Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib.

Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles' heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization.

Correction to: Paragangliomas arise through an autonomous vasculo-angio-neurogenic program inhibited by imatinib.

The given and family names of two co-authors were incorrect in the published article. The correct spelling should read as: Sampath Chandra Prasad and Vinagolu K Rajasekhar.

Magnetic susceptibility increases as diamagnetic molecules breakdown: Myelin digestion during multiple sclerosis lesion formation contributes to increase on QSM.

BACKGROUND: The pathological processes in the first weeks of multiple sclerosis (MS) lesion formation include myelin digestion that breaks chemical bonds in myelin lipid layers. This can increase lesion magnetic susceptibility, which is a potentially useful biomarker in MS patient management, but not yet investigated. PURPOSE: To understand and quantify the effects of myelin digestion on quantitative susceptibility mapping (QSM) of MS lesions. STUDY TYPE: Histological and QSM analyses on in vitro models of myelin breakdown and MS lesion formation in vivo. POPULATION/SPECIMENS: Acutely demyelinating white matter lesions from MS autopsy tissue were stained with the lipid dye oil red O. Myelin basic protein (MBP), a major membrane protein of myelin, was digested with trypsin. Purified human myelin was denatured with sodium dodecyl sulfate (SDS). QSM was performed on phantoms containing digestion products and untreated controls. In vivo QSM was performed on five MS patients with newly enhancing lesions, and then repeated within 2 weeks. FIELD STRENGTH/SEQUENCE: 3D T 2 * -weighted spoiled multiecho gradient echo scans performed at 3T. ASSESSMENT: Region of interest analyses were performed by a biochemist and a neuroradiologist to determine susceptibility changes on in vitro and in vivo QSM images. STATISTICAL TESTS: Not applicable. RESULTS: MBP degradation by trypsin increased the QSM measurement by an average of 112 ± 37 ppb, in excellent agreement with a theoretical estimate of 111 ppb. Degradation of human myelin by SDS increased the QSM measurement by 23 ppb. As MS lesions changed from gadolinium enhancing to nonenhancing over an average of 15.8 ± 3.7 days, their susceptibility increased by an average of 7.5 ± 6.3 ppb. DATA CONCLUSION: Myelin digestion in the early stages of MS lesion formation contributes to an increase in tissue susceptibility, detectable by QSM, as a lesion evolves from gadolinium enhancing to nonenhancing. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 3 J. Magn. Reson. Imaging 2018;47:1281-1287.

Intraspecific and interspecific variation in thermotolerance and photoacclimation in Symbiodinium dinoflagellates.

Light and temperature are major drivers in the ecology and biogeography of symbiotic dinoflagellates living in corals and other cnidarians. We examined variations in physiology among 11 strains comprising five species of clade A Symbiodinium We grew cultures at 26°C (control) and 32°C (high temperature) over a duration of 18 days while measuring growth and photochemical efficiency (Fv /Fm ). Responses to thermal stress ranged from susceptible to tolerant across species and strains. Most strains exhibited a decrease in cell densities and Fv /Fm when grown at 32°C. Tolerance to high temperature (T32) was calculated for all strains, ranging from 0 (unable to survive at high temperature) to 1 (able survive at high temperature). There was substantial variation in thermotolerance across species and among strains. One strain had a T32 close to 1, indicating that growth was not reduced at 32°C for only this one strain. To evaluate the combined effect of temperature and light on physiological stress, we selected three strains with different levels of thermotolerance (tolerant, intermediate and susceptible) and grew them under five different light intensities (65, 80, 100, 240 and 443 µmol quanta m-2 s-1) at 26 and 32°C. High irradiance exacerbated the effect of high temperature, particularly in strains from thermally sensitive species. This work further supports the recognition that broad physiological differences exist not only among species within Symbiodinium clades, but also among strains within species demonstrating that thermotolerance varies widely between species and among strains within species.

Head and neck paragangliomas: Recent advances in translational and clinical research and guidelines for patient care.

