Comparison of quantitative and qualitative anti-dsDNA assays.

OBJECTIVE: In evaluation of systemic lupus erythematosus (SLE), anti-double-stranded DNA antibodies (anti-dsDNA) play a significant role in diagnosis, monitoring SLE activity, and assessing prognosis. However, evaluations of the performance and limitations for recently developed methods for anti-dsDNA assessment are sparse. METHODS: Specimens used for antinuclear antibody testing (n = 129) were evaluated for anti-dsDNA assay comparability across 4 medical centers in the United States. The methods compared were Werfen Quanta Lite dsDNA, Zeus Scientific dsDNA Enzyme Immunoassay, Bio-Rad multiplex immunoassay (MIA) dsDNA, ImmunoConcepts Crithidia, and Bio-Rad Laboratories Crithidia. RESULTS: For quantitative anti-dsDNA measurements, Spearman's correlation coefficient was highest between Zeus and Werfen (ρ = 0.86; CI, 0.81-0.90; P < .0001). Comparison of MIA to Werfen or Zeus yielded similar results to each other (ρ = 0.58; CI, 0.44-0.68; P < .0001; and ρ = 0.59; CI, 0.46-0.69; P < .0001, respectively), but lower than the correlation between Zeus and Werfen. Positive concordance between assays ranged from 31.4% to 97.1%, and negative concordance between assays ranged from 58.5% to 100%. The detection of anti-dsDNA in those with SLE diagnosis ranged from 50.9% to 77.4% for quantitative assays and 15.1% to 24.5% for Crithidia assays. CONCLUSION: Current quantitative anti-dsDNA assays are not interchangeable for patient follow-up. Crithidia-based assays demonstrate high negative concordance and lack positive concordance among the methods.

Identification of a targetable JAK-STAT enriched androgen receptor and androgen receptor splice variant positive triple-negative breast cancer subtype.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no targeted therapeutics. The luminal androgen receptor (LAR) subtype constitutes 15% of TNBC and is enriched for androgen receptor (AR) and AR target genes. Here, we show that a cohort of TNBC not only expresses AR at a much higher rate (∼80%) but also expresses AR splice variants (AR-SVs) (∼20%), further subclassifying LAR-TNBC. Higher AR and AR-SV expression and corresponding aggressive phenotypes are observed predominantly in specimens obtained from African American women. LAR TNBC specimens are enriched for interferon, Janus kinase (JAK)-signal activator and transducer (STAT), and androgen signaling pathways, which are exclusive to AR-expressing epithelial cancer cells. AR- and AR-SV-expressing TNBC cell proliferation and xenograft and patient-tumor explant growth are inhibited by AR N-terminal domain-binding selective AR degrader or by a JAK inhibitor. Biochemical analysis suggests that STAT1 is an AR coactivator. Collectively, our work identifies pharmacologically targetable TNBC subtypes and identifies growth-promoting interaction between AR and JAK-STAT signaling.

Wheat Susceptibility Genes TaCAMTA2 and TaCAMTA3 Negatively Regulate Post-Penetration Resistance against Blumeria graminis forma specialis tritici.

Blumeria graminis forma specialis tritici (B.g. tritici) is the airborne fungal pathogen that causes powdery mildew disease on hexaploid bread wheat. Calmodulin-binding transcription activators (CAMTAs) regulate plant responses to environments, but their potential functions in the regulation of wheat-B.g. tritici interaction remain unknown. In this study, the wheat CAMTA transcription factors TaCAMTA2 and TaCAMTA3 were identified as suppressors of wheat post-penetration resistance against powdery mildew. Transient overexpression of TaCAMTA2 and TaCAMTA3 enhanced the post-penetration susceptibility of wheat to B.g. tritici, while knockdown of TaCAMTA2 and TaCAMTA3 expression using transient- or virus-induced gene silencing compromised wheat post-penetration susceptibility to B.g. tritici. In addition, TaSARD1 and TaEDS1 were characterized as positive regulators of wheat post-penetration resistance against powdery mildew. Overexpressing TaSARD1 and TaEDS1 confers wheat post-penetration resistance against B.g. tritici, while silencing TaSARD1 and TaEDS1 enhances wheat post-penetration susceptibility to B.g. tritici. Importantly, we showed that expressions of TaSARD1 and TaEDS1 were potentiated by silencing of TaCAMTA2 and TaCAMTA3. Collectively, these results implicated that the Susceptibility genes TaCAMTA2 and TaCAMTA3 contribute to the wheat-B.g. tritici compatibility might via negative regulation of TaSARD1 and TaEDS1 expression.

