Large-scale genomic analyses with machine learning uncover predictive patterns associated with fungal phytopathogenic lifestyles and traits.

Invasive plant pathogenic fungi have a global impact, with devastating economic and environmental effects on crops and forests. Biosurveillance, a critical component of threat mitigation, requires risk prediction based on fungal lifestyles and traits. Recent studies have revealed distinct genomic patterns associated with specific groups of plant pathogenic fungi. We sought to establish whether these phytopathogenic genomic patterns hold across diverse taxonomic and ecological groups from the Ascomycota and Basidiomycota, and furthermore, if those patterns can be used in a predictive capacity for biosurveillance. Using a supervised machine learning approach that integrates phylogenetic and genomic data, we analyzed 387 fungal genomes to test a proof-of-concept for the use of genomic signatures in predicting fungal phytopathogenic lifestyles and traits during biosurveillance activities. Our machine learning feature sets were derived from genome annotation data of carbohydrate-active enzymes (CAZymes), peptidases, secondary metabolite clusters (SMCs), transporters, and transcription factors. We found that machine learning could successfully predict fungal lifestyles and traits across taxonomic groups, with the best predictive performance coming from feature sets comprising CAZyme, peptidase, and SMC data. While phylogeny was an important component in most predictions, the inclusion of genomic data improved prediction performance for every lifestyle and trait tested. Plant pathogenicity was one of the best-predicted traits, showing the promise of predictive genomics for biosurveillance applications. Furthermore, our machine learning approach revealed expansions in the number of genes from specific CAZyme and peptidase families in the genomes of plant pathogens compared to non-phytopathogenic genomes (saprotrophs, endo- and ectomycorrhizal fungi). Such genomic feature profiles give insight into the evolution of fungal phytopathogenicity and could be useful to predict the risks of unknown fungi in future biosurveillance activities.

Inhibition of casein kinase 2 sensitizes mantle cell lymphoma to venetoclax through MCL-1 downregulation.

BCL-2 family proteins are frequently aberrantly expressed in mantle cell lymphoma (MCL). Recently, the BCL-2-specific inhibitor venetoclax has been approved by the US Food and Drug Administration for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). In MCL, venetoclax has shown promising efficacy in early clinical trials; however, a significant subset of patients is resistant. By conducting a kinome-centered CRISPR-Cas9 knockout sensitizer screen, we identified casein kinase 2 (CK2) as a major regulator of venetoclax resistance in MCL. Interestingly, CK2 is over-expressed in MCL and high CK2 expression is associated with poor patient survival. Targeting of CK2, either by inducible short hairpin RNA (shRNA)-mediated knockdown of CK2 or by the CK2-inhibitor silmitasertib, did not affect cell viability by itself, but strongly synergized with venetoclax in both MCL cell lines and primary samples, also if combined with ibrutinib. Furthermore, targeting of CK2 reduced MCL-1 levels, which involved impaired MCL-1 translation by inhibition of eIF4F complex assembly, without affecting BCL-2 and BCL-XL expression. Combined, this results in enhanced BCL-2 dependence and, consequently, venetoclax sensitization. In cocultures, targeting of CK2 overcame stroma-mediated venetoclax resistance of MCL cells. Taken together, our findings indicate that targeting of CK2 sensitizes MCL cells to venetoclax through downregulation of MCL-1. These novel insights provide a strong rationale for combining venetoclax with CK2 inhibition as therapeutic strategy for MCL patients.

An Unbiased CRISPR-Cas9 Screening Method for the Identification of Positive and Negative Regulatory Proteins of Cell Adhesion.

Mature B-cell lymphomas are highly dependent upon the protective lymphoid organ microenvironment for their growth and survival. Targeting integrin-mediated homing and retention of the malignant B cells in the lymphoid organs, using the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, is a highly efficacious FDA-approved therapy for chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and Waldenström macroglobulinemia (WM). Unfortunately, a significant subset of patients is intrinsically resistant to ibrutinib or will develop resistance upon prolonged treatment. Here, we describe an unbiased functional genomic CRISPR-Cas9 screening method to identify novel proteins involved in B-cell receptor-controlled integrin-mediated adhesion, which provides novel therapeutic targets to overcome ibrutinib resistance. This screening method is highly flexible and can be easily adapted to identify cell adhesion-regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types. Graphical abstract.

A loss-of-adhesion CRISPR-Cas9 screening platform to identify cell adhesion-regulatory proteins and signaling pathways.

