Engineered Agrobacterium improves transformation by mitigating plant immunity detection.

Agrobacterium tumefaciens microbe-associated molecular pattern elongation factor Tu (EF-Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating pattern-triggered immunity (PTI) that causes reduced T-DNA-mediated transient expression. We altered EF-Tu in A. tumefaciens to reduce PTI and improved transformation efficiency. A robust computational pipeline was established to detect EF-Tu protein variation in a large set of plant bacterial species and identified EF-Tu variants from bacterial pathogen Pseudomonas syringae pv. tomato DC3000 that allow the pathogen to escape EFR perception. Agrobacterium tumefaciens strains were engineered to substitute EF-Tu with DC3000 variants and examined their transformation efficiency in plants. Elongation factor Tu variants with rarely occurred amino acid residues were identified within DC3000 EF-Tu that mitigates recognition by EFR. Agrobacterium tumefaciens strains were engineered by expressing DC3000 EF-Tu instead of native agrobacterial EF-Tu and resulted in decreased plant immunity detection. These engineered A. tumefaciens strains displayed an increased efficiency in transient expression in both Arabidopsis thaliana and Camelina sativa. The results support the potential application of these strains as improved vehicles to introduce transgenic alleles into members of the Brassicaceae family.

The plant vampire diaries: a historic perspective on Cuscuta research.

The angiosperm genus Cuscuta lives as an almost achlorophyllous root- and leafless holoparasite and has therefore occupied scientists for more than a century. The 'evolution' of Cuscuta research started with early studies that established the phylogenetic framework for this unusual genus. It continued to produce groundbreaking cytological, morphological, and physiological insight throughout the second half of the 20th century and culminated in the last two decades in exciting discoveries regarding the molecular basis of Cuscuta parasitism that were facilitated by the modern 'omics' tools and traceable fluorescent marker technologies of the 21st century. This review will show how present activities are inspired by those past breakthroughs. It will describe significant milestones and recurring themes of Cuscuta research and connect these to the remaining as well as newly evolving questions and future directions in this research field that is expected to sustain its strong growth in the future.

Mechanisms of resistance and virulence in parasitic plant-host interactions.

Parasitic plants pose a major biotic threat to plant growth and development and lead to losses in crop productivity of billions of USD annually. By comparison with "normal" autotrophic plants, parasitic plants live a heterotrophic lifestyle and rely on water, solutes and to a greater (holoparasitic plants) or lesser extent (hemiparasitic plants) on sugars from other host plants. Most hosts are unable to detect an infestation by plant parasites or unable to fend off these parasitic invaders. However, a few hosts have evolved defense strategies to avoid infestation or protect themselves actively post-attack often leading to full or partial resistance. Here, we review the current state of our understanding of the defense strategies to plant parasitism used by host plants with emphasis on the active molecular resistance mechanisms. Furthermore, we outline the perspectives and the potential of future studies that will be indispensable to develop and breed resistant crops.

Histological differences between lumbar and tail intervertebral discs in mice.

Both the lumbar and tail intervertebral discs (IVD) of mice serve as models for the pathogenesis and histologic progression of degenerative disc disease. Recent studies in mature mice, however, demonstrate that the mechanics and physical attributes of lumbar and tail IVD-endplate (EP)-interfaces are strikingly different. We hypothesized that these structural disparities are associated with differences in the composition and organization of soft tissue elements that influence the biomechanical properties of the spine. Lumbar and tail vertebral segments and discs were collected from the same C57BL/6N and C57BL/6JRj mice, respectively for histological comparison of coronal sections at the ages of 4 weeks (weaned, both strains, C57BL/6N: n = 7; C57BL/6JRj: n = 4), three (mature, C57BL/6N: n = 7; C57BL/6JRj: n = 4), twelve (middle aged, C57BL/6JRj only: n = 3) and eighteen (old, C57BL/6JRj only: n = 3) months old. The histology of lumbar and tail IVD-EP-interfaces of mature mice differed markedly. The lumbar IVD-EP-interphase was characterized by a broad cartilaginous EP, while the tail IVD-EP-interphase comprised a thin layer of cartilage cells adjacent to a broad bony layer abutting the vertebral growth plate. Furthermore, the composition of the nuclei pulposi (NP) of lumbar and tail IVD in mature mice differed greatly. Lumbar NP consisted of a compact cluster of mainly large, uni-vacuolated cells centered in an amorphous matrix, while tail NP were composed of a loose aggregate of vacuolated and non-vacuolated cells. The anuli fibrosi also differed, with more abundant and sharply defined lamellae in tail compared to lumbar discs. The observed histological differences in the EP were even most prominent in weaned mice but were still discernible in middle-aged and old mice. An appreciation of the histological differences between lumbar and tail IVD components in mice, including nucleus pulposus, annulus fibrosus, and endplates, is essential to our understanding of spinal biomechanics in these animals and should inform the design and interpretation of future IVD-studies.