Prediction of very early major bleeding risk in acute pulmonary embolism: an independent external validation of the Pulmonary Embolism-Syncope, Anemia, and Renal Dysfunction (PE-SARD) bleeding score.

BACKGROUND: The Pulmonary Embolism-Syncope, Anemia, and Renal Dysfunction (PE-SARD) bleeding score was derived to predict very early major bleeding (MB) in patients with acute pulmonary embolism (PE). Before adoption into practice, the score requires external validation in different populations. OBJECTIVES: We independently validated the PE-SARD score in a prospective multicenter Swiss cohort of 687 patients aged ≥65 years with acute PE. METHODS: The PE-SARD score uses 3 variables (syncope, anemia, and renal dysfunction) to classify patients into 3 categories of increasing bleeding risk. The outcomes were very early MB at 7 days (primary) and MB at later time points (secondary). We calculated the PE-SARD score for each patient and classified the proportion of patients as being at low, intermediate, and high risk. To assess discrimination and calibration, we calculated the area under the receiver operating characteristic curve and the Hosmer-Lemeshow goodness-of-fit test, respectively. RESULTS: The prevalence of MB was 2.0% (14/687) at 7 days and 14.0% (96/687) after a median follow-up of 30 months. The PE-SARD score classified 40.2%, 42.2%, and 17.6% of patients as low, intermediate, and high risk for MB, respectively. The frequency of observed very early MB at 7 days was 1.8% in low-, 2.1% in intermediate-, and 2.5% in high-risk patients. The area under the receiver operating characteristic curve was 0.52 (95% CI, 0.48-0.56) at 7 days and increased to 0.60 (95% CI, 0.56-0.64) at the end of follow-up. Score calibration was adequate (p > .05) over the entire follow-up. CONCLUSION: In our independent validation, the PE-SARD score did not accurately predict very early MB and may not be transportable to older patients with PE.

Evaluation of Antinuclear Antibody and Subserology Reflex Testing for the Diagnosis of Systemic Autoimmune Rheumatic Disorders in an Academic Teaching Hospital.

OBJECTIVE: The aim of this study was to examine appropriate utilization of antinuclear antibody (ANA) screening tests with follow-up subserology tests (reflex testing) for diagnosing systemic autoimmune rheumatic disorder (SARD). METHODS: We conducted a retrospective chart review of 3003 SARD-test orders at an academic teaching hospital from January to December 2019. Testing patterns were categorized as American College of Rheumatology (ACR)-recommended reflex testing, panel testing, or single subserology testing. We described testing patterns, assessed their diagnostic accuracy, and explored factors associated with reflex testing. RESULTS: Reflex testing accounted for 79.7% of SARD test-ordering, whereas improper testing (panel or single subserology) accounted for the other 20.3%. Reflex testing was associated with significantly more SARD diagnoses than improper testing (P = .004). Testing patterns were significantly associated with race/ethnicity (P = .008), with reflex testing being less frequent than improper testing in Hispanics and Whites. CONCLUSION: In summary, one-fifth (20.3%) of testing patterns for suspected SARD did not follow the ACR-recommended guidelines for using reflex testing. Use of reflex testing was associated with an increased frequency of SARD diagnosis.

The Kelch-F-box protein SMALL AND GLOSSY LEAVES 1 (SAGL1) negatively influences salicylic acid biosynthesis in Arabidopsis thaliana by promoting the turn-over of transcription factor SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1).