Head and neck paragangliomas (HNPGLs), rare neuroendocrine tumors that mainly arise from parasympathetic ganglia along the cranial nerves, are challenging due to anatomic origin, tendency to aggressive neurovascular and skull base infiltration, unpredictable metastatic potential, radio-chemoresistance, and risk of multiplicity. Symptoms range from mild to life threatening depending on location/size, but rarely relate to catecholamine excess. Risk factors include female sex and pathogenic germline variants in genes affecting hypoxia signaling (foremost succinate dehydrogenase genes). Diagnostic work-up relies on imaging, measurements of plasma free metanephrines/methoxytyramine, genetic testing, and pathology/immunohistochemistry. Management is tailored to patient/tumor characteristics and encompasses wait-scan, upfront surgery, debulking surgery, and radiotherapy. Presurgical embolization is recommended, except for small tympanic and tympanomasoid tumors. Presurgical stenting is required for internal carotid artery involvement, and two-stage surgery for intradural extension. Current treatments for metastatic/inoperable HNPGL are non-curative, and long-term follow-up should be recommended for all patients to monitor local recurrence and new tumors.

Efficacy of water filters for dental chair units: assessment of the filtration action versus Coxsackievirus B5.

INTRODUCTION: The microbiological safety and control of the water used in dental practice has a critical importance for avoiding cross-linked infections in the dental office. The aim of this study was to establish coxsackie virus filtration of the water applied to a dental unit. METHODS: A specific water filter-system was used, to verify the viral load in the outgoing water. The statistical analysis was performedusing the Shapiro-Wilk and t-Student test. RESULTS: The outcome of the evaluation of the virologic tests shows an excellent capability of virus filtration that attested 99.9999% in the volume analyzed. A statistical difference was found in the bacterial water contamination parameter before and after filtration. (P = 0.000000). CONCLUSIONS: According to the tests, medical devices applied to a dental unit are able to filter viruses and therefore reduce risk of contamination in the dental office.

A relapsing inflammatory syndrome and active human herpesvirus 8 infection.

We describe an immunocompetent 61-year-old woman who was negative for human immunodeficiency virus and who had recurrent human herpesvirus 8 (HHV-8) infection associated with a relapsing systemic inflammatory syndrome characterized by fever, lymphadenopathy, splenomegaly, edema, arthrosynovitis, and rash. Kaposi's sarcoma developed 10 months after the initial clinical presentation. A correlation was documented between the recurrent clinical manifestations and the HHV-8 load in plasma and peripheral-blood mononuclear cells. Histologic examination of an enlarged lymph node heavily infected with HHV-8 revealed an atypical lymphoproliferative disorder characterized by paracortical hyperplasia and collapsed primary and secondary follicles.

A 5' splice site-proximal enhancer binds SF1 and activates exon bridging of a microexon.

Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3' and 5' splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5' splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3' splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon.

Monolithic zirconia and digital impression: case report.

AIMS: The aim of this study is to present a clinical case of a full arch prosthetic rehabilitation on natural teeth, combining both digital work-flow and monolithic zirconia. PATIENTS AND METHODS: Digital impression was taken with an intraoral optical scanner (CS3500, Carestream Dental, Atlanta, GA, USA). A prosthetic rehabilitation was realized on natural teeth using monolithic zirconia from 1.6 to 1.4 and from 2.7 to 2.4 frameworks, while in the aesthetic area (from 2.3 to 1.3), technicians left on the structure a 0.8 mm vestibular space for ceramic layering. DISCUSSION: The combination of digital impression technology and the use of the monolithic zirconia had demonstrated the delivery of the final prosthetic device in a quick time without the need to remodel functional or aesthetic areas. The digital work-flow combines intraoral optical impression techniques and CAD/CAM technology, in order to achieve a fully digital and successful way to deliver prosthetic restorations to patients, providing aesthetics and function in shorter intervals of time. The clinical outcome of this study was satisfactory but a long-term evaluation is needed.

Naive HIV/HCV-coinfected patients have higher intrahepatic pro-inflammatory cytokines than coinfected patients treated with antiretroviral therapy.

