A Pan-plant Protein Complex Map Reveals Deep Conservation and Novel Assemblies.

Plants are foundational for global ecological and economic systems, but most plant proteins remain uncharacterized. Protein interaction networks often suggest protein functions and open new avenues to characterize genes and proteins. We therefore systematically determined protein complexes from 13 plant species of scientific and agricultural importance, greatly expanding the known repertoire of stable protein complexes in plants. By using co-fractionation mass spectrometry, we recovered known complexes, confirmed complexes predicted to occur in plants, and identified previously unknown interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical for agriculture. We also observed plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers a cross-species view of conserved, stable protein assemblies shared across plant cells and provides a mechanistic, biochemical framework for interpreting plant genetics and mutant phenotypes.

Targeted DNA insertion in plants.

Conventional methods of DNA sequence insertion into plants, using Agrobacterium-mediated transformation or microprojectile bombardment, result in the integration of the DNA at random sites in the genome. These plants may exhibit altered agronomic traits as a consequence of disruption or silencing of genes that serve a critical function. Also, genes of interest inserted at random sites are often not expressed at the desired level. For these reasons, targeted DNA insertion at suitable genomic sites in plants is a desirable alternative. In this paper we review approaches of targeted DNA insertion in plant genomes, discuss current technical challenges, and describe promising applications of targeted DNA insertion for crop genetic improvement.

What Is the Prevalence of Clinically Important Findings Among Incidentally Found Osseous Lesions?

BACKGROUND: Patients with incidentally found musculoskeletal lesions are regularly referred to orthopaedic oncology. Most orthopaedic oncologists understand that many incidental findings are nonaggressive and can be managed nonoperatively. However, the prevalence of clinically important lesions (defined as those indicated for biopsy or treatment, and those found to be malignant) remains unknown. Missing clinically important lesions can result in harm to patients, but needless surveillance may exacerbate patient anxiety about their diagnosis and accrue low-value costs to the payor. QUESTIONS/PURPOSES: (1) What percentage of patients with incidentally discovered osseous lesions referred to orthopaedic oncology had lesions that were clinically important, defined as those receiving biopsy or treatment or those found to be malignant? (2) Using standardized Medicare reimbursements as a surrogate for payor expense, what is the value of reimbursements accruing to the hospital system for the imaging of incidentally found osseous lesions performed during the initial workup period and during the surveillance period, if indicated? METHODS: This was a retrospective study of patients referred to orthopaedic oncology for incidentally found osseous lesions at two large academic hospital systems. Medical records were queried for the word "incidental," and matches were confirmed by manual review. Patients evaluated at Indiana University Health between January 1, 2014, and December 31, 2020, and those evaluated at University Hospitals between January 1, 2017, and December 31, 2020, were included. All patients were evaluated and treated by the two senior authors of this study and no others were included. Our search identified 625 patients. Sixteen percent (97 of 625) of patients were excluded because their lesions were not incidentally found, and 12% (78 of 625) were excluded because the incidental findings were not bone lesions. Another 4% (24 of 625) were excluded because they had received workup or treatment by an outside orthopaedic oncologist, and 2% (10 of 625) were excluded for missing information. A total of 416 patients were available for preliminary analysis. Among these patients, 33% (136 of 416) were indicated for surveillance. The primary indication for surveillance included lesions with a benign appearance on imaging and low clinical suspicion of malignancy or fracture. A total of 33% (45 of 136) of these patients had less than 12 months of follow-up and were excluded from further analysis. No minimum follow-up criteria were applied to patients not indicated for surveillance because this would artificially inflate our estimated rate of clinically important findings. A total of 371 patients were included in the final study group. Notes from all clinical encounters with orthopaedic and nonorthopaedic providers were screened for our endpoints (biopsy, treatment, or malignancy). Indications for biopsy included lesions with aggressive features, lesions with nonspecific imaging characteristics and a clinical picture concerning for malignancy, and lesion changes seen on imaging during the surveillance period. Indications for treatment included lesions with increased risk of fracture or deformity, certain malignancies, and pathologic fracture. Diagnoses were determined using biopsy results if available or the documented opinion of the consulting orthopaedic oncologist. Imaging reimbursements were obtained from the Medicare Physician Fee Schedule for 2022. Because imaging charges vary across institutions and reimbursements vary across payors, this method was chosen to enhance the comparability of our findings across multiple health systems and studies. RESULTS: Seven percent (26 of 371) of incidental findings were determined to be clinically important, as previously defined. Five percent (20 of 371) of lesions underwent tissue biopsy, and 2% (eight of 371) received surgical intervention. Fewer than 2% (six of 371) of lesions were malignant. Serial imaging changed the treatment of 1% (two of 136) of the patients, corresponding to a rate of one in 47 person-years. Median reimbursements to work up the incidental findings analyzed was USD 219 (interquartile range USD 0 to 404), with a range of USD 0 to 890. Among patients indicated for surveillance, the median annual reimbursement was USD 78 (IQR USD 0 to 389), with a range of USD 0 to 2706. CONCLUSION: The prevalence of clinically important findings among patients referred to orthopaedic oncology for incidentally found osseous lesions is modest. The likelihood of surveillance resulting in a change of management was low, but the median reimbursements associated with following these lesions was also low. We conclude that after appropriate risk stratification by orthopaedic oncology, incidental lesions are rarely clinically important, and judicious follow-up with serial imaging can be performed without incurring high costs. LEVEL OF EVIDENCE: Level III, therapeutic study.