Salicylic acid (SA) is a key phytohormone regulating plant immunity. Although the transcriptional regulation of SA biosynthesis has been well-studied, its post-translational regulation is largely unknown. We report that a Kelch repeats-containing F-box (KFB) protein, SMALL AND GLOSSY LEAVES 1 (SAGL1), negatively influences SA biosynthesis in Arabidopsis thaliana by mediating the proteolytic turnover of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1), a master transcription factor that directly drives SA biosynthesis during immunity. Loss of SAGL1 resulted in characteristic growth inhibition. Combining metabolomic, transcriptional and phenotypic analyses, we found that SAGL1 represses SA biosynthesis and SA-mediated immune activation. Genetic crosses to mutants that are deficient in SA biosynthesis blocked the SA overaccumulation in sagl1 and rescued its growth. Biochemical and proteomic analysis identified that SAGL1 interacts with SARD1 and promotes the degradation of SARD1 in a proteasome-dependent manner. These results unravelled a critical role of KFB protein SAGL1 in maintaining SA homeostasis via controlling SARD1 stability.

Drought attenuates plant defence against bacterial pathogens by suppressing the expression of CBP60g/SARD1 during combined stress.

In nature, plants are frequently exposed to drought and bacterial pathogens simultaneously. However, information on how the drought and defence pathways interact and orchestrate global transcriptional regulation is limited. Here, we show that moderate drought stress enhances the susceptibility of Arabidopsis thaliana to Pseudomonas syringae pv. tomato DC3000. Using transcriptome meta-analysis, we found that drought and bacterial stress antagonistically modulate a large set of genes predominantly involved in salicylic acid (SA) and abscisic acid (ABA) signalling networks. We identified that the levels of SA and ABA are dynamically regulated during the course of stress. Importantly, under combined stress, drought through the ABA pathway downregulates the induction of Calmodulin-binding Protein 60 g (CBP60g) and Systemic Acquired Resistance Deficient 1 (SARD1), two transcription factors crucial for SA production upon bacterial infection. We also identified an important role of NPR1-LIKE PROTEIN 3 and 4 (NPR3/4) transcriptional repressors in the drought-mediated negative regulation of CBP60g/SARD1 expression. Using a genetic approach, we show that CBP60g/SARD1 expression is the key determinant of plant defence against bacterial pathogens under combined stress. Thus, these transcription factors act as critical nodes for the crosstalk between drought and bacterial stress signalling under combined stress in plants.

Arabidopsis CALMODULIN-BINDING PROTEIN 60b plays dual roles in plant immunity.

Arabidopsis SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g) are two master transcription factors that regulate many defense-related genes in plant immunity. They are required for immunity downstream of the receptor-like protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2). Constitutive defense responses in the gain-of-function autoimmune snc2-1D mutant are modestly affected in either sard1 or cbp60g single mutants but completely suppressed in the sard1 cbp60g double mutant. Here we report that CBP60b, another member of the CBP60 family, also functions as a positive regulator of SNC2-mediated immunity. Loss-of-function mutations of CBP60b suppress the constitutive expression of SARD1 and enhanced disease resistance in cbp60g-1 snc2-1D, whereas overexpression of CBP60b leads to elevated SARD1 expression and constitutive defense responses. In addition, transient expression of CBP60b in Nicotiana benthamiana activates the expression of the pSARD1::luciferase reporter gene. Chromatin immunoprecipitation assays further showed that CBP60b is recruited to the promoter region of SARD1, suggesting that it directly regulates SARD1 expression. Interestingly, knocking out CBP60b in the wild-type background leads to ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-dependent autoimmunity, suggesting that CBP60b is required for the expression of a guardee/decoy or a negative regulator of immunity mediated by receptors carrying an N-terminal Toll-interleukin-1 receptor-like domain.

Association Between a History of Dengue Fever and the Risk of Systemic Autoimmune Rheumatic Diseases: A Nationwide, Population-Based Case-Control Study.