Transcaval transcatheter aortic valve implantation in bicuspid aortic valve: A step-by-step procedural guidance.

We report the case of a 78-year-old female with Sapien 3 transcatheter heart valve implantation in the transcaval approach. In this setting, we describe the step-by-step management and technique of the transcaval transcatheter aortic valve implantation.

Outcomes of transcatheter mitral valve replacement for degenerated bioprostheses, failed annuloplasty rings, and mitral annular calcification.

Aims: We sought to evaluate the outcomes of transcatheter mitral valve replacement (TMVR) for patients with degenerated bioprostheses [valve-in-valve (ViV)], failed annuloplasty rings [valve-in-ring (ViR)], and severe mitral annular calcification [valve-in-mitral annular calcification (ViMAC)]. Methods and results: From the TMVR multicentre registry, procedural and clinical outcomes of ViV, ViR, and ViMAC were compared according to Mitral Valve Academic Research Consortium (MVARC) criteria. A total of 521 patients with mean Society of Thoracic Surgeons score of 9.0 ± 7.0% underwent TMVR (322 patients with ViV, 141 with ViR, and 58 with ViMAC). Trans-septal access and the Sapien valves were used in 39.5% and 90.0%, respectively. Overall technical success was excellent at 87.1%. However, left ventricular outflow tract obstruction occurred more frequently after ViMAC compared with ViR and ViV (39.7% vs. 5.0% vs. 2.2%; P < 0.001), whereas second valve implantation was more frequent in ViR compared with ViMAC and ViV (12.1% vs. 5.2% vs. 2.5%; P < 0.001). Accordingly, technical success rate was higher after ViV compared with ViR and ViMAC (94.4% vs. 80.9% vs. 62.1%; P < 0.001). Compared with ViMAC and ViV groups, ViR group had more frequent post-procedural mitral regurgitation ≥moderate (18.4% vs. 13.8% vs. 5.6%; P < 0.001) and subsequent paravalvular leak closure (7.8% vs. 0.0% vs. 2.2%; P = 0.006). All-cause mortality was higher after ViMAC compared with ViR and ViV at 30 days (34.5% vs. 9.9% vs. 6.2%; log-rank P < 0.001) and 1 year (62.8% vs. 30.6% vs. 14.0%; log-rank P < 0.001). On multivariable analysis, patients with failed annuloplasty rings and severe MAC were at increased risk of mortality after TMVR [ViR vs. ViV, hazard ratio (HR) 1.99, 95% confidence interval (CI) 1.27-3.12; P = 0.003; ViMAC vs. ViV, HR 5.29, 95% CI 3.29-8.51; P < 0.001]. Conclusion: The TMVR provided excellent outcomes for patients with degenerated bioprostheses despite high surgical risk. However, ViR and ViMAC were associated with higher rates of adverse events and mid-term mortality compared with ViV.

Vascular complications after percutaneous mitral valve repair and venous access closure using suture or closure device.

