The small molecule Zaractin activates ZAR1-mediated immunity in Arabidopsis

Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis. Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system.

Gram-negative bacterial plant pathogens such as Pseudomonas syringae use the type III secretion system (T3SS) to inject type III secreted effectors (T3SEs) into plant cells (1). A major function of T3SEs is to suppress plant immunity by targeting host proteins involved in disease resistance (2). However, plant nucleotide-binding leucine-rich-repeat (NLR) proteins can directly or indirectly recognize T3SEs and activate a response known as effector-triggered immunity (ETI), which can be accompanied by a hypersensitive cell death response (2, 3). The Arabidopsis thaliana NLR ZAR1 recognizes T3SE-induced complexes of ZED1-related kinases (ZRKs; RLCK XII-2 family) and PBS1-like (PBL) kinases (RLCK VII family) to activate ETI (4–7). T3SEs modify either ZRK and/or PBL kinases and promote their interaction, which activates ZAR1-mediated ETI (5, 6). For example, the Xanthomonas campestris AvrAC, uridylates PBL2 kinase, promoting an interaction with ZRK1 (also known as RKS1) that then acts as a nucleotide exchange factor to activate the ZAR1 resistosome (8, 9).In addition to AvrAC, five P. syringae effector families have been shown to trigger ZAR1/ZRK-mediated immunity in Arabidopsis (4, 10–13). ZAR1-mediated immunity triggered by the acetyltransferase HopZ1a requires the ZRK kinase ZED1 and the functionally redundant PBL kinases SZE1 and SZE2 (4, 7). Interestingly, acetylation of ZED1 by HopZ1a can activate immunity, suggesting that modification of either ZRK or PBL kinases can activate the ZAR1 resistosome (4, 6). HopX1i-recognition also requires ZED1 and SZE1, but not SZE2 (13). HopO1c- and HopF1r-mediated immunity requires ZRK3, whereas HopBA1a recognition requires ZRK2, but no PBL kinase requirement has been identified (11, 13). Based on the genetic requirements of ZAR1-mediated ETI against P. syringae effectors and the model of AvrAC recognition, a general mechanism of ZAR1 activation likely involves T3SE perturbations of ZRK and/or PBL kinases that promote their interaction, which in turn activates the ZAR1 resistosome (8, 9).We hypothesized that small molecules that mimic ETI-promoting effector perturbations would represent a powerful, targeted approach to activate plant immunity. Given the model of ZAR1 activation described above, we developed a chemical screen to identify small molecules that enhance ETI-inducing ZRK/PBL interactions. First, we show that the P. syringae T3SE HopF1r can enhance the interaction between ZRK3 and PBL27 and that PBL27 is a required component of ZAR1-mediated recognition of HopF1r. We then used our chemical screen to identify a small molecule (Zaractin) that can enhance the ZRK3/PBL27 interaction and activate ZAR1-dependent immunity. Overall, our results demonstrate that chemical mimicry of type III effector function can activate an NLR-mediated immune response, providing an approach to identify chemical immunomodulators.     

Study of Safety and Efficacy of Novel Sirolimus-Eluting Stent Incorporating Properties of Drug Coating Balloon Among Real World Patients Focusing Younger Population (<35 years)