In the era of antiretroviral therapy, liver disease has emerged as an important cause of morbidity and mortality in HIV/hepatitis C virus (HCV) coinfected patients. It is believed that HCV is a non-cytopathic virus and that T-cell-mediated events (including the production of pro-inflammatory cytokines) have an important role in promoting both liver damage and viral clearance. Whether HIV coinfection or antiretroviral therapies influence such events is still unclear. In the current study, we compared the expression of NKp46 (a natural killer cell marker), CD3 (a T-cell marker), interferon-gamma (IFN-gamma), tumour-necrosis factor-alpha (TNF-alpha; pro-inflammatory cytokines) and interleukin-10 (IL-10; an anti-inflammatory cytokine) mRNA in the liver of naive HIV/HCV-coinfected patients (group one, n=14), coinfected patients treated with antiretroviral therapy (group two, n=23) and naive HCV mono-infected patients (group three, n=24). All three groups had comparable HCV viremia, with coinfected patients showing similar and relatively high CD4+ T-cell counts and significantly different HIV vireamia. Interestingly, when compared to groups two and three, group one showed significantly higher intrahepatic mRNA levels for CD3, IFN-gamma and TNF-alpha, whereas the expression of NKp46 and IL-10 were comparable in all three groups. Further, higher histopathological grading scores within each group were independently associated with higher mRNA contents for CD3 and IFN-gamma and higher serum alanine aminotransferase levels at the time of liver biopsy. Together, these results suggest that HIV infection may exacerbate the immune-mediated inflammatory response in the liver of patients chronically infected with HCV and antiretroviral therapy may prevent this effect.

Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless.

A multibranched hierarchy of regulatory genes controls all aspects of somatic sexual development in Drosophila melanogaster. One branch of this hierarchy is headed by the fruitless (fru) gene and functions in the central nervous system, where it is necessary for male courtship behavior as well as the differentiation of a male-specific abdominal structure, the muscle of Lawrence (MOL). A preliminary investigation of several of the mutations described here showed that the fru gene also has a sex-nonspecific vital function. The fru gene produces a complex set of transcripts through the use of four promoters and alternative splicing. Only the primary transcripts produced from the most distal (P1) promoter are sex-specifically spliced under direction of the sex-determination hierarchy. We have analyzed eight new fru mutations, created by X-ray mutagenesis and P-element excision, to try to gain insight into the relationship of specific transcript classes to specific fru functions. Males that lack the P1-derived fru transcripts show a complete absence of sexual behavior, but no other defects besides the loss of the MOL. Both males and females that have reduced levels of transcripts from the P3 promoter develop into adults but frequently die after failing to eclose. Analysis of the morphology and behavior of adult escapers showed that P3-encoded functions are required for the proper differentiation and eversion of imaginal discs. Furthermore, the reduction in the size of the neuromuscular junctions on abdominal muscles in these animals suggests that one of fru's sex-nonspecific functions involves general aspects of neuronal differentiation. In mutants that lack all fru transcripts as well as a small number of adjacent genes, animals die at an early pupal stage, indicating that fru's function is required only during late development. Thus, fru functions both in the sex-determination regulatory hierarchy to control male sexual behavior through sex-specific transcripts and sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during adult development through sex-nonspecific transcript classes.

Development of lamellar gel phase emulsion containing marigold oil (Calendula officinalis) as a potential modern wound dressing.

Appropriate therapeutics for wound treatments can be achieved by studying the pathophysiology of tissue repair. Here we develop formulations of lamellar gel phase (LGP) emulsions containing marigold (Calendula officinalis) oil, evaluating their stability and activity on experimental wound healing in rats. LGP emulsions were developed and evaluated based on a phase ternary diagram to select the best LGP emulsion, having a good amount of anisotropic structure and stability. The selected LGP formulation was analyzed according to the intrinsic and accelerated physical stability at different temperatures. In addition, in vitro and in vivo studies were carried out on wound healing rats as a model. The LGP emulsion (15.0% marigold oil; 10.0% of blend surfactants and 75.0% of purified water [w/w/w]) demonstrated good stability and high viscosity, suggesting longer contact of the formulation with the wound. No cytotoxic activity (50-1000 µg/mL) was observed in marigold oil. In the wound healing rat model, the LGP (15 mg/mL) showed an increase in the leukocyte recruitment to the wound at least on days 2 and 7, but reduced leukocyte recruitment after 14 and 21 days, as compared to the control. Additionally, collagen production was reduced in the LGP emulsion on days 2 and 7 and further accelerated the process of re-epithelialization of the wound itself. The methodology utilized in the present study has produced a potentially useful formulation for a stable LGP emulsion-containing marigold, which was able to improve the wound healing process.

