Human papillomavirus type 16 E6 and E7 oncoproteins interact with the nuclear p53-binding protein 1 in an in vitro reconstructed 3D epithelium: new insights for the virus-induced DNA damage response.

BACKGROUND: Despite vaccination and screening measures, anogenital cancer, mainly promoted by HPV16 oncoproteins, still represents the fourth tumor and the second cause of death among women. Cell replication fidelity is the result of the host DNA damage response (DDR). Unlike many DNA viruses that promote their life cycle through the DDR inactivation, HR-HPVs encourage cells proliferation despite the DDR turned on. Why and how it occurs has been only partially elucidated. During HPV16 infection, E6 links and degrades p53 via the binding to the E6AP LXXLL sequence; unfortunately, E6 direct role in the DDR response has not clearly identified yet. Similarly, E7 increases DDR by competing with E2F1-pRb interaction, thus leading to the inactivation of pRb, and promotion, E2F1 mediated, of DDR genes translation, by binding to the pRb-like proteins CBP/p300 and p107, that also harbour LXXLL sequence, and via the interaction and activation of several DDR proteins. METHODS: To gain information regarding E6 and E7 contribution in DDR activation, we produced an in vitro 3D HPV16-E6E7 infected epithelium, already consolidated study model for HPVs, and validated it by assessing H&E staining and BrdU, HPV16 DNA, E6E7 proteins and γH2A.X/53BP1 double-strand break (DSBs) sensors expression; then we made an immuno-colocalization of E6 and E7 with cyclin E2 and B1. Since 53BP1, like E6 and E7, also binds p53 and pRb, we supposed their possible direct binding. To explore this hypothesis, we performed a double immunofluorescence of E6 and E7 with 53BP1, a sequence analysis of 53BP1 within its BRCT2 domain and then an in situ PLA within CaSki, E6E7HPV16 NHEKs and the 3D model. RESULTS: The in vitro epithelium resembled the histology and the events typical of in vivo infected tissues. E6E7HPV16 were both expressed in basal and differentiated strata and induced H2A.X phosphorylation and 53BP1 increment into nuclear foci. After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the bindings via the PLA technique. CONCLUSIONS: Our results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus.

Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome).

We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of α-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome.

[Oral manifestations in Crohn's disease - Two patient cases from the clinic]. / Orale Manifestationen bei Morbus Crohn

Crohn's disease (CD) is an inflammatory disease of the gastrointestinal system. It is suspected to be caused by a combination of genetic and environmental factors and changes in the patient's intestinal microbiome. As opposed to colitis ulcerosa, which usually only affects the large intestine, CD may affect various parts of the gastrointestinal tract. Many patients show oral lesions related to CD, which sometimes can precede the first gastrointestinal symptoms. Specific oral lesions include granulomatous cheilitis, cobblestone-like mucosal tags and linear ulcerations. Non-specific lesions occur in the form of angular cheilitis and opportunistic candidiasis, aphthous ulcerations, reduced salivation, dental caries and many more. We present two cases of patients suffering from Crohn's disease with different oral manifestations and discuss the dentist's role in early detection of specific oral lesions and the importance of the follow-up care of affected patients.

Monitoring jaw osteomyelitis therapy with single-photon emission computed tomography/computed tomography.

OBJECTIVE: The aim of the study was to evaluate the value of single-photon emission computed tomography/computed tomography (SPECT/CT) for therapy response assessment of jaw osteomyelitis. MATERIALS AND METHODS: Thirty-four baseline and 74 follow-up SPECT/CT examinations for therapy response assessment were performed in 34 patients with jaw osteomyelitis. SPECT/CT and planar late-phase bone scintigraphy images were assessed at baseline and follow-up, according to the following criteria: tracer uptake grade (0 = no uptake, 1 = low uptake, 2 = moderate uptake and 3 = high uptake); and morphologic signs (osteolysis, sequestration, sclerosis, periosteal reaction and pathologic fracture). RESULTS: At baseline, SPECT/CT showed marked (grade 2 or 3) uptake in 91% (31/34) of the patients, osteolysis in 85% (29/34), sclerosis in 71% (24/34), periosteal reaction in 44% (15/34) and a sequestrum in 24% (8/34). In 24 patients with clinically complete remission during or after at least 12 months' therapy, bone scintigraphy showed grade 0 or 1 uptake in 100% (24/24) and SPECT/CT in 91% (22/24) of the patients. Sclerosis with the disappearance of osteolysis, sequestration and periosteal reactions was the predominant morphologic finding in complete responders (68%; 16/24). In 10 patients with symptoms of exacerbation of the osteomyelitis, 80% (8/10) showed increasing uptake, 90% (9/10) sclerosis, 80% osteolysis (8/10) and 40% (4/10) osteolysis and periosteal reactions. CONCLUSION: SPECT/CT is a valuable tool to accurately assess therapy response, disease exacerbation and complications of jaw osteomyelitis. Low-grade (grade 1) residual tracer uptake is common in patients with clinically complete remission and is suggestive of ongoing bone remodeling and healing.

