N6L pseudopeptide interferes with nucleophosmin protein-protein interactions and sensitizes leukemic cells to chemotherapy.

NPM1 is a multifunctional nucleolar protein implicated in several processes such as ribosome maturation and export, DNA damage response and apoptotic response to stress stimuli. The NPM1 gene is involved in human tumorigenesis and is found mutated in one third of acute myeloid leukemia patients, leading to the aberrant cytoplasmic localization of NPM1. Recent studies indicated that the N6L multivalent pseudopeptide, a synthetic ligand of cell-surface nucleolin, is also able to bind NPM1 with high affinity. N6L inhibits cell growth with different mechanisms and represents a good candidate as a novel anticancer drug for a number of malignancies of different histological origin. In this study we investigated whether N6L treatment could drive antitumor effect in acute myeloid leukemia cell lines. We found that N6L binds NPM1 at the N-terminal domain, co-localizes with cytoplasmic, mutated NPM1, and interferes with its protein-protein associations. N6L toxicity appears to be p53 dependent but interestingly, the leukemic cell line harbouring the mutated form of NPM1 is more resistant to treatment, suggesting that NPM1 cytoplasmic delocalization confers protection from p53 activation. Moreover, we show that N6L sensitizes AML cells to doxorubicin and cytarabine treatment. These studies suggest that N6L may be a promising option in combination therapies for acute myeloid leukemia treatment.

Serum from differently exercised subjects induces myogenic differentiation in LHCN-M2 human myoblasts.

Myogenesis is the formation of muscle tissue from muscle precursor cells. Physical exercise induces satellite cell activation in muscle. Currently, C2C12 murine myoblast cells are used to study myogenic differentiation. Herein, we evaluated whether human LHCN-M2 myoblasts can differentiate into mature myotubes and express early (myotube formation, creatine kinase activity and myogenin) and late (MyHC-ß) muscle-specific markers when cultured in differentiation medium (DM) for 2, 4 and 7 days. We demonstrate that treatment of LHCN-M2 cells with DM supplemented with 0.5% serum from long-term (3 years) differently exercised subjects for 4 days induced myotube formation and significantly increased the early (creatine kinase activity and myogenin) and late (MyHC-ß expression) differentiation markers versus cells treated with serum from untrained subjects. Interestingly, serum from aerobic exercised subjects (swimming) had a greater positive effect on late-differentiation marker (MyHC-ß) expression than serum from anaerobic (body building) or from mixed exercised (soccer and volleyball) subjects. Moreover, p62and anti-apoptotic Bcl-2 protein expression was lower in LHCN-M2 cells cultured with human sera from differently exercised subjectst han in cells cultured with DM. In conclusion, LHCN-M2 human myoblasts represent a species-specific system with which to study human myogenic differentiation induced by serum from differently exercised subjects.

A novel miR-371a-5p-mediated pathway, leading to BAG3 upregulation in cardiomyocytes in response to epinephrine, is lost in Takotsubo cardiomyopathy.

Molecular mechanisms protecting cardiomyocytes from stress-induced death, including tension stress, are essential for cardiac physiology and defects in these protective mechanisms can result in pathological alterations. Bcl2-associated athanogene 3 (BAG3) is expressed in cardiomyocytes and is a component of the chaperone-assisted autophagy pathway, essential for homeostasis of mechanically altered cells. BAG3 ablation in mice results in a lethal cardiomyopathy soon after birth and mutations of this gene have been associated with different cardiomyopathies including stress-induced Takotsubo cardiomyopathy (TTC). The pathogenic mechanism leading to TTC has not been defined, but it has been suggested that the heart can be damaged by excessive epinephrine (epi) spillover in the absence of a protective mechanism. The aim of this study was to provide more evidence for a role of BAG3 in the pathogenesis of TTC. Therefore, we sequenced BAG3 gene in 70 TTC patients and in 81 healthy donors with the absence of evaluable cardiovascular disease. Mutations and polymorphisms detected in the BAG3 gene included a frequent nucleotide change g2252c in the BAG3 3'-untranslated region (3'-UTR) of Takotsubo patients (P<0.05), resulting in loss of binding of microRNA-371a-5p (miR-371a-5p) as evidenced by dual-luciferase reporter assays and argonaute RNA-induced silencing complex catalytic component 2/pull-down assays. Moreover, we describe a novel signaling pathway in cardiomyocytes that leads to BAG3 upregulation on exposure to epi through an ERK-dependent upregulation of miR-371a-5p. In conclusion, the presence of a g2252c polymorphism in the BAG3 3'-UTR determines loss of miR-371a-5p binding and results in an altered response to epi, potentially representing a new molecular mechanism that contributes to TTC pathogenesis.

miR-205-5p-mediated downregulation of ErbB/HER receptors in breast cancer stem cells results in targeted therapy resistance.

