Genetically engineered two-warhead evasins provide a method to achieve precision targeting of disease-relevant chemokine subsets

Both CC and CXC-class chemokines drive inflammatory disease. Tick salivary chemokine-binding proteins (CKBPs), or evasins, specifically bind subsets of CC- or CXC-chemokines, and could precisely target disease-relevant chemokines. Here we have used yeast surface display to identify two tick evasins: a CC-CKBP, P1243 from Amblyomma americanum and a CXC-CKBP, P1156 from Ixodes ricinus. P1243 binds 11 CC-chemokines with Kd < 10 nM, and 10 CC-chemokines with Kd between 10 and 100 nM. P1156 binds two ELR + CXC-chemokines with Kd < 10 nM, and four ELR + CXC-chemokines with Kd between 10 and 100 nM. Both CKBPs neutralize chemokine activity with IC50 < 10 nM in cell migration assays. As both CC- and CXC-CKBP activities are desirable in a single agent, we have engineered "two-warhead" CKBPs to create single agents that bind and neutralize subsets of CC and CXC chemokines. These results show that tick evasins can be linked to create non-natural proteins that target subsets of CC and CXC chemokines. We suggest that "two-warhead" evasins, designed by matching the activities of parental evasins to CC and CXC chemokines expressed in disease, would achieve precision targeting of inflammatory disease-relevant chemokines by a single agent

Goishi tea consumption inhibits airway hyperresponsiveness in BALB/c mice

<p>Abstract</p> <p>Background</p> <p>Airway hyperresponsiveness (AHR) is one of the important traits that characterize bronchial asthma. Goishi tea is a post-heating fermented tea that has been reported to have higher free radical scavenging activity. In this study, we evaluated the prophylactic effects of Goishi tea on AHR in BALB/c mice.</p> <p>Results</p> <p>The number of inflammatory cells in BAL fluid was considerably reduced in Goishi tea/<it>Der f </it>and Gallic acid/<it>Der f </it>groups as compared with Tap water/<it>Der f </it>group. Regarding inflammatory cells in BAL, a significant reduction of eosinophils and neutrophils was observed in Goishi tea-treated mice (p < 0.01), as well as in the Gallic acid/<it>Der f </it>group (p < 0.05), as compared with Tap water/<it>Der f </it>group. In asthmatic mice (Tap water/<it>Der f </it>group), the intensity of airway resistance increased simultaneously with the increase in acetylcholine concentration in a dose-dependant way. AHR was significantly inhibited in Goishi tea/<it>Der f </it>and Gallic acid/<it>Der f </it>(p < 0.01) groups as compared with the Tap water/<it>Der f </it>group. Regarding serum specific-IgG₁, significantly lower levels of this antibody were observed in Goishi tea/<it>Der f </it>and Gallic acid/<it>Der f </it>groups as compared with the Tap water/<it>Der f </it>group (p < 0.05). In addition, adiponectin level was significantly higher in the Goishi tea group as compared with the Tap water treated mice (p < 0.01).</p> <p>Conclusions</p> <p>The results suggest that Goishi tea consumption exerted an inhibitory effect on eosinophilic and neutrophilic infiltration in the lung, attenuated the increase in airway resistance and increased the production of adiponectin; thus reducing Der f induced allergic inflammatory process in mice.</p

Multiplexed epigenetic memory editing using CRISPRoff sensitizes glioblastoma to chemotherapy

BackgroundGlioblastoma (GBM) carries a poor prognosis, and new therapeutic strategies are necessary to improve outcomes for patients with this disease. Alkylating chemotherapies including temozolomide (TMZ) and lomustine (CCNU) are critical for treating GBM, but resistance mechanisms, including hypomethylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter, undermine treatment. CRISPRoff is a programmable epigenetic memory editor that can induce stable and heritable gene silencing after transient delivery, and we hypothesize that CRISPRoff could potentiate the activity of TMZ and CCNU through long term suppression of target genes.MethodsWe transiently delivered CRISPRoff mRNA along with sgRNAs against target genes using both electroporation and lipid nanoparticles (LNPs) into established GBM cell lines, patient-derived primary GBM cultures, and orthotopic GBM xenografts. Gene repression, specificity, and stability was measured by RT-qPCR, Western blot, bisulfite sequencing, and RNA-sequencing. Sensitivity to chemotherapies was measured by cell viability dose response, microscopy, and bioluminescence imaging. Genome-wide mapping of CCNU sensitizers was performed using CRISPRi screens.ResultsCRISPRoff induced complete suppression of MGMT and sensitization to TMZ that were stable for over 8 months of continuous cell propagation. GBM orthotopic tumors treated with CRISPRoff against MGMT demonstrated sensitivity to TMZ in vivo, and CRISPRoff delivery resulted in chemosensitivity in patient-derived primary GBM. Genome-wide CRISPRi screens identified combinatorial genetic vulnerabilities (BRIP1, FANCE) that were targetable by multiplexed CRISPRoff to achieve sensitization to CCNU.ConclusionTransient delivery of a site-specific epigenetic memory can induce stable, complete, and multiplexed suppression of target genes for therapeutic application in GBM

