Architectural Unit Testing

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Insights into the safety and versatility of 4D printed intravesical drug delivery systems

This paper focuses on recent advancements in the development of 4D printed drug delivery systems (DDSs) for the intravesical administration of drugs. By coupling the effectiveness of local treatments with major compliance and long-lasting performance, they would represent a promising innovation for the current treatment of bladder pathologies. Being based on a shape-memory pharmaceutical-grade polyvinyl alcohol (PVA), these DDSs are manufactured in a bulky shape, can be programmed to take on a collapsed one suitable for insertion into a catheter and re-expand inside the target organ, following exposure to biological fluids at body temperature, while releasing their content. The biocompatibility of prototypes made of PVAs of different molecular weight, either uncoated or coated with Eudragit®-based formulations, was assessed by excluding relevant in vitro toxicity and inflammatory response using bladder cancer and human monocytic cell lines. Moreover, the feasibility of a novel configuration was preliminarily investigated, targeting the development of prototypes provided with inner reservoirs to be filled with different drug-containing formulations. Samples entailing two cavities, filled during the printing process, were successfully fabricated and showed, in simulated urine at body temperature, potential for controlled release, while maintaining the ability to recover about 70% of their original shape within 3 min

Drp1 overexpression induces desmin disassembling and drives kinesin-1 activation promoting mitochondrial trafficking in skeletal muscle

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Reversal of Defective Mitochondrial Biogenesis in Limb-Girdle Muscular Dystrophy 2D by Independent Modulation of Histone and PGC-1α Acetylation

Mitochondrial dysfunction occurs in many muscle degenerative disorders. Here, we demonstrate that mitochondrial biogenesis was impaired in limb-girdle muscular dystrophy (LGMD) 2D patients and mice and was associated with impaired OxPhos capacity. Two distinct approaches that modulated histones or peroxisome proliferator-activated receptor-gamma coactivator 1 \u3b1 (PGC-1\u3b1) acetylation exerted equivalent functional effects by targeting different mitochondrial pathways (mitochondrial biogenesis or fatty acid oxidation[FAO]). The histone deacetylase inhibitor Trichostatin A (TSA) changed chromatin assembly at the PGC-1\u3b1 promoter, restored mitochondrial biogenesis, and enhanced muscle oxidative capacity. Conversely, nitric oxide (NO) triggered post translation modifications of PGC-1\u3b1 and induced FAO, recovering the bioenergetics impairment of muscles but shunting the defective mitochondrial biogenesis. In conclusion, a transcriptional blockade of mitochondrial biogenesis occurred in LGMD-2D and could be recovered by TSA changing chromatin conformation, or it could be overcome by NO activating a mitochondrial salvage pathway

Nitric Oxide Generated by Tumor-Associated Macrophages Is Responsible for Cancer Resistance to Cisplatin and Correlated With Syntaxin 4 and Acid Sphingomyelinase Inhibition

Tumor microenvironment is fundamental for cancer progression and chemoresistance. Among stromal cells tumor-associated macrophages (TAMs) represent the largest population of infiltrating inflammatory cells in malignant tumors, promoting their growth, invasion, and immune evasion. M2-polarized TAMs are endowed with the nitric oxide (NO)-generating enzyme inducible nitric oxide synthase (iNOS). NO has divergent effects on tumors, since it can either stimulate tumor cells growth or promote their death depending on the source of it; likewise the role of iNOS in cancer differs depending on the cell type. The role of NO generated by TAMs has not been investigated. Using different tumor models in vitro and in vivo we found that NO generated by iNOS of M2-polarized TAMs is able to protect tumor cells from apoptosis induced by the chemotherapeutic agent cisplatin (CDDP). Here, we demonstrate that the protective effect of NO depends on the inhibition of acid sphingomyelinase (A-SMase), which is activated by CDDP in a pathway involving the death receptor CD95. Mechanistic insights indicate that NO actions occur via generation of cyclic GMP and activation of protein kinase G (PKG), inducing phosphorylation of syntaxin 4 (synt4), a SNARE protein responsible for A-SMase trafficking and activation. Noteworthy, phosphorylation of synt4 at serine 78 by PKG is responsible for the proteasome-dependent degradation of synt4, which limits the CDDP-induced exposure of A-SMase to the plasma membrane of tumor cells. This inhibits the cytotoxic mechanism of CDDP reducing A-SMase-triggered apoptosis. This is the first demonstration that endogenous NO system is a key mechanism through which TAMs protect tumor cells from chemotherapeutic drug-induced apoptosis. The identification of the pathway responsible for A-SMase activity downregulation in tumors leading to chemoresistance warrants further investigations as a means to identify new anti-cancer molecules capable of specifically inhibiting synt4 degradation

