M6A reduction relieves FUS-associated ALS granules.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by aggregation of mutant proteins, among which the RNA binding protein FUS. Here we show that, in neuronal cells and in iPSC-derived MN expressing mutant FUS, such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished. Interestingly, stress granules formed in ALS conditions showed a distinctive transcriptome with respect to control cells, which reverted to similar to control after m6A downregulation. Notably, cells expressing mutant FUS were characterized by higher m6A levels suggesting a possible link between m6A homeostasis and pathological aggregates. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, an inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

CircAFF1 Is a Circular RNA with a Role in Alveolar Rhabdomyosarcoma Cell Migration.

Circular RNAs (circRNAs), covalently closed RNAs that originate from back-splicing events, participate in the control of several processes, including those that occur in the development of pathological conditions such as cancer. Hereby, we describe circAFF1, a circular RNA overexpressed in alveolar rhabdomyosarcoma. Using RH4 and RH30 cell lines, a classical cell line models for alveolar rhabdomyosarcoma, we demonstrated that circAFF1 is a cytoplasmatic circRNA and its depletion impacts cell homeostasis favouring cell migration through the downregulation of genes involved in cell adhesion pathways. The presented data underline the importance of this circular RNA as a new partial suppressor of the alveolar rhabdomyosarcoma tumour progression and as a putative future therapeutic target.

The m6A reader YTHDC1 and the RNA helicase DDX5 control the production of rhabdomyosarcoma-enriched circRNAs.

N6-Methyladenosine (m6A) is well-known for controlling different processes of linear RNA metabolism. Conversely, its role in the biogenesis and function of circular RNAs (circRNAs) is still poorly understood. Here, we characterize circRNA expression in the pathological context of rhabdomyosarcoma (RMS), observing a global increase when compared to wild-type myoblasts. For a set of circRNAs, such an increase is due to the raised expression of the m6A machinery, which we also find to control the proliferation activity of RMS cells. Furthermore, we identify the RNA helicase DDX5 as a mediator of the back-splicing reaction and as a co-factor of the m6A regulatory network. DDX5 and the m6A reader YTHDC1 are shown to interact and to promote the production of a common subset of circRNAs in RMS. In line with the observation that YTHDC1/DDX5 depletion reduces RMS proliferation, our results provide proteins and RNA candidates for the study of rhabdomyosarcoma tumorigenicity.

Role of New Anatomy, Biliopancreatic Reflux, and Helicobacter Pylori Status in Postgastrectomy Stump Cancer.

Distal gastrectomy for benign gastroduodenal peptic disease has become rare, but it still represents a widely adopted procedure for advanced and, in some countries, even for early distal gastric cancer. Survival rates following surgery for gastric malignancy are constantly improving, hence the residual mucosa of the gastric stump is exposed for a prolonged period to biliopancreatic reflux and, possibly, to Helicobacter pylori (HP) infection. Biliopancreatic reflux and HP infection are considered responsible for gastritis and metachronous carcinoma in the gastric stump after oncologic surgery. For gastrectomy patients, in addition to eradication treatment for cases that are already HP positive, endoscopic surveillance should also be recommended, for prompt surveillance and detection in the residual mucosa of any metaplastic-atrophic-dysplastic features following surgery.

Enhanced amphiphilic profile of a short ß-stranded peptide improves its antimicrobial activity.

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a ß-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH2, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH2, ß-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular ß-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, ß-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state 19F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and ß-SB056-lin adopt a ß-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized ß-SB056-lin forms well-ordered ß-strands once electro-neutrality is approached, and it aggregates into more extended ß-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended ß-sheets, which also leads to a dramatic alteration of membrane integrity as shown by 31P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic ß-strands.

Extent of lymphadenectomy and perioperative therapies: two open issues in gastric cancer.