Antivirulence Bispecific Monoclonal Antibody-Mediated Protection against Pseudomonas aeruginosa Ventilator-Associated Pneumonia in a Rabbit Model.

Ventilator-associated pneumonia is an important clinical manifestation of the nosocomial pathogen Pseudomonas aeruginosa. We characterized the correlates of protection with MEDI3902, a bispecific human IgG1 monoclonal antibody that targets the P. aeruginosa type 3 secretion system PcrV protein and the Psl exopolysaccharide, in a rabbit model of ventilator-associated pneumonia using lung-protective, low-tidal-volume mechanical ventilation. Rabbits infused with MEDI3902 prophylactically were protected, whereas those pretreated with irrelevant isotype-matched control IgG (c-IgG) succumbed between 12 and 44 h postinfection (100% survival [8/8 rabbits] versus 0% survival [8/8 rabbits]; P < 0.01 by log rank test). Lungs from rabbits pretreated with c-IgG, but not those pretreated with MEDI3902, had bilateral, multifocal areas of marked necrosis, hemorrhage, neutrophilic inflammatory infiltrate, and diffuse fibrinous edema in alveolar spaces. All rabbits pretreated with c-IgG developed worsening bacteremia that peaked at the time of death, whereas only 38% of rabbits pretreated with MEDI3902 (3/8 rabbits) developed such high-grade bacteremia (two-sided Fisher's exact test, P = 0.026). Biomarkers associated with acute respiratory distress syndrome were evaluated longitudinally in blood samples collected every 2 to 4 h to assess systemic pathophysiological changes in rabbits pretreated with MEDI3902 or c-IgG. Biomarkers were sharply increased or decreased in rabbits pretreated with c-IgG but not those pretreated with MEDI3902, including the ratio of arterial oxygen partial pressure to the fraction of inspired oxygen of <300, hypercapnia or hypocapnia, severe lactic acidosis, leukopenia, and neutropenia. Cytokines and chemokines associated with acute respiratory distress syndrome were significantly downregulated in lungs from rabbits pretreated with MEDI3902, compared with c-IgG. These results suggest that MEDI3902 prophylaxis could have potential clinical utility for decreasing the severity of P. aeruginosa ventilator-associated pneumonia.

Differential regulation of TNL-mediated immune signaling by redundant helper CNLs.

Higher plants utilize nucleotide-binding leucine-rich repeat domain proteins (NLRs) as intracellular immune receptors to recognize pathogen-derived effectors and trigger a robust defense. The Activated Disease Resistance 1 (ADR1) family of coiled-coil NLRs (CNLs) have evolved as helper NLRs that function downstream of many TIR-type sensor NLRs (TNLs). Close homologs of ADR1s form the N REQUIREMENT GENE 1 (NRG1) family in Arabidopsis, the function of which is unclear. Through CRISPR/Cas9 gene editing methods, we discovered that the tandemly repeated NRG1A and NRG1B are functionally redundant and operate downstream of TNLs with differential strengths. Interestingly, ADR1s and NRG1s function in two distinct parallel pathways contributing to TNL-specific immunity. Synergistic effects on basal and TNL-mediated defense were detected among ADR1s and NRG1s. An intact P-loop of NRG1s is not required for mediating signals from sensor TNLs, whereas auto-active NRG1A exhibits autoimmunity. Importantly, NRG1s localize to the cytosol and endomembrane network regardless of the presence of effectors, suggesting a cytosolic activation mechanism. Taken together, different sensor TNLs differentially use two groups of helper NLRs, ADR1s and NRG1s, to transduce downstream defense signals.

