Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4.

The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters the pre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1.

A Proximity Complementation Assay to Identify Small Molecules That Enhance the Traffic of ABCA4 Misfolding Variants.

ABCA4-related retinopathy is the most common inherited Mendelian eye disorder worldwide, caused by biallelic variants in the ATP-binding cassette transporter ABCA4. To date, over 2200 ABCA4 variants have been identified, including missense, nonsense, indels, splice site and deep intronic defects. Notably, more than 60% are missense variants that can lead to protein misfolding, mistrafficking and degradation. Currently no approved therapies target ABCA4. In this study, we demonstrate that ABCA4 misfolding variants are temperature-sensitive and reduced temperature growth (30 °C) improves their traffic to the plasma membrane, suggesting the folding of these variants could be rescuable. Consequently, an in vitro platform was developed for the rapid and robust detection of ABCA4 traffic to the plasma membrane in transiently transfected cells. The system was used to assess selected candidate small molecules that were reported to improve the folding or traffic of other ABC transporters. Two candidates, 4-PBA and AICAR, were identified and validated for their ability to enhance both wild-type ABCA4 and variant trafficking to the cell surface in cell culture. We envision that this platform could serve as a primary screen for more sophisticated in vitro testing, enabling the discovery of breakthrough agents to rescue ABCA4 protein defects and mitigate ABCA4-related retinopathy.

Enhancing the light yield of He:CF 4 based gaseous detector.

The CYGNO experiment aims to build a large ( O ( 10 ) m 3 ) directional detector for rare event searches, such as nuclear recoils (NRs) induced by dark matter (DM), such as weakly interactive massive particles (WIMPs). The detector concept comprises a time projection chamber (TPC), filled with a He:CF 4 60/40 scintillating gas mixture at room temperature and atmospheric pressure, equipped with an amplification stage made of a stack of three gas electron multipliers (GEMs) which are coupled to an optical readout. The latter consists in scientific CMOS (sCMOS) cameras and photomultipliers tubes (PMTs). The maximisation of the light yield of the amplification stage plays a major role in the determination of the energy threshold of the experiment. In this paper, we simulate the effect of the addition of a strong electric field below the last GEM plane on the GEM field structure and we experimentally test it by means of a 10 × 10 cm 2 readout area prototype. The experimental measurements analyse stacks of different GEMs and helium concentrations in the gas mixture combined with this extra electric field, studying their performances in terms of light yield, energy resolution and intrinsic diffusion. It is found that the use of this additional electric field permits large light yield increases without degrading intrinsic characteristics of the amplification stage with respect to the regular use of GEMs.

Antisense oligonucleotide therapy corrects splicing in the common Stargardt disease type 1-causing variant ABCA4 c.5461-10T>C.

Stargardt disease type 1 (STGD1) is the most common hereditary form of maculopathy and remains untreatable. STGD1 is caused by biallelic variants in the ABCA4 gene, which encodes the ATP-binding cassette (type 4) protein (ABCA4) that clears toxic byproducts of the visual cycle. The c.5461-10T>C p.[Thr1821Aspfs∗6,Thr1821Valfs∗13] variant is the most common severe disease-associated variant, and leads to exon skipping and out-of-frame ABCA4 transcripts that prevent translation of functional ABCA4 protein. Homozygous individuals typically display early onset STGD1 and are legally blind by early adulthood. Here, we applied antisense oligonucleotides (AONs) to promote exon inclusion and restore wild-type RNA splicing of ABCA4 c.5461-10T>C. The effect of AONs was first investigated in vitro using an ABCA4 midigene model. Subsequently, the best performing AONs were administered to homozygous c.5461-10T>C 3D human retinal organoids. Isoform-specific digital polymerase chain reaction revealed a significant increase in correctly spliced transcripts after treatment with the lead AON, QR-1011, up to 53% correct transcripts at a 3 µM dose. Furthermore, western blot and immunohistochemistry analyses identified restoration of ABCA4 protein after treatment. Collectively, we identified QR-1011 as a potent splice-correcting AON and a possible therapeutic intervention for patients harboring the severe ABCA4 c.5461-10T>C variant.