Clinical effects of interdental cleansing on supragingival biofilm formation and development of experimental gingivitis.

PURPOSE: The aim of the present study was to test the effects of interdental cleansing with dental floss on supragingival biofilm removal in natural dentition during a 3-week period of experimental biofilm accumulation. MATERIALS AND METHODS: The present study was performed as a single-blind, parallel, randomised, controlled clinical trial using the experimental gingivitis model (Löe et al, 1965). Thirty-two students were recruited and assigned to one of the following experimental or control groups: Group A used a fluoride-containing dentifrice (NaF dentifrice) on a toothbrush for 60 s twice a day, Group B used an unwaxed dental floss twice a day, Group C used a waxed dental floss twice a day in every interproximal space and Group D rinsed twice a day for 60 s with drinking water (control). RESULTS: During 21 days of abolished oral hygiene, the groups developed various amounts of plaque and gingivitis. Neither of the cleansing protocols alone allowed the prevention of gingivitis development. Toothbrushing alone yielded better outcomes than did any of the flossing protocols. Interdental cleansing with a waxed floss had better biofilm removal effects than with unwaxed floss. CONCLUSIONS: Toothbrushing without interdental cleansing using dental floss and interdental cleansing alone cannot prevent the development of gingivitis.

Deficits in temporal order memory induced by interferon-alpha (IFN-α) treatment are rescued by aerobic exercise.

Patients receiving cytokine immunotherapy with IFN-α frequently present with neuropsychiatric consequences and cognitive impairments, including a profound depressive-like symptomatology. While the neurobiological substrates of the dysfunction that leads to adverse events in IFN-α-treated patients remains ill-defined, dysfunctions of the hippocampus and prefrontal cortex (PFC) are strong possibilities. To date, hippocampal deficits have been well-characterised; there does however remain a lack of insight into the nature of prefrontal participation. Here, we used a PFC-supported temporal order memory paradigm to examine if IFN-α treatment induced deficits in performance; additionally, we used an object recognition task to assess the integrity of the perirhinal cortex (PRH). Finally, the utility of exercise as an ameliorative strategy to recover temporal order deficits in rats was also explored. We found that IFN-α-treatment impaired temporal order memory discriminations, whereas recognition memory remained intact, reflecting a possible dissociation between recognition and temporal order memory processing. Further characterisation of temporal order memory impairments using a longitudinal design revealed that deficits persisted for 10 weeks following cessation of IFN-α-treatment. Finally, a 6 week forced exercise regime reversed IFN-α-induced deficits in temporal order memory. These data provide further insight into the circuitry involved in cognitive impairments arising from IFN-α-treatment. Here we suggest that PFC (or the hippocampo-prefrontal pathway) may be compromised whilst the function of the PRH is preserved. Deficits may persist after cessation of IFN-α-treatment which suggests that extended patient monitoring is required. Aerobic exercise may be restorative and could prove beneficial for patients treated with IFN-α.

Illness Severity, Social and Cognitive Ability, and EEG Analysis of Ten Patients with Rett Syndrome Treated with Mecasermin (Recombinant Human IGF-1).

Rett Syndrome (RTT) is a severe neurodevelopmental disorder characterized by an apparently normal development followed by an arrest and subsequent regression of cognitive and psychomotor abilities. At present, RTT has no definitive cure and the treatment of RTT represents a largely unmet clinical need. Following partial elucidation of the underlying neurobiology of RTT, a new treatment has been proposed, Mecasermin (recombinant human Insulin-Like Growth Factor 1), which, in addition to impressive evidence from preclinical murine models of RTT, has demonstrated safety in human studies of patients with RTT. The present clinical study examines the disease severity as assessed by clinicians (International Scoring System: ISS), social and cognitive ability assessed by two blinded, independent observers (RSS: Rett Severity Score), and changes in brain activity (EEG) parameters of ten patients with classic RTT and ten untreated patients matched for age and clinical severity. Significant improvement in both the ISS (p = 0.0106) and RSS (p = 0.0274) was found in patients treated with IGF1 in comparison to untreated patients. Analysis of the novel RSS also suggests that patients treated with IGF1 have a greater endurance to social and cognitive testing. The present clinical study adds significant preliminary evidence for the use of IGF-1 in the treatment of RTT and other disorders of the autism spectrum.