Osa-miR398b boosts H2 O2 production and rice blast disease-resistance via multiple superoxide dismutases.

miRNAs contribute to plant resistance against pathogens. Previously, we found that the function of miR398b in immunity in rice differs from that in Arabidopsis. However, the underlying mechanisms are unclear. In this study, we characterized the mutants of miR398b target genes and demonstrated that multiple superoxide dismutase genes contribute to miR398b-regulated rice immunity against the blast fungus Magnaporthe oryzae. Out of the four target genes of miR398b, mutations in Cu/Zn-Superoxidase Dismutase1 (CSD1), CSD2 and Os11g09780 (Superoxide DismutaseX, SODX) led to enhanced resistance to M. oryzae and increased hydrogen peroxide (H2 O2 ) accumulation. By contrast, mutations in Copper Chaperone for Superoxide Dismutase (CCSD) resulted in enhanced susceptibility. Biochemical studies revealed that csd1, csd2 and sodx displayed altered expression of CSDs and other superoxide dismutase (SOD) family members, leading to increased total SOD enzyme activity that positively contributed to higher H2 O2 production. By contrast, the ccsd mutant showed CSD protein deletion, resulting in decreased CSD and total SOD enzyme activity. Our results demonstrate the roles of different SODs in miR398b-regulated resistance to rice blast disease, and uncover an integrative regulatory network in which miR398b boosts total SOD activity to upregulate H2 O2 concentration and thereby improve disease resistance.

Arabidopsis TAF15b Localizes to RNA Processing Bodies and Contributes to snc1-Mediated Autoimmunity.

In both animals and plants, messenger (m)RNA export has been shown to contribute to immune response regulation. The Arabidopsis nuclear protein MOS11, along with the nucleoporins MOS3/Nup96/SAR3 and Nup160/SAR1 are components of the mRNA export machinery and contribute to immunity mediated by nucleotide binding leucine-rich repeat immune receptors (NLR). The human MOS11 ortholog CIP29 is part of a small protein complex with three additional members: the RNA helicase DDX39, ALY, and TAF15b. We systematically assessed the biological roles of the Arabidopsis homologs of these proteins in toll interleukin 1 receptor-type NLR (TNL)-mediated immunity using reverse genetics. Although mutations in ALY and DDX39 did not result in obvious defects, taf15b mutation partially suppressed the autoimmune phenotypes of a gain-of-function TNL mutant, snc1. An additive effect on snc1 suppression was observed in mos11-1 taf15b snc1 triple mutant plants, suggesting that MOS11 and TAF15b have independent functions. TAF15b-GFP fusion protein, which fully complemented taf15b mutant phenotypes, localized to nuclei similarly to MOS11. However, it was also targeted to cytosolic granules identified as processing bodies. In addition, we observed no change in SNC1 mRNA levels, whereas less SNC1 protein accumulated in taf15b mutant, suggesting that TAF15b contributes to SNC1 homeostasis through posttranscriptional mechanisms. In summary, this study highlights the importance of posttranscriptional RNA processing mediated by TAF15b in the regulation of TNL-mediated immunity.

TNL-mediated immunity in Arabidopsis requires complex regulation of the redundant ADR1 gene family.