Purpose: To determine the association between a history of clinically diagnosed dengue infection and the risk of systemic autoimmune rheumatic diseases (SARDs). Methods: Using claims data from the 1997-2013 Taiwanese National Health Insurance Research Database, we included 74,422 patients who were diagnosed with SARDs and 297,688 patients without SARDs who were matched (in a 1:4 ratio) for age, sex, year of SARDs index date, and city of residence. The associations between the development of SARDs and a history of dengue infection (International Classification of Diseases, Ninth Revision, Clinical Modification code 061) were investigated using conditional logistic regression analysis shown as odds ratios (ORs) with 95% confidence intervals (CIs) after adjusting for potential confounders. Results: We included 17,126 patients with systemic lupus erythematosus (SLE), 15,531 patients with Sjogren's syndrome (SS), 37,685 patients with rheumatoid arthritis (RA), 1,911 patients with systemic sclerosis (SSc), 1,277 patients with dermatomyositis (DM), and 892 patients with polymyositis (PM). SLE (OR, 4.55; 95% CI, 2.77-7.46; p <0.001) risk was significantly associated with a history of dengue infection. However, no statistically significant association was found between dengue infection and SS (OR, 1.41; 95% CI, 0.88-2.26; p = 0.155), RA (OR, 1.03; 95% CI, 0.70-1.50; p = 0.888), SSc (OR, 1.97; 95% CI, 0.38-10.29; p = 0.420), DM (OR, 0.54; 95% CI, 0.04-7.27; p = 0.641), or PM (OR, 2.08; 95% CI, 0.23-18.79; p = 0.513). Conclusion: This study revealed that a history of dengue infection was significantly associated with the risk of SLE, but not SS, RA, SSc, DM, or PM.

Outcomes of renal replacement therapy in the critically ill with COVID-19 / Resultados de la terapia de reemplazo renal en pacientes críticamente enfermos con COVID-19

Objective To describe outcomes of critically ill patients with COVID-19, particularly the association of renal replacement therapy to mortality. Design A single-center prospective observational study was carried out. Setting ICU of a tertiary care center. Patients Consecutive adults with COVID-19 admitted to the ICU. Intervention Renal replacement therapy. Main variables of interest Demographic data, medical history, illness severity, type of oxygen therapy, laboratory data and use of renal replacement therapy to generate a logistic regression model describing independent risk factors for mortality. Results Of the total of 166 patients, 51% were mechanically ventilated and 26% required renal replacement therapy. The overall hospital mortality rate was 36%, versus 56% for those requiring renal replacement therapy, and 68% for those with both mechanical ventilation and renal replacement therapy. The logistic regression model identified four independent risk factors for mortality: age (adjusted OR 2.8 [95% CI 1.8–4.4] for every 10-year increase), mechanical ventilation (4.2 [1.7–10.6]), need for continuous venovenous hemofiltration (2.3 [1.3–4.0]) and C-reactive protein (1.1 [1.0–1.2] for every 10mg/L increase). Conclusions In our cohort, acute kidney injury requiring renal replacement therapy was associated to a high mortality rate similar to that associated to the need for mechanical ventilation, while multiorgan failure necessitating both techniques implied an extremely high mortality risk. (AU)