ObjectiveAim of study was to evaluate safety and efficacy of abluminal Mitigator DES + Sirolimus Eluting Stent (Envision Scientific, Surat, India) incorporating novel technology of fusion coating of bioresorbable polymer on both abluminal surface of stent and exposed parts of balloon among real world patients specially focusing younger patients (<35 years).Method1293 patients received Mitigator DES + at LPS Institute of Cardiology, Kanpur, India. Primary outcome was target lesion failure (TLF)- composite of cardiovascular death, target vessel myocardial infarction (TVMI), and target lesion revascularization (TLR) and secondary end points including peri-procedural device failure (failure of stent delivery, change of stent, stent fracture), target vessel failure (TVF), and patient oriented composite end point (POCE)-composite of all deaths, MI, and revascularization and stent thrombosis (ST) at 1-year follow-up.ResultYounger population comprised of 374 (29%) patients. Various indications of interventions were STEMI (n = 614; 47.4%), NSTEMI (n = 416; 32.2%), UA (n = 161; 12.5%), and CCS (n = 102; 7.9%). TLF at 1 year in young and overall population were 3.4% and 3.5% respectively which was driven by TVMI and TLR in 1.3% and 1.1% patients respectively. POCE was observed in 9.5% in each group mainly contributed by any revascularization (3.9%). Device failure was significantly lower in young group than overall population (1.3% vs. 2.2%; p = 0.04) which was mainly driven by stent delivery (1.1%) and edge dissection (0.5%). Definite and probable ST was 1.3% and 1.7% respectively which was not significant. Young patients showed insignificantly lower TLF, TVF, ST and POCE and significantly lower device failure (1.3% vs. 2.6%; p = 0.04) when compared to patients >35 years. On multivariate regression analysis, complex lesion, in-stent restenosis, failure of stent delivery and edge dissection were independent predictors of events or device success rate.ConclusionMitigator DES+™ is safe among real world patients, including young population.     

Coherent neural oscillations inform early stroke motor recovery

Neural oscillations may contain important information pertaining to stroke rehabilitation. This study examined the predictive performance of electroencephalography‐derived neural oscillations following stroke using a data‐driven approach. Individuals with stroke admitted to an inpatient rehabilitation facility completed a resting‐state electroencephalography recording and structural neuroimaging around the time of admission and motor testing at admission and discharge. Using a lasso regression model with cross‐validation, we determined the extent of motor recovery (admission to discharge change in Functional Independence Measurement motor subscale score) prediction from electroencephalography, baseline motor status, and corticospinal tract injury. In 27 participants, coherence in a 1–30 Hz band between leads overlying ipsilesional primary motor cortex and 16 leads over bilateral hemispheres predicted 61.8% of the variance in motor recovery. High beta (20–30 Hz) and alpha (8–12 Hz) frequencies contributed most to the model demonstrating both positive and negative associations with motor recovery, including high beta leads in supplementary motor areas and ipsilesional ventral premotor and parietal regions and alpha leads overlying contralesional temporal–parietal and ipsilesional parietal regions. Electroencephalography power, baseline motor status, and corticospinal tract injury did not significantly predict motor recovery during hospitalization (R ² = 0–6.2%). Findings underscore the relevance of oscillatory synchronization in early stroke rehabilitation while highlighting contributions from beta and alpha frequency bands and frontal, parietal, and temporal–parietal regions overlooked by traditional hypothesis‐driven prediction models.     

Protein kinase N2 mediates flow-induced endothelial NOS activation and vascular tone regulation

Formation of NO by endothelial NOS (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation, and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and induce the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and G_q/G₁₁-mediated signaling, as well as by Ca²⁺ and phosphoinositide-dependent protein kinase 1 (PDK1), plays a pivotal role in this process. Active PKN2 promoted the phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved the phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation, whereas active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone, and blood pressure.     

The impact of humanoid robot presence in the paediatric emergency department waiting room: A prospective cohort study

BackgroundThe waiting room represents families’ first point of contact with the emergency department (ED). We wished to study if a humanoid robot technology presence in the waiting room would improve satisfaction and decrease anxiety for caregivers in the paediatric ED. MethodsThis observational cohort study was conducted from September to December 2018 at a Canadian paediatric ED. All caregivers and children >11 years in the ED waiting room were eligible. We compared a robotic intervention (RI) to standard of care (SOC) education in the waiting room. The RI was a 5-minute psychoeducational program describing the ED process and flow. Specific days were designated for RI or SOC. An anonymous survey was administered twice, at the same times, on both SOC and RI days. The primary outcomes were (a) caregiver satisfaction with waiting room experience using a 5-point Likert scale; and (b) caregiver-reported anxiety in the waiting room, as measured by the State Trait Anxiety Inventory – State Scale. ResultsSix hundred and thirty-three caregivers participated, with a median age of 37 years (IQR 32 to 42); 80 children participated, with a median age of 15 years (IQR 13 to 16). Caregivers reported greater overall satisfaction in the RI cohort (174/200, 87.0%) compared to the SOC cohort (144/229, 62.9%; P<0.0001). Caregivers also reported lower anxiety in the RI cohort (39.38±11.38) compared to the SOC cohort (42.04±11.99; P=0.009).ConclusionsA humanoid robot-based psycho educational intervention in the paediatric ED waiting room has a positive impact on caregiver satisfaction and anxiety.     

