H4K5 Butyrylation Coexist with Acetylation during Human Spermiogenesis and Are Retained in the Mature Sperm Chromatin.

Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.

Histone H4 acetylation is dysregulated in active seminiferous tubules adjacent to testicular tumours.

STUDY QUESTION: Is histone H4 acetylation (H4ac) altered in the seminiferous tubules of patients affected by testicular tumours? SUMMARY ANSWER: A considerable dysregulation of H4ac was detected in the cells of the seminiferous tubules adjacent to testicular tumours of different aetiology and prior to any treatment, while no comparable alterations were observed in patients with disrupted spermatogenesis. WHAT IS KNOWN ALREADY: Altered H4ac levels have been associated with a variety of testicular pathological conditions. However, no information has been available regarding potential alterations in the spermatogenic cells adjacent to the neoplasia in testicular tumour patients. STUDY DESIGN, SIZE, DURATION: A retrospective analysis using testicular sections from 33 men aged between 21 and 74 years old was performed. Three study groups were defined and subjected to double-blind evaluation: a control group with normal spermatogenesis (n = 6), patients with testicular tumours (n = 18) and patients with spermatogenic impairments (n = 8). One additional sample with normal spermatogenesis was used as a technical internal control in all evaluations. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immunohistochemistry against H4ac and, when needed, Placental-like alkaline phosphatase and CD117, was performed on testicular sections. The H4ac H-score, based on the percentage of detection and signal intensity, was used as the scoring method for statistical analyses. Protein expression data from the Human Protein Atlas were used to compare the expression levels of predicted secreted proteins from testicular tumours with those present in the normal tissue. MAIN RESULTS AND THE ROLE OF CHANCE: We revealed, for the first time, a dramatic disruption of the spermatogenic H4ac pattern in unaffected seminiferous tubule cells from different testicular tumour patients prior to any antineoplastic treatment, as compared to controls (P < 0.05). Since no similar alterations were associated with spermatogenic impairments and the in silico analysis revealed proteins potentially secreted by the tumour to the testicular stroma, we propose a potential paracrine effect of the neoplasia as a mechanistic hypothesis for this dysregulation. LIMITATIONS, REASONS FOR CAUTION: Statistical analyses were not performed on the hypospermatogenesis and Leydig cell tumour groups due to limited availability of samples. WIDER IMPLICATIONS OF THE FINDINGS: To the best of our knowledge, this is the first report showing an epigenetic alteration in cells from active seminiferous tubules adjacent to tumour cells in testicular tumour patients. Our results suggest that, despite presenting spermatogenic activity, the global epigenetic dysregulation found in the testicular tumour patients could lead to molecular alterations of the male germ cells. Since testicular tumours are normally diagnosed in men at reproductive age, H4ac alterations might have an impact when these testicular tumour patients express a desire for fatherhood. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the European Union Marie Curie European Training Network actions and by grants to R.O. from the 'Ministerio de Economía y Competividad (Spain)' (fondos FEDER 'una manera de hacer Europa', PI13/00699, PI16/00346 and PI20/00936) and from EU-FP7-PEOPLE-2011-ITN289880. J.C. was supported by the Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109. J.C. is a Serra Húnter fellow (Universitat de Barcelona, Generalitat de Catalunya). F.B. has received grants from the Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario (Spain) (FPU15/02306). A.d.l.I. is supported by a fellowship of the Ministerio de Economía, Industria y Competitividad (Spain) (PFIS, FI17/00224). M.J. is supported by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Stable-protein Pair Analysis as A Novel Strategy to Identify Proteomic Signatures: Application To Seminal Plasma From Infertile Patients.

