Assessment of Therapeutic Potential of a Dual AAV Approach for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a yet incurable rare genetic disease that affects the skeletal and cardiac muscles, leading to progressive muscle wasting and premature death. DMD is caused by the lack of dystrophin, a muscle protein essential for the biochemical support and integrity of muscle fibers. Gene replacement strategies for Duchenne muscular dystrophy (DMD) employing the adeno-associated virus (AAV) face the challenge imposed by the limited packaging capacity of AAV, only allowing the accommodation of a short version of dystrophin (µDys) that is still far removed from correcting human disease. The need to develop strategies leading to the expression of a best performing dystrophin variant led to only few studies reporting on the use of dual vectors, but none reported on a method to assess in vivo transgene reconstitution efficiency, the degree of which directly affects the use of safe AAV dosing. We report here on the generation of a dual AAV vector approach for the expression of a larger dystrophin version (quasidystrophin) based on homologous recombination, and the development of a methodology employing a strategic droplet digital PCR design, to determine the recombination efficiency as well as the occurrence of unwanted concatemerization events or aberrant expression from the single vectors. We demonstrated that, upon systemic delivery in the dystrophic D2.B10-Dmdmdx/J (DBA2mdx) mice, our dual AAV approach led to high transgene reconstitution efficiency and negligible Inverted Terminal Repeats (ITR)-dependent concatemerization, with consequent remarkable protein restoration in muscles and improvement of muscle pathology. This evidence supports the suitability of our system for gene therapy application and the potential of this methodology to assess and improve the feasibility for therapeutic translation of multiple vector approaches.

Colocalization of senescent biomarkers in deep, superficial, and ovarian endometriotic lesions: a pilot study.

Endometriosis is a prevalent gynecological condition with deleterious effects on women's quality of life in terms of physical, emotional, and social compromise. It is an inflammatory disease characterized by the presence of endometrial-like tissue outside the uterus, and its presentation varies from superficial peritoneal lesions to deep infiltrative endometriosis and ovarian endometrioma. In our previous study, endometriotic lesions were implicated in cellular senescence as their inflammatory pattern could potentially compromise surrounding tissue integrity, thereby inducing a senescent state in cells. P16Ink4a and lamin b1 are biomarkers used to assess cellular senescence. Indirect immunofluorescence staining is a broad technique used to assess cellular structure and behavior driven by protein-protein interactions that provide valuable information about cell functioning. The etiopathogeny of endometriosis is not completely understood and diagnostic approaches still rely on invasive methods; therefore, it is important to use validated methods to increase our understanding of the disease and the development of novel diagnostic tools. However, indirect immunofluorescence protocols are often tissue specific and, if neglected, can lead to misinterpretation of results. Moreover, no valid endometriotic tissue-specific colocalization immunofluorescence protocols have been established. Thus, we have validated a well-funded and suitable protocol to allow precise evaluation of the three presentations of endometriosis lesions using indirect immunofluorescence aiming to support further investigations in endometriosis lesions.

Suprapatellar intramedullary nailing of tibial shaft fractures in pregnancy. A report of two cases.

BACKGROUND: Treatment of closed tibial shaft fractures in the 3rd trimester of pregnancy is controversial. Since there are few case reports published in literature, there is no consensus on the appropriate management of these fractures. This case report proposes intramedullary nailing throught the suprapatellar approach for the treatment of tibial shaft fracture in pregnant women, never described before in literature. CASE PRESENTATION: We report 2 cases of a tibial diaphyseal fracture treated by intramedullary nailing in women at the 3rd trimester of pregnancy. CONCLUSION: Surgical treatment of tibial shaft fracture of pregnant women in the 3rd trimester of pregnancy with intramedullary nailing seems to be safe. The use of the specific suprapatellar approach helps in the intra-operative management of the pregnant patients.

Evaluation of Second-Hand Exposure to Electronic Cigarette Vaping under a Real Scenario: Measurements of Ultrafine Particle Number Concentration and Size Distribution and Comparison with Traditional Tobacco Smoke.

The present study aims to evaluate the impact of e-cig second-hand aerosol on indoor air quality in terms of ultrafine particles (UFPs) and potential inhalation exposure levels of passive bystanders. E-cig second-hand aerosol characteristics in terms of UFPs number concentration and size distribution exhaled by two volunteers vaping 15 different e-liquids inside a 49 m3 room and comparison with tobacco smoke are discussed. High temporal resolution measurements were performed under natural ventilation conditions to simulate a realistic exposure scenario. Results showed a systematic increase in UFPs number concentration (part cm-3) related to a 20-min vaping session (from 6.56 × 103 to 4.01 × 104 part cm-3), although this was one up to two order of magnitude lower than that produced by one tobacco cigarette consumption (from 1.12 × 105 to 1.46 × 105 part cm-3). E-cig second-hand aerosol size distribution exhibits a bimodal behavior with modes at 10.8 and 29.4 nm in contrast with the unimodal typical size distribution of tobacco smoke with peak mode at 100 nm. In the size range 6-26 nm, particles concentration in e-cig second-hand aerosol were from 2- (Dp = 25.5 nm) to 3800-fold (Dp = 9.31 nm) higher than in tobacco smoke highlighting that particles exhaled by users and potentially inhaled by bystanders are nano-sized with high penetration capacity into human airways.

Is laparotomy the unavoidable step to diagnose caecal volvulus?

Caecal volvulus is the axial twist of the caecum, ascending colon and terminal ileum around the mesenteric pedicle. This infrequently encountered clinical entity is responsible for 1-1.5% of all intestinal obstruction with a mortality of 10-40% depending on the presence of colon viability or intestinal gangrene. Many factors have been referred as correlated to caecal volvulus development, mainly anatomical predisposition and previous abdominal operations. Pre-operative diagnosis plays an important role in the management of such patients. Unfortunately, clinical signs, symptoms and laboratory tests are never specific enough to lead to a prompt diagnosis. Abdominal radiography and computed tomography may allow a diagnosis if typical signs are present. However, up to 30% of patients do not show these radiographic peculiarities, making the diagnosis difficult or impossible. Moreover, the low incidence of this disease is often responsible of a wrong or imprecise diagnosis, especially for radiologists who work with low volume of patients. We report a case of a patient with caecal volvulus, in which laparotomy was unavoidable to reach a diagnosis.