Nucleotide-binding leucine-rich repeat proteins (NLRs) serve as intracellular immune receptors in animals and plants. Sensor NLRs perceive pathogen-derived effector molecules and trigger robust host defense. Recent studies revealed the role of three coiled-coil-type NLRs (CNLs) of the ADR1 family - ADR1, ADR1-L1 and ADR1-L2 - as redundant helper NLRs, whose function is required for defense mediated by multiple sensor NLRs. From a mutant snc1-enhancing (MUSE) forward genetic screen in Arabidopsis targeted to identify negative regulators of snc1 that encodes a TIR-type NLR (TNL), we isolated two alleles of muse15, both carrying mutations in ADR1-L1. Interestingly, loss of ADR1-L1 also enhances immunity-related phenotypes in other autoimmune mutants including cpr1, bal and lsd1. This immunity-enhancing effect is not mediated by increased SNC1 protein stability, nor is it fully dependent on the accumulation of the defense hormone salicylic acid (SA). Transcriptional analysis revealed an upregulation of ADR1 and ADR1-L2 in the adr1-L1 background, which may overcompensate the loss of ADR1-L1, resulting in enhanced immunity. Interestingly, autoimmunity of snc1 and chs2, which encode typical TNLs, is fully suppressed by the adr1 triple mutant, suggesting that the ADRs are required for TNL downstream signaling. This study extends our knowledge on the interplay among ADRs and reveals their complexity in defense regulation.

HSP90s are required for NLR immune receptor accumulation in Arabidopsis.

Heat shock proteins (HSPs) serve as molecular chaperones for diverse client proteins in many biological processes. In plant immunity, cytosolic HSP90s participate in the assembly, stability control and/or activation of immune receptor complexes. In this paper we report that in addition to the well-established positive roles that HSP90 isoforms play in plant immunity, they are also involved in the negative regulation of immune receptor accumulation. Point mutations in two HSP90 genes, HSP90.2 and HSP90.3, were identified from a forward genetic screen designed to isolate mutants with enhanced disease resistance. We found that specific mutations in HSP90.2 and HSP90.3 lead to heightened accumulation of immune receptors, including SNC1, RPS2 and RPS4. HSP90s may assist SGT1 in the formation of SCF E3 ubiquitin ligase complexes that target immune receptors for degradation. Such regulation is critical for maintaining appropriate levels of immune receptor proteins to avoid autoimmunity.

The MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates immunity mediated by a mitogen-activated protein kinase kinase kinase in Arabidopsis.

In Arabidopsis thaliana, the MEKK1-MKK1/MKK2-MPK4 mitogen-activated protein (MAP) kinase cascade represses cell death and immune responses. In mekk1, mkk1 mkk2, and mpk4 mutants, programmed cell death and defense responses are constitutively activated, but the mechanism by which MEKK1, MKK1/MKK2, and MPK4 negatively regulate cell death and immunity was unknown. From a screen for suppressors of mkk1 mkk2, we found that mutations in suppressor of mkk1 mkk2 1 (summ1) suppress the cell death and defense responses not only in mkk1 mkk2 but also in mekk1 and mpk4. SUMM1 encodes the MAP kinase kinase kinase MEKK2. It interacts with MPK4 and is phosphorylated by MPK4 in vitro. Overexpression of SUMM1 activates cell death and defense responses that are dependent on the nucleotide binding-leucine-rich repeat protein SUMM2. Taken together, our data suggest that the MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates MEKK2 and activation of MEKK2 triggers SUMM2-mediated immune responses.

Heterotrimeric G proteins serve as a converging point in plant defense signaling activated by multiple receptor-like kinases.

In fungi and metazoans, extracellular signals are often perceived by G-protein-coupled receptors (GPCRs) and transduced through heterotrimeric G-protein complexes to downstream targets. Plant heterotrimeric G proteins are also involved in diverse biological processes, but little is known about their upstream receptors. Moreover, the presence of bona fide GPCRs in plants is yet to be established. In Arabidopsis (Arabidopsis thaliana), heterotrimeric G protein consists of one Gα subunit (G protein α-subunit1), one Gß subunit (Arabidopsis G protein ß-subunit1 [AGB1]), and three Gγs subunits (Arabidopsis G protein γ-subunit1 [AGG1], AGG2, and AGG3). We identified AGB1 from a suppressor screen of BAK1-interacting receptor-like kinase1-1 (bir1-1), a mutant that activates cell death and defense responses mediated by the receptor-like kinase (RLK) suppressor of BIR1-1. Mutations in AGB1 suppress the cell death and defense responses in bir1-1 and transgenic plants overexpressing suppressor of BIR1-1. In addition, agb1 mutant plants were severely compromised in immunity mediated by three other RLKs, flagellin-sensitive2 (FLS2), Elongation Factor-TU RECEPTOR (EFR), and chitin elicitor receptor kinase1 (CERK1), respectively. By contrast, G protein α-subunit1 is not required for either cell death in bir1-1 or pathogen-associated molecular pattern-triggered immunity mediated by FLS2, EFR, and CERK1. Further analysis of agg1 and agg2 mutant plants indicates that AGG1 and AGG2 are also required for pathogen-associated molecular pattern-triggered immune responses mediated by FLS2, EFR, and CERK1, as well as cell death and defense responses in bir1-1. We hypothesize that the Arabidopsis heterotrimeric G proteins function as a converging point of plant defense signaling by mediating responses initiated by multiple RLKs, which may fulfill equivalent roles to GPCRs in fungi and animals.