The clinical introduction of the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which targets B-cell antigen-receptor (BCR)-controlled integrin-mediated retention of malignant B cells in their growth-supportive lymphoid organ microenvironment, provided a major breakthrough in lymphoma and leukemia treatment. Unfortunately, a significant subset of patients is intrinsically resistant or acquires resistance against ibrutinib. Here, to discover novel therapeutic targets, we present an unbiased loss-of-adhesion CRISPR-Cas9 knockout screening method to identify proteins involved in BCR-controlled integrin-mediated adhesion. Illustrating the validity of our approach, several kinases with an established role in BCR-controlled adhesion, including BTK and PI3K, both targets for clinically applied inhibitors, are among the top hits of our screen. We anticipate that pharmacological inhibitors of the identified targets, e.g. PAK2 and PTK2B/PYK2, may have great clinical potential as therapy for lymphoma and leukemia patients. Furthermore, this screening platform is highly flexible and can be easily adapted to identify cell adhesion-regulatory proteins and signaling pathways for other stimuli, adhesion molecules, and cell types.

Flexible ureteroscopy in extreme elderly patients (80 years of age and older) is feasible and safe.

PURPOSE: To compare the safety and efficacy of RIRS in patients ≥ 80 years to a younger population. METHODS: We retrospectively compared the data from patients ≥ 80 years of age undergoing RIRS with the data of a group of patients from 18 and < 80 years. Perioperative outcomes, complications and emergency department visits were compared between two groups. RESULTS: A total of 173 patients were included in the study. Mean age was 44 (27-79) and 81 years-old (80-94), for younger and elderly group, respectively. Elderly patients had higher ASA scores (≥ 3) (28.6% vs 75.8%; p = 0.0001) and Charlson comorbidity index (1.99 vs 7.86; p = 0.0001), more diabetes (p = 0.006) and respiratory comorbidities (p = 0.002). No statistical difference was found between two groups in stone size (p = 0.614) and number (p = 0.152). Operative time (74.48 vs 102.96 min; p = 0.0001) and duration of hospitalisation (1.7 vs 2.9 days; p = 0.001) were longer for the elderly. Intraoperative complication rate did not show differences between the two groups (p = 0.166). Postoperative complications rates were similar between the cohorts (7.7% vs 9.5%; p = 0.682). The success rates were 67.5% in the younger group and 71.4% in the elderly group (p = 0.584). No difference was seen in stone recurrence (p = 0.73). A higher rate of visits to the emergency department was found in younger cohort (23.6% vs 11.6%; p = 0.046), mostly duo to stent-related symptoms. CONCLUSIONS: Despite the higher rate of comorbidity in the elderly group, RIRS was a safe procedure with similar complication rate and outcomes at an expense of higher operative time and hospital stay.

Fragmentation targeted at preferred discontinuities: A new concept in endolithotripsy with Holmium laser:YAG. / Fragmentación dirigida a discontinuidades preferentes: un nuevo concepto en endolitotricia con láser Holmium:YAG.

INTRODUCTION: There are currently 3holmium laser, YAG (Ho:YAG) endolithotripsy procedures that are considered basic (fragmentation, pulverisation, "pop-corn" technique). We present the technique of fragmentation targeted at preferred discontinuities (FTPD), a new concept of endolithotripsy by Ho:YAG laser. MATERIAL AND METHODS: The FTPD technique is based on the selective application of energy (targeting a specific preselected point) to an area that is visually prone to the formation of a fracture line or preferred discontinuity (conditioned by the anisotropy of the urolithiasis). The ideal energy regimen (setting) is a high range of working energy (2-3J) with a very low frequency range (5-8Hz) and short pulse width. Between January 2015 to February 2017, the FTPD technique was used in 37 procedures (7 NLP, 16 RIRS, 12 URS, 2 cystolithotomies), with a Ho:YAG laser (Lumenis Pulse 120H®, Tel-Aviv, Israel). Maximum power used: 24W (3J/8Hz) with fibres of 365µ and 273µ (URS, RIRS), and 32W (4J/8Hz) with fibres of 550µ (NLP, cystolithotomy). RESULTS: Strategic improvement was achieved in all cases using the TFPD technique to continue the endolithotripsy or remove fragments. No complications were recorded after the use of this method. CONCLUSIONS: FTPD can be considered a complementary option in combination with the basic methods of fragmentation and pulverisation. In our experience, it constitutes significant progress in optimising the performance of Ho:YAG laser endolithotripsy.