Gastric cancer is one of the leading causes of death for cancer worldwide, although geographical variations in incidence exist. Over the last decades, its incidence and mortality have gradually decreased in Western countries, while these have increased, or remained stable, in the other world regions. Gastric cancer is often diagnosed at an advanced stage, with the only notable exception of Japan, where nationwide screening programs are enforced, due to local high incidence. Curative- intent surgery (i.e., gastrectomy, total or partial, and lymphadenectomy) remains the cornerstone of treatment of gastric cancer. Much has been debated about the extent of lymph node dissection and, although it is a valuable contribution to staging and cure, operative treatment only represents one aspect of overall effective management, as the risk of both locoregional and distant recurrences are high, and bear a poor prognosis. As a matter of fact, surgery, as a single modality treatment, has probably achieved its maximum efficacy for local control and survival, while other accompanying nonsurgical treatment modalities have to be taken into account, although their role is still the subject of considerable debate. The authors in this review present an update on the outcome of treatment of gastric cancer in relation to the extent of lymphadenectomy and of various nonsurgical preoperative, intraoperative, and postoperative strategies.

Inhibition of human respiratory syncytial virus infectivity by a dendrimeric heparan sulfate-binding peptide.

Respiratory syncytial virus (RSV) interacts with cell surface heparan sulfate proteoglycans (HSPGs) to initiate infection. The interaction of RSV with HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In the present study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was screened with the aim of identifying peptides able to bind HSPGs and thus block RSV attachment and infectivity. Of the compounds identified, the dendrimer SB105-A10 was the most potent inhibitor of RSV infectivity, with 50% inhibitory concentrations (IC(50)s) of 0.35 µM and 0.25 µM measured in Hep-2 and A549 cells, respectively. SB105-A10 was found to bind to both cell types via HSPGs, suggesting that its antiviral activity is indeed exerted by competing with RSV for binding to cell surface HSPGs. SB105-A10 prevented RSV infection when added before the viral inoculum, in line with its proposed HSPG-binding mechanism of action; moreover, antiviral activity was also exhibited when SB105-A10 was added postinfection, as it was able to reduce the cell-to-cell spread of the virus. The antiviral potential of SB105-A10 was further assessed using human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. SB105-A10 strongly reduced RSV infectivity in this model and exhibited no signs of cytotoxicity or proinflammatory effects. Together, these features render SB105-A10 an attractive candidate for further development as a RSV inhibitor to be administered by aerosol delivery.

Inhibition of herpes simplex virus type 1 and type 2 infections by peptide-derivatized dendrimers.

In response to the need for new antiviral agents, dendrimer-based molecules have been recognized as having a large number of potential therapeutic applications. They include peptide-derivatized dendrimers, which are hyperbranched synthetic well-defined molecules which consist of a peptidyl branching core and covalently attached surface functional peptides. However, few studies have addressed their applications as direct-acting antiviral agents. Here, we report on the ability of the peptide dendrimer SB105 and its derivative, SB105_A10, to directly inhibit herpes simplex virus 1 (HSV-1) and HSV-2 in vitro replication, with favorable selective indexes discerned for both compounds. An analysis of their mode of action revealed that SB105 and SB105_A10 prevent HSV-1 and HSV-2 attachment to target cells, whereas SB104, a dendrimer with a different amino acid sequence within the functional group and minimal antiviral activity, was ineffective in blocking HSV attachment. Moreover, both SB105 and SB105_A10 retained their ability to inhibit HSV adsorption at pH 3.0 and 4.0 and in the presence of 10% human serum proteins, conditions mimicking the physiological properties of the vagina, a potential therapeutic location for such compounds. The inhibition of HSV adsorption is likely to stem from the ability of SB105_A10 to bind to the glycosaminoglycan moiety of cell surface heparan sulfate proteoglycans, thereby blocking virion attachment to target cells. Finally, when combined with acyclovir in checkerboard experiments SB105_A10 exhibited highly synergistic activity. Taken together, these findings suggest that SB105 and SB105_A10 are promising candidates for the development of novel topical microbicides for the prevention of HSV infections.

Endoluminal metastasis of colon cancer to the urinary bladder via the ureter: report of a case.

A 67-year-old woman was admitted for intermittent gross hematuria. Her medical history included a right colectomy for cancer of the ascending colon and removal of metastatic nodes adjacent to the right internal iliac vessels, respectively at 63 and 65 years of age. Cystoscopy detected a semi-pedunculated, nonpapillary (3.5-4 cm diameter) tumor situated above the right ureteral orifice. The histological evaluation of the resected specimen revealed metastatic colonic adenocarcinoma. The history and pathological findings were consistent with a mechanism of endoluminal implantation of adenocarcinoma of the large bowel to the bladder via the right ureter.