Diffusion of laparoscopic appendectomies in Italy: a national audit.

A national audit focused on laparoscopic appendectomy was promoted by the Italian Association of Hospital Surgeons (ACOI). Four-hundred and sixty surgical practices received an e-mail questionnaire. Data concerning epidemiology, timetable, surgeon's age, selection of patients, laparotomic conversion, behaviour in the case of a normal appendix, and technical aspects were investigated. The response rate was 51.7%. The median number of appendectomies performed is 50-100 each year in a surgery ward. Laparoscopic operations are very common (93%), but mostly performed in less than 50% of the observed cases. There is no significant difference between the number of operations during the day vs. at night, and they are performed by a limited (<30%) group of surgeons, equally composed of physicians aged above and below 40. The majority of surgeons adopt an "all comers" policy regarding laparoscopic appendectomy, including selected older patients (>70 years old). There are no standard indications for conversion, while the behaviour in the presence of a normal appendix is generally removal. Even if laparoscopic appendectomy is not yet considered a gold standard, it is widely diffused in Italy, and the audit's data show different behaviours between subgroups.

miR-181a/b downregulation: a mutation-independent therapeutic approach for inherited retinal diseases.

Inherited retinal diseases (IRDs) are a group of diseases whose common landmark is progressive photoreceptor loss. The development of gene-specific therapies for IRDs is hampered by their wide genetic heterogeneity. Mitochondrial dysfunction is proving to constitute one of the key pathogenic events in IRDs; hence, approaches that enhance mitochondrial activities have a promising therapeutic potential for these conditions. We previously reported that miR-181a/b downregulation boosts mitochondrial turnover in models of primary retinal mitochondrial diseases. Here, we show that miR-181a/b silencing has a beneficial effect also in IRDs. In particular, the injection in the subretinal space of an adeno-associated viral vector (AAV) that harbors a miR-181a/b inhibitor (sponge) sequence (AAV2/8-GFP-Sponge-miR-181a/b) improves retinal morphology and visual function both in models of autosomal dominant (RHO-P347S) and of autosomal recessive (rd10) retinitis pigmentosa. Moreover, we demonstrate that miR-181a/b downregulation modulates the level of the mitochondrial fission-related protein Drp1 and rescues the mitochondrial fragmentation in RHO-P347S photoreceptors. Overall, these data support the potential use of miR-181a/b downregulation as an innovative mutation-independent therapeutic strategy for IRDs, which can be effective both to delay disease progression and to aid gene-specific therapeutic approaches.

Detailed Phenotyping and Therapeutic Strategies for Intronic ABCA4 Variants in Stargardt Disease.

Stargardt disease is a progressive retinal disorder caused by bi-allelic mutations in the ABCA4 gene that encodes the ATP-binding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease.

Differences in electromyographic activity of biceps brachii and brachioradialis while performing three variants of curl.

BACKGROUND: Dumbbell curl (DC) and barbell curl in its two variants, straight (BC) or undulated bar (EZ) are typical exercises to train the elbow flexors. The aim of the study was to verify if the execution of these three variants could induce a selective electromyographic (EMG) activity of the biceps brachii (BB) and brachioradialis (BR). METHODS: Twelve participants performed one set of ten repetitions at 65% of their 1-RM for each variant of curl. Pre-gelled electrodes were applied with an inter-electrode distance of 24 mm on BB and BR. An electrical goniometer was synchronously recorded with EMG signals to determine the concentric and eccentric phases of each variant of curl. RESULTS: We detected higher activation profile of both BB (P < 0.05) and BR (P < 0.01) during the EZ compared to the DC. Higher levels of activation was found during the concentric phase for only the BR performed with an EZ compared to DC (P < 0.001) and performing BC compared to DC (P < 0.05). The eccentric phase showed a higher activation of the BB muscle in EZ compared to DC (P < 0.01) and in BC compared to DC (P < 0.05). The BR muscle showed a higher activation performing EZ compared to DC (P < 0.01). DISCUSSION: The EZ variant may be preferred over the DC variant as it enhances BB and BR EMG activity during the whole range of motion and only in the eccentric phase. The small difference between BC and EZ variants of the BB and BR EMG activity makes the choice between these two exercises a matter of subjective comfort.