Our aim was to define seminal plasma proteome signatures of infertile patients categorized according to their seminal parameters using TMT-LC-MS/MS. To that extent, quantitative proteomic data was analyzed following two complementary strategies: (1) the conventional approach based on standard statistical analyses of relative protein quantification values; and (2) a novel strategy focused on establishing stable-protein pairs. By conventional analyses, the abundance of some seminal plasma proteins was found to be positively correlated with sperm concentration. However, this correlation was not found for all the peptides within a specific protein, bringing to light the high heterogeneity existing in the seminal plasma proteome because of both the proteolytic fragments and/or the post-translational modifications. This issue was overcome by conducting the novel stable-protein pairs analysis proposed herein. A total of 182 correlations comprising 24 different proteins were identified in the normozoospermic-control population, whereas this proportion was drastically reduced in infertile patients with altered seminal parameters (18 in patients with reduced sperm motility, 0 in patients with low sperm concentration and 3 in patients with no sperm in the ejaculate). These results suggest the existence of multiple etiologies causing the same alteration in seminal parameters. Additionally, the repetition of the stable-protein pair analysis in the control group by adding the data from a single patient at a time enabled to identify alterations in the stable-protein pairs profile of individual patients with altered seminal parameters. These results suggest potential underlying pathogenic mechanisms in individual infertile patients, and might open up a window to its application in the personalized diagnostic of male infertility.

Prognostic microRNAs in upper tract urothelial carcinoma: multicenter and international validation study.

OBJECTIVE: To validate previously discovered miRNAs (miR-31-5p and miR-149-5p) as prognostic factors for UTUC in an independent cohort of UTUC patients. PATIENTS AND METHODS: Multicenter, international and retrospective study of formalin-fixed paraffin-embedded tissue samples from 103 UTUC patients (45 progressing and 58 non-progressing) who underwent radical nephroureterectomy. Total RNA was isolated and reverse transcribed. The expression of target miRNAs (miR-31-5p and miR-149-5p) and the endogenous control miR-218-5p was evaluated in all samples by reverse transcription quantitative PCR. Normalized miRNA expression values were evaluated by multivariate forward stepwise Cox regression analysis. Kaplan Meier curves were used to discriminate between two groups of patients with a different probability of tumour progression. RESULTS: The mean age (range) of the series was 67 (33-94) years. Overall, 45 patients (43.7%) developed tumour progression and 32 patients (31.2%) died, 20 of these (62.5%) due to their UTUC, after a median follow-up of 36 months. The mean time for tumour progression and cancer-specific survival were 15 and 20 months, respectively. Five year tumour progression free survival and cancer-specific survival were 58% for ≤ pT2, 36% for pT3 and 0% for pT4 and 67.8% for ≤ pT2, 50.6% for pT3 and 0% for pT4, respectively. In the multivariate analysis, expression of miR-31-5p was found to be an independent prognostic factor of tumour progression (HR 1.1; 95% CI 1.039-1.273; p=0.02). Kaplan Meier curve shows that miR-31-5p expression values are able to discriminate between two groups of UTUC patients with a different probability of tumour progression (p=0.007). CONCLUSIONS: We have been able to validate our previous results in an independent multicentre international cohort of UTUC patients, suggesting that miRNA-31-5p could be a useful prognostic marker of UTUC progression. The application of miRNA expression values to clinical practice could refine the currently used clinicopathological-based approach for predicting UTUC patients' outcome.

The role of contrast agents in the diagnosis of renal diseases.

The behavior of dynamic enhancement using imaging modalities such as enhanced computed tomography, enhanced magnetic resonance, and contrast-enhanced ultrasound is one of the most important features of imaging characterization of kidney diseases. The enhancement pattern depends on the macrovasculature and microvasculature and is correlated with specific pathologic features. This review focuses on the enhancement pattern of different renal conditions including inflammatory, vascular, and oncologic diseases, with emphasis on the pathophysiological bases of altered perfusion.

Prospective evaluation of CT indeterminate renal masses using US and contrast-enhanced ultrasound.

