Corrigendum to "Circulating Cancer Stem Cell-Derived Extracellular Vesicles as a Novel Biomarker for Clinical Outcome Evaluation".

[This corrects the article DOI: 10.1155/2019/5879616.].

Circulating Cancer Stem Cell-Derived Extracellular Vesicles as a Novel Biomarker for Clinical Outcome Evaluation.

The recent introduction of the "precision medicine" concept in oncology pushed cancer research to focus on dynamic measurable biomarkers able to predict responses to novel anticancer therapies in order to improve clinical outcomes. Recently, the involvement of extracellular vesicles (EVs) in cancer pathophysiology has been described, and given their release from all cell types under specific stimuli, EVs have also been proposed as potential biomarkers in cancer. Among the techniques used to study EVs, flow cytometry has a high clinical potential. Here, we have applied a recently developed and simplified flow cytometry method for circulating EV enumeration, subtyping, and isolation from a large cohort of metastatic and locally advanced nonhaematological cancer patients (N = 106); samples from gender- and age-matched healthy volunteers were also analysed. A large spectrum of cancer-related markers was used to analyse differences in terms of peripheral blood circulating EV phenotypes between patients and healthy volunteers, as well as their correlation to clinical outcomes. Finally, EVs from patients and controls were isolated by fluorescence-activated cell sorting, and their protein cargoes were analysed by proteomics. Results demonstrated that EV counts were significantly higher in cancer patients than in healthy volunteers, as previously reported. More interestingly, results also demonstrated that cancer patients presented higher concentrations of circulating CD31+ endothelial-derived and tumour cancer stem cell-derived CD133 + CD326- EVs, when compared to healthy volunteers. Furthermore, higher levels of CD133 + CD326- EVs showed a significant correlation with a poor overall survival. Additionally, proteomics analysis of EV cargoes demonstrated disparities in terms of protein content and function between circulating EVs in cancer patients and healthy controls. Overall, our data strongly suggest that blood circulating cancer stem cell-derived EVs may have a role as a diagnostic and prognostic biomarker in cancer.

Application of a chemiluminescence immunoassay system and GC/MS for toxicological investigations on skeletonized human remains.

Hair, larvae and cardiac muscle, the only biological samples present on a skeletonized human body found in a rural area, were used for forensic toxicological analyses in order to determine possible causes of death. Since no information about the victim or the circumstances of death was available (except for the place where the corpse was found, known to be a gathering place for drug addicts), the first approach for the analysis of non-conventional matrices involved the screening of different classes of active principles, using a chemiluminescence-based screening assay designed for whole blood. The immunoassay test results showed positivity to amphetamines, cocaine and opiates on water/methanol extract from cardiac tissue, larvae and hair samples. Gas chromatography-mass spectrometry (GC/MS) analyses confirmed the immunoassay results, except for amphetamines. The minimal sample preparation (hydration and extraction in an ultrasonic bath), the reduced sample volume required for the analyses, together with the correctness of results as confirmed by GC/MS, showed the suitability of the screening test for forensic applications on non-conventional matrices. Quantitative analyses in GC/MS allowed the cause of death to be ascertained on the basis of the ratio between parent drugs and metabolites.

Physical Activity Among Organ Recipients: Data Collected From the Latin American Transplant Games.

BACKGROUND: Cardiovascular complications after transplantation are an important cause of non-transplant-related deaths. Depression and anxiety are not unusual among organ recipients. Physical activity reduces cardiovascular risk and promotes a sensation of well-being. The aims of the study were to examine how exercise affects psychological well-being sensation in organ recipients and to describe the physician's role in promoting and controlling safe sport training in transplanted patients. METHODS: A descriptive study was conducted. A questionnaire was answered by participants of the "2012 Latin American Transplant Games." RESULTS: One hundred sixty-six organ recipients completed the questionnaire. Eleven percent heard about the transplant games from a physician. Seventy percent had not received a proper medical pre-competitive evaluation. Only 39% trained in a supervised manner and 53% had experienced some kind of sport-related injury. Self-perception of global health was reported as excellent by 19.75%, very good by 43.95%, good by 30.67%, and regular or poor by 5.73%. An excellent or very good health perception was reported by 47.8% of those who practiced only one kind of sport versus 71.5% of those who practiced more than one sport and by 89.6% of those who performed isometric activity versus 59.3% of those who did not perform isometric activity. CONCLUSIONS: Diversity of practiced sports and isometric activity are associated with a better self-reported health condition. Most participants had not received a proper medical pre-competitive evaluation; they trained in an unsupervised manner, and injuries were common.