Impact of Patient-Reported Allergies on Post-operative Complications and Healthcare Utilization Following Carpal Tunnel Release.

INTRODUCTION: Open carpal tunnel release (O-CTR) is associated with high patient satisfaction and low complication rates. Risk factors for complications are well-established. Recent studies have found that patient-reported allergies (PRAs) and psychiatric comorbidities may be associated with increased complication rates. The impact of these factors after elective hand surgery has not been evaluated. This study sought to identify whether PRAs and psychiatric comorbidities are associated with complications after O-CTR and to evaluate their association with prolonged follow-up and the need for post-operative occupational therapy (OT). METHODS: Patient demographics, PRAs, Patient Health Questionnaire-2 score, Charlson Comorbidity Index, Carpal Tunnel Symptoms-6 score, postoperative complications, OT utilization, and time to final follow-up were recorded for patients who underwent elective O-CTR between 2014 and 2022. Multivariable binomial logistic regression analysis was used to determine pre-operative variables associated with increased risk for complication. RESULTS: About 250 patients met the inclusion criteria. Fifty-one (20.4%) patients developed minor complications, including scar tenderness (N=34, 13.6%), superficial wound dehiscence (N=9, 3.6%), and superficial infection (N=8, 3.2%). There were no major complications. Independent risk factors for complications included PRAs (OR 1.80, p<0.01) and PHQ-2 score (OR 1.39, p=0.04). Five or more PRAs and PHQ-2 score ≥3 are significant independent risk factors for increased post-operative complications. Increased PRAs and PHQ-2 scores were associated with longer follow-up (p=0.01 and p<0.01, respectively) but not increased OT utilization. CONCLUSION: An increased number of PRAs and higher PHQ-2 scores are significant, independent risk factors for minor complications following O-CTR. Risk adjustment and peri-operative counseling should incorporate and account for these variables.

Femoral interference screw divergence as a result of anteromedial portal insertion and outside-in FlipCutter femoral tunnel drilling: A cadaveric study.

Aims and objectives: To investigate whether interference screw fixation through an anteromedial portal into an outside-in drilled femoral tunnel via a flip cutter results in acceptable hardware position. Materials & methods: 10 cadaveric knees underwent ACL-reconstruction with patellar BTB autograft. Femoral tunnel drilling was performed utilizing an outside-in flip cutter drill and interference screws for femoral fixation. Lateral and anterior-posterior (AP) fluoroscopic images were taken to measure screw divergence within the femoral tunnel. The means of AP and lateral divergence angles were compared using two-tailed t-tests. Results: Using the flip cutter, the AP and lateral divergence angles were 7.3° ± 4.5° and 9.3° ± 9.3°, respectively, while the total divergence angles were 16.6° ± 11.8°. Divergence angles using a cannulated reamer were found to be 14.4° ± 2.5° and 6.8° ± 2.8° for AP and lateral, respectively and 21.1° ± 5.2° for the total divergence. The AP divergence angles using the flip cutter were significantly less than those reported using a cannulated reamer (p = 0.001). Conclusions: The flip cutter method resulted in significantly reduced divergence angle between the screw and graft when compared to previous cadaveric studies in the coronal plane. There was no significant difference in divergence angle in the sagittal plane. Both methods appear to result in divergence angles below the threshold which would be considered to significantly decrease pull-out strength. Large standard deviations also reflect limited sample size but may also suggest more variability in divergence when compared to historical control set. This study clearly establishes the outside-in technique using a retrograde reamer as a viable independent femoral drilling solution for ACL reconstruction when using a BTB autograft with a femoral interference screw.

MOS11: a new component in the mRNA export pathway.