Akut exazerbierende primär chronische Osteomyelitis der Mandibula im Kindesalter.

Inflammation of bone is caused either by bacterial infection or occasionally by physical stimulus. Primary chronic osteomyelitis of mandibular bone is a chronic inflammation of an unknown cause. Pain, swelling, limited mouth opening, regional lymphadenopathy and hypaesthesia are clinical symptoms at initial presentation. Results of biopsy, computed tomography and scintigraphy reveal the diagnosis of a primary chronic osteomyelitis. Its management is long-term antibiotic therapy, hyperbaric oxygen and surgical therapy, even bisphophonate treatement may be a good option. The case report presents a primary progressive chronic osteomyelitis of the manibular bone of a ten year old boy. Clinical and radiological signs are discussed as well as diagnosis, management and follow-up.

Engineering mammalian cell factories with SINEUP noncoding RNAs to improve translation of secreted proteins.

Whenever the function of a recombinant protein depends on post-translational processing, mammalian cells become an indispensable tool for their production. This is particularly true for biologics and therapeutic monoclonal antibodies (MAbs). Despite some drawbacks, Chinese Hamster Ovary (CHO) cells are the workhorse for MAbs production in academia and industry. Several methodologies have been adopted to improve expression and stability, including methods based on selective pressure or cell engineering. We have previously identified SINEUPs as a new functional class of natural and synthetic long non-coding RNAs that through the activity of an inverted SINEB2 element are able to promote translation of partially overlapping sense coding mRNAs. Here we show that by taking advantage of their modular structure, synthetic SINEUPs can be designed to increase production of secreted proteins. Furthermore, by experimentally validating antisense to elastin (AS-eln) RNA as a natural SINEUP, we show that SINEUP-mediated control may target extracellular proteins. These results lead us to propose synthetic SINEUPs as new versatile tools to optimize production of secreted proteins in manufacturing pipelines and natural SINEUPs as new regulatory RNAs in the secretory pathways.

Metastatic breast cancer in the mandibular condyle mimicking temporomandibular joint (TMJ) disease.

Metastases or tumour to the jaws are rare and those to the temporomandibular joint (TMJ) are even rarer. The symptoms like preauricular pain, swelling and clicking are generally associated with TMJ disease. But the same symptoms are also found in tumours of the jaws or other diseases. We report on the case of a 48-year-old woman with a 12-year history of breast cancer who was referred to our department for clarification of preauricular swelling and pain. The possible aetiology of TMJ disorders and the frequency and localization of metastases to the jaws are discussed.

Age-related declines in delayed non-match-to-sample performance (DNMS) are reversed by the novel 5HT6 receptor antagonist SB742457.

Alterations in synaptic plasticity and neurocognitive function with age have been well documented in the literature. These changes are accompanied by modifications of neurotransmitter systems in the central nervous system (CNS). The serotonergic system in particular plays an important role in attention, alertness and cognition. Disturbances in serotonergic function have been implicated in differing neurological and neuropsychiatric disorders including depression, psychosis aggression and dementia. The serotonin receptor subtype 5HT6 is distributed within CNS regions relevant to learning and memory, including the striatum, cortex and hippocampus. We examined here the effects of acute and chronic administration of the 5HT6 receptor antagonist SB742457 on performance in a delayed non-matching-to-sample task (DNMS), which was used to identify neurocognitive differences between middle-aged (MA, 13 months) and young adult (YG, 3 months) rats. We found that MA rats have significantly lower performance in the DNMS task compared to YG rats. Acute administration of SB742457 (3 mg/kg/po) significantly improved performance of the MA rats. Chronic administration of SB742457 (3 mg/kg) reversed the age-related deficit of the MA to match their performance to that of YG rats. Furthermore, these improvements were observed for 1 week post-SB742457 treatment cessation. The acute and chronic effects of this treatment suggest that there is both an immediate effect on neurotransmitter action and potentially a longer-term modification of synaptic plasticity. Together these data indicate a role for modulation of the serotonergic system in the development of cognition-enhancing agents.