[Considerations regarding the role of arterial embolization with an angiographic approach in the treatment of upper gastrointestinal bleeding: personal experience]. / Considerazioni sul ruolo dell'embolizzazione celiaco-mesenterica con approccio arteriografico nel trattamento delle emorragie gastrointestinali alte: esperienza personale.

Upper gastrointestinal bleeding can be produced by varicose, inflammatory-ulcerative or neoplastic lesions of the eosophageal-gastric-duodenal anatomical district. The aim of this study was to define the role of arterial embolotherapy with an angiographic approach in the treatment of these conditions, starting from our personal experience and a review of the literature. The treatment of upper gastrointestinal bleeding is based on a multimodal approach in which arterial embolotherapy has its place alongside endoscopy and surgery.

[Rupture of the spleen in angiosarcoma: a case report and review of the literature]. / Rottura di milza in angiosarcoma: considerazioni su di un caso clinico e revisione della letteratura.

Splenic angiosarcoma is a rare neoplasm originating from endothelial cells of the blood vessels. Its incidence is about 0.14-0.25 per million. We report the case of a patient admitted in a state of hypovolaemic shock with haemoperitoneum due to rupture of the spleen. Splenectomy was performed with evacuation of the haemorrhagic effusion. The blood was aspirated and in part instilled during the operation through intraoperative blood salvage due to the large haemoperitoneum. Histological examination revealed a splenic angiosarcoma. Splenic angiosarcoma should be suspected in cases of splenomegaly with unknown anaemia and no lymphoma, leukaemia or myelofibrosis, because of its neoplastic aggressiveness and its invariably fatal outcome. It is important to perform a splenectomy before splenic rupture owing to its negative impact on long-term survival.

Pancreatic pseudocysts following acute pancreatitis: risk factors influencing therapeutic outcomes.

CONTEXT: The natural history of pancreatic pseudocysts has become well known in recent years, but the choice of a proper treatment still remains controversial. OBJECTIVE: This study aims at establishing whether predictive factors influencing therapeutic outcomes exist. SETTING: Patients with pancreatic pseudocysts following an episode of acute pancreatitis treated from January 1980 to December 2001 at the Department of General Surgery and Organ Transplantation of the University of Parma, Italy. PATIENTS: Seventy-four patients were studied: 12 had a spontaneous resolution, 37 patients were treated surgically, 15 were treated endoscopically and in 10, percutaneous drainage was used. MAIN OUTCOME MEASURES: Epidemiological, clinical and pathological characteristics of patients with pancreatic pseudocysts were related to morbidity, recurrence rates and hospital stay. RESULTS: At univariate logistic regression, our data reveal a significant increase in morbidity related to age (P=0.013), etiology (alcoholic vs. biliary, P=0.024), Ranson score of previous pancreatitis (P=0.006), nutritional assessment (P=0.001), residual necrosis (P<0.001) and modality of treatment (P=0.009), whereas none of these parameters has been shown to be significantly correlated to recurrence. At multivariate logistic regression, only residual necrosis was significantly related to morbidity. CONCLUSIONS: Some factors, such as epidemiological (age, etiology), clinical (severity of previous pancreatitis, malnourishment), pathological (residual necrosis), and therapeutical factors (emergency/urgency treatment) are predictive of worse outcomes for invasive treatment of pseudocysts. In particular residual necrosis appeared to be the most important factor influencing invasive treatment outcomes, confirming that this pathological aspect deserves particular attention from surgeons. No risk factors predicting pancreatic pseudocyst recurrence emerged.