Objetivo Describir los resultados de pacientes críticamente enfermos con COVID-19, especialmente la asociación de la terapia de reemplazo renal con la mortalidad. Diseño Estudio observacional, prospectivo y unicéntrico. Ámbito En la unidad de cuidados intensivos (UCI) de un centro de atención terciaria. Pacientes Pacientes adultos con COVID-19 ingresados de forma consecutiva en la UCI. Intervención Administración de terapia de reemplazo renal. Variables de interés principales Datos demográficos, antecedentes médicos, gravedad de la enfermedad, tipo de oxigenoterapia, datos analíticos y uso de terapia de reemplazo renal para generar un modelo de regresión logística que describa factores de riesgo independientes de la mortalidad. Resultados De los 166 pacientes, el 51% recibieron ventilación mecánica (VM) y el 26% requirió terapia de reemplazo renal (TRR). La mortalidad hospitalaria global fue del 36%, frente al 56% en el caso de los pacientes que requirieron TRR y el 68% en el subconjunto de pacientes que necesitó tanto VM como RTT. Un modelo de regresión logística señala cuatro factores de riesgo independientes de la mortalidad: edad (OR ajustada: 2,8 [IC del 95%: 1,8-4,4] por cada incremento de 10 años), ventilación mecánica (4,2 [1,7-10,6]), necesidad de hemofiltración venovenosa continua (HVVC) (2,3 [1,3-4,0]), y proteína C reactiva (1,1 [1,0-1,2] por cada incremento de 10mg/L). Conclusiones En nuestra cohorte, la lesión renal aguda que necesita TRR se asocia con una mortalidad similarmente elevada a la de los pacientes que requieren VM, y la insuficiencia multiorgánica que hace necesarias ambas intervenciones se asocia con un riesgo de mortalidad extremadamente alta. (Au)

Outcomes of renal replacement therapy in the critically ill with COVID-19.

OBJECTIVE: To describe outcomes of critically ill patients with COVID-19, particularly the association of renal replacement therapy to mortality. DESIGN: A single-center prospective observational study was carried out. SETTING: ICU of a tertiary care center. PATIENTS: Consecutive adults with COVID-19 admitted to the ICU. INTERVENTION: Renal replacement therapy. MAIN VARIABLES OF INTEREST: Demographic data, medical history, illness severity, type of oxygen therapy, laboratory data and use of renal replacement therapy to generate a logistic regression model describing independent risk factors for mortality. RESULTS: Of the total of 166 patients, 51% were mechanically ventilated and 26% required renal replacement therapy. The overall hospital mortality rate was 36%, versus 56% for those requiring renal replacement therapy, and 68% for those with both mechanical ventilation and renal replacement therapy. The logistic regression model identified four independent risk factors for mortality: age (adjusted OR 2.8 [95% CI 1.8-4.4] for every 10-year increase), mechanical ventilation (4.2 [1.7-10.6]), need for continuous venovenous hemofiltration (2.3 [1.3-4.0]) and C-reactive protein (1.1 [1.0-1.2] for every 10mg/L increase). CONCLUSIONS: In our cohort, acute kidney injury requiring renal replacement therapy was associated to a high mortality rate similar to that associated to the need for mechanical ventilation, while multiorgan failure necessitating both techniques implied an extremely high mortality risk.

WRKY54 and WRKY70 positively regulate SARD1 and CBP60g expression in plant immunity.

SARD1 and CBP60g are two master regulators in plant immunity. They are required for the constitutive defense responses in the Arabidopsis snc2-1D mutant, which carries a gain-of-function mutation in a receptor-like protein. Here we report that WRKY54 and WRKY70 are required for activation of SARD1 and CBP60g expression and defense responses in snc2-1D. In addition, the induction of SARD1 and CBP60g by the bacterial pathogen Pseudomonas syringae pv. maculicola is significantly reduced in sid2 wrky54 wrky70 triple mutants compared to the sid2 single mutants, suggesting that WRKY54 and WRKY70 positively regulate the SID2-independent expression of SARD1 and CBP60g during pathogen infection. Our study revealed WRKY54 and WRKY70 as positive regulators of SARD1 and CBP60g expression in plant defense.

Presence of anti-TIF-1γ, anti-Ro52, anti-SSA/Ro60 and anti-Su/Ago2 antibodies in breast cancer: a cross-sectional study.

