The coronatine toxin of Pseudomonas syringae is a multifunctional suppressor of Arabidopsis defense.

The phytotoxin coronatine (COR) promotes various aspects of Pseudomonas syringae virulence, including invasion through stomata, growth in the apoplast, and induction of disease symptoms. COR is a structural mimic of active jasmonic acid (JA) conjugates. Known activities of COR are mediated through its binding to the F-box-containing JA coreceptor CORONATINE INSENSITIVE1. By analyzing the interaction of P. syringae mutants with Arabidopsis thaliana mutants, we demonstrate that, in the apoplastic space of Arabidopsis, COR is a multifunctional defense suppressor. COR and the critical P. syringae type III effector HopM1 target distinct signaling steps to suppress callose deposition. In addition to its well-documented ability to suppress salicylic acid (SA) signaling, COR suppresses an SA-independent pathway contributing to callose deposition by reducing accumulation of an indole glucosinolate upstream of the activity of the PEN2 myrosinase. COR also suppresses callose deposition and promotes bacterial growth in coi1 mutant plants, indicating that COR may have multiple targets inside plant cells.

Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV-CATCHER): A customizable purification assay designed for small-RNA biomarker identification and evaluation of circulating small-EVs.

Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory-based purification techniques rely on the physical properties of small-EVs rather than their inherited cellular fingerprints. We established a highly-selective purification assay, termed EV-CATCHER, initially designed for high-throughput analysis of low-abundance small-RNA cargos by next-generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small-RNAs from mouse small-EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small-EVs. As proof-of-principle for sensitive detection of circulating miRNAs, we compared small-RNA sequencing data from a subset of small-EVs serum-purified with EV-CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid-19 patients. We identified and validated, only in small-EVs, hsa-miR-146a and hsa-miR-126-3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid-19 patients with high anti-spike IgG titers, we confirmed the neutralizing properties, against SARS-CoV-2 in vitro, of a subset of small-EVs serum-purified by EV-CATCHER, as initially observed with ultracentrifuged small-EVs. Altogether our data highlight the sensitivity and versatility of EV-CATCHER.

Prolactin Induced Protein (PIP) is a potential biomarker for early stage and malignant breast cancer.

OBJECTIVES: Breast cancer (BC) is the second leading cause of cancer-related mortality in women. Bioinformatic analysis and expression screening showed that Prolactin Induced Protein (PIP) was differentially expressed in BC. The objective of this investigation was to characterize the expression pattern of PIP, an aspartyl proteinase, in malignant and non-malignant breast tissues. MATERIALS AND METHODS: Real time quantitative PCR was employed to analyze PIP and androgen receptor (AR) mRNA levels in BC cell lines and 190 normal tissues and tumor samples. The tumor specimens were categorized based on TNM classification, anatomic stage, histologic grade and molecular subtype and expression pattern evaluated. To detect protein levels, immunohistochemistry followed by semi quantitative scoring was employed in the examination of 517 normal, benign, and invasive BC tissues. RESULTS: We observed substantial downregulation of PIP transcription in cancer samples compared to normal breast tissue. mRNA levels were significantly downregulated (93 fold, P < 0.005) in advanced grades compared to lower grades. Transcript levels were also significantly lower (22 fold, P < 0.05) in triple negative tumors compared to hormone receptor positive tumors. Significant downregulation was observed in early stage samples of triple negative and hormone receptor positive tumors. Though PIP protein showed a wide range of expression levels in BC, early stage samples showed significant downregulation. CONCLUSIONS: PIP mRNA is significantly downregulated in early stage BC compared to normal breast tissue. Consequently, low PIP mRNA expression in BC tissues could potentially be used as a tissue based biomarker to assist pathologists in confirmation of early stage BC diagnosis.

Protein calorie malnutrition, nutritional intervention and personalized cancer care.

Cancer patients often experience weight loss caused by protein calorie malnutrition (PCM) during the course of the disease or treatment. PCM is expressed as severe if the patient has two or more of the following characteristics: obvious significant muscle wasting, loss of subcutaneous fat; nutritional intake of <50% of recommended intake for 2 weeks or more; bedridden or otherwise significantly reduced functional capacity; weight loss of >2% in 1 week, 5% in 1 month, or 7.5% in 3 months. Cancer anorexia-cachexia syndrome (CACS) is a multifactorial condition of advanced PCM associated with underlying illness (in this case cancer) and is characterized by loss of muscle with or without loss of fat mass. Cachexia is defined as weight loss of more than 5% of body weight in 12 months or less in the presence of chronic disease. Hence with a chronic illness on board even a small amount of weight loss can open the door to cachexia. These nutritional challenges can lead to severe morbidity and mortality in cancer patients. In the clinic, the application of personalized medicine and the ability to withstand the toxic effects of anti-cancer therapies can be optimized when the patient is in nutritional homeostasis and is free of anorexia and cachexia. Routine assessment of nutritional status and appropriate intervention are essential components of the effort to alleviate effects of malnutrition on quality of life and survival of patients.