OBJECTIVE: The aim of this study was to assess the accuracy of Ultrasound (US) and contrast-enhanced ultrasound (CEUS) in the characterization of renal nodules indeterminate on CT by identifying benign cystic lesions not requiring further examination. METHODS: 72 patients with 83 indeterminate renal nodules on CT underwent baseline US and CEUS that classified lesions as benign (Bosniak I, II or IIF cysts) or potentially malignant (Bosniak III or IV cysts, solid nodules). The accuracy of US and CEUS in the differentiation between benign cysts and potentially malignant nodules was analyzed and compared with the final diagnosis obtained by histology or follow-up of at least 23 months with CEUS ± a conclusive CT/MR study. RESULTS: Final diagnoses comprised 50 benign complex cysts, 1 focal nephritis, 1 multilocular cystic nephroma, 3 oncocytomas, 1 transitional cell carcinoma and 27 renal cell carcinomas. Unenhanced US correctly classified 18/50 (36%) benign cysts and 17/33 (51.5%) of the potentially malignant lesions obtaining a sensitivity of 36%, specificity of 51.5%, and overall accuracy of 42.2%. The addition of CEUS allowed a correct diagnosis of 48 /50 (96%) benign cysts and of 31/33 (93.9%) nodules as potentially malignant, with a sensitivity of 96%, specificity of 93.9%, and overall accuracy of 95.2%. CONCLUSION: CEUS is very useful in the differentiation between benign complex cysts and other lesions that require further investigation in non-conclusive renal nodules detected on CT, improving the accuracy of baseline US from 42.2 to 95.2%.

Interface membrane is the best sample for histological study to diagnose prosthetic joint infection.

The objective of our study was to study which is the most accurate specimen for histological diagnosis of prosthetic joint infections (pseudocapsule or interface membrane). This is a prospective study including hip revision arthroplasties performed from January 2007 to June 2009. Specimens from pseudocapsule and from interface membrane were obtained from each patient. The histology was considered positive for infection when ≥5 neutrophils per high-power field ( × 40) were found. Definitive diagnosis of infection was considered when ≥2 cultures were positive for the same microorganism. According to the definition of infection, patients were classified in two groups: (A) patients with aseptic loosening in whom cultures obtained during surgery were negative and (B) patients with prosthetic joint infection. A total of 69 revisions were included in the study; 57 were classified in group A and 12 in group B. In group B, the percentage of positive interface membrane histology was significantly higher than the percentage of positive pseudocapsule histology (83 vs 42%, P=0.04, Fisher's exact test). The results suggest that periprosthetic interface membrane is the best specimen for the histological diagnosis of prosthetic joint infection.

Adhesion molecules alpha, beta and gamma-catenin as prognostic factors of tumour progression in upper urinary tract urothelial tumours: the role of AKT-P/GSK-3beta/beta-catenin pathway.

OBJECTIVES To evaluate alpha, beta and gamma-catenin expression in upper urinary tract urothelial tumours (UUTC) and determine their value as prognostic factors; to investigate the correlation between the catenin complex and the AKT pathway. PATIENTS AND METHODS We retrospectively analysed 114 consecutive patients treated at our institution from 1990 to 2004; the mean follow-up was 54 months. Tumour samples were available from 70 patients, and included in tissue microarrays for immunohistochemical analysis. The antibodies used were anti-alpha, -beta and gamma-catenin, and antiphospho-AKT. The prognostic value of the expression of these molecules was analysed using tumour progression and cancer-specific survival as end-points. RESULTS Of the 114 patients, 27% developed tumour progression; the cancer-specific and overall survival were 77% and 60.6%, respectively. Abnormal alpha, beta and gamma-catenin expression was found in 44 (63%), 22 (31%) and 28 (41%) patients, respectively; the abnormal catenin expression patterns correlated with each other. Positive cytoplasm phospho-AKT expression was found in 27 (39%) patients. Three of them were found to have cytoplasmic beta-catenin accumulation and none of them nuclear expression. beta-catenin expression was the only one that was an independent marker of tumour progression, with a hazard ratio (95% confidence interval) of 3.1(1.2-8.6), together with grade (7.1, 1.2-55.8) and stage (4.6, 2.1-10). In the cancer-specific survival analysis, again beta-catenin was an independent prognostic factor (3.4, 1-11.5) together with stage (4.6, 2.2-9.8). CONCLUSIONS The loss of the normal membrane beta-catenin expression constitutes an independent factor of tumour progression and cancer-specific survival. Our data suggest that the AKT/GSK3beta/beta-catenin signalling pathway is not activated in the UUTC carcinogenesis.

Neutrophils in frozen section and type of microorganism isolated at the time of resection arthroplasty for the treatment of infection.