Effect of cellulose structure and morphology on the properties of poly(butylene succinate-co-butylene adipate) biocomposites.

Composites based on poly(butylene succinate-co-butylene adipate) (PBSA) containing amorphized and crystalline cellulose reinforcements have been prepared and characterized. In order to improve the polymer/filler interfacial adhesion, an efficient compatibilizing agent has been synthesized by chemical modification of PBSA and characterized by FT-IR, FT-NIR and (1)H NMR spectroscopy. Uncompatibilized and compatibilized composites have been tested through morphological, mechanical, calorimetric and thermogravimetric analysis. Moreover, water vapor permeability and biodegradation kinetics of composites have been investigated. The addition to PBSA of cellulose fillers differing from each other by crystallinity degree and morphology, and the use of a compatibilizing agent have allowed modulating tensile and thermal properties, water vapor transmission rate and biodegradation kinetic of the composites.

Association of low-penetrance alleles with male breast cancer risk and clinicopathological characteristics: results from a multicenter study in Italy.

It is well-known that male breast cancer (MBC) susceptibility is mainly due to high-penetrance BRCA1/2 mutations. Here, we investigated whether common low-penetrance breast cancer (BC) susceptibility alleles may influence MBC risk in Italian population and whether variant alleles may be associated with specific clinicopathological features of MBCs. In the frame of the Italian Multicenter Study on MBC, we genotyped 413 MBCs and 745 age-matched male controls at 9 SNPs annotating known BC susceptibility loci. By multivariate logistic regression models, we found a significant increased MBC risk for 3 SNPs, in particular, with codominant models, for rs2046210/ESR1 (OR = 1.71; 95 % CI: 1.43-2.05; p = 0.0001), rs3803662/TOX3 (OR = 1.59; 95 % CI: 1.32-1.92; p = 0.0001), and rs2981582/FGFR2 (OR = 1.26; 95 % CI: 1.05-1.50; p = 0.013). Furthermore, we showed that the prevalence of the risk genotypes of ESR1 tended to be higher in ER- tumors (p = 0.062). In a case-case multivariate analysis, a statistically significant association between ESR1 and ER- tumors was found (OR = 1.88; 95 % CI: 1.03-3.49; p = 0.039). Overall, our data, based on a large and well-characterized MBC series, support the hypothesis that common low-penetrance BC susceptibility alleles play a role in MBC susceptibility and, interestingly, indicate that ESR1 is associated with a distinct tumor subtype defined by ER-negative status.

BAG3: a multifaceted protein that regulates major cell pathways.

Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110-124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression.

Leukocyte immunotherapy improves live delivery rates following embryo transfer in women with at least two previous failures: a retrospective review.

PURPOSE: To determine whether leukocyte immunotherapy (LIT) could improve live delivery rate following embryo transfer (ET) in women who were not successful in prior attempts. METHODS: Paternal leukocytes were intradermally injected in some women who had failed to have a successful pregnancy following at least two prior ETs approximately two weeks prior to fresh or frozen ET and repeated at the time of the 3rd rising serum beta human chorionic gonadotropin level and at eight weeks if a pregnancy occurred. Clinical pregnancy and live pregnancy rates (PRs) were compared to those women having ETs during the same time period not receiving LIT. RESULTS: Thirty-six of 94 (38.3%) patients receiving LIT (group 1) conceived following fresh or frozen ET vs 98 of 341 (28.7%) for women not receiving LIT (group 2) (p = NS). The live delivery rate per ET cycle was 30.8% (39/94) vs 19.7% for group 2 (p = .02). For the subset of women failing despite five previous ETs 17 of 37 (45.9%) group 1 women had a clinical pregnancy vs 18 of 64 (28.1%) group 2 women (p = .07%) and live delivery rates were 35.1% (13/37) vs 15.6% (10/64) (p = .024). CONCLUSIONS: These retrospective data encourage a prospective study of LIT combined with progesterone vs controls receiving progesterone only for recalcitrant patients having ETs.

A comparison of luteal phase support in graduated estradiol/progesterone replacement cycles using intramuscular progesterone alone versus combination with vaginal suppositories on outcome following frozen embryo transfer.