Rosiglitazone enhances learning, place cell activity, and synaptic plasticity in middle-aged rats.

As an antidiabetic agent, rosiglitazone (ROSI) binds and activates peroxisome proliferator-activator receptor gamma (PPARγ), altering the expression of genes involved in glucose uptake and disposal, ultimately affecting glucose regulation. ROSI might therefore be a potential treatment to ameliorate age-related decline in cognitive function, particularly on an insulin-resistant background, where improvements in peripheral insulin sensitivity and central nervous system (CNS) glucose utilization may facilitate recovery of cognitive function. We therefore examined the amelioration potential of ROSI for neurocognitive deficits resulting from aging in an animal model. Behaviorally, acute and chronic ROSI treatments enhanced acquisition of learning in the water plus maze, a modified version of the Morris water maze task. In parallel, restoration of synaptic plasticity in the dentate gyrus of ROSI-treated middle-aged rats was evident after a single dose intake. Additionally, the spatial receptive fields of hippocampal CA1 place cells were significantly improved by chronic ROSI administration. ROSI treatment reversed basal plasma insulin abnormalities and increased hippocampal glucose transporter (GLUT)-3 expression in middle-aged rats. Taken together, these results suggest that ROSI modulates hippocampal circuitry effectively to promote an improvement in cognitive function, possibly via a glucose transporter-3 mechanism.

IGF1 as a Potential Treatment for Rett Syndrome: Safety Assessment in Six Rett Patients.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder that affects one in ten thousand girls and has no cure. The majority of RTT patients display mutations in the gene that codes for the methyl-CpG-binding protein 2 (MeCP2). Clinical observations and neurobiological analysis of mouse models suggest that defects in the expression of MeCP2 protein compromise the development of the central nervous system, especially synaptic and circuit maturation. Thus, agents that promote brain development and synaptic function, such as insulin-like growth factor 1 (IGF1), are good candidates for ameliorating the symptoms of RTT. IGF1 and its active peptide, (1-3) IGF1, cross the blood brain barrier, and (1-3) IGF1 ameliorates the symptoms of RTT in a mouse model of the disease; therefore they are ideal treatments for neurodevelopmental disorders, including RTT. We performed a pilot study to establish whether there are major risks associated with IGF1 administration in RTT patients. Six young girls with classic RTT received IGF1 subcutaneous injections twice a day for six months, and they were regularly monitored by their primary care physicians and by the unit for RTT in Versilia Hospital (Italy). This study shows that there are no risks associated with IGF1 administration.

Dissociation of dorsal hippocampal regional activation under the influence of stress in freely behaving rats.

Stress has deleterious effects on brain, body, and behavior in humans and animals alike. The present work investigated how 30-min acute photic stress exposure impacts on spatial information processing in the main sub-regions of the dorsal hippocampal formation [CA1, CA3, and dentate gyrus (DG)], a brain structure prominently implicated in memory and spatial representation. Recordings were performed from spatially tuned hippocampal and DG cells in rats while animals foraged in a square arena for food. The stress procedure induced a decrease in firing frequencies in CA1 and CA3 place cells while sparing locational characteristics. In contrast to the CA1-CA3 network, acute stress failed to induce major changes in the DG neuronal population. These data demonstrate a clear dissociation of the effects of stress on the main hippocampal sub-regions. Our findings further support the notion of decreased hippocampal excitability arising from behavioral stress in areas CA1 and CA3, but not in DG.

The polyunsaturated fatty acids, EPA and DPA exert a protective effect in the hippocampus of the aged rat.

Age is characterized by deficits in synaptic function identified by decreased performance of aged animals in spatial learning tasks and reduced ability of animals to sustain long term potentiation (LTP). Several cellular and molecular events are correlated with these deficits, many of which are indicative of age-related neuroinflammatory and oxidative cell stress. It is significant that agents which decrease microglial activation are commonly associated with restoration of function. We set out to examine whether the n-3 polyunsaturated fatty acid docosapentaenoic acid (DPA), which is a metabolite of eicosapentaenoic acid (EPA), could modulate the age-related increase in microglial activation and the associated increase in oxidative changes and therefore impact on synaptic function in aged rats. We demonstrate that docosapentaenoic acid possesses neurorestorative effects and is capable of downregulating microglial activation. The data show that it also decreases the coupled activation of sphingomyelinase and caspase 3, probably because of its ability to decrease age-related oxidative changes, and consequently attenuates the age-related decrease in spatial learning and long-term potentiation.