INTRODUCTION: The histology of periprosthetic tissue is a gold standard for the diagnosis of periprosthetic joint infection. However, the specificity and sensitivity of histology has never been 100%. In the present study we hypothesized that the type of microorganism could be related to the effectiveness of histology in the detection of infection. MATERIAL AND METHODS: Frozen sections and cultures from periprosthetic tissue of 38 revision arthroplasties taken at the time of resection arthroplasty for the treatment of infection were retrospectively reviewed. Frozen sections were evaluated following Feldman's criteria. A culture was considered positive when the same microorganism was isolated in at least two samples or when pus was present around the prosthesis. The literature providing information on histology and microbiology of arthroplasty-associated infection was reviewed. RESULTS: Coagulase-negative staphylococcus (CNS) was the etiology in 13 cases, Gram-negative bacilli in 8, Staphylococcus aureus in 7, Candida sp in 2, Peptococcus sp in 2, Enterococcus sp in 1 and S. pneumoniae in 1. No microorganism was isolated in four cases. Almost all the frozen sections in the 38 cases were positive except in 2 of the 13 that were caused by CNS (15.3%). The articles reviewed supported our findings. CONCLUSION: Frozen section is a useful test to intraoperatively confirm an infection when preoperative septic loosening is suspected. However, histology has false-negative results when the infection is due to low-virulence microorganisms.

Usefulness of histological analysis for predicting the presence of microorganisms at the time of reimplantation after hip resection arthroplasty for the treatment of infection.

BACKGROUND: Appropriate interpretation of a frozen section has a relatively high specificity and sensitivity for the diagnosis of infection when septic loosening of a prosthesis is suspected. However, its usefulness for predicting the presence of microorganisms at the time of reimplantation after hip resection arthroplasty for the treatment of infection is not well defined. The aim of the present study was to evaluate the usefulness of histological analysis in this situation. METHODS: From January 2002 to February 2006, a total of twenty-one patients underwent reimplantation after hip resection arthroplasty for the treatment of infection. Histological studies and cultures of specimens of periprosthetic tissue that had been obtained at the time of reimplantation were retrospectively reviewed. The results of culture were considered positive when the same microorganism was isolated in at least two samples. Two histological criteria were used to diagnose infection: (1) Criterion A (the Feldman criterion), defined as the presence of at least five neutrophils per high-power field (x400) in at least five separate microscopic fields and (2) Criterion B (the Athanasou criterion), defined as the presence of at least one neutrophil per high-power field (x400), on average, after examination of ten microscopic fields. The sensitivity, specificity, positive predictive value, and negative predictive value of each of these criteria were calculated with use of microbiological results as the gold standard for defining infection. RESULTS: Seven of the twenty-one patients had a positive result on culture, and the most common microorganism was coagulase-negative staphylococcus. The sensitivity, specificity, positive predictive value, and negative predictive value of frozen-section analysis were 28.5%, 100%, 100%, and 73.6%, respectively, according to the Feldman criterion and 71.4%, 64.2%, 50%, and 81.8%, respectively, according to the Athanasou criterion. The numbers of lymphocytes and plasma cells did not help in the diagnosis of infection. Fibrosis was more common in patients without an infection. CONCLUSIONS: The probability of infection is high when at least five neutrophils per high-power field are found in the periprosthetic tissue, but it is not possible to rule out infection when the number of neutrophils is less than five. LEVEL OF EVIDENCE: Diagnostic Level I.

Low sensitivity of histology to predict the presence of microorganisms in suspected aseptic loosening of a joint prosthesis.

Intraoperative histology has a high specificity and sensitivity when a septic prosthesis loosening is suspected. However, its usefulness to predict the presence of microorganisms when aseptic loosening is suspected is not well defined. Intraoperative histology and cultures from periprosthetic tissue of 61 revision arthroplasties performed owing to suspected aseptic loosening were retrospectively reviewed. Frozen sections were evaluated following Mirra's criteria (adapted by Feldman). Culture was considered positive when the same microorganism was isolated in at least two samples. The cultures were positive in 12 cases and coagulase-negative staphylococci were the most common microorganisms (11 cases). In six out of 12 cases (50%), the histology revealed more than five polymorphonuclear leukocytes per high-power field. The sensitivity, specificity, positive and negative predictive value of histology to detect the presence of microorganisms was 50, 81, 40 and 86%, respectively. In conclusion, intraoperative histology using Mirra's criteria had a low sensitivity to predict the presence of microorganisms in samples from suspected aseptic prosthetic loosening.