PURPOSE: To compare pregnancy outcome following frozen embryo transfer according to type of progesterone (P) support given in the luteal phase. METHODS: Retrospective cohort analysis of frozen embryo transfer (ET) cycles in which ovulation was suppressed by graduated estradiol in the follicular phase. Two P regimens in the luteal phase were compared: P vaginal suppositories and intramuscular P vs intramuscular alone. RESULTS: The clinical and viable pregnancy rates were significantly higher for the women receiving only intramuscular P (57.6% and 43.7%) vs those receiving combined therapy (45.9% and 35.6%, respectively). The implantation rates were not significantly different (22.6% vs 19.5%). CONCLUSION: The increased pregnancy rates with intramuscular P may have been related to a higher number of embryos transferred (3.69 vs 3.26). Nevertheless, intramuscular P alone is at least as effective, if not more effective, than combined therapy for frozen embryo transfers.

Effect of an abnormal sperm chromatin structural assay (SCSA) on pregnancy outcome following (IVF) with ICSI in previous IVF failures.

A high DNA fragmentation index (DFI) when performing the sperm chromatin structural (SCSA) assay was claimed to be so specific for male subfertility that even IVF and ICSI did not result in live pregnancies. The present study was designed to corroborate or refute these findings. The SCSA test was performed on the male partner from couples failing to have a successful pregnancy despite at least 2 previous IVF attempts. In contrast to the aforementioned studies, ongoing pregnancies were found despite working with a group of recalcitrant patients. Nevertheless, a high DFI score was associated with a trend for lower ongoing pregnancy rates especially related to a high miscarriage rate. Other more recent studies seem to support our conclusions. A high DFI score should influence a patient to choose IVF as a therapeutic modality sooner, especially with ICSI.

Neither sildenafil nor vaginal estradiol improves endometrial thickness in women with thin endometria after taking oral estradiol in graduating dosages.

PURPOSE: To determine if sildenafil improves endometrial thickness better than vaginal estradiol (E2) in women with a history of thin endometria. METHODS: Women failing to attain an 8 mm endometrial thickness on either the oocyte retrieval cycle or their first frozen embryo transfer (ET) despite an oral graduated E2 regimen were treated again with graduated oral E2 and were also randomly assigned to vaginal sildenafil or vaginal E2 therapy. Endometrial thickness was compared between the groups. RESULTS: Neither vaginal E2 nor sildenafil significantly improved endometrial thickness or blood flow in the subsequent frozen ET-cycle. CONCLUSIONS: These data fail to corroborate previous claims that 25 mg sildenafil four times daily intravaginally can improve endometrial thickness.

Effect of treating antibody-coated sperm with chymotrypsin on pregnancy rates following IUI as compared to outcome of IVF/ICSI.

Males with 100% of their sperm coated by antisperm antibody have a very small chance of achieving a pregnancy by intercourse or conventional intrauterine insemination (IUI). A previous study found that treatment of the sperm with the protein digestive enzyme chymotrypsin improved the efficacy of IUI. The present study was designed to corroborate or refute this previous study and compare efficacy to IVF with ICSI. This time the subjects were an even more difficult group with 100% of the sperm coated by autoantibodies.

Structure of a yeast hypothetical protein selected by a structural genomics approach.

Yeast hypothetical protein YBL036C (SWISS-PROT P38197), initially thought to be a member of an 11-protein family, was selected for crystal structure determination since no structural or functional information was available. The structure has been determined independently by MIR and MAD methods to 2.0 A resolution. The MAD structure was determined largely through automated model building. The protein folds as a TIM barrel beginning with a long N-terminal helix, in contrast to the classic triose phosphate isomerase (TIM) structure, which begins with a beta-strand. A cofactor, pyridoxal 5'-phosphate, is covalently bound near the C-terminal end of the barrel, the usual active site in TIM-barrel folds. A single-domain monomeric molecule, this yeast protein resembles the N-terminal domain of alanine racemase or ornithine decarboxylase, both of which are two-domain dimeric proteins. The yeast protein has been shown to have amino-acid racemase activity. Although selected as a member of a protein family having no obvious relationship to proteins of known structure, the protein fold turned out to be a well known and widely distributed fold. This points to the need for a more comprehensive base of structural information and better structure-modeling tools before the goal of structure prediction from amino-acid sequences can be realised. In this case, similarity to a known structure allowed inferences to be made about the structure and function of a widely distributed protein family.

Characterization of the solution complex between the interferon-induced, double-stranded RNA-activated protein kinase and HIV-I trans-activating region RNA.