The serine protease prostasin (PRSS8) is a potential biomarker for early detection of ovarian cancer.

BACKGROUND: Ovarian cancer (OVC) is the deadliest of all gynecologic cancers, primarily as a consequence of asymptomatic progression. The complex nature of OVC creates challenges for early detection, and there is a lack of specific and sensitive biomarkers suitable for screening and detecting early stage OVC. METHODS: Potential OVC biomarkers were identified by bioinformatic analysis. Candidates were further screened for differential expression in a library of OVC cell lines. OVC-specific overexpression of a candidate gene, PRSS8, which encodes prostasin, was confirmed against 18 major human cancer types from 390 cancer samples by qRT-PCR. PRSS8 expression profiles stratified by OVC tumor stage-, grade- and subtype were generated using cDNA samples from 159 OVC samples. Cell-specific expression and localization of prostasin was determined by immunohistological tissue array analysis of more than 500 normal, benign, and cancerous ovarian tissues. The presence of prostasin in normal, benign, and OVC serum samples was also determined. RESULTS: Gene expression analysis indicated that PRSS8 was expressed in OVC at levels more than 100 fold greater than found in normal or benign ovarian lesions. This overexpression signature was found in early stages of OVC and was maintained in higher stages and grades of OVC. The PRSS8 overexpression signature was specific for OVC and urinary bladder cancer among 18 human cancer types. The majority of ovarian cell lines overexpressed PRSS8. In situ hybridization and histopathology studies of OVC tissues indicated that overexpression of prostasin was largely localized to tumor epithelium and was absent in neighboring stroma. Significantly higher levels of prostasin were found in early stage OVC serum samples compared to benign ovarian and normal donor samples. CONCLUSIONS: The abundant amounts of secreted prostasin found in sera of early stage OVC can potentially be used as a minimally invasive screening biomarker for early stage OVC. Overexpression of PRSS8 mRNA and high levels of prostasin in multiple subtypes of early stage ovarian tumors may provide clinical biomarkers for early detection of OVC, which can potentially be used with CA125 and HE4.

The Pseudomonas syringae pv. tomato type III effector HopM1 suppresses Arabidopsis defenses independent of suppressing salicylic acid signaling and of targeting AtMIN7.

Pseudomonas syringae pv tomato strain DC3000 (Pto) delivers several effector proteins promoting virulence, including HopM1, into plant cells via type III secretion. HopM1 contributes to full virulence of Pto by inducing degradation of Arabidopsis proteins, including AtMIN7, an ADP ribosylation factor-guanine nucleotide exchange factor. Pseudomonas syringae pv phaseolicola strain NPS3121 (Pph) lacks a functional HopM1 and elicits robust defenses in Arabidopsis thaliana, including accumulation of pathogenesis related 1 (PR-1) protein and deposition of callose-containing cell wall fortifications. We have examined the effects of heterologously expressed HopM1Pto on Pph-induced defenses. HopM1 suppresses Pph-induced PR-1 expression, a widely used marker for salicylic acid (SA) signaling and systemic acquired resistance. Surprisingly, HopM1 reduces PR-1 expression without affecting SA accumulation and also suppresses the low levels of PR-1 expression apparent in SA-signaling deficient plants. Further, HopM1 enhances the growth of Pto in SA-signaling deficient plants. AtMIN7 contributes to Pph-induced PR-1 expression. However, HopM1 fails to degrade AtMIN7 during Pph infection and suppresses Pph-induced PR-1 expression and callose deposition in wild-type and atmin7 plants. We also show that the HopM1-mediated suppression of PR-1 expression is not observed in plants lacking the TGA transcription factor, TGA3. Our data indicate that HopM1 promotes bacterial virulence independent of suppressing SA-signaling and links TGA3, AtMIN7, and other HopM1 targets to pathways distinct from the canonical SA-signaling pathway contributing to PR-1 expression and callose deposition. Thus, efforts to understand this key effector must consider multiple targets and unexpected outputs of its action.

Identification of immunity-related genes in Arabidopsis and cassava using genomic data.

Recent advances in genomic and post-genomic technologies have provided the opportunity to generate a previously unimaginable amount of information. However, biological knowledge is still needed to improve the understanding of complex mechanisms such as plant immune responses. Better knowledge of this process could improve crop production and management. Here, we used holistic analysis to combine our own microarray and RNA-seq data with public genomic data from Arabidopsis and cassava in order to acquire biological knowledge about the relationships between proteins encoded by immunity-related genes (IRGs) and other genes. This approach was based on a kernel method adapted for the construction of gene networks. The obtained results allowed us to propose a list of new IRGs. A putative function in the immunity pathway was predicted for the new IRGs. The analysis of networks revealed that our predicted IRGs are either well documented or recognized in previous co-expression studies. In addition to robust relationships between IRGs, there is evidence suggesting that other cellular processes may be also strongly related to immunity.