Functional redundancy and compensation: Deletion of multiple murine Crisp genes reveals their essential role for male fertility.

Mammalian Cysteine-RIch Secretory Protein (CRISP) family includes four members present in sperm and reported to regulate Ca2+ channels and fertilization. Based on our previous observations using single knockouts models and suggesting the existence of functional compensation among CRISP proteins, we investigated their relevance for male fertility by generating multiple Crisp gene mutants by CRISPR/Cas9 technology. Whereas targeting of Crisp1 and Crisp3 yielded subfertile males with early embryo developmental defects, the same deletion in zygotes from fertile Crisp2-/- .Crisp4-/- mice led to the generation of both triple and quadruple knockout mice exhibiting a complete or severe disruption of male fertility due to a combination of sperm transport, fertilization, and embryo developmental defects linked to intracellular Ca2+ dysregulation. These observations reveal that CRISP proteins are essential for male fertility and organize in functional modules that contribute distinctly to fertility success, bringing insights into the mechanisms underlying functional redundancy/compensation in protein families and emphasizing the importance of generating multiple and not just single knockout which might be masking the true functional relevance of family genes.

Impacto de la anemia sobre la supervivencia de los pacientes intervenidos de cistectomía radical por tumor vesical / Impact of anemia on the survival of patients undergoing radical cystectomy for bladder cancer

OBJETIVO: Evaluar la prevalencia de anemia preoperatoria y su impacto sobre los resultados oncológicos de pacientes intervenidos de cistectomía radical (CR) por tumor vesical. MATERIAL Y MÉTODOS: Estudio retrospectivo de 176 CR realizadas entre mayo de 2008 y julio de 2018 en un mismo centro. La anemia fue definida según los criterios de la OMS (hemoglobina < 130 mg/dl en hombres y < 120 mg/dl en mujeres). Mediante el método de Kaplan-Meier analizamos la supervivencia global, la supervivencia específica de cáncer y la supervivencia libre de recurrencia. Utilizamos la regresión logística multivariante para identificar los factores pronósticos de mortalidad global. RESULTADOS: Del total, 89 (50,6%) pacientes eran anémicos preoperatoriamente y 44 de ellos (49,4%) recibieron quimioterapia neoadyuvante. Los pacientes anémicos tuvieron un ASA mayor (ASA > 2: 54,6 vs. 27,5%, p = 0,003), más ectasia prequirúrgica (41,6 vs. 19,5%; p = 0,002), peor estadio patológico (pT > 2: 49,4 vs. 33,3%; p = 0,03), realizaron más quimioterapia neoadyuvante (49,4 vs. 19,5%; p < 0,001) y requirieron más transfusiones sanguíneas (25,8 vs. 11,5%; p = 0,015). La mediana de seguimiento fue de 27,2 meses (RIC 11,12-72,28). La supervivencia global (105 vs. 34 meses; p = 0,001), la supervivencia específica de cáncer (89 vs. 61 meses; p = 0,004) y la supervivencia libre de recurrencia (85 vs. 57 meses; p = 0,002) fueron peores en las CR anémicas. En el estudio multivariante, la anemia, un estadio pT > 2 y tener afectación ganglionar fueron identificados como factores predictores independientes de mortalidad. CONCLUSIÓN: La anemia previa a CR es común y asocia un peor pronóstico oncológico. Siendo esta una variable modificable, la implementación de programas de Patient Blood Management durante la prehabilitación puede tener un papel importante para mejorar la supervivencia de estos pacientes

