HPr prevents FruR-mediated facilitation of RNA polymerase binding to the fru promoter in Vibrio cholerae.

Phosphorylation state-dependent interactions of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) components with transcription factors play a key role in carbon catabolite repression (CCR) by glucose in bacteria. Glucose inhibits the PTS-dependent transport of fructose and is preferred over fructose in Vibrio cholerae, but the mechanism is unknown. We have recently shown that, contrary to Escherichia coli, the fructose-dependent transcriptional regulator FruR acts as an activator of the fru operon in V. cholerae and binding of the FruR-fructose 1-phosphate (F1P) complex to an operator facilitates RNA polymerase (RNAP) binding to the fru promoter. Here we show that, in the presence of glucose, dephosphorylated HPr, a general PTS component, binds to FruR. Whereas HPr does not affect DNA-binding affinity of FruR, regardless of the presence of F1P, it prevents the FruR-F1P complex from facilitating the binding of RNAP to the fru promoter. Structural and biochemical analyses of the FruR-HPr complex identify key residues responsible for the V. cholerae-specific FruR-HPr interaction not observed in E. coli. Finally, we reveal how the dephosphorylated HPr interacts with FruR in V. cholerae, whereas the phosphorylated HPr binds to CcpA, which is a global regulator of CCR in Bacillus subtilis and shows structural similarity to FruR.

Protein stability governed by α1-2 helices in Pvr4 is essential for localization and cell death.

Plant nucleotide-binding domain leucine-rich-repeat receptor (NLR) confers disease resistance to various pathogens by recognizing effectors derived from the pathogen. Previous studies have shown that overexpression of the CC domain in several NLRs triggers cell death, implying that the CC domain plays an important role as a signaling module. However, how CC domain transduces immune signals remains largely unknown. A Potyvirus-resistant NLR protein, Pvr4, possesses a CC domain (CCPvr4 ) that induces cell death upon transient overexpression in Nicotiana benthamiana. In this study, loss-of-function mutants were generated by error-prone PCR-based random mutagenesis to understand the molecular mechanisms underlying CCPvr4 -mediated cell death. Cell biology and biochemical studies revealed that M16 and Q52 in the α1 and α2 helices, respectively, are crucial for protein stability, and mutation of these residues disrupts localization to the plasma membrane and oligomerization activity. The increase of the protein stability of these mutants by tagging a green fluorescent protein (GFP) variant led to restoration of cell death-inducing activity and plasma membrane localization. Another mutant, I7E in the very N-terminal region, lost cell death-inducing activity by weakening the interaction with plasma membrane H+ -ATPase compared to CCPvr4 , although the protein remained in the plasma membrane. Moreover, most of the mutated residues are on the outer surface of the funnel shape in the predicted pentameric CCPvr4 , implying that the disordered N-terminal region plays a crucial role in association with PMA as well as targeting to the plasma membrane. This work could provide insights into the molecular mechanisms of cell death induced by NLR immune receptors.

A Zwitterionic Detergent and Catalyst-Based Single-Cell Proteomics Using a Loss-Free Microhole-Collection Disc.

Recent advances in single-cell proteomics have solved many bottlenecks, such as throughput, sample recovery, and scalability via nanoscale sample handling. In this study, we aimed for a sensitive mass spectrometry (MS) analysis capable of handling single cells with a conventional mass spectrometry workflow without additional equipment. We achieved seamless cell lysis and TMT labeling in a micro-HOLe Disc (microHOLD) by developing a mass-compatible single solution based on a zwitterionic detergent and a catalyst for single-cell lysis and tandem mass tag labeling without a heat incubation step. This method was developed to avoid peptide loss by surface adsorption and buffer or tube changes by collecting tandem mass tag-labeled peptide through microholes placed in the liquid chromatography injection vials in a single solution. We successfully applied the microHOLD single-cell proteomics method for the analysis of proteome reprogramming in hormone-sensitive prostate cells to develop castration-resistant prostate cancer cells. This novel single-cell proteomics method is not limited by cutting-edge nanovolume handling equipment and achieves high throughput and ultrasensitive proteomics analysis of limited samples, such as single cells.

Oomycete effector AVRblb2 targets cyclic nucleotide-gated channels through calcium sensors to suppress pattern-triggered immunity.