In silico prediction of monovalent and chimeric tetravalent vaccines for prevention and treatment of dengue fever

Reverse vaccinology method was used to predict the monovalent peptide vaccine candidate to produce antibodies for therapeutic purpose and to predict tetravalent vaccine candidate to act as a common vaccine to cover all the dengue virus serotypes. Envelope (E)-proteins of DENV-1-4 serotypes were used for vaccine prediction using NCBI, Uniprot/Swissprot, Swiss-prot viewer, VaxiJen V2.0, TMHMM, BCPREDS, Propred-1, Propred and MHC Pred. Eproteins of DENV-1-4 serotypes were identified as antigen from which T cell epitopes, through B cell epitopes, were predicted to act as peptide vaccine candidates. Each selected T cell epitope of E-protein was confirmed to act as vaccine and to induce complementary antibody against particular serotype of dengue virus. Chimeric tetravalent vaccine was formed by the conjugation of four vaccines, each from four dengue serotypes to act as a common vaccine candidate for all the four dengue serotypes. It can be justifiably concluded that the monovalent 9-mer T cell epitope for each DENV serotype can be used to produce specific antibody against dengue virus and a chimeric common tetravalent vaccine candidate to yield a comparative vaccine to cover any of the four dengue virus serotype. This vaccine is expected to be highly immunogenic against dengue fever.     

Clinical Outcomes of Patients With Recurrent Lung Cancer Reirradiated With Proton Therapy on the Proton Collaborative Group and University of Florida Proton Therapy Institute Prospective Registry Studies

PurposeWe sought to assess clinical outcomes and toxicities of patients with recurrent lung cancer reirradiated with proton beam therapy (PBT) who were enrolled in 2 prospective registry trials.Methods and MaterialsSeventy-nine consecutive patients were reirradiated with PBT at 8 institutions. Conventionally fractionated radiation therapy was used to treat the previous lung cancer in 68% of patients (median equivalent dose in 2 Gy fractions [EQD₂], 60.2 Gy) and hypofractionated/stereotactic body radiation therapy in 32% (median EQD₂, 83.3 Gy). Nine patients (11%) received ≥2 courses of thoracic irradiation before PBT. Eastern Cooperative Oncology Group (ECOG) performance status was 2 to 3 in 13%. Median time from prior radiation therapy to PBT was 19.9 months. PBT was delivered with conventional fractionation in 58% (median EQD₂, 60 Gy), hyperfractionation in 3% (median EQD₂, 62.7 Gy), and hypofractionation in 39% (median EQD₂, 60.4 Gy). Twenty-four patients (30%) received chemotherapy concurrently with PBT.ResultsAll patients completed PBT as planned. At a median follow-up of 10.7 months after PBT, median overall survival (OS) and progression-free survival (PFS) were 15.2 months and 10.5 months, respectively. Acute and late grade 3 toxicities occurred in 6% and 1%, respectively. Three patients died after PBT from possible radiation toxicity. On multivariate analysis, ECOG performance status ≤1 was associated with OS (hazard ratio, 0.35; 95% confidence interval, 0.15-0.80; P = .014) and PFS (hazard ratio, 0.32; 95% confidence interval, 0.14-0.73; P = .007).ConclusionsThis is the largest series to date of PBT reirradiation for recurrent lung cancer and indicates that reirradiation with PBT is well tolerated with acceptable toxicity and encouraging efficacy. ECOG performance status was associated with OS and PFS.