Architectural unit testing in a robot teleoperation case study

A formal testing methodology is outlined in this paper, that proves applicable to validation of architectural units in object-oriented models, and its use is illustrated in the context of the design of a robot teleoperation architecture. Automated generation of test cases to validate the functionality of the robot trajectory generation unit showcases the key features of this methodology. A disciplined use of UML state diagrams, to model the unit’s dynamics consistently with its static properties as modeled by class diagrams, enables one to provide such models with IOLTS semantics, whence a rich machinery of testing theories and tools based on those theories become readily available. Our case study tells that, besides black-box testing of final implementation units, white-box analysis of architectural units may greatly benefit from the flexibility of parameterized I/Oconformance relations. Test purposes turn out to be a useful methodological link between functional requirements, which they are drawn from, and conformance relations, which they help one to instantiate, thereby delimiting test selection to purposeful tests. Contingent aspects of our methodology include: a mechanical translation of state diagrams in Basic LOTOS, a non-mechanical, use-case driven synthesis of test purposes, expressed in the same language, and the use of the TGV tool for automated test case generation. Other choices in these respects are well possible, without affecting the characteristic trait of the proposed methodology, that is rather to be found in the combination of object-oriented architectural modeling with IOLTS semantics

Cryopreserved placental biopsies maintain mitochondrial activity for high-resolution respirometry

Abstract Background High-resolution respirometry (HRR) of human biopsies can provide useful metabolic, diagnostic, and mechanistic insights for clinical research and comparative medical studies. Fresh tissues analysis offers the potential best condition, the drawback being the need to use them shortly after dissection for mitochondrial respiratory experiments. The development of effective long-term storage protocols for biopsies that allow the assessment of key Electron Transport System (ETS) parameters at later stages is thus a major need. Methods We optimised a cryopreservation protocol that preserves mitochondrial membranes intactness, otherwise affected by direct tissue freezing. The protocol is based on a gradual freezing step from on-ice to liquid nitrogen and − 80 °C storage using a specific DMSO-based buffer. Results Placenta is a suitable tissue to design and test the effectiveness of long-term storage protocols being metabolically active foetal tissue with mitochondrial dysfunctions contributing to placental disease and gestational disorders. Here we designed and tested the effectiveness of the cryopreservation protocol using human placenta biopsies; we measured the ETS activity by HRR of placenta specimens comparing fresh, cryopreserved, and snap frozen conditions. Conclusions By this protocol, Oxygen Consumption Rate (OCR) measurements of fresh and cryopreserved placental specimens are comparable whereas snap frozen procedure impairs mitochondrial activity. Graphical Abstrac

Encapsulating peritoneal sclerosis in an Italian center: thirty year experience

BACKGROUND: Encapsulating peritoneal sclerosis (EPS) is a rare but life-threatening complication of peritoneal dialysis (PD). Its incidence and prevalence are still not clearly defined. No data exist on the prevalence of EPS in Italy. OBJECTIVES: To evaluate the incidence and prevalence of EPS, and identify potential factors useful for prevention or early diagnosis of EPS. METHODS: A retrospective study in patients starting PD between 1979 and 2013 in one Italian center. Data on demographics, occurrence of EPS, time on PD, peritoneal equilibration test, and therapy for EPS were gathered. RESULTS: EPS occurred in 26/920 patients with a prevalence of 2.8 % and incidence of 1/105 patient-years. The prevalence increased with the time spent on PD: 0.4 % for PD duration 14 years). EPS prevalence was not higher in PD patients transplanted: 5/172 (2.9 %); only two of them (1.2 %) were diagnosed while with a functioning graft. In only one patient (0.6 %) was the diagnosis made during hemodialysis; the other 23 were diagnosed while still on PD. Mortality due to EPS was 38.5 %, and was associated with PD duration. Therapy with steroids reduced mortality [hazard ratio 0.047 (95 % CI: 0.008-0.273); p < 0.001]. CONCLUSIONS: In our experience the prevalence of EPS is low, but increases progressively with the duration of PD. The transfer to hemodialysis or transplantation does not appear to be a key factor for EPS. Therapy with steroids significantly improves the outcom