The ErbB tyrosine kinase receptor family has been shown to have an important role in tumorigenesis, and the expression of its receptor members is frequently deregulated in many types of solid tumors. Various drugs targeting these receptors have been approved for cancer treatment. Particularly, in breast cancer, anti-Her2/EGFR molecules represent the standard therapy for Her2-positive malignancies. However, in a number of cases, the tumor relapses or progresses thus suggesting that not all cancer cells have been targeted. One possibility is that a subset of cells capable of regenerating the tumor, such as cancer stem cells (CSCs), may not respond to these therapeutic agents. Accumulating evidences indicate that miR-205-5p is significantly downregulated in breast tumors compared with normal breast tissue and acts as a tumor suppressor directly targeting oncogenes such as Zeb1 and ErbB3. In this study, we report that miR-205-5p is highly expressed in BCSCs and represses directly ERBB2 and indirectly EGFR leading to resistance to targeted therapy. Furthermore, we show that miR-205-5p directly regulates the expression of p63 which is in turn involved in the EGFR expression suggesting a miR-205/p63/EGFR regulation.

Breast cancer stem cells rely on fermentative glycolysis and are sensitive to 2-deoxyglucose treatment.

A number of studies suggest that cancer stem cells are essential for tumour growth, and failure to target these cells can result in tumour relapse. As this population of cells has been shown to be resistant to radiation and chemotherapy, it is essential to understand their biology and identify new therapeutic approaches. Targeting cancer metabolism is a potential alternative strategy to counteract tumour growth and recurrence. Here we applied a proteomic and targeted metabolomic analysis in order to point out the main metabolic differences between breast cancer cells grown as spheres and thus enriched in cancer stem cells were compared with the same cells grown in adherent differentiating conditions. This integrated approach allowed us to identify a metabolic phenotype associated with the stem-like condition and shows that breast cancer stem cells (BCSCs) shift from mitochondrial oxidative phosphorylation towards fermentative glycolysis. Functional validation of proteomic and metabolic data provide evidences for increased activities of key enzymes of anaerobic glucose fate such as pyruvate kinase M2 isoform, lactate dehydrogenase and glucose 6-phopshate dehydrogenase in cancer stem cells as well as different redox status. Moreover, we show that treatment with 2-deoxyglucose, a well known inhibitor of glycolysis, inhibits BCSC proliferation when used alone and shows a synergic effect when used in combination with doxorubicin. In conclusion, we suggest that inhibition of glycolysis may be a potentially effective strategy to target BCSCs.

FLASH is essential during early embryogenesis and cooperates with p73 to regulate histone gene transcription.

Replication-dependent histone gene expression is a fundamental process occurring in S-phase under the control of the cyclin-E/CDK2 complex. This process is regulated by a number of proteins, including Flice-Associated Huge Protein (FLASH) (CASP8AP2), concentrated in specific nuclear organelles known as HLBs. FLASH regulates both histone gene transcription and mRNA maturation, and its downregulation in vitro results in the depletion of the histone pull and cell-cycle arrest in S-phase. Here we show that the transcription factor p73 binds to FLASH and is part of the complex that regulates histone gene transcription. Moreover, we created a novel gene trap to disrupt FLASH in mice, and we show that homozygous deletion of FLASH results in early embryonic lethality, owing to arrest of FLASH(-/-) embryos at the morula stage. These results indicate that FLASH is an essential, non-redundant regulator of histone transcription and cell cycle during embryogenesis.

[Treatment of obstructive defecation syndrome related to hedrocele. Our experience]. / Trattamento chirurgico della sindrome da ostruita defecazione correlata alla presenza di hedrocele. Nostra esperienza.

Hedrocele represents an unusual variant of the rare posterior perineal hernia and results from a defect in the rectogenital septum. We report two cases of obstructive defecation syndrome (ODS) related to presence of hedrocele successfully treated by laparoscopy-assisted transanal surgery.

FLASH degradation in response to UV-C results in histone locus bodies disruption and cell-cycle arrest.

Eucaryotic cell nuclei contain a number of different organelles that are highly dynamic structures and respond to a variety of stimuli. Here we investigated the effect of UV irradiation on a recently identified group of organelles, Histone Locus Bodies. Histone Locus Bodies contain at least two main proteins, FLASH and NPAT, and have been shown to be involved in replication-dependent histone gene transcription. We show that these organelles are disrupted after sublethal irradiation and both FLASH and NPAT are degraded, which in turn results in cell-cycle arrest at the S/G2 transition. The effect on the cell cycle is due to reduced transcription of histone genes and restoring normal histone protein levels by stabilizing histone mRNA allows cells to progress through the cell cycle. This provides a novel mechanism of S-phase arrest in response to DNA damage that potentially allows DNA repair before cells continue into mitosis, and thus prevents transmission of genomic alterations.