OBJECTIVES: The presence of myositis-specific antibodies (MSA), was recently reported in healthy individuals, cancer patients without myopathy and paraneoplastic rheumatic syndromes. We sought to analyze the frequency of MSA, myositis-associated antibodies (MAA) and autoantibodies related to systemic autoimmune rheumatic diseases (SARD) in breast cancer patients. METHODS: One hundred fifty-two breast cancer patients were enrolled in a cross-sectional study. Clinical information was collected, and autoantibodies tested by immunoprecipitation of an 35S-methionine-labeled K562 cell extract, enzyme-linked immunosorbent assay (ELISA) and Western blot when indicated. All statistical tests were performed using the software statistical package for the social science (SPSS) ver. 19.0 (IBM Inc., NYSE, USA). RESULTS: Autoantibodies associated with SARD: anti-52 kD ribonucleoprotein/tripartite motif-containing 21 (anti-Ro52/TRIM21) was found in 5.9% (9/152), anti-Sjögren syndrome-related antigen A/60 kD ribonucleoprotein antibody (anti-SSA/Ro60) in 3.9% (6/152) and anti-Su antigen/Argonaute 2 antibody (anti-Su/Ago2) in 2.6% (4/152). Meanwhile, anti-transcription intermediary factor-1γ (anti-TIF-1γ, p155/140) antibody was positive in 2 cases and anti-polymyositis/scleroderma antibody was detected in one case. As a whole, 14.47% (22/152) of breast cancer patients showed autoantibodies associated with SARD. These specific autoantibodies were not associated with the presence of rheumatic diseases except one rheumatoid arthritis patient positive for anti-Ro52/TRIM21. CONCLUSIONS: Autoantibodies to TIF-1γ were found in two patients with breast cancer without dermatomyositis (DM). More common specificities were autoantibodies anti-SSA/Ro60, anti-Ro52/TRIM21 and anti-Su/Ago2. More studies are needed in order to establish the biological meaning of the presence of SARD-associated autoantibodies in breast cancer.

How social intimidation interferred with the increasing number of improper accesses at the E.R.: a first-month COVID19 outbreak evaluation.

ABSTRACT: SARS Cov 2 pandemic outbreak caused countless changes in the daily habits among people in the entire World. National Health Systems were highly stressed and at severe risk of collapse. In the first months of 2020, it was expected a worsening of the typical overcrowding working flow. Quite the opposite, we found out an unexpected decrease throughout the daily ED visits. We evaluated the ER visits of a University Metropolitan Hospital in Rome in March 2020, comparing them with the same month in 2019. We highlight the sharp decline in ED visits for deferrable urgencies, considered among the leading causes of ED overcrowding. On the contrary, the rate of visits for "time-dependent" pathologies is superimposable for those pathologies mainly centralized through the Out-of-Hospital Emergency System. In a historical period where significant outpatient activity restrictions took place, we expected an increase in ED visits for deferred emergencies. On the contrary, it was undergoing a considerable decrease. The critical decrease in accesses recorded in March 2020 can be considered an indicator of ED's improper use. Probably, the fear of a possible coronavirus-related infection might have to lead the population to refer to the ED just in case of real emergency condition or severe medical issues, as it should "normally" be. The critical decrease in accesses recorded in March 2020 can be considered an indicator of ED's improper use. We would highlight the need to sensitize people to proper use of Emergency Medical Services, avoiding overcrowding and overuse. This unexpected event, lead by a global pandemic, could help reorganize the whole Health System.

An Overview of Next-Generation Androgen Receptor-Targeted Therapeutics in Development for the Treatment of Prostate Cancer.

Traditional endocrine therapy for prostate cancer (PCa) has been directed at suppression of the androgen receptor (AR) signaling axis since Huggins et al. discovered that diethylstilbestrol (DES; an estrogen) produced chemical castration and PCa tumor regression. Androgen deprivation therapy (ADT) still remains the first-line PCa therapy. Insufficiency of ADT over time leads to castration-resistant PCa (CRPC) in which the AR axis is still active, despite castrate levels of circulating androgens. Despite the approval and use of multiple generations of competitive AR antagonists (antiandrogens), antiandrogen resistance emerges rapidly in CRPC due to several mechanisms, mostly converging in the AR axis. Recent evidence from multiple groups have defined noncompetitive or noncanonical direct binding sites on AR that can be targeted to inhibit the AR axis. This review discusses new developments in the PCa treatment paradigm that includes the next-generation molecules to noncanonical sites, proteolysis targeting chimera (PROTAC), or noncanonical N-terminal domain (NTD)-binding of selective AR degraders (SARDs). A few lead compounds targeting each of these novel noncanonical sites or with SARD activity are discussed. Many of these ligands are still in preclinical development, and a few early clinical leads have emerged, but successful late-stage clinical data are still lacking. The breadth and diversity of targets provide hope that optimized noncanonical inhibitors and/or SARDs will be able to overcome antiandrogen-resistant CRPC.