OBJECTIVE: To evaluate the prevalence of preoperative anemia and its effect on oncological outcomes in patients undergoing radical cystectomy (RC) due to bladder cancer. MATERIAL AND METHODS: Retrospective single-center study with 176 RCs between May 2008 and July 2018. Anemia was defined according to the WHO classification (male < 130 mg/dL, female < 120 mg/dL). Kaplan-Meier test was used to estimate recurrence-free, cancer-specific and overall survival rates. Multivariate logistic regression was used to identify factors associated with overall mortality rates. RESULTS: Overall, 89 (50.6%) patients had preoperative anemia, and 44 of them (49.4%) received neoadjuvant chemotherapy. Anemic patients resulted in higher rates of ASA (ASA > 2: 54.6 vs. 27.5%; P = .003), ectasia rate previous to RC (41.6 vs. 19.5%; P = .002), treatment with neoadjuvant chemotherapy (49.4 vs. 19.5%; P < .001), blood transfusion rate (25.8 vs. 11.5%; P = .015) and pathological stage (pT > 2: 49.4 vs. 33.3%; P = .03) compared to non-anemic patients. Median follow-up was 27.2 months (IQR 11.12-72.28). Median overall survival (105 vs. 34 months, log-rank; P = .001), cancer-specific survival (89 vs. 61 months; P = .004) and recurrence-free survival (85 vs. 57 months; P = .002) were significantly lower in anemic patients compared to the non-anemic group. In multivariable Cox analysis, preoperative anemia, pT > 2 and N ≥ 1 were independently associated with overall mortality. CONCLUSION: Preoperative anemia was common in patients undergoing RC for bladder cancer, and it is related with a worse cancer prognosis. Anemia is a preoperative modifiable factor; we believe that the implementation of Patient Blood Management programs during prehabilitation may have a relevant role in improving the oncological outcomes in these patients

Impact of anemia on the survival of patients undergoing radical cystectomy for bladder cancer. / Impacto de la anemia sobre la supervivencia de los pacientes intervenidos de cistectomía radical por tumor vesical.

OBJECTIVE: To evaluate the prevalence of preoperative anemia and its effect on oncological outcomes in patients undergoing radical cystectomy (RC) due to bladder cancer. MATERIAL AND METHODS: Retrospective single-center study with 176 RCs between May 2008 and July 2018. Anemia was defined according to the WHO classification (male<130mg/dL, female<120mg/dL). Kaplan-Meier test was used to estimate recurrence-free, cancer-specific and overall survival rates. Multivariate logistic regression was used to identify factors associated with overall mortality rates. RESULTS: Overall, 89 (50.6%) patients had preoperative anemia, and 44 of them (49.4%) received neoadjuvant chemotherapy. Anemic patients resulted in higher rates of ASA (ASA>2: 54.6 vs. 27.5%; P=.003), ectasia rate previous to RC (41.6 vs. 19.5%; P=.002), treatment with neoadjuvant chemotherapy (49.4 vs. 19.5%; P<.001), blood transfusion rate (25.8 vs. 11.5%; P=.015) and pathological stage (pT>2: 49.4 vs. 33.3%; P=.03) compared to non-anemic patients. Median follow-up was 27.2 months (IQR 11.12-72.28). Median overall survival (105 vs. 34 months, log-rank; P=.001), cancer-specific survival (89 vs. 61 months; P=.004) and recurrence-free survival (85 vs. 57 months; P=.002) were significantly lower in anemic patients compared to the non-anemic group. In multivariable Cox analysis, preoperative anemia, pT>2 and N≥1 were independently associated with overall mortality. CONCLUSION: Preoperative anemia was common in patients undergoing RC for bladder cancer, and it is related with a worse cancer prognosis. Anemia is a preoperative modifiable factor; we believe that the implementation of Patient Blood Management programs during prehabilitation may have a relevant role in improving the oncological outcomes in these patients.

Sectional anatomic and tomographic study of the feline abdominal cavity for obtaining a three-dimensional vascular model.

BACKGROUND: Unlike dogs, feline abdominal studies are rare. Note that anatomical estudies in felines are scarce and almost unique using feline cadaver by means of sectional anatomy and computed tomography (CT) or magnetic resonance imaging (MRI). Aims: In this study, a non-pathological vascularization model of feline abdomen was conducted on three adult cats was using anatomical and diagnostic imaging techniques. METHODS: A live pet cat and two cat cadavers were used in this study. Cat cadavers were injected with colored latex to show well-differentiated vascular structures and serial sections of cat abdomen were then provided. Computed tomography was performed by injecting an iodinated contrast medium through the cephalic vein of a live cat immediately before scanning. The CT images showed the arterial and venous vascular formations hyper-attenuated with two tomographic windows. The correlation between anatomical sections and their CTs was studied to identify vascular and and visceral structures. RESULTS: Hyper-attenuated vascular structures with the contrast medium were identified and marked along their path in the series of Dicom images with the Amira program. In this approach, sequentially and semiautomatically, vascular volumetric reconstruction was obtained without visceral formations. With the OsiriX program, volumetric reconstruction was automatic and maintained the fidelity of all visceral and vascular formations. CONCLUSION: We conclude that these improved prototypes could be used in veterinary clinics as normal vascular models and as a basis for obtaining future 3D models of vascular anomalies such as portosystemic shunts.