OBJECTIVE: The aim of this study was to assess the impact of different access-site closure strategies, suture or closure device (Proglide, Abbott Vascular), on vascular and bleeding complications after percutaneous mitral valve repair (MitraClip, Abbott Vascular). BACKGROUND: Considering the high-risk profile in patients receiving percutaneous mitral valve repair, complications related to the large 24 Fr access sheath and its relation to the closure technique have not been evaluated so far. METHODS AND RESULTS: Between 2009 and 2015, 277 consecutive high-risk patients with severe mitral valve regurgitation (MR) underwent percutaneous mitral valve repair at our institution using Z-suture (n = 150) or closure device (n = 127) to close the access-site. Duplex sonography was performed in all patients. The primary endpoint was access-site related complications according to the Valve Academic Research Consortium (VARC) criteria. Secondary outcomes were the incidence of bleeding complications and mortality. Access-site related VARC2 major and minor complications were comparable after closure with Z-suture or closure device (2,7% vs 3.1%, P = 0.81 and 15,3% vs 15.7%, P = 0.92). Three patients (2%) in the suture and four patients (3.1%) in the closure device group experienced unplanned endovascular intervention at the access site. Access-site related major bleeding was observed in 4 (2.7%) suture and 4 (3.1%) closure device treated patients (P = 0.81). No access site related mortality occurred. CONCLUSION: Both Z-suture and closure device use after percutaneous mitral valve repair are feasible and safe. However, there is no benefit of one strategy over the other according to VARC2 major and minor complications.

Dexmedetomidine versus propofol-opioid for sedation in transcatheter aortic valve implantation patients: a retrospective analysis of periprocedural gas exchange and hemodynamic support.

PURPOSE: Different sedation regimens have been described for use during transfemoral transcatheter aortic valve implantation (tf-TAVI) for treatment in patients with severe aortic stenosis. The purpose of this study was to compare dexmedetomidine (DEX) with a combination of propofol-opioid (PO) with respect to periprocedural gas exchange and hemodynamic support. METHODS: Data from a cohort of patients sedated with either DEX or PO for tf-TAVI were retrospectively analyzed from a prospectively maintained TAVI registry. Operative risk was determined from comorbidities and risk scores. Periprocedural partial pressure of carbon dioxide (PaCO2) was chosen as the primary endpoint. Other differences in gas exchange, need for catecholamine therapy, the frequency of conversion to general anesthesia, and need for sedative "rescue therapy" (in DEX patients) were secondary endpoints. Inverse probability of treatment weighting (IPTW) was used for analysis to minimize any selection bias. RESULTS: Of the 297 patients (140 PO, 157 DEX) included, the median [interquartile range] periprocedural PaCO2 values of DEX patients were significantly lower than in PO patients (40 [36-45] mmHg vs 44 [40-49] mmHg, respectively; median difference -4 mmHg; 95% confidence interval, -5 to -3 mmHg; P < 0.001). Hypercapnia (PaCO2 > 45 mmHg) was significantly less frequent in DEX patients compared with the PO group (25% vs 42%, respectively; P = 0.005). Vasopressor support was more frequent in the PO group compared with DEX (68% vs 25%, respectively; P < 0.001). Conversion to general anesthesia was not different between groups (9%, PO vs 3%, DEX; P = 0.051). Additional sedatives/opioids were required in 25 (16%) of the DEX patients. CONCLUSIONS: In sedated TAVI patients, DEX was associated with lower PaCO2 values and reduced requirements for vasopressor support, making it a promising alternative to PO for sedation during TAVI. TRIAL REGISTRATION: www.ClinicalTrials.gov (NCT01390675). Registered 11 July 2011.

Tools and Strategies to Match Peptide-Ligand Receptor Pairs.

Peptide signals have emerged as an important class of regulators in cell-to-cell communication in plants. Several families of small, secreted proteins with a conserved C-terminal Pro-rich motif have been identified as functional peptide signals in Arabidopsis thaliana. These proteins are presumed to be trimmed proteolytically and undergo posttranslational modifications, such as hydroxylation of Pro residues and glycosylation, to form mature, bioactive signals. Identification and matching of such ligands with their respective receptors remains a major challenge since the genes encoding them often show redundancy and low expression restricted to a few cells or particular developmental stages. To overcome these difficulties, we propose the use of ectopic expression of receptor genes in suitable plant cells like Nicotiana benthamiana for testing ligand candidates in receptor output assays and in binding studies. As an example, we used the IDA peptide HAE/HSL2 receptor signaling system known to regulate floral organ abscission. We demonstrate that the oxidative burst response can be employed as readout for receptor activation by synthetic peptides and that a new, highly sensitive, nonradioactive labeling approach can be used to reveal a direct correlation between peptide activity and receptor affinity. We suggest that these approaches will be of broad value for the field of ligand-receptor studies in plants.

The receptor-like protein ReMAX of Arabidopsis detects the microbe-associated molecular pattern eMax from Xanthomonas.