Corinebacterium urealyticum: increased incidence of infection and encrusted uropathy.

INTRODUCTION: Corynebacterium urealyticum (CU) affects patients who are immunosuppressed, chronically ill or have undergone numerous operations. Obstructive uropathy (OU) is a complication of infection. STUDY OBJECTIVE: To demonstrate the growing increase in cases of infection by CU and OU in the past 5 years. MATERIAL AND METHODS: A descriptive study was conducted of urological patients with CU-positive urine cultures (January 2009-December 2014). We calculated the annual distribution and clinical characteristics of infection by CU and OU. Minimum follow-up: 6 months. We obtained the statistical means and ranges of clinical parameters pre/post-therapy. RESULTS: The total number of patients with CU was 115 (men, 87; women, 28). The mean age was 67.9 years (range, 6-95 years), and the annual distribution of cases for 2009, 2010, 2011, 2012, 2013 and 2014 was 9 (7.8%), 13 (11.3%), 9 (7.8%), 20 (17.4%), 31 (27%) and 33 (28.7%), respectively. The increase in cases for 2009-2014 was 300%. Multiple urological surgeries were performed in 89 cases (77.3%), with surgical complications in 77 cases (66.9%). Eighteen (15.6%) patients had OU (men, 13; women, 5), 12 had pyelitis (66.7%), 3 had cystopathy (16.6%), 2 had prostatic capsule disease (11.2%) and 1 had mesh calcification (5.5%). The analysis of the 18 cases with OU showed pre/postantibiotic therapy urine pHs of 8 (r, 6-9) vs. 6 (r, 5-7). All postantibiotic cultures were negative. Acidifying solution was applied in 5 cases, and surgery was performed in 13 cases (72.2%). The results from before/after the multimodal therapy showed renal impairment in 12 (66.6%) vs. 9 cases (50%) and glomerular filtration rates (GFR) of 45.8 (r, 6->90) vs. 52.7 (r, 13->90). The improvement in GFR was 6.94 points (T Wilcoxon; P=.102). The radiology results (incrustations) showed improvement in 13 patients (72.2%) and no change in 5 (27.8%). There was no specific mortality for CU. CONCLUSIONS: The prevalence of infection by CU and OU is increasing. Antibiotic treatment is highly effective. Acidifying solutions are an acceptable option for reducing calcifications.

Implications of vitamin D deficiency in lithiasic patient and in general population.

CONTEXT AND OBJECTIVE: Vitamin D deficiency causes problems in mineral metabolism but also overall health. In first place a review of the topic was carried out. Then, in order to contextualize it in lithiasic patient, a study on Vitamin D deficiency and its possible relationship with impaired PTH levels is performed. EVIDENCES ACQUISITION: A review of topics such as metabolism, epidemiology and the relationship of vitamin D deficiency with several pathologies was performed. Besides a multivariate analysis and a correlation study between vitamin D and PTH levels was conducted in 100 lithiasic patients. EVIDENCES SYNTHESIS: We present a review of Vitamin D metabolism, receptors and functions, as well as about its valuation methodology and the treatment of its deficiency. Lithiasic patients show a higher vitamin D deficiency than general population. Vitamin D deficiency has been significantly associated with increased PTH levels. In addition, there is enough literature showing a relationship between vitamin D deficiency not only with bone disease, but also with multiple diseases. CONCLUSION: vitamin D levels should be measured in all lithiasic patients, and those with vitamin D deficiency should be treated.

Extraction of a bladder stone in a child as described by the renaissance physician Cristóbal Méndez.

INTRODUCTION: in his Libro del exercicio y de sus provechos (Book of exercise and profits), the Spanish Renaissance physician Christopher Mendez (1500-1553) describes extracting a bulk stone from the bladder of a child younger than 5 years in the land of colonial Mexico. This is the first description of a surgical procedure in America. MATERIAL AND METHODS: Biographical data were collected on Christopher Mendez. The electronic facsimile of the Book of exercise and profits was read. The historical aspects of perineal lithotomy and etiology of bladder stones were analyzed. RESULTS: In chapter seven of the third treatise (page 120), Mendez speaks about the removal of a bladder stone in a boy named «Villaseñor¼. It uses the word «open¼ to describe the procedure, corresponding to a lithotomy more than a necropsy. It attributes the etiology of excess movements after ingestion and suggests a possible hereditary etiology. DISCUSSION: Perineal lithotomy was a common practice in ancient times for children due to the high incidence of bladder stones. The technique was very invasive and was improved over the centuries. CONCLUSIONS: The surgery described by Mendez for the child called Villaseñor most likely corresponds to a perineal lithotomy. A congenital cause could play a role in its etiology.