The effects of prebiotics on microbial dysbiosis, butyrate production and immunity in HIV-infected subjects.

Altered interactions between the gut mucosa and bacteria during HIV infection seem to contribute to chronic immune dysfunction. A deeper understanding of how nutritional interventions could ameliorate gut dysbiosis is needed. Forty-four subjects, including 12 HIV+ viremic untreated (VU) patients, 23 antiretroviral therapy-treated (ART+) virally suppressed patients (15 immunological responders and 8 non-responders) and 9 HIV- controls (HIV-), were blindly randomized to receive either prebiotics (scGOS/lcFOS/glutamine) or placebo (34/10) over 6 weeks in this pilot study. We assessed fecal microbiota composition using deep 16S rRNA gene sequencing and several immunological and genetic markers involved in HIV immunopathogenesis. The short dietary supplementation attenuated HIV-associated dysbiosis, which was most apparent in VU individuals but less so in ART+ subjects, whose gut microbiota was found more resilient. This compositional shift was not observed in the placebo arm. Significantly, declines in indirect markers of bacterial translocation and T-cell activation, improvement of thymic output, and changes in butyrate production were observed. Increases in the abundance of Faecalibacterium and Lachnospira strongly correlated with moderate but significant increases of butyrate production and amelioration of the inflammatory biomarkers soluble CD14 and high-sensitivity C-reactive protein, especially among VU. Hence, the bacterial butyrate synthesis pathway holds promise as a viable target for interventions.

De novo SCN1A mutations in migrating partial seizures of infancy.

OBJECTIVE: To determine the genetic etiology of the severe early infantile onset syndrome of malignant migrating partial seizures of infancy (MPSI). METHODS: Fifteen unrelated children with MPSI were screened for mutations in genes associated with infantile epileptic encephalopathies: SCN1A, CDKL5, STXBP1, PCDH19, and POLG. Microarray studies were performed to identify copy number variations. RESULTS: One patient had a de novo SCN1A missense mutation p.R862G that affects the voltage sensor segment of SCN1A. A second patient had a de novo 11.06 Mb deletion of chromosome 2q24.2q31.1 encompassing more than 40 genes that included SCN1A. Screening of CDKL5 (13/15 patients), STXBP1 (13/15), PCDH19 (9/11 females), and the 3 common European mutations of POLG (11/15) was negative. Pathogenic copy number variations were not detected in 11/12 cases. CONCLUSION: Epilepsies associated with SCN1A mutations range in severity from febrile seizures to severe epileptic encephalopathies including Dravet syndrome and severe infantile multifocal epilepsy. MPSI is now the most severe SCN1A phenotype described to date. While not a common cause of MPSI, SCN1A screening should now be considered in patients with this devastating epileptic encephalopathy.

Morphometry of the canine intestine with reference to the use of double balloon endoscopy.

Double balloon endoscopy (DBE) enables the diagnosis and treatment of small intestinal disease. The dog is a potential animal model for DBE training and also a candidate for the clinical application of this technique. However, the anatomy of the canine small intestine may limit the use of DBE by restricting the push-and-pull manoeuvres required for DBE. To establish what these limitations are, the length and width of different portions of the small intestine as well as the mesenteric dependences were measured in 55 dogs. Several external parameters related to body size plus the age, weight, sex and breed were recorded. Potential restriction of the progress of the enteroscope due to narrow intestinal diameter is expected in the jejunum of mid- and small size dogs.

Autologous mesenchymal stem cell treatment increased T regulatory cells with no effect on disease activity in two systemic lupus erythematosus patients.