Screening of Helicobacter pylori infection after gastrectomy for cancer or peptic ulcer: results of a cohort study.

BACKGROUND: Gastric cancer commonly follows a long-standing inflammation, mainly due to Helicobacter pylori (HP) infection. After resection, the stump develops precancerous alterations. DESIGN: Prospective study of patients undergoing endoscopy from April 1, 2000, through March 31, 2006. SETTING: University departments of Surgery and Experimental Medicine and Pathology. PATIENTS: One hundred eighty-seven patients receiving upper gastrointestinal tract endoscopy many years after surgery for duodenal ulcer or gastric cancer. Ten to 12 postoperative endoscopic biopsy samples were taken from the remnant stomach. MAIN OUTCOME MEASURE: The risk of gastric cancer precursor lesions associated with HP infection. RESULTS: The gastric cancer precursor lesions were more common in the entire HP-positive population (odds ratio [OR], 2.37; 95% confidence interval [CI], 1.25-4.49; P = .007). However, HP-positive patients undergoing resection for cancer had a higher risk of the precursor lesions compared with HP-negative patients in the same diagnostic group (OR, 4.20; 95% CI, 1.10-15.96) and all patients undergoing resection for duodenal ulcer (OR, 1.59; 95% CI, 0.44-5.73). CONCLUSION: The results of this investigation support the role of HP in gastric carcinogenesis and suggest that the HP eradication therapy might prevent the development of metachronous gastric cancer after gastric resection.

Identification of a dendrimeric heparan sulfate-binding peptide that inhibits infectivity of genital types of human papillomaviruses.

Peptide dendrimers consist of a peptidyl branching core and/or covalently attached surface functional units. They show a variety of biological properties, including antiviral activity. In this study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was evaluated for in vitro activity against human papillomaviruses (HPVs). The peptide dendrimer SB105-A10 was found to be a potent inhibitor of genital HPV types (i.e., types 16, 18, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 2.8 and 4.2 µg/ml (0.59 and 0.88 µM), and no evidence of cytotoxicity was observed. SB105-A10 interacts with immobilized heparin and with heparan sulfates exposed on the cell surface, most likely preventing virus attachment. The findings from this study indicate SB105-A10 to be a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections.

Quantitative investigation of desmoplasia as a prognostic indicator in colorectal cancer.

BACKGROUND: The role of desmoplastic reaction (DR) in colorectal cancer invasion is still an open question. The presence of fibrous connective tissue may represent a barrier against cancer diffusion or a stroma to build up and support the tumor. Aims of the present study were to evaluate the influence of DR on long-term survival and to validate a reliable quantitative method to measure the desmoplastic tissue. METHODS: This retrospective study included 86 patients who underwent radical colorectal resection for cancer, from a database of 429 patients. To achieve a quantitative histochemical measurement of DR, digital images were analyzed by a computerized image analysis program. DR was related to the overall survival and the quantitative method was related to the traditional one. RESULTS: By using the Kaplan-Meier analysis, DR was found to be significantly associated with overall survival. Patients with a higher value of DR survived longer than those with smaller DR and the quantitative results were in accordance with those obtained by using the traditional methods. CONCLUSIONS: Desmoplasia seems to be a protective factor for survival in patients with colorectal carcinoma. The quantitative technique is easily standardized and can be routinely performed, so that DR may be a useful prognostic indicator. Notwithstanding, the conflicting outcomes reported in literature about DR need further biological and molecular studies to achieve definitive conclusions.

Synthesis, characterization, antimicrobial activity and LPS-interaction properties of SB041, a novel dendrimeric peptide with antimicrobial properties.