A program of balanced physical fitness in the preventive care of elderly ambulatory patients.

A program of balanced physical fitness incorporated into a medical "team" approach to treatment contributed significantly to the general health of elderly ambulatory patients. Each week for eleven months, 13 patients were given one hour of balanced exercise and encouraged to perform it for two additional hours per week. Initial differences favoring the control group. Physicians' single-blind assessment of the two groups resulted in significantly better health ratings for the patients who had received the therapeutic exercise program than for those who did not participate.

The Gruppo Otologico experience of endolymphatic sac tumor.

OBJECTIVE: Endolymphatic sac tumor (ELST) is a rare low grade adenocarcinoma of the skull base. During the past decade the number of the reported cases has increased. This study exposes our experience in the management of ELST with a review of the literature. STUDY DESIGN: Retrospective study of patients with ELST at a quaternary referral otology and skull base center. METHODS: A review of the records from the Gruppo Otologico revealed 7 patients treated for ELST. All papers containing series of three or more cases of ELST published in the English literature were selected for analysis. RESULTS: Hearing loss and tinnitus were present in almost all our cases. All of them were evaluated with audiometric tests, computed tomography and magnetic resonance imaging. All the patients were treated surgically with preservation of the facial nerve and preoperative embolization was performed in 5 patients. Genetic study was performed on all our cases and revealed the presence of von Hippel-Lindau syndrome in one patient who had the tumor as the initial manifestation of his syndrome. None of the patients received postoperative radiotherapy and one of them had recurrence of the tumor 13 years following surgery. CONCLUSIONS: Complete surgical resection with preoperative embolization of large tumors is the mainstay treatment for ELST. The facial nerve should not be sacrificed unless it is totally invaded by the tumor. A long term follow up is recommended and the role of postoperative adjunctive radiotherapy is still controversial.

Aspirin-triggered resolvin D1 reduces mucosal inflammation and promotes resolution in a murine model of acute lung injury.

Acute lung injury (ALI) is a severe illness with excess mortality and no specific therapy. Protective actions were recently uncovered for docosahexaenoic acid-derived mediators, including D-series resolvins. Here, we used a murine self-limited model of hydrochloric acid-induced ALI to determine the effects of aspirin-triggered resolvin D1 (AT-RvD1; 7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) on mucosal injury. RvD1 and its receptor ALX/FPR2 were identified in murine lung after ALI. AT-RvD1 (~0.5-5 µg kg(-1)) decreased peak inflammation, including bronchoalveolar lavage fluid (BALF) neutrophils by ~75%. Animals treated with AT-RvD1 had improved epithelial and endothelial barrier integrity and decreased airway resistance concomitant with increased BALF epinephrine levels. AT-RvD1 inhibited neutrophil-platelet heterotypic interactions by downregulating both P-selectin and its ligand CD24. AT-RvD1 also significantly decreased levels of BALF pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6, Kupffer cells, and tumor necrosis factor-α, and decreased nuclear factor-κB-phosphorylated p65 nuclear translocation. Taken together, these findings indicate that AT-RvD1 displays potent mucosal protection and promotes catabasis after ALI.

Malignancy in vestibular schwannoma after stereotactic radiotherapy: a case report and review of the literature.