[Low rectovaginal fistula treated with platelet-rich plasma (PRP)]. / Fistola retto-vaginale bassa post-radioterapia trattata con l'ausilio di PRP (gel piastrinico autologo).

Rectovaginal fistula (RVF) is an abnormal communication between vagina and anorectum. Most frequent causes are iatrogenic. Often surgery is the therapy of choice. The suggested treatments include trans-anal access or combined trans-anal and vaginal access. We present the case of a woman with complicated iatrogenic fistula treated by a combined trans-anal and vaginal access, interposition of buccal mucosa and opposition of PRP (platelet rich plasma).

[Gastric lipoma presenting as intestinal obstruction]. / Occlusione intestinale alta da lipoma gastrico.

Gastric lipoma is a rare benign tumor. The symptoms are correlated with the size and the dimensions of neoplasm. It can be the cause of bleeding, gastroduodenal intussusception and intestinal obstruction, as in case reported and surgically treated. The Authors make a literature review to define the better diagnostic and surgical approach.

Tumor-induced hypophosphatemic osteomalacia associated with tertiary hyperparathyroidism: a case report.

BACKGROUND: Tumor-induced hypophosphatemic osteomalacia is a syndrome characterized by urinary phosphate wasting related to the presence of a slowly-growing tumor of mesenchymal origin. The characteristic laboratory findings are normal serum calcium, marked hypophosphatemia, increased serum alkaline phosphatase, markedly reduced renal tubular reabsorption of phosphorus and inappropriately low levels of 1,25-dihydroxyvitamin D [1,25-(OH)2D]. CASE PRESENTATION: A 65-year-old woman presented with a 17-year clinical history of musculoskeletal pain, muscular weakness in the pelvic girdle, spontaneous fractures and difficulty in walking. Over the ensuing years the patient suffered other multiple spontaneous fractures, surgically treated, and the muscular pains worsened until she became bedridden. During the years before hospital admission the patient received treatment with clodronate, oral calcium salts and vitamin D therapy. Standard laboratory, ultrasonography and scintigraphic findings provided a "convenient" diagnosis of primary hyperparathyroidism, but the low plasma level of phosphorus induced to perform an Indium111-octreotide scintigraphy. Scintigraphy visualized an area of pathologic increased signal uptake in the left groin, consistent with a mass containing a high density of somatostatin receptors. After surgery, histologic examination and immunostaining of the resected specimen indicated an hemangiopericytoma. Nevertheless, the persistently low blood phosphorus level, in association with the increased serum calcium and PTH levels, were attributed to the prolonged phosphate therapy the patient underwent over the years, and the persisting abnormal laboratory indexes indicated the development of a tertiary hyperparathyroidism. We performed a subtotal parathyroidectomy and intraoperative assay of serum PTH showed that levels had diminished by more than 80% from preoperative values. Over the ensuing months Ca+2, PTH and serum phosphorus values returned to normal, and the pain symptoms disappeared. CONCLUSIONS: Tumour-induced osteomalacia is a very rare syndrome associated in 5% of cases with tertiary hyperparathyroidism due to long-term therapy with phosphorus and vitamin D. The initial diagnosis of primary hyperparathyroidism, confirmed by the parathyroid MIBI-scintigraphy, would lead us to an inappropriate surgical treatment. Therefore we want to stress the importance of In111-octreotide scintigraphy in detecting tumours, rich in somatostatin receptors, in presence of an hypophosphatemic syndrome.

[Rectal stenosis after stapler hemorrhoidopexy]. / Stenosi rettale dopo proctopessi con stapler per patologia emorroidaria.

We report a case of a 52-year-old woman with hemorrhoids submitted to Procedure for Prolapse and Hemorrhoids (PPH), i.e. stapled hemorrhoidopexy, using a modified Longo's technique (double purse string) to treat an associated rectocele and rectal prolapse; the STARR technique (Stapled Trans Anal Rectal Resection) was not yet codified. The post-operative course was uneventful and the patient is discharged in II p.o. day. In fifth post-operative day the patient came back with an unusual rectal stenosis due to the superior purse string. The stricture was easily resolved by 'opening' the purse string with a transanal approach in outpatient setting.

[Adhesion ileus in patient with previous diagnosis of Ogilvie's syndrome]. / Occlusione intestinale su briglia in paziente con pregressa diagnosi di sindrome di Ogilvie.

A case of a 72-year-old man with abdominal pain and ileus is reported. Previous surgery for Ogilvie's syndrome had been performed. Despite conservative therapy, the occlusive symptoms worsen. Therefore the patient was submitted to surgery. At laparotomy two abdominal adhesions were found and sectioned. The differential diagnosis between mechanical ileus and pseudoobstruction for neuro-mechanics dissociation (Ogilvie's syndrome) is difficult, particularly in patients with neurodegenerative diseases.