Mesenchymal stem cells (MSCs) exert suppressive effects in several disease models including lupus prone mice. However, autologous MSC therapy has not been tested in human systemic lupus erythematosus (SLE). We evaluate the safety and efficacy of bone marrow (BM)-derived MSCs in two SLE patients; the suppressor effect of these cells in-vitro and the change in CD4+CD25+FoxP3+ T regulatory (Treg) cells in response to treatment. Two females (JQ and SA) of 19 and 25 years of age, fulfilling the 1997 American College of Rheumatology (ACR) criteria for SLE were infused with autologous BM-derived MSCs. Disease activity indexes and immunological parameters were assessed at baseline, 1, 2, 7 and 14 weeks. Peripheral blood lymphocyte (PBL) subsets and Treg cells were quantitated by flow cytometry, and MSCs tested for in-vitro suppression of activation and proliferation of normal PBLs. No adverse effects or change in disease activity indexes were noted during 14 weeks of follow-up, although circulating Treg cells increased markedly. Patient MSCs effectively suppressed in-vitro PBL function. However, JQ developed overt renal disease 4 months after infusion. MSC infusion was without adverse effects, but did not modify initial disease activity in spite of increasing CD4+CD25+FoxP3+ cell counts. One patient subsequently had a renal flare. We speculate that the suppressive effects of MSC-induced Treg cells might be dependent on a more inflammatory milieu, becoming clinically evident in patients with higher degrees of disease activity.

Analysis of risk factors associated with nosocomial bacteraemias.

A prospective study of 2676 blood cultures was performed to identify the factors associated with clinically, significant nosocomial bacteraemia that occurred during a one year period in the Malaga University Clinical Hospital. Three hundred and fifty-five episodes of bacteraemia were considered clinically significant. The overall incidence of bacteraemia was 19.5/1000 admissions, of which 46% were hospital-acquired. A multivariate model showed that only six factors were significantly, and independently, responsible for nosocomial bacteraemias: intravascular catheterization (P < 0.0001, OR = 18.37), invasive procedures (P < 0.0001, OR = 10.38), malignancy (P = 0.035, OR = 3.11), indwelling devices (P = 0.005, OR = 3.05), stay in intensive care or surgical departments (P = 0.05, OR = 2.63) and length of hospital stay (P = 0.051, OR = 1.02). These results show that the factors which had most influence on the development of nosocomial bacteraemias were those factors associated with the treatment received by patients during their hospital stay.

Structural biology of chemokine receptors.

Chemokine receptors are G protein-coupled receptors that mediate migration and activation of leukocytes as an important part of a protective immune response to injury and infection. In addition, chemokine receptors are used by HIV-1 to infect CD4 positive cells. The structural bases of chemokine receptor recognition and signal transduction are currently being investigated. High-resolution X-ray diffraction and NMR spectroscopy of chemokines indicate that all these peptides exhibit a common folding pattern, in spite of its low degree of primary-sequence homology. Chemokines' functional motifs have been identified by mutagenesis studies, and a possible mechanism for receptor recognition and activation is proposed, but high-resolution structure data of chemokine receptors is not yet available. Studies with receptor chimeras have identified the putative extracellular domains as the major selectivity determinants. Single-amino acid substitutions in the extracellular domains produce profound changes in receptor specificity, suggesting that motifs in these domains operate as a restrictive barrier to a common activation motif. Similarly HIV-1 usage of chemokine receptors involve interaction of one or more extracellular domains of the receptor with conserved and variable domains on the viral envelope protein gp 120, indicating a highly complex interaction. Elucidating the structural requirements for receptor interaction with chemokines and with HIV-1 will provide important insights into understanding the mechanisms of chemokine recognition and receptor activation. In addition, this information can greatly facilitate the design of effective immunomodulatory and anti-HIV-1 therapeutic agents.

[Transformation of glandular cystitis into bladder transitional carcinoma with adenocarcinoma areas]. / Conversión de cistitis glandular a carcinoma transicional vesical con áreas de adenocarcinoma.

OBJECTIVE: The association of cystitis glandularis with primary bladder tumors is well known. A case of cystitis glandularis progressing to transitional cell carcinoma is described and the literature briefly reviewed. METHODS/RESULTS: We report on a case of diffuse cystitis glandularis in whom progression to transitional cell carcinoma with areas of adenocarcinoma had been discovered. Treatment was by radical cystectomy with urinary diversion. CONCLUSION: Extensive and diffuse cystitis glandularis can be a potentially malignant lesion. Patients with diffuse cystitis glandularis should have a regular cystoscopic follow-up for early detection of progression to a bladder tumor.

[Ureteral stenosis secondary to Churg-Strauss allergic granulomatous vasculitis]. / Estenosis ureteral producida por vasculitis alérgica granulomatosa de churg-strauss.