The antiviral activity of the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (PKR) is mediated through dsRNA binding leading to PKR autophosphorylation and subsequent inhibition of protein synthesis. Previous biochemical studies have suggested that autophosphorylation of PKR occurs via a protein-protein interaction and that PKR can form dimers in vitro. Using four independent biophysical and biochemical methods, we have characterized the solution complex formed between PKR and trans-activating region (TAR) RNA, a 57-nucleotide RNA species with double-stranded secondary structure derived from the human immunodeficiency virus type I genome. Chemical cross-linking and gel filtration analyses of PKR.TAR RNA complexes reveals that TAR RNA addition increases PKR dimerization and results in the formation of a solution complex with a molecular weight of approximately 150,000. Addition of TAR RNA to PKR results in a quenching of tryptophan fluorescence, indicative of a conformational shift. Through small angle neutron scattering analysis, we show that PKR exists in solution predominantly as a dimer, and has an elongated solution structure. Addition of TAR RNA to PKR causes a significant conformational shift in the protein at a 2:1 stoichiometric ratio of protein to RNA. Taken together, these data indicate that the PKR activation complex consists of a protein dimer bound cooperatively to one dsRNA molecule.

Identification of two DNA-binding sites on the globular domain of histone H5.

The nature of the complexes of histones H1 and H5 and their globular domains (GH1 and GH5) with DNA suggested two DNA-binding sites which are likely to be the basis of the preference of H1 and H5 for the nucleosome, compared with free DNA. More recently the X-ray and NMR structures of GH5 and GH1, respectively, have identified two basic clusters on opposite sides of the domains as candidates for these sites. Removal of the positive charge at either location by mutagenesis impairs or abolishes the ability of GH5 to assemble cooperatively in 'tramline' complexes containing two DNA duplexes, suggesting impairment or loss of its ability to bind two DNA duplexes. The mutant forms of GH5 also fail to protect the additional 20 bp of nucleosomal DNA that are characteristically protected by H1, H5 and wild-type recombinant GH5. They still bind to H1/H5-depleted chromatin, but evidently inappropriately. These results confirm the existence of, and identify the major components of, two DNA-binding sites on the globular domain of histone H5, and they strongly suggest that both binding sites are required to position the globular domain correctly on the nucleosome.

Neutron scattering studies on chromatin higher-order structure.

We have been engaged in studies of the structure and condensation of chromatin into the 30 nm filament using small-angle neutron scattering. We have also used deuterated histone H1 to determine its location in the chromatin 30 nm filament. Our studies indicate that chromatin condenses with increasing ionic strength to a limiting structure that has a mass per unit length of 6-7 nucleosomes/11 nm. They also show that the linker histone H1/H5 is located in the interior of the chromatin filament, in a position compatible with its binding to the inner face of the nucleosome. Analysis of the mass per unit length as a function of H5 stoichiometry suggests that 5-7 contiguous nucleosomes need to have H5 bound before a stable higher order structure can exist.

Prokaryotic translation initiation factor IF3 is an elongated protein consisting of two crystallizable domains.

We show that translation initiation factor IF3 can be split into two fragments of nearly equal size by the Escherichia coli outer membrane protease omptin. Circular dichroism and small-angle neutron scattering show that the two fragments are structured as domains. Each domain is relatively compact, and they are separated by about 45 A in intact IF3. Thus IF3 is an elongated protein that consists of two well-separated domains. We suggest that these two domains are involved in ribosome binding across the cleft of the 30S ribosome. We also report the crystallization of each domain of IF3.

Expression of chicken linker histones in E. coli: sources of problems and methods for overcoming some of the difficulties.

Expression of histones in Escherichia coli is important in structural studies on chromatin, because it allows isotopic labeling such as deuteration and replacement of methionines with selenomethionine as well as expression of specific domains of histones. We show that full-length H5 cannot be expressed in E. coli. We have determined that the problem is translational rather than transcriptional. Pulse-labeling studies show that protein turnover is not the reason for lack of accumulation. On dissecting the gene, we find that the problem lies in expressing the highly charged C-terminal tail of H5. We can make progressively increasing amounts of the tail, but at the point where over two-thirds of this region is transcribed, the protein ceases to be made. Surprisingly, full-length H1 is made. In vitro studies show that the H5 gene can be translated in a rabbit reticulocyte system but not in an E. coli system, suggesting that there may be a difference in the ability of eukaryotic and prokaryotic ribosomes to translate this message. The expression of the globular domains of H5 and H1 posed a different problem. There was little or no expression of some of the constructs, even though they were fragments of larger constructs that were well made. Replacement of the first five codons downstream of the initiating ATG codon with those optimized for E. coli, and which were AT rich, restored expression. This may have general implications for expression of eukaryotic proteins in E. coli.