[Gangrene of Meckel's diverticulum in strangulated left inguinal hernia]. / Ernia inguinale sinistra strozzata con necrosi d'ansa e di diverticolo di Meckel.

We report a case of a 57-year-old woman admitted for abdominal pain and a not reducible mass in left inguino-abdominal region. With a diagnosis of strangulated inguinal hernia, the patient underwent urgent surgery. The surgical exploration showed a gangrenous intestinal loop with a Meckel's necrotic diverticulum. A small bowel resection (20 cm) was performed. The post-operative course was uneventful. This seems the first case reported in the literature of woman with a Meckel's diverticulum involved in a strangulated left inguinal hernia.

The complexity of pathways for protein import into thylakoids: it's not easy being green.

Numerous proteins are transported into or across the chloroplast thylakoid membrane. To date, two major pathways have been identified for the transport of luminal proteins (the Sec- and Tat-dependent pathways) and it is now clear that these protein translocases use fundamentally different transport mechanisms. Integral membrane proteins are inserted by means of at least two further pathways. One involves the input of numerous targeting factors, including SRP (signal recognition particle), FtsY and Albino3. Surprisingly, the other pathway does not involve any of the known chloroplastic targeting factors, and insertion is energy-independent, raising the possibility of an unusual 'spontaneous' insertion mechanism.

[Strangulated abdominal herniation by Ladd's band]. / Ernia intestinale strozzata su briglia di Ladd.

A case of a 64-year-old man with abdominal pain since three days, with vomiting and ileus and without previous surgery is reported. Faecal material was aspirated by a nasogastric tube; TC showed dilatation of the small bowel with the walls of the loops thickened. The patient was submitted to emergency surgery with diagnosis of intestinal infarction. At laparotomy an extensive necrosis (two meters) of small bowel was present, caused by visceral herniation by a Ladd's band. Intestinal resection was performed with latero-lateral stapled anastomosis. The postoperative course was uneventful and the patient was transferred in VII p.o. day to an hospital of his Country.

[Prevention of post-operative pain and haemorrhage in PPH (Procedure for Prolapse and Hemorrhoids) and STARR (Stapled Trans-Anal Rectal Resection). Preliminary results in 261 cases]. / La prevenzione del dolore e dell'emorragia post-operatoria nella PPH (Procedure for Prolapse and Hemorrhoids) e nella STARR (Stapled Trans-Anal Rectal Resection). Risultati su una serie di 261 pazienti.

Intra- and early (first week) post-operative haemorrhages are the most common complications in stapled hemorrhoidectomy PPH (Procedure for Prolapse and Hemorrhoids) and in circumferential resection of the rectal prolapse STARR (Stapled Trans Anal Rectal Resection). Performing PPH and STARR we employed a gelatin based haemostatic sealant with thrombin component (FloSeal) to control intra-operative bleeding and to reduce post-operative bleeding avoiding haemostatic stitches on suture line. We report the preliminary results on 197 PPH and 64 STARR; 44 PPH (22.4%) and 27 STARR (42.2%) were treated by FloSeal. No major post-operative bleeding was observed in all patients treated by FloSeal, compared to 1.3% and 2.7% of hemorrhage respectively in PPH and STARR patients treated without sealant. Post-operative pain was less severe in patients treated by FloSeal, without a difference statistically significant. The data are preliminary and must be confirmed in prospective randomized trials in larger series.

Ricin A chain without its partner B chain is degraded after retrotranslocation from the endoplasmic reticulum to the cytosol in plant cells.

When expressed in tobacco cells, the catalytic subunit of the dimeric ribosome inactivating protein, ricin, is first inserted into the endoplasmic reticulum (ER) and then degraded in a manner that can be partially inhibited by the proteasome inhibitor clasto-lactacystin beta-lactone. Consistent with the implication of cytosolic proteasomes, degradation of ricin A chain is brefeldin A-insensitive and the polypeptides that accumulate in the presence of the proteasome inhibitor are not processed in a vacuole-specific fashion. Rather, these stabilized polypeptides are in part deglycosylated by a peptide:N-glycanase-like activity. Taken together, these results indicate that ricin A chain, albeit a structurally native protein, can behave as a substrate for ER to cytosol export, deglycosylation in the cytosol, and proteasomal degradation. Furthermore, retrotranslocation of this protein is not tightly coupled to proteasomal activity. These data are consistent with the hypothesis that ricin A chain can exploit the ER-associated protein degradation pathway to reach the cytosol. Although well characterized in mammalian and yeast cells, the operation of a similar pathway to the cytosol of plant cells has not previously been demonstrated.