As part of their immune system, plants have pattern recognition receptors (PRRs) that can detect a broad range of microbe-associated molecular patterns (MAMPs). Here, we identified a PRR of Arabidopsis thaliana with specificity for the bacterial MAMP eMax from xanthomonads. Response to eMax seems to be restricted to the Brassicaceae family and also varied among different accessions of Arabidopsis. In crosses between sensitive accessions and the insensitive accession Shakhdara, eMax perception mapped to receptor-like protein1 (RLP1). Functional complementation of rlp1 mutants required gene constructs that code for a longer version of RLP1 that we termed ReMAX (for receptor of eMax). ReMAX/RLP1 is a typical RLP with structural similarity to the tomato (Solanum lycopersicum) RLP Eix2, which detects fungal xylanase as a MAMP. Attempts to demonstrate receptor function by interfamily transfer of ReMAX to Nicotiana benthamiana were successful after using hybrid receptors with the C-terminal part of ReMAX replaced by that of Eix2. These results show that ReMAX determines specificity for eMax. They also demonstrate hybrid receptor technology as a promising tool to overcome problems that impede interfamily transfer of PRRs to enhance pathogen detection in crop plants.

Contamination risks in work with synthetic peptides: flg22 as an example of a pirate in commercial peptide preparations.

The pattern recognition receptor FLAGELLIN SENSING2 (FLS2) renders plant cells responsive to subnanomolar concentrations of flg22, the active epitope of bacterial flagellin. We recently observed that a preparation of the peptide IDL1, a signal known to regulate abscission processes via the receptor kinases HAESA and HAESA-like2, apparently triggered Arabidopsis thaliana cells in an FLS2-dependent manner. However, closer investigation revealed that this activity was due to contamination by a flg22-type peptide, and newly synthesized IDL1 peptide was completely inactive in FLS2 signaling. This raised alert over contamination events occurring in the process of synthesis or handling of peptides. Two recent reports have suggested that FLS2 has further specificities for structurally unrelated peptides derived from CLV3 and from Ax21. We thus scrutinized these peptides for activity in Arabidopsis cells as well. While responding to <1 nM flg22, Arabidopsis cells proved blind even to 100 µM concentrations of CLV3p and axY(s)22. Our results confirm the exquisite sensitivity and selectivity of FLS2 for flg22. They also show that inadvertent contaminations with flg22-type peptides do occur and can be detected even in trace amounts by FLS2.

Chimeric FLS2 receptors reveal the basis for differential flagellin perception in Arabidopsis and tomato.

The flagellin receptor of Arabidopsis thaliana, At-FLAGELLIN SENSING2 (FLS2), has become a model for mechanistic and functional studies on plant immune receptors. Here, we started out with a comparison of At-FLS2 and the orthologous tomato (Solanum lycopersicum) receptor Sl-FLS2. Both receptors specifically responded to picomolar concentrations of the genuine flg22 ligand but proved insensitive to >10(6)-fold higher concentrations of CLV3 peptides that have recently been reported as a second type of ligand for At-FLS2. At-FLS2 and Sl-FLS2 exhibit species-specific differences in the recognition of shortened or sequence-modified flg22 ligands. To map the sites responsible for these species-specific traits on the FLS2 receptors, we performed domain swaps, substituting subsets of the 28 leucine-rich repeats (LRRs) in At-FLS2 with the corresponding LRRs from Sl-FLS2. We found that the LRRs 7 to 10 of Sl-FLS2 determine the high affinity of Sl-FLS2 for the core part RINSAKDD of flg22. In addition, we discovered importance of the LRRs 19 to 24 for the responsiveness to C-terminally modified flagellin peptides. These results indicate that ligand perception in FLS2 is a complex molecular process that involves LRRs from both the outermost and innermost LRRs of the FLS2 ectodomain.

A two-hybrid-receptor assay demonstrates heteromer formation as switch-on for plant immune receptors.