Nucleocytoplasmic trafficking is emerging as an important aspect of plant immunity. The three related pathways affecting plant immunity include Nuclear Localization Signal (NLS)-mediated nuclear protein import, Nuclear Export Signal (NES)-dependent nuclear protein export, and mRNA export relying on MOS3, a nucleoporin belonging to the Nup107-160 complex. Here we report the characterization, identification, and detailed analysis of Arabidopsis modifier of snc1, 11 (mos11). Mutations in MOS11 can partially suppress the dwarfism and enhanced disease resistance phenotypes of snc1, which carries a gain-of-function mutation in a TIR-NB-LRR type Resistance gene. MOS11 encodes a conserved eukaryotic protein with homology to the human RNA binding protein CIP29. Further functional analysis shows that MOS11 localizes to the nucleus and that the mos11 mutants accumulate more poly(A) mRNAs in the nucleus, likely resulting from reduced mRNA export activity. Epistasis analysis between mos3-1 and mos11-1 revealed that MOS11 probably functions in the same mRNA export pathway as MOS3, in a partially overlapping fashion, before the mRNA molecules pass through the nuclear pores. Taken together, MOS11 is identified as a new protein contributing to the transfer of mature mRNA from the nucleus to the cytosol.

Short-term outcome of unicortical, intramedullary repair of distal biceps ruptures - A retrospective cohort study.

Background: Modern distal biceps reconstruction techniques generally have satisfactory outcomes, but are not without complications. Posterior interosseous nerve (PIN) palsy is a rare but potentially devastating complication of bicortical metal button fixation. Recently, a unicortical, intramedullary, repair technique utilizing a suture anchor has been described. The primary aim of this study was to compare short-term functional and patient-reported outcomes and complication rates in patients receiving unicortical intramedullary repair (UR) with suture anchor against those receiving bicortical repair (BR) with metallic button. We hypothesized that UR would have equally satisfactory outcomes without the complication profile. Methods: Retrospective chart review was conducted for all patients undergoing operative fixation of distal biceps tendon ruptures from 2015 to 2021 at our tertiary referral center. Twenty patients received BR, and eight patients received UR. Patient demographics and surgical complications were compared. QuickDASH scores at two-month and latest in-person and telehealth postoperative visits, as well as elbow and forearm range of motion at last clinical visit, were collected and analyzed. Results: Average patient age in the BR & UR cohorts were 49.3 ± 9.3 and 42.1 ± 6.2 years, respectively, with a male predominance. There was no statistical difference in patient age, sex, hand dominance, injury laterality, injury chronicity, and follow-up duration. Range of motion was comparable and excellent in both groups. Latest follow-up was 3.0 ± 0.5 years in the BR and 1.5 ± 0.4 years in the UR cohorts. QuickDASH scores improved between the two-month and latest time points in each cohort however did not differ significantly in head-to-head comparison. Complications included a case of PIN palsy, distal biceps tendon rerupture, and lateral antebrachial cutaneous nerve (LABC) neuropraxia in the BR group and two cases of LABC neuropraxia in the UR group. The number needed to treat (NNT) for the prevention of one additional case of PIN palsy using UR is 22 patients. Discussion: Short-term functional and patient-reported outcomes in traditional BR and newly reported UR of distal biceps tendon ruptures are comparable and excellent. UR did not have higher failure rate despite follow-up periods beyond what is typically reported for tendon reruptures. In this limited retrospective cohort study, UR also did not encounter postoperative PIN palsy and had an NNT of 22 patients. In the appropriate clinical setting, this provides early evidence supporting the utilization of unicortical intramedullary suture anchor fixation of distal biceps tendon ruptures as well as associated perioperative interventions such as preoperative nerve blocks.

Development and validation of a rabbit model of Pseudomonas aeruginosa non-ventilated pneumonia for preclinical drug development.