Transient and rapid increase in cytosolic Ca2+ plays a crucial role in plant-pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Cyclic nucleotide-gated channels (CNGCs) have been implicated in mediating this Ca2+ influx; however, their regulatory mechanisms remain poorly understood. Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with the NbCNGCs, resulting in the formation of AVRblb2-CaM/CML-NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18. Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine-tuning of cytosolic Ca2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs. Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection.

Agrobacterium-mediated Cuscuta campestris transformation as a tool for understanding plant-plant interactions.

Cuscuta campestris, a stem parasitic plant, has served as a valuable model plant for the exploration of plant-plant interactions and molecular trafficking. However, a major barrier to C. campestris research is that a method to generate stable transgenic plants has not yet been developed. Here, we describe the development of a Cuscuta transformation protocol using various reporter genes (GFP, GUS, or RUBY) and morphogenic genes (CcWUS2 and CcGRF/GIF), leading to a robust protocol for Agrobacterium-mediated C. campestris transformation. The stably transformed and regenerated RUBY C. campestris plants produced haustoria, the signature organ of parasitic plants, and these were functional in forming host attachments. The locations of T-DNA integration in the parasite genome were confirmed through TAIL-PCR. Transformed C. campestris also produced flowers and viable transgenic seeds exhibiting betalain pigment, providing proof of germline transmission of the RUBY transgene. Furthermore, RUBY is not only a useful selectable marker for the Agrobacterium-mediated transformation, but may also provide insight into the movement of molecules from C. campestris to the host during parasitism. Thus, the protocol for transformation of C. campestris reported here overcomes a major obstacle to Cuscuta research and opens new possibilities for studying parasitic plants and their interactions with hosts.

The Phytophthora capsici RxLR effector CRISIS2 triggers cell death via suppressing plasma membrane H+-ATPase in the host plant.

Pathogen effectors can suppress various plant immune responses, suggesting that they have multiple targets in the host. To understand the mechanisms underlying plasma membrane-associated and effector-mediated immunity, we screened the Phytophthora capsici RxLR cell death-inducer suppressing immune system (CRISIS). We found that the cell death induced by the CRISIS2 effector in Nicotiana benthamiana was inhibited by the irreversible plasma membrane H+-ATPase (PMA) activator fusicoccin. Biochemical and gene-silencing analyses revealed that CRISIS2 physically and functionally associated with PMAs and induced host cell death independent of immune receptors. CRISIS2 induced apoplastic alkalization by suppressing PMA activity via its association with the C-terminal regulatory domain. In planta expression of CRISIS2 significantly enhanced the virulence of P. capsici, whereas host-induced gene-silencing of CRISIS2 compromised the disease symptoms and the biomass of the pathogen. Thus, our study has identified a novel RxLR effector that plays multiple roles in the suppression of plant defense and in the induction of cell death to support the pathogen hemibiotrophic life cycle in the host plant.

Practical delta check limits for tumour markers in different clinical settings.

OBJECTIVES: Few studies have reported on delta checks for tumour markers, even though these markers are often evaluated serially. Therefore, this study aimed to establish a practical delta check limit in different clinical settings for five tumour markers: alpha-fetoprotein, cancer antigen 19-9, cancer antigen 125, carcinoembryonic antigen, and prostate-specific antigen. METHODS: Pairs of patients' results (current and previous) for five tumour markers between 2020 and 2021 were retrospectively collected from three university hospitals. The data were classified into three subgroups, namely: health check-up recipient (subgroup H), outpatient (subgroup O), and inpatient (subgroup I) clinics. The check limits of delta percent change (DPC), absolute DPC (absDPC), and reference change value (RCV) for each test were determined using the development set (the first 18 months, n=179,929) and then validated and simulated by applying the validation set (the last 6 months, n=66,332). RESULTS: The check limits of DPC and absDPC for most tests varied significantly among the subgroups. Likewise, the proportions of samples requiring further evaluation, calculated by excluding samples with both current and previous results within the reference intervals, were 0.2-2.9% (lower limit of DPC), 0.2-2.7% (upper limit of DPC), 0.3-5.6% (absDPC), and 0.8-35.3% (RCV99.9%). Furthermore, high negative predictive values >0.99 were observed in all subgroups in the in silico simulation. CONCLUSIONS: Using real-world data, we found that DPC was the most appropriate delta-check method for tumour markers. Moreover, Delta-check limits for tumour markers should be applied based on clinical settings.