Multimeric peptides offer several advantages with respect to their monomeric counterparts, as increased activity and greater stability to peptidases and proteases. SB041 is a novel antimicrobial peptide with dendrimeric structure; it is a tetramer of pyrEKKIRVRLSA linked by a lysine core, with an amino valeric acid chain. Here, we report on its synthesis, NMR characterization, antimicrobial activity, and LPS-interaction properties. The peptide was especially active against Gram-negative strains, with a potency comparable (on molar basis) to that of lipopeptides colistin and polymixin B, but it also displayed some activity against selected Gram-positive strains. Following these indications, we investigated the efficacy of SB041 in binding Escherichia coli and Pseudomonas aeruginosa LPS in vitro and counteracting its biological effects in RAW-Blue cells, derived from RAW 264.7 macrophages. SB041 strongly bound purified LPS, especially that of E. coli, as proved by fluorescent displacement assay, and readily penetrated into LPS monolayers. However, the killing activity of SB041 against E. coli was not inhibited by increasing concentrations of LPS added to the medium. Checking the SB041 effect on LPS-induced activation of pattern recognition receptors (PRRs) in Raw-Blue cells revealed that while the peptide gave a statistically significant decrease in PRRs stimulation when RAW-Blue cells were challenged with P. aeruginosa LPS, the same was not seen when E. coli LPS was used to activate innate immune defense-like responses. Thus, as previously seen for other antimicrobial peptides, also for SB041 binding to LPS did not translate necessarily into LPS-neutralizing activity, suggesting that SB041-LPS interactions must be of complex nature.

Peptide-derivatized dendrimers inhibit human cytomegalovirus infection by blocking virus binding to cell surface heparan sulfate.

Dendrimers are hyperbranched synthetic well-defined molecules with a number of potential applications, especially in relation to the need for new antiviral agents. One subclass of dendrimers are peptide-derivatized dendrimers which consist of a peptidyl branching core and covalently attached surface peptide functional units. Few studies have addressed the potential uses of peptide dendrimers as direct-acting antiviral agents. Here, we report on the ability of two peptide dendrimers, SB105 and SB105_A10, to directly and almost completely inhibit human cytomegalovirus (HCMV) replication in both primary fibroblasts and endothelial cells; the agents were also found to inhibit murine CMV replication, whereas they were not able to inhibit adenovirus or vesicular stomatitis virus. The peptide dendrimers prevented adsorption of the HCMV to cells at 4 degrees C, whereas SB104, a dendrimer with a different amino acid sequence within the functional group and minimal anticytomegaloviral activity, was ineffective in blocking HCMV attachment. In effect, SB105_A10 bound to human cells through an interaction with cell surface heparan sulfate and thereby blocked virion attachment to target cells. These results indicate that the SB105 and SB105_A10 dendrimers could provide a useful starting point for the development of novel molecules to block HCMV infection.

Characterization of the branched antimicrobial peptide M6 by analyzing its mechanism of action and in vivo toxicity.

We analyzed functional activity of the antimicrobial peptide M6 in vitro and in vivo. The peptide was identified by our group by phage library selection, rational modification and synthesis in a tetrabranched form (Pini et al., Antimicrob. Agents Chemother. 2005; 49: 2665-72). We found that it binds lipopolysaccharide, causes perforation of cell membranes without destroying external cell morphology and strongly binds DNA. The latter feature suggests that it could inhibit metabolic pathways, blocking DNA replication and/or transcription. We also observed that M6 does not stimulate humoral immune response when repeatedly administered to animals. We also analyzed M6 toxicity when administered to animals by intraperitoneal or by intravenous injection, determining a preliminary LD50 (125 and 37.5 mg/kg, respectively), which suggested that M6 could be used in vivo. These features make the antimicrobial branched peptide M6 a promising candidate for the development of a new antibacterial drug.

Silent colon carcinoma presenting as a hepatic abscess.

Penetration and abscess formation in an adjacent parenchymal organ as presentation of a colon cancer is very uncommon. We report a rare case of pyogenic liver abscess as the first manifestation of an infiltrative and penetrating hepatic flexure colon carcinoma without liver metastases. A 50-year-old woman was admitted with right abdominal pain, fever and chills. The initial diagnosis was a pyogenic liver abscess. Subsequent CT scan and colonoscopy evidenced a hepatic flexure colon cancer abscessed within segment 6 of the liver. Eight months after a right colectomy and liver resection there was no evidence of disease. The occurrence of a pyogenic liver abscess should raise the suspicion of a silent colon cancer.