Mujer de 70 años con dolor en el talón izquierdo, febrícula y afección del estado general / A 70-years-old woman with low-grade fever, malaise and pain in left heel

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Fluorescence in situ hybridization analysis of matched primary tumour and lymph-node metastasis of D1 (pT2-3pN1M0) prostate cancer.

OBJECTIVE: To describe the chromosomal numerical changes present in primary prostate tumours and their matched lymph-node metastases, to identify a clonal cell migration process which could account for the metastatic behaviour. MATERIALS AND METHODS: Twenty-eight cases of unsuspected stage D1 (pT2-3pN1M0) prostate cancer were detected among patients who had a radical prostatectomy for clinically localized prostate cancer. Fluorescence in situ hybridization (FISH), using centromeric probes to enumerate chromosomes 7, 8, 10 and 12, was used to assess numerical chromosomal changes. FISH analysis was used on isolated nuclei obtained from matched primary tumours and their lymph node metastases. RESULTS: Of the 28 suitable cases it was possible to complete the study in 18 pairs of matched tissues; the remainder were excluded because of insufficient tissue or poor preservation of at least one of the tissues. There was cytogenetic change (aneuploidy) in 16 of the 18 primary tumours, the most common being monosomy 8, detected in 14, followed by trisomy 7, in 13 aneuploid tumours. All lymph node metastases were aneuploid by FISH. As in the primary tumours, monosomy 8 and trisomy 7 were the most common cytogenetic alterations, in 13 and 15 of the lymph node tissues. FISH analysis showed a high correlation (83%) in the cytogenetic pattern of changes between the primary tumours and their lymph node metastases. Moreover, a similar number of cells had the most common aneusomies when comparing prostate and the lymph node tissues. CONCLUSIONS: These results show a similar pattern of cytogenetic alteration in the primary tumour and its lymph node metastasis, characterized by the frequent presence of trisomy 7 and monosomy 8, suggesting that clonal cell selection is not involved in the metastatic process.

C-kit expression in sarcomatoid renal cell carcinoma: potential therapy with imatinib.

PURPOSE: Sarcomatoid (S) renal cell carcinoma (RCC) is an uncommon subtype of RCC with a poor prognosis because of its local aggressiveness and high metastatic rate. Currently, there is no specific, effective treatment for it. A relatively nontoxic tyrosine kinase inhibitor, imatinib (STI-571) has been approved as a target therapy in neoplasms that express c-Kit. We investigated c-Kit expression in this type of tumor, which to our knowledge has not been previously described. MATERIALS AND METHODS: We reviewed 215 cases of RCC diagnosed at our department from 1995 to 2002. Of the cases 20 (9.3%) were SRCC. Formalin fixed, paraffin embedded material was available in 19 cases. We performed immunohistochemical staining against c-Kit using rabbit polyclonal antihuman antibody (CD117, Dako Corp., Carpinteria, California), diluted 1:100. Its expression was evaluated in the epithelial and the spindle components. RESULTS: Two of the 20 SRCC cases (10%) showed no epithelial differentiation. The epithelial component was conventional RCC in 10 cases (50%), papillary RCC in 5 (25%) and chromophobe RCC in 3 (15%). A total of 16 cases (80%) presented at an advanced stage at diagnosis, namely T3 or T4 and/or metastatic disease. Immunohistochemical study showed positivity in the epithelial component only in the 3 chromophobe SRCCs. The sarcomatoid component was positive for c-Kit in 18 cases (94.7%). CONCLUSIONS: High c-Kit expression in SRCC in our series and the existence of a target therapy, imatinib (STI-571), against cells that express this receptor open the possibility of using this treatment for these tumors, especially in cases of advanced disease.