Receptor kinases sense extracellular signals and trigger intracellular signaling and physiological responses. However, how does signal binding to the extracellular domain activate the cytoplasmic kinase domain? Activation of the plant immunoreceptor Flagellin sensing2 (FLS2) by its bacterial ligand flagellin or the peptide-epitope flg22 coincides with rapid complex formation with a second receptor kinase termed brassinosteroid receptor1 associated kinase1 (BAK1). Here, we show that the receptor pair of FLS2 and BAK1 is also functional when the roles of the complex partners are reversed by swapping their cytosolic domains. This reciprocal constellation prevents interference by redundant partners that can partially substitute for BAK1 and demonstrates that formation of the heteromeric complex is the molecular switch for transmembrane signaling. A similar approach with swaps between the Elongation factor-Tu receptor and BAK1 also resulted in a functional receptor/coreceptor pair, suggesting that a "two-hybrid-receptor assay" is of more general use for studying heteromeric receptor complexes.

Peptides as triggers of plant defence.

Plants are confronted with several biotic stresses such as microbial pathogens and other herbivores. To defend against such attackers, plants possess an array of pattern recognition receptors (PRRs) that sense the danger and consequently initiate a defence programme that prevents further damage and spreading of the pest. Characteristic pathogenic structures, so-called microbe-associated molecular patterns (MAMPs), serve as signals that allow the plant to sense invaders. Additionally, pathogens wound or damage the plant and the resulting release of damage-associated molecular patterns (DAMPs) serves as a warning signal. This review focuses on peptides that serve as triggers or amplifiers of plant defence and thus follow the definition of a MAMP or a DAMP.

Histopathological evaluation of infertility: Lessons from laboratory rodents.

Infertility is a growing challenge globally with emerging risk factors. There are effective laboratory tests to evaluate infertility in humans, nevertheless, some measures, especially histopathological evaluations, are invasive due to the pain inflicted when accessing the reproductive organs and obtaining samples; hence, their relevance may be limited in humans. However, these histopathological evaluations provide essential information on the etiopathogenesis of infertility and the likely mechanisms of action of potential therapeutic candidates. Also, non-invasive methods are available, such as the assay of testosterone in the blood and semen analysis, both of which are predictors of testicular functions. This review provides detailed information on the available histopathological investigations of infertility, such as qualitative and quantitative histopathological assessments of gonadal tissues, specific cell counts, and sperm morphology characterization, with a focus on the procedures, interpretation, and pathophysiological basis. Data from the literature revealed that histopathological examinations of the reproductive organs, as well as spermatozoa, are useful in understanding the pathogenesis of incident infertility. Histopathological evaluation may range from basic hematoxylin and eosin stains to some special stains. Also, histopathological findings (such as spermatogenic cells and planimetric variables, like seminiferous tubule diameter and theca cell and corpus luteum thickness) may be quantified and analyzed for comparison. Some skill is required for these investigations, which may be a limiting factor; however, they are important tools in translational medicine.

"Puncture-to-Loop" Technique to Retrieve Embolized Patent Foramen Ovale Occluder.

A 62-year-old man experienced embolization of a patent foramen ovale (PFO) occlusion device in the pulmonary artery. The device was successfully retrieved using "puncture to loop" technique, without the need of specific materials. This is a challenging retrieval situation, confirming the feasibility and flexibility of the technique. (Level of Difficulty: Advanced.).

Genotyping-by-sequencing-based identification of Arabidopsis pattern recognition receptor RLP32 recognizing proteobacterial translation initiation factor IF1.

Activation of plant pattern-triggered immunity (PTI) relies on the recognition of microbe-derived structures, termed patterns, through plant-encoded surface-resident pattern recognition receptors (PRRs). We show that proteobacterial translation initiation factor 1 (IF1) triggers PTI in Arabidopsis thaliana and related Brassicaceae species. Unlike for most other immunogenic patterns, IF1 elicitor activity cannot be assigned to a small peptide epitope, suggesting that tertiary fold features are required for IF1 receptor activation. We have deployed natural variation in IF1 sensitivity to identify Arabidopsis leucine-rich repeat (LRR) receptor-like protein 32 (RLP32) as IF1 receptor using a restriction site-associated DNA sequencing approach. RLP32 confers IF1 sensitivity to rlp32 mutants, IF1-insensitive Arabidopsis accessions and IF1-insensitive Nicotiana benthamiana, binds IF1 specifically and forms complexes with LRR receptor kinases SOBIR1 and BAK1 to mediate signaling. Similar to other PRRs, RLP32 confers resistance to Pseudomonas syringae, highlighting an unexpectedly complex array of bacterial pattern sensors within a single plant species.