Egress of CD19(+)CD5(+) cells into peripheral blood following treatment with the Bruton tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma patients.

Ibrutinib (PCI-32765) is a highly potent oral Bruton tyrosine kinase (BTK) inhibitor in clinical development for treating B-cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often show marked, transient increases of circulating CLL cells following ibrutinib treatments, as seen with other inhibitors of the B-cell receptor (BCR) pathway. In a phase 1 study of ibrutinib, we noted similar effects in patients with mantle cell lymphoma (MCL). Here, we characterize the patterns and phenotypes of cells mobilized among patients with MCL and further investigate the mechanism of this effect. Peripheral blood CD19(+)CD5(+) cells from MCL patients were found to have significant reduction in the expression of CXCR4, CD38, and Ki67 after 7 days of treatment. In addition, plasma chemokines such as CCL22, CCL4, and CXCL13 were reduced 40% to 60% after treatment. Mechanistically, ibrutinib inhibited BCR- and chemokine-mediated adhesion and chemotaxis of MCL cell lines and dose-dependently inhibited BCR, stromal cell, and CXCL12/CXCL13 stimulations of pBTK, pPLCγ2, pERK, or pAKT. Importantly, ibrutinib inhibited migration of MCL cells beneath stromal cells in coculture. We propose that BTK is essential for the homing of MCL cells into lymphoid tissues, and its inhibition results in an egress of malignant cells into peripheral blood. This trial was registered at www.clinicaltrials.gov as #NCT00114738.

Two methods to determine the prevalence of overweight and obesity in 8-9 year-old-children in Seville, Spain.

OBJECTIVE: The prevalence of overweight and obesity in Spanish children has increased considerably in the last decades. Obesity has been recognized as a public health problem in developed countries because its association with increased risk for several pathologies. Obesity in children and, specifically, in adolescents, is a major concern. Actually, in Spain, the prevalence rates of childhood overweight and obesity are among the highest in European countries. The objective of this study was to measure the prevalence of overweight and obesity in children from Seville. METHOD: A cross-sectional study was performed on 990 children, aged 8-9 years old (51.5% 8-year old and 48.5% 9-year old). Overweight and obesity were defined according to two methods: Spanish standards, applying the criterion of BMI-specific percentiles for age and sex, and the international standards established by Cole et al. (IOTF). RESULTS: A high prevalence of overweight (11%) and obesity (22%) was observed. The prevalence of obesity as well as the prevalence of overall excess weight was significantly higher in 9-year old children (28% and 41%, respectively) than in 8-year old children (17% and 27%, respectively). CONCLUSION: The prevalence of overweight and obesity among Sevillian school children is high, and increases in the studied range of age.

The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia.

Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α(4)ß(1)-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.

The cocaine- and amphetamine-regulated transcript mediates ligand-independent activation of ERα, and is an independent prognostic factor in node-negative breast cancer.

Personalized medicine requires the identification of unambiguous prognostic and predictive biomarkers to inform therapeutic decisions. Within this context, the management of lymph node-negative breast cancer is the subject of much debate with particular emphasis on the requirement for adjuvant chemotherapy. The identification of prognostic and predictive biomarkers in this group of patients is crucial. Here, we demonstrate by tissue microarray and automated image analysis that the cocaine- and amphetamine-regulated transcript (CART) is expressed in primary and metastatic breast cancer and is an independent poor prognostic factor in estrogen receptor (ER)-positive, lymph node-negative tumors in two separate breast cancer cohorts (n=690; P=0.002, 0.013). We also show that CART increases the transcriptional activity of ERα in a ligand-independent manner via the mitogen-activated protein kinase pathway and that CART stimulates an autocrine/paracrine loop within tumor cells to amplify the CART signal. Additionally, we demonstrate that CART expression in ER-positive breast cancer cell lines protects against tamoxifen-mediated cell death and that high CART expression predicts disease outcome in tamoxifen-treated patients in vivo in three independent breast cancer cohorts. We believe that CART profiling will help facilitate stratification of lymph node-negative breast cancer patients into high- and low-risk categories and allow for the personalization of therapy.