Age-related changes in human testicular function

Main testicular functions are spermatogenesis and steroidogenesis. Androgen production, Testosterone (T) in particular, stimulates the differentiation of germ cells under the strict endocrine control of the pituitary gland via the two gonadotropins LH and FSH. Today we know that the reduction in serum T concentration (late onset hypogonadism) observed with age can be associated with an overall unhealthy status of the body. Moreover, the prevalence of obesity, metabolic syndrome and diabetes increases with age, and these conditions are significantly associated with hypogonadism, as assessed by sexual symptoms and low serum T levels in spite of normal LH. There is few information about mechanisms underlying androgen deficiency with age and little is known about human Leydig cell (LC) aging, in terms of number and function, mainly because of the scarce availability of the tissue. In this study, we evaluated the age-related changes in testicular function in healthy human testicular tissues of different ages (19-85 years) obtained from heart-beating organ donors and from patients referred to the andrology clinic having normal spermatogenesis and hormone levels. We demonstrated that aging is associated with changes in testicular morphology which are variable in different areas of the parenchyma. Changes included a significative reduction of the area occupied by seminiferous tubules coupled with an increased area occupied by interstitium, mainly composed by fibrotic tissue. In elderly donors, we noticed also peritubular thickening and the appearance of sclerotic tubules and empty tubules. Another morphological parameter evaluated was the LC micronodules frequency and size, since these aggregates composed by more than 15 LCs have been associated with a lower Testosterone/LH ratio. We did not find any correlation between age and micronodules distribution or size, pointing to a normal Hypothalamus-Pituitary-Gonad axis in our group of donors. We found a significative reduction of both LC and Sertoli cell (SC) number with age indicating that cellular senescence generally observed in many tissues with advancing age affects also the testis. Moreover, we evidenced a significative positive correlation between LC and SC number at all ages, which has not been described before in men. Concerning the function of LCs, we analysed gene expression of steroidogenic pathway enzymes and other LC markers by qRT-PCR. We did not find a significative correlation between enzyme gene expression and age but a negative trend was observed for 17β-HSD3, StAR and CYP11A1 genes. In contrast, INSL3 transcript, which also reflects LC number, significantly declined in aged men, consistent with our observation that LC population size changed during aging. Using the in vitro organ culture model previously developed in our laboratory, we demonstrated that three hours in vitro culture of both fresh and cryopreserved testis fragments allows to analyse the LC androgen production (testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA) and 17-Hydroxyprogesterone (17OHP)) secreted into the culture media and simultaneously measured by mass spectrometry both in basal conditions and under recombinant gonadotropin stimulation. We did not find any correlation between androgens secreted in basal conditions and age, indicating that LCs of the donors analysed were able to produce T, A, DHEA and 17OHP in an age- independent manner. The response to gonadotropin stimulation was found greatly variable among donors of different ages and not related to the age. The response to hCG was higher than that to LH when fresh tissue was used. Tissue cryopreservation did not alter interstitial compartment morphology but caused a significative reduction in T concentration, whereas A, DHEA and 17-OH-P levels increased, pointing to a particular vulnerability of specific enzymes to the freezing condition. These data are in line with the finding that the response to hCG was lost when the fragments were cultured after cryopreservation, reinforcing the idea that cryopreservation represents a stressor to the testicular tissue. In conclusion, our data point to a cellular senescence of the aging human testis which is not associated to the in vitro ability to produce androgens. Thus, our results support the idea that LC dysfunction is largely driven by aging of the whole testicular microenvironment rather than aging of LC population alone

Mutation patterns of amino acid tandem repeats in the human proteome

BACKGROUND: Amino acid tandem repeats are found in nearly one-fifth of human proteins. Abnormal expansion of these regions is associated with several human disorders. To gain further insight into the mutational mechanisms that operate in this type of sequence, we have analyzed a large number of mutation variants derived from human expressed sequence tags (ESTs). RESULTS: We identified 137 polymorphic variants in 115 different amino acid tandem repeats. Of these, 77 contained amino acid substitutions and 60 contained gaps (expansions or contractions of the repeat unit). The analysis showed that at least about 21% of the repeats might be polymorphic in humans. We compared the mutations found in different types of amino acid repeats and in adjacent regions. Overall, repeats showed a five-fold increase in the number of gap mutations compared to adjacent regions, reflecting the action of slippage within the repetitive structures. Gap and substitution mutations were very differently distributed between different amino acid repeat types. Among repeats containing gap variants we identified several disease and candidate disease genes. CONCLUSION: This is the first report at a genome-wide scale of the types of mutations occurring in the amino acid repeat component of the human proteome. We show that the mutational dynamics of different amino acid repeat types are very diverse. We provide a list of loci with highly variable repeat structures, some of which may be potentially involved in disease