Mycorrhizal symbioses and tree diversity in global forest communities

Unraveling the mechanisms underlying the maintenance of species diversity is a central pursuit in ecology. It has been hypothesized that ectomycorrhizal (EcM) in contrast to arbuscular mycorrhizal fungi can reduce tree species diversity in local communities, which remains to be tested at the global scale. To address this gap, we analyzed global forest inventory data and revealed that the relationship between tree species richness and EcM tree proportion varied along environmental gradients. Specifically, the relationship is more negative at low latitudes and in moist conditions but is unimodal at high latitudes and in arid conditions. The negative association of EcM tree proportion on species diversity at low latitudes and in humid conditions is likely due to more negative plant-soil microbial interactions in these regions. These findings extend our knowledge on the mechanisms shaping global patterns in plant species diversity from a belowground view

Relationship between peripheral airway function and patient-reported outcomes in COPD: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Health status, dyspnea and psychological status are important clinical outcomes in chronic obstructive pulmonary disease (COPD). However, forced expiratory volume in one second (FEV₁) measured by spirometry, the standard measurement of airflow limitation, has only a weak relationship with these outcomes in COPD. Recently, in addition to spirometry, impulse oscillometry (IOS) measuring lung resistance (R) and reactance (X) is increasingly being used to assess pulmonary functional impairment.</p> <p>Methods</p> <p>We aimed to identify relationships between IOS measurements and patient-reported outcomes in 65 outpatients with stable COPD. We performed pulmonary function testing, IOS, high-resolution computed tomography (CT), and assessment of health status using the St. George's Respiratory Questionnaire (SGRQ), dyspnea using the Medical Research Council (MRC) scale and psychological status using the Hospital Anxiety and Depression Scale (HADS). We then investigated the relationships between these parameters. For the IOS measurements, we used lung resistance at 5 and 20 Hz (R5 and R20, respectively) and reactance at 5 Hz (X5). Because R5 and R20 are regarded as reflecting total and proximal airway resistance, respectively, the fall in resistance from R5 to R20 (R5-R20) was used as a surrogate for the resistance of peripheral airways. X5 was also considered to represent peripheral airway abnormalities.</p> <p>Results</p> <p>R5-R20 and X5 were significantly correlated with the SGRQ and the MRC. These correlation coefficients were greater than when using other objective measurements of pulmonary function, R20 on the IOS and CT instead of R5-R20 and X5. Multiple regression analyses showed that R5-R20 or X5 most significantly accounted for the SGRQ and MRC scores.</p> <p>Conclusions</p> <p>IOS measurements, especially indices of peripheral airway function, are significantly correlated with health status and dyspnea in patients with COPD. Therefore, in addition to its simplicity and non-invasiveness, IOS may be a useful clinical tool not only for detecting pulmonary functional impairment, but also to some extent at least estimating the patient's quality of daily life and well-being.</p

Rapid Accumulation of Polymorphonuclear Neutrophils in the Corpus luteum during Prostaglandin F2α-Induced Luteolysis in the Cow

Prostaglandin F2α (PGF2α) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF2α administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF2α injection. The number of PMNs was increased at 5 min after PGF2α administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF2α directly stimulated P-selectin protein expression at 5–30 min in luteal endothelial cells (LECs). Moreover, PGF2α enhanced PMN adhesion to LECs, and this enhancement by PGF2α was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF2α is crucial in PMN migration. In conclusion, PGF2α rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF2α may involve an acute inflammatory-like response due to rapidly infiltrated PMNs

Mycorrhizal symbioses and tree diversity in global forest communities

Unraveling the mechanisms underlying the maintenance of species diversity is a central pursuit in ecology. It has been hypothesized that ectomycorrhizal (EcM) in contrast to arbuscular mycorrhizal fungi can reduce tree species diversity in local communities, which remains to be tested at the global scale. To address this gap, we analyzed global forest inventory data and revealed that the relationship between tree species richness and EcM tree proportion varied along environmental gradients. Specifically, the relationship is more negative at low latitudes and in moist conditions but is unimodal at high latitudes and in arid conditions. The negative association of EcM tree proportion on species diversity at low latitudes and in humid conditions is likely due to more negative plant-soil microbial interactions in these regions. These findings extend our knowledge on the mechanisms shaping global patterns in plant species diversity from a belowground view

Co-limitation towards lower latitudes shapes global forest diversity gradients

The latitudinal diversity gradient (LDG) is one of the most recognized global patterns of species richness exhibited across a wide range of taxa. Numerous hypotheses have been proposed in the past two centuries to explain LDG, but rigorous tests of the drivers of LDGs have been limited by a lack of high-quality global species richness data. Here we produce a high-resolution (0.025° × 0.025°) map of local tree species richness using a global forest inventory database with individual tree information and local biophysical characteristics from ~1.3 million sample plots. We then quantify drivers of local tree species richness patterns across latitudes. Generally, annual mean temperature was a dominant predictor of tree species richness, which is most consistent with the metabolic theory of biodiversity (MTB). However, MTB underestimated LDG in the tropics, where high species richness was also moderated by topographic, soil and anthropogenic factors operating at local scales. Given that local landscape variables operate synergistically with bioclimatic factors in shaping the global LDG pattern, we suggest that MTB be extended to account for co-limitation by subordinate drivers