Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners.

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase delta assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair

Integrin Beta 1 Is Crucial for Urinary Concentrating Ability and Renal Medulla Architecture in Adult Mice

Integrins are heterodimers anchoring cells to the surrounding extracellular matrix (ECM), an active and complex process mediating a series of inside-out and outside-in stimuli regulating cellular turn-over, tissue growth and architecture. Itgb1 is the main subunit of the renal integrins and it is critical for renal development. This study aims to investigate the role of Itgb1 in the adult renal epithelial cells by knocking down Itgb1 in PAX8 expressing cells. Itgb1-Pax8 cKO mice develop a progressively worsening proteinuria and renal abnormalities leading to severe renal failure and hypertension. This phenotype is also associated with severe dysfunction of distal nephron and polyuria. To further investigate whether distal nephron involvement was primarily related to Itgb1 suppression or secondary to renal failure, an Itgb1-AQP2 cKO mouse model was generated. These mice lack Itgb1 expression in AQP2 expressing cells. They do not show any developmental alteration, but 1 month old mice are resistant to dDAVP administration and finally, at 2 months of age, they develop overt polyuria. This phenotype is due to primary collecting duct (CD) cells anoikis. The entire architecture of the outer medulla is altered, with loss of the typical organization pattern of vascular and tubular bundles alternation. Indeed, even though not primarily affected by genetic ablation, the TAL is secondarily affected in this model. It is sufficient to suppress Itgb1 expression in the CD in order to stimulate proliferation and then disappearance of neighboring TAL cells. This study shows that cell to cell interaction through the ECM is critical for architecture and function maintenance of the outer medulla and that Itgb1 is crucial for this process

Multidisciplinary management of neuroendocrine neoplasia: a real-world experience from a referral center

Purpose: Multidisciplinary approach is widely advised for an effective care of patients with neuroendocrine neoplasia (NEN). Since data on efficacy of multidisciplinary management of NENs patients in referral centers are scanty, this study aimed at analyzing the modality of presentation and clinical outcome of patients with NENs managed by a dedicated multidisciplinary team. Methods. In this prospective observational study, we included all consecutive new patients visiting the Sant'Andrea Hospital in Rome (ENETS-Center of Excellence) between January 2014 and June 2018. Results. A total of 195 patients were evaluated. The most frequent sites were pancreas (38.5%), small bowel (22%), and lung (9.7%). Median Ki67 was 3%. After the first visit at the center, additional radiological and/or nuclear medicine procedures were requested in 163 patients (83.6%), whereas histological data revision was advised in 84 patients (43.1%) (revision of histological slides: 27.7%, new bioptic sampling: 15.4%). After that, disease imaging staging and grading was modified in 30.7% and 17.9% of patients, respectively. Overall, a change in therapeutic management was proposed in 98 patients (50.3%). Conclusions. Multidisciplinary approach in a dedicated team may lead to change of disease imaging staging and grading in a significant proportion of patients. Enhancing referral routes to dedicated-NEN center should be promoted, since it may improve patients' clinical outcome

Evidence of Common Isolates of Streptococcus agalactiae in Bovines and Humans in Emilia Romagna Region (Northern Italy)

Streptococcus agalactiae (group B Streptococcus, GBS) is one of the most important agents of bovine mastitis and causes remarkable direct and indirect economic losses to the livestock sector. Moreover, this species can cause severe human diseases in susceptible individuals. To investigate the zoonotic potential of S. agalactiae, 203 sympatric isolates from both humans and cattle, isolated in the same time frame (2018) and in the same geographic area (Emilia Romagna region, Northern Italy), were characterized by molecular capsular typing (MCT), pilus island typing (PI), and multi-locus sequence typing (MLST). In addition, antibiotic-resistant phenotypes were investigated. The distribution of the allelic profiles obtained by combining the three genotyping methods (MCT-PI-MLST) resulted in 64 possible genotypes, with greater genetic variability among the human compared to the bovine isolates. Although the combined methods had a high discriminatory power (>96,2%), five genotypes were observed in both species (20,9% of the total isolates). Furthermore, some of these strains shared the same antibiotic resistance profiles. The finding of human and bovine isolates with common genotypes and antibiotic resistance profiles supports the hypothesis of interspecies transmission of S. agalactiae between bovines and humans