OBJECTIVES/HYPOTHESIS: A relation between conventional radiotherapy and the development of intracranial neoplasma is well known, but radiation-associated tumor following stereotactic radiotherapy of vestibular schwannoma is underestimated. In this article we will study this relation by doing a complete literature review on all the malignant intracranial tumors that appeared following radiosurgery and adding a case of malignant vestibular schwannoma following stereotactic radiotherapy in a Neurofibromatosis type 2 patient. METHODS: Literature review and discussion. RESULTS: We found 26 cases of malignant brain tumor following stereotactic radiotherapy including our case. In 13 cases the tumor occurred in context of Neurofibromatosis type 2. None of the patients had a tumor size less than 2.5 cm. and the mean latency period between the radiotherapy and malignant tumor development was 5.8 years. CONCLUSION: Patients with vestibular schwannoma should be made aware of the low incidence of the radiation-induced malignant changes and long-term follow-up is mandatory.

Architectural limits on split genes.

Exon/intron architecture varies across the eukaryotic kingdom with large introns and small exons the rule in vertebrates and the opposite in lower eukaryotes. To investigate the relationship between exon and intron size in pre-mRNA processing, internally expanded exons were placed in vertebrate genes with small and large introns. Both exon and intron size influenced splicing phenotype. Intron size dictated if large exons were efficiently recognized. When introns were large, large exons were skipped; when introns were small, the same large exons were included. Thus, large exons were incompatible for splicing if and only if they were flanked by large introns. Both intron and exon size became problematic at approximately 500 nt, although both exon and intron sequence influenced the size at which exons and introns failed to be recognized. These results indicate that present-day gene architecture reflects at least in part limitations on exon recognition. Furthermore, these results strengthen models that invoke pairing of splice sites during recognition of pre-mRNAs, and suggest that vertebrate consensus sequences support pairing across either introns or exons.

An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon.

The average length of a vertebrate axon is approximately 130 nt. Decreasing the size of an internal axon to less than 51 nt induces axon skipping, implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt axon from chicken cardiac troponin T (cTNT). In vivo, the cTNT micro-exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the micro-exon in the downstream intron. Increasing the length of the micro-exon alleviated the requirement for the intron element, indicating that the lack of inclusion of the micro-exon in the absence of a facilitating sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt sequence. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The entire intron element activated inclusion of a heterologous 7-nt exon, suggesting that the intron element is a general enhancer for the splicing of micro-exons. In vitro, the intron element and the repeated sequence facilitated splicing of a heterologous exon. Because of the ability of the cTNT intron element to facilitate the splicing of heterologous exons, we have termed the element an intron splicing enhancer (ISE). Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed either upstream or downstream of the micro-exon. In vitro, the ISE or copies of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE thus becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target axon.

Spatial, temporal, and sexually dimorphic expression patterns of the fruitless gene in the Drosophila central nervous system.

The fruitless (fru) gene of Drosophila produces both sex-specifically and non-sex-specifically spliced transcripts. Male-specific fru products are believed to regulate male courtship. To further an understanding of this gene's behavioral role, we examined the central nervous system (CNS) for temporal, spatial, and sexually dimorphic expression patterns of sex-specific fru products by in situ hybridization and immunohistochemistry. For the latter, antibodies were designed to detect only male-specific forms of the protein (FRU(M)) or amino acid sequences that are in common among all translated products (FRU(COM)). Sex-specific mRNAs and male-specific proteins were first observed in mature larvae and peaked in their apparent abundances during the first half of the pupal period. At later stages and in adults, faint mRNA signals were seen in only a few neural clusters; in contrast, relatively strong FRU(M) signals persisted into adulthood. Twenty neuronal groups composed of 1700 fru-expressing neurons were identified in the midpupal CNS. These groups overlap most of the neural sites known to be involved in male courtship. Anti-FRU(COM) led to widespread labeling of neural and nonneural tissues in both sexes, but in the female CNS, only in developing ganglia in a pattern different from that of the male's FRU(M) cells. Expression of sex-specific fru mRNAs in the CNS of males analyzed from the earliest pupal stages indicated that sex-specific alternative splicing is not the exclusive mechanism regulating expression of fruitless transcripts.