OBJECTIVE: Ureteral stenosis secondary to vasculitis is a rare disease. The etiology and treatment of this unusual cause of ureteral obstruction are discussed. METHODS/RESULTS: We report a case of ureteral obstruction secondary to Churg-Strauss vasculitis in a 45-year-old man. The patient was treated with prednisone and cyclophosphamide. Subsequently ureteral resection and reanastomosis were performed. CONCLUSIONS: Vasculitis of the ureter should be considered in patients with connective tissue disorders who present with ureteral dilatation. In some cases ureteral stenosis may require surgery in combination with steroid and/or immunosuppressive therapy.

[Intrascrotal aggressive angiomyxoma]. / Angiomixoma agresivo intraescrotal.

OBJECTIVE: To describe a case of aggressive scrotal angiomyxoma in a 55-year old man. METHODS: Ultrasound examination revealed a tumor on the scrotum. Surgical resection and histopathological study of the tumor was performed. The literature is briefly reviewed. RESULTS: Pathological analysis disclosed spindle-shaped neoplastic cells widely separated by a myxoid stroma rich in collagen fibers and prominent irregular-shaped blood vessels. Immunohistochemically, the stromal cells stained consistently for vimentin but not for desmin or S-100 protein. The patient had no symptom or signs of systemic disease and remains symptom-free 15 months later. CONCLUSIONS: Aggressive angiomyxoma is a rare condition that can be successfully treated surgically and has a great tendency to local recurrence.

Cloning, expression and properties of the alpha' subunit of casein kinase 2 from zebrafish (Danio rerio).

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits (alpha and/or alpha') and regulatory subunits (beta 2). The alpha and alpha' subunits are encoded by different genes but are very similar in amino acid sequence, except that alpha' is normally considerably shorter. There have been extensive biochemical studies with recombinant alpha and beta subunits of many species, but only one previous description of the activity of an isolated recombinant alpha' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (alpha, alpha', and beta), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur. J. Biochem. 220, 263-273). In the present work, the isolation and bacterial expression of a cDNA coding for the alpha' subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the alpha' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2 alpha subunit of zebrafish. The recombinant alpha' subunit has apparent K(m) values for ATP (6 microM), GTP (20 microM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant alpha subunit of Xenopus laevis. The alpha' subunit kcat was 7.2 min-1 which is again similar to that of Xenopus laevis alpha subunit (7.5 min-1). The alpha' subunit also behaved similarly to CK2 alpha with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide. However alpha' kinase activity was less sensitive to poly(U) inhibition than alpha, it was more heat stable than alpha, and alpha' was slightly more sensitive to KCl inhibition than alpha. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory beta subunit which shifted the optimal salt concentration of the phosphorylating activity. The alpha' 2 beta 2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the alpha 2 beta 2 enzyme was stimulated by KCl concentrations up to 150 mM and required 180 mM for inhibition. Another important difference between alpha and alpha' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory beta subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2 beta produced maximal stimulation of both alpha' (D. rerio) and alpha (X. laevis), however the activity levels with alpha' were stimulated 20-fold by beta while the addition of beta stimulated alpha (X. laevis) only 7-8-fold.

[Submucous urethral angioma]. / Angioma uretral submucoso.

OBJECTIVES: Urethral hemangioma is a rare benign vascular tumor. The clinical features of this tumor type and the therapeutic options are discussed. METHODS/RESULTS: We report on a 55-year-old male with urethral hemangioma who consulted for urethral hemorrhage. CONCLUSIONS: To our knowledge, approximately 40 cases of this rare tumor type have been reported in the literature. Urethroscopy is the best diagnostic procedure. Treatment depends on tumor site, size and number. Transurethral resection of the tumor, urethrectomy, arterial embolization, radiotherapy or ablation with Nd:YAG laser can be utilized.

[Ureteral stenosis secondary to methacrylate used in total hip arthroplasty]. / Estenosis ureteral secundaria a metacrilato utilizado en una artoplastia total de cadera.

OBJECTIVES: The urological complications in orthopedic surgery, the mechanisms of injury and its treatment are discussed. METHODS/RESULTS: Herein we describe a case of ureterohydronephrosis arising from extrinsic ureteral compression by cement utilized in total hip arthroplasty in a patient with a solitary kidney. CONCLUSIONS: The risk of acetabular perforation and subsequent migration of the cement into the pelvic cavity must be taken into account in patients undergoing hip replacement surgery. Conservative endourological management should first be attempted in the event of ureteral involvement, although resolution is by surgical repair in most cases.