CHYR1 ubiquitinates the phosphorylated WRKY70 for degradation to balance immunity in Arabidopsis thaliana.

It is critically important for plants to control the trade-off between normal growth and pathogen immunity. However, the underlying molecular mechanism remains largely unknown. Here we report such a mechanism controlled by WRKY70 and its partner CHYR1 in Arabidopsis. We found that both levels of the WRKY70 target gene SARD1 and the phosphorylated forms of WRKY70 were increased in WRKY70OE plants upon Pst DC3000 infection. Mechanistically, phosphorylation of WRKY70 at Thr22 and Ser34 occurs, which then activates SARD1 expression through binding to a WT box. Phosphorylated WRKY70 is degraded by 26S proteasome via CHYR1 when resuming normal growth after infection. In addition, nonphosphorylated WRKY70 represses SARD1 expression by binding to both W (inhibitory activity site) and WT (active activity site) boxes. The binding of WRKY70 to alternative cis-elements of SARD1 through a phosphorylation-mediated switch controlled by CHYR1 contributes to modulating the balance between immunity and growth.

Calcium-dependent protein kinase 5 links calcium signaling with N-hydroxy-l-pipecolic acid- and SARD1-dependent immune memory in systemic acquired resistance.

Systemic acquired resistance (SAR) prepares infected plants for faster and stronger defense activation upon subsequent attacks. SAR requires an information relay from primary infection to distal tissue and the initiation and maintenance of a self-maintaining phytohormone salicylic acid (SA)-defense loop. In spatial and temporal resolution, we show that calcium-dependent protein kinase CPK5 contributes to immunity and SAR. In local basal resistance, CPK5 functions upstream of SA synthesis, perception, and signaling. In systemic tissue, CPK5 signaling leads to accumulation of SAR-inducing metabolite N-hydroxy-L-pipecolic acid (NHP) and SAR marker genes, including Systemic Acquired Resistance Deficient 1 (SARD1) Plants of increased CPK5, but not CPK6, signaling display an 'enhanced SAR' phenotype towards a secondary bacterial infection. In the sard1-1 background, CPK5-mediated basal resistance is still mounted, but NHP concentration is reduced and enhanced SAR is lost. The biochemical analysis estimated CPK5 half maximal kinase activity for calcium, K50 [Ca2+ ], to be c. 100 nM, close to the cytoplasmic resting level. This low threshold uniquely qualifies CPK5 to decode subtle changes in calcium, a prerequisite to signal relay and onset and maintenance of priming at later time points in distal tissue. Our data explain why CPK5 functions as a hub in basal and systemic plant immunity.

Serum immunoglobulin free light chain levels in systemic autoimmune rheumatic diseases.

Several reports have highlighted the abnormal increments of serum immunoglobulin free light chains (FLCs) in the course of systemic autoimmune rheumatic diseases (SARD), but a comparative analysis among different conditions is still lacking. A strong association between elevated FLC and hepatitis C virus (HCV)-related mixed cryoglobulinaemia (HCVMC) has been well established. Here, we aimed to analyse serum FLC levels in patients with four different SARD in comparison with HCVMC. Using a turbidimetric assay, free κ and λ chains were quantified in sera from 198 SARD patients (37 rheumatoid arthritis, RA; 47 systemic lupus erythematosus, SLE; 52 anti-phospholipid syndrome, APS; 62 primary Sjogren's syndrome, pSS), 62 HCVMC and 50 healthy blood donors (HD). All patient groups showed increased κ levels when compared to HD: 33·5 ± 2·6 mg/l in HCVMC, 26·7 ± 2·3 mg/l in RA, 29·7 ± 1·9 mg/l in SLE, 23·8 ± 1·1 mg/l in APS, 24·2 ± 1·1 mg/l in pSS; 10·1 ± 0·6 mg/l in HD. Free λ levels displayed a significant increase only for HCVMC (20·4 ± 1·4 mg/l) and SLE (18·4 ± 1·0 mg/l) compared to HD (13·6 ± 0·9 mg/l). The increase of κ compared to λ takes into account a κ /λ ratio of 1·6 for all groups. Our results substantially analyse and strengthen the association between FLC and SARD focusing the questions regarding their role in the pathogenesis and diagnosis of human diseases. Unfortunately, the biochemical differences distinguishing normal from pathological FLC have not been identified. Production of different isotypes is probably connected to still-unknown pathways.