Involvement of sperm acetylated histones and the nuclear isoform of Glutathione peroxidase 4 in fertilization

We previously demonstrated that the nuclear form of Glutathione peroxidase 4 (nGPx4) has a peculiar distribution in sperm head, being localized to nuclear matrix and acrosome and that sperm lacking nGPx4 are more prone to decondensation in vitro. In this study we have hypothesized that sperm retained acetylated histones and nGPx4 are implicated in paternal chromatin decondensation and male pronucleus formation at fertilization. Indeed, significant higher amounts of acetylated histone H4 and acetylated histone H3 were observed by both immunofluorescence and western blotting in nGPx4-KO sperm vs WT ones. In vitro fertilization of zona pellucida- deprived oocytes by WT sperm in the presence of trichostatin (TSA) also demonstrated that paternal histone acetylation was inversely related to the timing of sperm nucleus decondensation at fertilization. In contrast, TSA had no effect on nGPx4-KO sperm, indicating they had a maximal level of histone acetylation. Moreover the paternally imprinted gene Igf2/H19 was hypomethylated in KO sperm compared to WT ones. The lack of nGPx4 negatively affected male fertility, causing a marked decrease in total pups and pregnancies with delivery, a significant reduction in pronuclei (PN) embryos in in vitro fertilization assays and an approximately 2 h delay in egg fertilization in vivo. Because the zona pellucida binding and fusion to oolemma of nGPx4-KO and WT sperm were similar, the subfertility of nGPx4 sperm reflected a decreased sperm progression through egg cumulus/zona pellucida, pinpointing a defective acrosome in line with acrosomal nGPx4 localization. We conclude that paternal acetylated histones and acrosomal nGPx4 are directly involved in fertilization

Beta-D-glucan in patients with haematological malignancies

(1-3)-beta-D-glucan (BDG) is an almost panfungal marker (absent in zygomycetes and most cryptococci), which can be successfully used in screening and diagnostic testing in patients with haematological malignancies if its advantages and limitations are known. The aim of this review is to report the data, particularly from the last 5 years, on the use of BDG in haematological population. Published data report mainly on the performance of the Fungitell™ assay, although several others are currently available, and they vary in method and cut-off of positivity. The sensitivity of BDG for invasive fungal disease (IFD) in haematology patients seems lower than in other populations, possibly because of the type of IFD (lower sensitivity was found in case of aspergillosis compared to candidiasis and pneumocystosis) or the use of prophylaxis. The specificity of the test can be improved by using two consecutive positive assays and avoiding testing in the case of the concomitant presence of factors associated with false positive results. BDG should be used in combination with clinical assessment and other diagnostic tests, both radiological and mycological, to provide maximum information. Good performance of BDG in cerebrospinal fluid (CSF) has been reported. BDG is a useful diagnostic method in haematology patients, particularly for pneumocystosis or initial diagnosis of invasive fungal infections

Cytoskeleton Dynamics in Peripheral T Cell Lymphomas: An Intricate Network Sustaining Lymphomagenesis.

Defects in cytoskeleton functions support tumorigenesis fostering an aberrant proliferation and promoting inappropriate migratory and invasive features. The link between cytoskeleton and tumor features has been extensively investigated in solid tumors. However, the emerging genetic and molecular landscape of peripheral T cell lymphomas (PTCL) has unveiled several alterations targeting structure and function of the cytoskeleton, highlighting its role in cell shape changes and the aberrant cell division of malignant T cells. In this review, we summarize the most recent evidence about the role of cytoskeleton in PTCLs development and progression. We also discuss how aberrant signaling pathways, like JAK/STAT3, NPM-ALK, RhoGTPase, and Aurora Kinase, can contribute to lymphomagenesis by modifying the structure and the signaling properties of cytoskeleton

Determinants of influenza vaccination among solid organ transplant recipients attending Sicilian reference center