Promotion of tumorigenesis by miR-1260b-targeting CASP8: Potential diagnostic and prognostic marker for breast cancer.

MicroRNAs are reported as promising biomarkers for the diagnosis and treatment of breast cancer. miR-1260b is identified as a tumor-associated noncoding microRNA in other cancers, although the role of miR-1260b and its clinical relevance in breast cancer remain unclear. In this study, miR-1260b as a potential prognostic biomarker was observed by univariate and multivariate Cox regression analyses in 102 breast tumor tissues. The tumorigenic role of miR-1260b in terms of proliferation, apoptosis, and migration of breast cancer cells was investigated using gain- and loss-of-function assays in vitro. Additionally, the potential early diagnosis and treatment monitoring marker of miR-1260b was validated in 129 plasma samples. We found that high miR-1260b expression was markedly associated with bulky tumor size, advanced stage, and lymph node invasion. Particularly, the high-miR-1260b-expression group showed shorter overall survival than the low-miR-1260b-expression group. The inhibition of oncogenic miR-1260b induced apoptosis and decreased migration and invasion of MDA-MB-231 cells. CASP8 was revealed as a direct target gene of miR-1260b, which is closely related to apoptosis. Furthermore, miR-1260b expression levels in plasma were significantly higher in patients with breast cancer than in healthy controls. The patients who tested positive for miR-1260b showed 16.3- and 18.2-fold higher risks in the early stage and locally advanced stage, respectively, compared with healthy controls, and the risk was decreased 6.2-fold after neoadjuvant chemotherapy. Taken together, miR-1260b may be a potential novel diagnostic, prognostic, and therapeutic target in breast cancer.

Plasma membrane-localized plant immune receptor targets H+ -ATPase for membrane depolarization to regulate cell death.

The hypersensitive response (HR) is a robust immune response mediated by nucleotide-binding, leucine-rich repeat receptors (NLRs). However, the early molecular event that links activated NLRs to cell death is unclear. Here, we demonstrate that NLRs target plasma membrane H+ -ATPases (PMAs) that generate electrochemical potential, an essential component of living cells, across the plasma membrane. CCA 309, an autoactive N-terminal domain of a coiled-coil NLR (CNL) in pepper, is associated with PMAs. Silencing or overexpression of PMAs reversibly affects cell death induced by CCA 309 in Nicotiana benthamiana. CCA 309-induced extracellular alkalization causes plasma membrane depolarization, followed by cell death. Coimmunoprecipitation analyses suggest that CCA 309 inhibits PMA activation by preoccupying the dephosphorylated penultimate threonine residue of PMA. Moreover, pharmacological experiments using fusicoccin, an irreversible PMA activator, showed that inhibition of PMAs contributes to CNL-type (but not Toll interleukin-1 receptor NLR-type) resistance protein-induced cell death. We suggest PMAs as primary targets of plasma membrane-associated CNLs leading to HR-associated cell death by disturbing the electrochemical gradient across the membrane. These results provide new insight into NLR-mediated cell death in plants, as well as innate immunity in higher eukaryotes.

Inter-laboratory comparison of semiquantitative allergen-specific Immunoglobulin E test: 7 years of experience in Korea.