Large size, villous content and distal location are associated with severe dysplasia in colorectal adenomas.

UNLABELLED: Adenomatous polyps are precursor lesions for colorectal carcinoma. The risk of cancer development has been associated with age and size, amount of villous component and high-grade dysplasia of adenomas. The subject-related and adenoma-related risk factors for severely dysplastic lesions were further investigated. PATIENTS AND METHODS: The study was performed in 474 men and 339 women undergoing endoscopic removal at index colonoscopy of 1217 polyps. RESULTS: The male gender, cases aged over 55 and cases examined for rectal bleeding, showed an increased risk of colorectal polyps (odds ratios, OR = 1.95, 5.1 and 2.99, respectively). Adenomas synchronous with hyperplastic polyps of larger diameter (>10 mm) showed an increased risk of severe dysplasia (OR = 6.94). Severe dysplasia occurred more significantly in younger subjects harbouring villous growths (OR = 4.28, p < 0.03) and in larger adenomas (OR = 3.91, p < 0.001). The risk for severe dysplasia in relation to gender, age, multiplicity and location was higher in adenomas of larger diameter and with villous content. Multivariate analysis showed that distal site (p < 0.02), large size (p < 0.001) and villous content (p < 0.001) were the independent risk factors for severe dysplasia. CONCLUSION: Large size, villous content and distal location are associated with severe dysplasia in colorectal adenomas. The risk for severe dysplasia does not appear to be correlated with age.

Gastric cancer precursor lesions and Helicobacter pylori infection in patients with partial gastrectomy for peptic ulcer.

The mucosa of the gastric stump is considered at greater risk of dysplastic and neoplastic changes than that of the intact stomach. The combination of enteric reflux and Helicobacter pylori infection may have a synergistic damaging effect on the mucosa of the gastric remnant, both producing and increasing mucosal proliferation. The aim of this study was to assess whether the occurrence of H. pylori infection in the remnant mucosa of partially gastrectomized subjects for peptic ulcer disease is associated with an increase of the mucosal precursor lesions of malignancy. A series of 151 subjects who underwent partial gastrectomy for peptic ulcer disease were submitted to upper digestive endoscopy for long-term surveillance. Biopsy specimens of the gastric stump were tested for the occurrence of H. pylori infection and for the presence of precancerous mucosal lesions. The prevalence of H. pylori colonization in the remnant stomach was less than 30% and similar in subjects with different time intervals between gastrectomy and endoscopy. Age at surgery (chi(2): p = 0.03) and H. pylori infection (chi(2): p = 0.002) were significantly associated with the grading of mucosal lesions. The prevalence of normal mucosa was 10 times higher in H. pylori-negative patients as in H. pylori-positive ones (22.0% vs. 2.4%), and the prevalence of intestinal metaplasia was four times higher in H. pylori-positive patients than in H. pylori-negative ones (19.6% vs. 4.6%). We concluded that H. pylori infection may play a causal role in the development of gastric lesions in the operated stomach.

Antimicrobial activity of novel dendrimeric peptides obtained by phage display selection and rational modification.

A large 10-mer phage peptide library was panned against whole Escherichia coli cells, and an antimicrobial peptide (QEKIRVRLSA) was selected. The peptide was synthesized in monomeric and dendrimeric tetrabranched form (multiple antigen peptide [MAP]), which generally allows a dramatic increase of peptide stability to peptidases and proteases. The antibacterial activity of the dendrimeric peptide against E. coli was much higher than that of the monomeric form. Modification of the original sequence, by residue substitution or sequence shortening, produced three different MAPs, M4 (QAKIRVRLSA), M5 (KIRVRLSA), and M6 (QKKIRVRLSA) with enhanced stability to natural degradation and antimicrobial activity against a large panel of gram-negative bacteria. The MICs of the most potent peptide, M6, were as low as 4 to 8 microg/ml against recent clinical isolates of multidrug-resistant Pseudomonas aeruginosa and members of the Enterobacteriaceae. The same dendrimeric peptides showed high stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells, making them promising candidates for the development of new antibacterial drugs.