[Therapy of post-necrotic pancreatic pseudocysts: invasive treatments and their results]. / Terapia delle pseudocisti pancreatiche postnecrotiche: trattamenti invasivi e loro risultati.

Pancreatic pseudocysts are the most common lesions of the pancreas. Endoscopic and US-endoscopic techniques are today the best minimally invasive diagnostic and therapeutic procedures available for this pathology. From January 1980 to December 2001 we observed a total of 74 patients with pancreatic pseudocysts secondary to acute pancreatitis. Twelve patients were treated by medical therapy, 37 with a surgical approach, 15 by endoscopic drainage and 10 by CT-guided drainage. The mean size of the pseudocysts was 12.9 cm (range: 3.4 to 24 cm) and 69.4% were larger than 10 cm. CT-guided drainage had a 50% complication rate (P = 0.00814), a 20% mortality rate (P = 0.00463) and a 10% relapse rate. The surgical approach was associated with a complication rate of 18.9% and a 5.4% relapse rate. The endoscopic approach presented a 13% morbidity rate and a 6.6% relapse rate. CT-guided drainage is the therapeutic approach we use in emergency cases, but endoscopic therapeutic technique is the best procedure and is a valid alternative to surgical and CT-guided drainage. The shortest mean hospital stay (4.8 days) was observed with the endoscopic approach.

Serous psammocarcinoma of the ovary: a case report and review of literature.

BACKGROUND: Serous psammocarcinoma is a form of ovarian carcinoma, characterized by massive psammoma body formation, invasiveness, and low-grade cytological features. We reported a new bilateral case of serous psammocarcinoma. We also reviewed the literature in order to delineate clinical, pathological, and prognostic features of this rare neoplasm. CASE HISTORY: A 66-year-old white woman was admitted to our Institution for a voluminous abdomino-pelvic mass, which was suspected to be a leiomyoma of the uterus. Abdominal computed tomography scan revealed a heavily calcified abdominopelvic mass with cystic areas. The origin of this lesion was unclear in diverse scanning of computed tomography. Elevated serum CA-125 levels suggested the eventuality of an ovarian neoplasm. Thus, the patient underwent an exploratory laparotomy. Intraoperatory findings showed two ovarian calcified lesions. The surface of omentum was covered with a white nodule. Pathological findings were consistent with serous psammocarcinoma with invasive implant of the omentum. CONCLUSION: Serous psammocarcinoma is a rare form of ovarian carcinoma with only 13 cases reported in literature. Patient's age can range from 18 to 76 years. Prognostic factors suggest that this neoplasm has more favorable prognosis than usual serous carcinomas. It is important to differentiate these two neoplasms and other pelvic mass, such as calcified leiomyoma.

Detection of Chlamydiae pneumoniae but not Helicobacter pylori DNA in atherosclerosis plaques.

Chronic infections have been associated with cardiovascular disease. We used bacterial culture, polymerase chain reaction (PCR), and immunohistochemical staining with anti-vacA and anticagA antibodies to search for Helicobacter pylori and Chlamydiae pneumoniae in atherosclerotic plaques obtained at endarterectomy. Serum IgG antibodies to H. pylori and C. pneumoniae were also determined. Thirty-two patients were enrolled. Anti-H. pylori and anti-C. pneumoniae IgG were present in 72% and 81%, respectively. Culture and PCR for H. pylori of vessel walls and plaques were negative. Atherosclerotic plaque and normal vessel sections from H. pylori-negative and- positive patients showed reactivity with anti-vacA and anti-cagA antibodies. C. pneumoniae DNA was amplified in three atherosclerotic lesions. These findings suggest that the association between H. pylori infection and atherosclerosis does not result from continuing direct effects of H. pylori antigens in the vessel walls. Antigens within vessel atherosclerotic plaques cross-react with H. pylori virulence factors and could act as cofactors in determining instability for the atherosclerotic plaques.