Redundant CAMTA Transcription Factors Negatively Regulate the Biosynthesis of Salicylic Acid and N-Hydroxypipecolic Acid by Modulating the Expression of SARD1 and CBP60g.

Two signal molecules, salicylic acid (SA) and N-hydroxypipecolic acid (NHP), play critical roles in plant immunity. The biosynthetic genes of both compounds are positively regulated by master immune-regulating transcription factors SARD1 and CBP60g. However, the relationship between the SA and NHP pathways is unclear. CALMODULIN-BINDING TRANSCRIPTION FACTOR 1 (CAMTA1), CAMTA2, and CAMTA3 are known redundant negative regulators of plant immunity, but the underlying mechanism also remains largely unknown. In this study, through chromatin immunoprecipitation and electrophoretic mobility shift assays, we uncovered that CBP60g is a direct target of CAMTA3, which also negatively regulates the expression of SARD1, presumably via an indirect effect. The autoimmunity of camta3-1 is suppressed by sard1 cbp60g double mutant as well as ald1 and fmo1, two single mutants defective in NHP biosynthesis. Interestingly, a suppressor screen conducted in the camta1/2/3 triple mutant background yielded various mutants blocking biosynthesis or signaling of either SA or NHP, leading to nearly complete suppression of the extreme autoimmunity of camta1/2/3, suggesting that the SA and NHP pathways can mutually amplify each other. Together, these results reveal that CAMTAs repress the biosynthesis of SA and NHP by modulating the expression of SARD1 and CBP60g, and that the SA and NHP pathways are coordinated to optimize plant immune response.

Pattern of antinuclear antibody and antiextractable nuclear antigen antibody test requisitions in Riyadh.

BACKGROUND: International guidelines for screening of systemic autoimmune rheumatic diseases (SARD) recommend antinuclear antibody (ANA) test as the first level test and antiextractable antigen (anti-ENA) along with anti-double-stranded DNA (anti-dsDNA) as second line tests following a reactive ANA test. This study was performed to assess adherence to international guidelines for investigation of SARD and to compare the requesting pattern of ANA and second level tests between rheumatology and nonrheumatology physicians in Riyadh. METHODOLOGY: This retrospective cross-sectional study comprising of 300 first time requests for investigation of SARD was performed in the immunology unit at King Khalid University Hospital (KKUH). Data were collected between April and May 2018. Information regarding the requesting physicians' specialty and the first time requested tests (ANA, anti-dsDNA, and anti-ENA) were extracted from the electronic medical records. Reasons for requisition of tests were also recorded. RESULTS: Of the total requests, 159 (53%) requests included ANA as a single first level test, whereas the rest of the requests (n = 141, 47%) included ANA test in conjunction with second level tests for the investigation of SARD. From the department of rheumatology, 14 (29.8%) initial requests were for ANA test as the only first line investigation that was significantly lower than 145 (57.3%) similar requests from the rest of the departments (P < 0.001). CONCLUSION: ANA and second level tests requests by physicians particularly among rheumatologists lacked compliance to international guidelines. The current study strongly suggests the need for strict compliance to international guidelines for screening of systemic autoimmune disorders among physicians.