INTRODUCTION: Multiple allergen simultaneous test (MAST) is widely used as a screening tool for allergic diseases and has the advantage of providing specific IgE (sIgE) results for various allergens in semiquantitative class. We have continuously conducted external quality assessment (EQA) since 2012 for clinical laboratories performing MAST using AdvanSure allergy screen test (LG CHEM, Korea). This study provides an account of the EQA experience. METHODS: Samples were prepared using pooled sera collected from patients with suspected allergic disease and sent to each laboratory twice a year. Each round included 4-6 serum samples with sIgE for 10-20 inhaled or food allergens. The acceptable class value was the most frequently reported MAST class ±1 titer that exceeded 80% of the total laboratory results. RESULTS: The average number of participating laboratories was 76 (49-90) and the average response rate was 97.3% during the entire survey period. The acceptable rates were consistently high at 97.7% ± 3.7%. Of the total 537 trials, 18 trials (3.4%) were regarded as nonconsensus results, in which acceptable answers did not exceed 80%. For unacceptable results, the false-negative rate (1.5% ± 2.8%) was higher than the false-positive rate (0.8% ± 2.7%) (p < 0.001). MAST class results were correlated with quantitative IgE results by ImmunoCAP (Spearman's correlation coefficient of 0.682 (p < 0.001) and gamma index of 0.777 (p < 0.001). CONCLUSION: Although EQA for MAST showed a high level of acceptable answer, some allergen assays require harmonization. Continuous performance of systematic EQA is needed to improve the accuracy of sIgE assays and quality control in clinical laboratories.

Doubly-robust methods for differences in restricted mean lifetimes using pseudo-observations.

In clinical studies or trials comparing survival times between two treatment groups, the restricted mean lifetime (RML), defined as the expectation of the survival from time 0 to a prespecified time-point, is often the quantity of interest that is readily interpretable to clinicians without any modeling restrictions. It is well known that if the treatments are not randomized (as in observational studies), covariate adjustment is necessary to account for treatment imbalances due to confounding factors. In this article, we propose a simple doubly-robust pseudo-value approach to effectively estimate the difference in the RML between two groups (akin to a metric for estimating average causal effects), while accounting for confounders. The proposed method combines two general approaches: (a) group-specific regression models for the time-to-event and covariate information, and (b) inverse probability of treatment assignment weights, where the RMLs are replaced by the corresponding pseudo-observations for survival outcomes, thereby mitigating the estimation complexities in presence of censoring. The proposed estimator is double-robust, in the sense that it is consistent if at least one of the two working models remains correct. In addition, we explore the potential of available machine learning algorithms in causal inference to reduce possible bias of the causal estimates in presence of a complex association between the survival outcome and covariates. We conduct extensive simulation studies to assess the finite-sample performance of the pseudo-value causal effect estimators. Furthermore, we illustrate our methodology via application to a dataset from a breast cancer cohort study. The proposed method is implementable using the R package drRML, available in GitHub.

Combined Analysis of HLA Class II Eplet Mismatch and Tacrolimus Levels for the Prediction of De Novo Donor Specific Antibody Development in Kidney Transplant Recipients.

We investigated whether HLA class II eplet mismatch was related to dnDSA development and analyzed its combined impact with tacrolimus levels for kidney transplantation outcomes. A total of 347 kidney transplants were included. HLA Matchmaker was used for the single molecular eplet, total eplet, antibody (Ab)-verified eplet mismatch analyses, and Ab-verified single molecular analysis to identify HLA-DR/DQ molecular thresholds for the risk of dnDSA development. A time-weighted tacrolimus trough level (TAC-C0) of 5 ng/mL and a TAC-C0 time-weighted coefficient variability (TWCV) of 20% were applied to find the combined effects on dnDSA development. A high level of mismatch for single molecular eplet (DQ ≥ 10), total eplet (DQ ≥ 12), Ab-verified eplet (DQ ≥ 4), and Ab-verified single molecular eplet (DQ ≥ 4) significantly correlated with HLA class II dnDSA development. Class II dnDSA developed mostly in patients with low TAC-C0 and high eplet mismatch. In the multivariable analyses, low TAC-C0 and high eplet mismatch showed the highest hazard ratio for the development of dnDSA. No significant combined effect was observed in dnDSA development according to TWCV. In conclusion, the determination of HLA class II eplet mismatch may improve the risk stratification for dnDSA development, especially in conjunction with tacrolimus trough levels.

Blood Test for Breast Cancer Screening through the Detection of Tumor-Associated Circulating Transcripts.

