliter and 86% of patients had detectable plasma HIV RNA and CSF JCV DNA levels. Seven patients died, all before month 4. The one-year survival estimate was 0.75 (95% confidence interval, 0.61 to 0.93). At month 6, JCV DNA was undetectable in the CSF of 81% of survivors. At month 12, 81% of patients had undetectable plasma HIV RNA, and the median CD4+ T-cell increment was 105 per microliter. In univariate analysis, higher total and naive CD4+ T-cell counts and lower CSF JCV DNA level at baseline were associated with better survival. JCV-specific functional memory CD4+ T-cell responses, based on a proliferation assay, were detected in 4% of patients at baseline and 43% at M12 (P = 0.008).

Conclusions

The early use of five-drug cART after PML diagnosis appears to improve survival. This is associated with recovery of anti-JCV T-cell responses and JCV clearance from CSF. A low CD4+ T-cell count (particularly naive subset) and high JCV DNA copies in CSF at PML diagnosis appear to be risk factors for death.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00120367","term_id":"NCT00120367"}}NCT00120367     

The influence of recent climate change on tree height growth differs with species and spatial environment

Tree growth has been reported to increase in response to recent global climate change in controlled and semi-controlled experiments, but few studies have reported response of tree growth to increased temperature and atmospheric carbon dioxide (CO₂) concentration in natural environments. This study addresses how recent global climate change has affected height growth of trembling aspen (Populus tremuloides Michx) and black spruce (Picea mariana Mill B.S.) in their natural environments. We sampled 145 stands dominated by aspen and 82 dominated by spruce over the entire range of their distributions in British Columbia, Canada. These stands were established naturally after fire between the 19^th and 20^th centuries. Height growth was quantified as total heights of sampled dominant and co-dominant trees at breast-height age of 50 years. We assessed the relationships between 50-year height growth and environmental factors at both spatial and temporal scales. We also tested whether the tree growth associated with global climate change differed with spatial environment (latitude, longitude and elevation). As expected, height growth of both species was positively related to temperature variables at the regional scale and with soil moisture and nutrient availability at the local scale. While height growth of trembling aspen was not significantly related to any of the temporal variables we examined, that of black spruce increased significantly with stand establishment date, the anomaly of the average maximum summer temperature between May-August, and atmospheric CO₂ concentration, but not with the Palmer Drought Severity Index. Furthermore, the increase of spruce height growth associated with recent climate change was higher in the western than in eastern part of British Columbia. This study demonstrates that the response of height growth to recent climate change, i.e., increasing temperature and atmospheric CO₂ concentration, did not only differ with tree species, but also their growing spatial environment.     

Alleviation of drought stress in faba bean (Vicia faba L.) by exogenous application of β-aminobutyric acid (BABA)

Physiology and Molecular Biology of Plants - Drought stress is one of the most prevalent environmental factors limiting faba bean (Vicia faba L.) crop productivity. β-aminobutyric acid (BABA)...