Rischi e opportunità connessi con la commercializzazione del latte crudo

In the European Community only a few EU member states have authorized the sale of raw milk for human consumption, including Italy where the phenomenon is strongly growing. Raw milk is richer in enzymes and vitamins than pasteurized milk, but some potential hazards are associated with the benefits because the product keeps the entire microbial flora that results from the health status of the animal intact. Most foodborne diseases caused by the consumption of dairy products are due to S. aureus, Salmonella spp., enterotoxigenic E. coli, Listeria monocytogenes and Campylobacter spp. Correct information for the costumer is very important because raw milk, if properly managed, monitored and evaluated, can be an opportunity for increasing the farmer’s income and an opportunity to consume genuine products without unjustified constraint

p21(waf1/cip1)-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites

The cyclin-dependent kinase inhibitor p21(waf1/cip1) is known to impair DNA synthesis by binding to PCNA, the co-factor of DNA polymerases delta and epsilon. However, a positive role for p21 in nucleotide excision repair (NER) has been suggested. In this study, the sensitivity to DNA damage and DNA repair efficiency were investigated in p21-null human fibroblasts obtained by targeted homologous recombination. After UV-C irradiation, p21-/- cells showed a threefold reduction in clonogenic survival and an increased susceptibility to apoptosis, as compared with parental p21+/+ cells. Removal of cyclobutane pyrimidine dimers was significantly reduced in p21-/- cells both in the whole genome, and at the level of the rDNA gene cluster, as determined by immunoassay and Southern blot, respectively. After DNA damage, the recruitment of PCNA as detergent-insoluble form associated to DNA repair sites in p21-/- fibroblasts, was comparable to that observed in parental p21+/+ cells. However, PCNA remained associated with DNA for a longer period in p21-/- than in p21+/+ cells. These results suggest that in human cells, p21 is required for NER at a step located downstream the recruitment of PCNA to DNA repair sites

E5 nucleotide polymorphisms suggest quasispecies occurrence in BPV-1 sub-clinically infected horses

BPV-1 is known as the main causative agent of equine sarcoid, but the virus has also been detected in skin and blood of healthy horses. Previous reports demonstrated the presence of E5 variants in sarcoids of donkeys and horses; we investigated whether this genetic variability might be also found in BPV-1, PBMC associated, of sub-clinically infected horses. With this aim, we analyzed the E5 gene of 21 BPV-1 strains from diseased and sub-clinically infected horses. Our analyses lead us to demonstrate that multiple sequence variants can be present in the blood of sub-clinically infected horses, with alternative bases corresponding to either synonymous or non-synonymous codons in the E5 oncogene sequences. The results give support to the proposed existence of "equine adapted" BPV-1 strains with the occurrence of viral variants, resembling quasispecies, in clinically healthy horses with viremia

Effects of anthocyanins on cell cycle progression in normal human fibroblasts

Anthocyanins are flavonoids and constitute the largest group of water–soluble pigment. It has been reported that anthocyanins exert several therapeutical activities, acting as anti-inflammatories, maintaining the normal vascular permeability, and exerting anticarcinogenic effects in vitro. The above effects have been related mainly to the antioxidant properties of anthocyanins, since they have been demonstrated to act as antioxidants both in vitro and in vivo. In this study, different anthocyanins namely delphinidin, (DP) cyanidin (CY), and their glycosilated and rutinosinated derivatives, i.e. delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside, and delphinidin-3-rutinoside, contained in blackcurrant, have been tested for their ability to influence cell cycle progression in normal human fibroblasts