Liquid biopsy has been emerging for early screening and treatment monitoring at each cancer stage. However, the current blood-based diagnostic tools in breast cancer have not been sufficient to understand patient-derived molecular features of aggressive tumors individually. Herein, we aimed to develop a blood test for the early detection of breast cancer with cost-effective and high-throughput considerations in order to combat the challenges associated with precision oncology using mRNA-based tests. We prospectively evaluated 719 blood samples from 404 breast cancer patients and 315 healthy controls, and identified 10 mRNA transcripts whose expression is increased in the blood of breast cancer patients relative to healthy controls. Modeling of the tumor-associated circulating transcripts (TACTs) is performed by means of four different machine learning techniques (artificial neural network (ANN), decision tree (DT), logistic regression (LR), and support vector machine (SVM)). The ANN model had superior sensitivity (90.2%), specificity (80.0%), and accuracy (85.7%) compared with the other three models. Relative to the value of 90.2% achieved using the TACT assay on our test set, the sensitivity values of other conventional assays (mammogram, CEA, and CA 15-3) were comparable or much lower, at 89%, 7%, and 5%, respectively. The sensitivity, specificity, and accuracy of TACTs were appreciably consistent across the different breast cancer stages, suggesting the potential of the TACTs assay as an early diagnosis and prediction of poor outcomes. Our study potentially paves the way for a simple and accurate diagnostic and prognostic tool for liquid biopsy.

Genome-wide functional analysis of hot pepper immune receptors reveals an autonomous NLR clade in seed plants.

Plants possess hundreds of intracellular immune receptors encoding nucleotide-binding domain leucine-rich repeat (NLR) proteins. Full-length NLRs or a specific domain of NLRs often induce plant cell death in the absence of pathogen infection. In this study we used genome-wide transient expression analysis to identify a group of NLRs (ANLs; ancient and autonomous NLRs) carrying autoactive coiled-coil (CCA ) domains in pepper (Capsicum annuum). CCA -mediated cell death mimics hypersensitive cell death triggered by the interaction between NLRs and pathogen effectors. Sequence alignment and mutagenesis analyses revealed that the intact α1 helix of CCA s is critical for both CCA - and ANL-mediated cell death. Cell death induced by CCA s does not require NRG1/ADR1 or NRC type helper NLRs, suggesting ANLs may function as singleton NLRs. We also found that CCA s localize to the plasma membrane, as demonstrated for Arabidopsis singleton NLR ZAR1. Extended studies revealed that autoactive CCA s are well conserved in other Solanaceae plants as well as in rice, a monocot plant. Further phylogenetic analyses revealed that ANLs are present in all tested seed plants (spermatophytes). Our study not only uncovers the autonomous NLR clade in plants but also provides powerful resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in plants.

Multilaboratory Evaluation of the MALDI-TOF Mass Spectrometry System, MicroIDSys Elite, for the Identification of Medically Important Filamentous Fungi.

With the increasing number of fungal infections and immunocompromised patients, rapid and accurate fungal identification is required in clinical microbiology laboratories. We evaluated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system, MicroIDSys Elite (ASTA Corp., South Korea) for the identification of medically important filamentous fungi. A total of 505 strains comprising 37 genera and 90 species collected from 11 Korean hospitals were sent to the microbiology laboratory of International St. Mary's Hospital. All isolates were tested using MicroIDSys Elite, and data were analyzed using the MoldDB v.1.22 database (ASTA). Correct identification rates were compared with the multigene sequencing results. MicroIDSys Elite correctly identified 86.5% (437/505) and 88.9% (449/505) of all tested isolates at the species and genus level, respectively. About 98.2% of Aspergillus isolates were identified at the species level, including cryptic and rare species of A. calidoustus, A. tamarii, A. lentulus, A. versicolor and A. aculeatus. MicroIDSys Elite identified 75.0% of basidiomycetes, including Schizophyllum commune, and 84.3% of the dermatophytes. It also distinguished Sprothrix globosa at the species level. The mean scores of total isolates corresponding to correct species identification were significantly higher than those obtained for genus-level identification (253.5 ± 50.7 vs. 168.6 ± 30.3, P < 0.001). MicroIDSys Elite showed high accuracy for the identification of filamentous fungi, including cryptic and rare Aspergillus species. It is suitable for use in clinical laboratories as a rapid and efficient tool for clinical mold identification. Further evaluations are recommended for MicroIDSys Elite as a rapid and efficient tool for the identification of medically important filamentous fungi.

BAFF Inhibition Effectively Suppresses the Development of Anti-HLA.A2 Antibody in the Highly Sensitized Mouse Model.