Background: New drugs targeting antimicrobial resistant pathogens, including Pseudomonas aeruginosa, have been challenging to evaluate in clinical trials, particularly for the non-ventilated hospital-acquired pneumonia and ventilator-associated pneumonia indications. Development of new antibacterial drugs is facilitated by preclinical animal models that could predict clinical efficacy in patients with these infections. Methods: We report here an FDA-funded study to develop a rabbit model of non-ventilated pneumonia with Pseudomonas aeruginosa by determining the extent to which the natural history of animal disease reproduced human pathophysiology and conducting validation studies to evaluate whether humanized dosing regimens of two antibiotics, meropenem and tobramycin, can halt or reverse disease progression. Results: In a rabbit model of non-ventilated pneumonia, endobronchial challenge with live P. aeruginosa strain 6206, but not with UV-killed Pa6206, caused acute respiratory distress syndrome, as evidenced by acute lung inflammation, pulmonary edema, hemorrhage, severe hypoxemia, hyperlactatemia, neutropenia, thrombocytopenia, and hypoglycemia, which preceded respiratory failure and death. Pa6206 increased >100-fold in the lungs and then disseminated from there to infect distal organs, including spleen and kidneys. At 5 h post-infection, 67% of Pa6206-challenged rabbits had PaO2 <60 mmHg, corresponding to a clinical cut-off when oxygen therapy would be required. When administered at 5 h post-infection, humanized dosing regimens of tobramycin and meropenem reduced mortality to 17-33%, compared to 100% for saline-treated rabbits (P<0.001 by log-rank tests). For meropenem which exhibits time-dependent bactericidal activity, rabbits treated with a humanized meropenem dosing regimen of 80 mg/kg q2h for 24 h achieved 100% T>MIC, resulting in 75% microbiological clearance rate of Pa6206 from the lungs. For tobramycin which exhibits concentration-dependent killing, rabbits treated with a humanized tobramycin dosing regimen of 8 mg/kg q8h for 24 h achieved Cmax/MIC of 9.8 ± 1.4 at 60 min post-dose, resulting in 50% lung microbiological clearance rate. In contrast, rabbits treated with a single tobramycin dose of 2.5 mg/kg had Cmax/MIC of 7.8 ± 0.8 and 8% (1/12) microbiological clearance rate, indicating that this rabbit model can detect dose-response effects. Conclusion: The rabbit model may be used to help predict clinical efficacy of new antibacterial drugs for the treatment of non-ventilated P. aeruginosa pneumonia.

An open source plant kinase chemogenomics set.

One hundred twenty-nine protein kinases, selected to represent the diversity of the rice (Oryza sativa) kinome, were cloned and tested for expression in Escherichia coli. Forty of these rice kinases were purified and screened using differential scanning fluorimetry (DSF) against 627 diverse kinase inhibitors, with a range of structures and activities targeting diverse human kinases. Thirty-seven active compounds were then tested for their ability to modify primary root development in Arabidopsis. Of these, 14 compounds caused a significant reduction of primary root length compared with control plants. Two of these inhibitory compounds bind to the predicted orthologue of Arabidopsis PSKR1, one of two receptors for PSK, a small sulfated peptide that positively controls root development. The reduced root length phenotype could not be rescued by the exogenous addition of the PSK peptide, suggesting that chemical treatment may inhibit both PSKR1 and its closely related receptor PSKR2. Six of the compounds acting as root growth inhibitors in Arabidopsis conferred the same effect in rice. Compound RAF265 (CHIR-265), previously shown to bind the human kinase BRAF (B-Raf proto-oncogene, serine/threonine kinase), also binds to nine highly conserved rice kinases tested. The binding of human and rice kinases to the same compound suggests that human kinase inhibitor sets will be useful for dissecting the function of plant kinases.

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9.

Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.

Individual components of paired typical NLR immune receptors are regulated by distinct E3 ligases.

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins serve as intracellular immune receptors. Defence signalling by NLRs often requires the formation of NLR heteropairs. Our knowledge of the molecular mechanism regulating this process is limited. In a reverse genetic screen to identify the partner of the Arabidopsis typical NLR, SUPRESSOR OF NPR1, CONSTITUTIVE 1 (SNC1), we discovered three NLRs that are redundantly required for SNC1-mediated defence, which were named SIDEKICK SNC1 1 (SIKIC1), SIKIC2 and SIKIC3. Immunoprecipitation-mass spectrometry analyses revealed that SIKIC2 physically associates with SNC1. We also uncovered that the protein level of SIKIC2 is under the control of two previously uncharacterized redundant E3 ubiquitin ligases MUSE1 and MUSE2. As SNC1 accumulation has previously been shown to be regulated by the E3 ubiquitin ligase SCFCPR1, this report provides evidence that the homeostasis of individual components of partnered typical NLRs is subjected to differential regulation via ubiquitin-mediated protein degradation.