Among solid organ transplant recipients, influenza infection is commonly associated with higher morbidity and mortality than immunocompetent hosts. Therefore, in these subjects influenza vaccination is of paramount importance. The main objective of the study was to assess compliance to vaccination and analyze factors associated with influenza vaccination of solid organ transplant recipients admitted to the Sicilian solid organ transplant Reference Center IRCCS-ISMETT in Palermo during 2014\u20132015 influenza season. Thirty one (37.8%) out of 82 solid organ transplant recipients were vaccinated against influenza. The main reason for vaccination refusal was fear of adverse reaction (n = 16, 31.4%), impaired health status (n = 14, 27.4%) and low vaccine efficacy (n = 10, 19.6%). Vaccinated solid organ transplant recipients compare with unvaccinated had smaller hospital admissions for infectious respiratory diseases (9.7% Vs 23.5%) during surveillance period. On multivariate analysis the factors positively associated with influenza vaccination were the advice of Reference Center physicians (OR 53.4, p < 0.001) and to perform vaccine against pneumococcus (OR 7.0, p = 0.016). This study showed that Reference Center physicians play a key role on vaccine communication and recommendation for patients at risk and it underlines the effectiveness of influenza vaccination in solid organ transplant recipients. However, it remains that, although physician advice resulted a strong determinant for vaccination, influenza vaccination coverage in this subset of population remains still unsatisfactory

HIV infection with viro-immunological dissociation in a patient with polycystic kidney disease: Candidate for transplantation?

Here we describe the case of a HIV-infected patient with polycystic kidney disease and end stage renal diseases not transplantable due to the persistence of a CD4 count &lt;200 notwithstanding a good virological response to highly active antiretroviral therapy and suggest that such limitation to kidney transplantation in such as cases might be bypassed

Combined burden and functional impact tests for cancer driver discovery using DriverPower

The discovery of driver mutations is one of the key motivations for cancer genome sequencing. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we describe DriverPower, a software package that uses mutational burden and functional impact evidence to identify driver mutations in coding and non-coding sites within cancer whole genomes. Using a total of 1373 genomic features derived from public sources, DriverPower's background mutation model explains up to 93% of the regional variance in the mutation rate across multiple tumour types. By incorporating functional impact scores, we are able to further increase the accuracy of driver discovery. Testing across a collection of 2583 cancer genomes from the PCAWG project, DriverPower identifies 217 coding and 95 non-coding driver candidates. Comparing to six published methods used by the PCAWG Drivers and Functional Interpretation Working Group, DriverPower has the highest F1 score for both coding and non-coding driver discovery. This demonstrates that DriverPower is an effective framework for computational driver discovery

Methanol fixation is the method of choice for droplet-based single-cell transcriptomics of neural cells

The main critical step in single-cell transcriptomics is sample preparation. Several methods have been developed to preserve cells after dissociation to uncouple sample handling from library preparation. Yet, the suitability of these methods depends on the cell types to be processed. In this project, we perform a systematic comparison of preservation methods for droplet-based single-cell RNA-seq on neural and glial cells derived from induced pluripotent stem cells. Our results show that while DMSO provides the highest cell quality in terms of RNA molecules and genes detected per cell, it strongly affects the cellular composition and induces the expression of stress and apoptosis genes. In contrast, methanol fixed samples display a cellular composition similar to fresh samples and provide a good cell quality and little expression biases. Taken together, our results show that methanol fixation is the method of choice for performing droplet-based single-cell transcriptomics experiments on neural cell populations. Methanol fixation appears to be the method of choice for droplet-based scRNA-seq on neural cell populations, based on a broad analysis of how various fixation or preservation methods impact the single-cell transcriptomes of hiPSC-derived neural cells

Discovery of Cancer Driver Long Noncoding RNAs across 1112 Tumour Genomes: New Candidates and Distinguishing Features

Long noncoding RNAs (lncRNAs) represent a vast unexplored genetic space that may hold missing drivers of tumourigenesis, but few such "driver lncRNAs" are known. Until now, they have been discovered through changes in expression, leading to problems in distinguishing between causative roles and passenger effects. We here present a different approach for driver lncRNA discovery using mutational patterns in tumour DNA. Our pipeline, ExInAtor, identifies genes with excess load of somatic single nucleotide variants (SNVs) across panels of tumour genomes. Heterogeneity in mutational signatures between cancer types and individuals is accounted for using a simple local trinucleotide background model, which yields high precision and low computational demands. We use ExInAtor to predict drivers from the GENCODE annotation across 1112 entire genomes from 23 cancer types. Using a stratified approach, we identify 15 high-confidence candidates: 9 novel and 6 known cancer-related genes, including MALAT1, NEAT1 and SAMMSON. Both known and novel driver lncRNAs are distinguished by elevated gene length, evolutionary conservation and expression. We have presented a first catalogue of mutated lncRNA genes driving cancer, which will grow and improve with the application of ExInAtor to future tumour genome projects