B cell activating factor (BAFF) is a cytokine that plays a role in the survival, proliferation and differentiation of B cells. We proposed to observe the effects of BAFF inhibition on the humoral immune responses of an allosensitized mouse model using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and were treated with anti-BAFF monoclonal antibody (mAb) (named Sandy-2) or control IgG1 antibody. HLA.A2-specific IgG was reduced in BAFF-inhibited mice compared to the control group (Δ-13.62 vs. Δ27.07, p < 0.05). BAFF inhibition also resulted in increased pre-pro and immature B cell proportions and decreased mature B cells in the bone marrow (p < 0.05 vs. control). In the spleen, an increase in transitional B cells was observed with a significant decrease in marginal and follicular B cells (p < 0.05 vs. control). There was no significant difference in the proportions of long-lived plasma and memory B cells. Microarray analysis showed that 19 gene probes were significantly up- (>2-fold, p < 0.05) or down-regulated (≤2-fold, p < 0.05) in the BAFF-inhibited group. BAFF inhibition successfully reduced alloimmune responses through the reduction in alloantibody production and suppression of B cell differentiation and maturation. Our data suggest that BAFF suppression may serve as a useful target in desensitization therapy.

Coronavirus Disease Outbreak in Call Center, South Korea.

We describe the epidemiology of a coronavirus disease (COVID-19) outbreak in a call center in South Korea. We obtained information on demographic characteristics by using standardized epidemiologic investigation forms. We performed descriptive analyses and reported the results as frequencies and proportions for categoric variables. Of 1,143 persons who were tested for COVID-19, a total of 97 (8.5%, 95% CI 7.0%-10.3%) had confirmed cases. Of these, 94 were working in an 11th-floor call center with 216 employees, translating to an attack rate of 43.5% (95% CI 36.9%-50.4%). The household secondary attack rate among symptomatic case-patients was 16.2% (95% CI 11.6%- 22.0%). Of the 97 persons with confirmed COVID-19, only 4 (1.9%) remained asymptomatic within 14 days of quarantine, and none of their household contacts acquired secondary infections. Extensive contact tracing, testing all contacts, and early quarantine blocked further transmission and might be effective for containing rapid outbreaks in crowded work settings.

Contact Tracing during Coronavirus Disease Outbreak, South Korea, 2020.

We analyzed reports for 59,073 contacts of 5,706 coronavirus disease (COVID-19) index patients reported in South Korea during January 20-March 27, 2020. Of 10,592 household contacts, 11.8% had COVID-19. Of 48,481 nonhousehold contacts, 1.9% had COVID-19. Use of personal protective measures and social distancing reduces the likelihood of transmission.

Exon splicing analysis of intronic variants in multigene cancer panel testing for hereditary breast/ovarian cancer.

The use of multigene panel testing for patients with a predisposition to breast/ovarian cancer is increasing as the identification of variants is useful for diagnosis and disease management. We identified pathogenic and likely pathogenic (P/LP) variants of high-and moderate-risk genes using a 23-gene germline cancer panel in 518 patients with hereditary breast and ovarian cancers (HBOC). The frequency of P/LP variants was 12.4% (64/518) for high- and moderate-penetrant genes, namely, BRCA2 (5.6%), BRCA1 (3.3%), CHEK2 (1.2%), MUTYH (0.8%), PALB2 (0.8%), MLH1 (0.4%), ATM (0.4%), BRIP1 (0.4%), TP53 (0.2%), and PMS2 (0.2%). Five patients possessed two P/LP variants in BRCA1/2 and other genes. We also compared the results from in silico splicing predictive tools and exon splicing patterns from patient samples by analyzing RT-PCR product sequences in six P/LP intronic variants and two intronic variants of unknown significance (VUS). Altered transcriptional fragments were detected for P/LP intronic variants in BRCA1, BRIP1, CHEK2, PARB2, and PMS2. Notably, we identified an in-frame deletion of the BRCA1 C-terminal (BRCT) domain by exon skipping in BRCA1 c.5152+6T>C-as known VUS-indicating a risk for HBOC. Thus, exon splicing analysis can improve the identification of veiled intronic variants that would aid decision making and determination of hereditary cancer risk.

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments.

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th ß-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 ß-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.