Characterization of six Merkel cell polyomavirus-positive Merkel cell carcinoma cell lines: Integration pattern suggest that large T antigen truncating events occur before or during integration

Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin tumor, is a polyomavirus-induced human cancer. To study the causal relationship of MCC carcinogenesis with the integrated Merkel cell polyomavirus (MCPyV) in detail, well-characterized MCC cell lines are needed. Consequently, in the current study, we established and characterized six MCPyV-positive MCC cell lines. Microarray-based comparative genomic hybridization revealed a stable genome carrying only a limited number of chromosomal gains and deletions. All cell lines expressed MCC markers Keratin-20 and neuron-specific enolase as well as truncated MCPyV-encoded large T antigen (LT). For five cell lines, we were able to identify the MCPyV-integration sites in introns of different genes. The LT-truncating stop codon mutations and integration sites were affirmed in the respective clinical patient samples. Inverse PCR suggested that three of the cell lines contained MCPyV genomes as concatemers. This notion was confirmed for the two cell lines with known integration sites. Importantly, our observation of distinct stop codon mutations in cell lines with concatemeric MCPyV integration indicates that these LT-truncating mutations occur before integration. In summary, we provide the detailed characterization of six MCPyV-positive MCC cell lines, which are likely to serve as valuable tools in future MCC research.     

The effect of oxygen in Sirt3-mediated myocardial protection: a proof-of-concept study in cultured cardiomyoblasts

Sirtuin 3 is a nicotinamide adenine dinucleotide dependent mitochondrial deacetylase that governs mitochondrial metabolism and oxidative defense. The demise in myocardial function following myocardial ischemia has been associated with mitochondrial dysfunction. Sirt3 maintains myocardial contractile function and protects from cardiac hypertrophy. The role of Sirt3 in ischemia is controversial. Our objective was to understand, under what circumstances Sirt3 is protective in different facets of ischemia, using an in vitro proof-of-concept approach based on simulated ischemia in cultured cardiomyoblasts. Cultured H9c2 cardiomyoblasts were subjected to hypoxia and/or serum deprivation, the combination of which we refer to as simulated ischemia. Apoptosis, as assessed by Annexin V staining in life-cell imaging and propidium-iodide inclusion in flow cytometry, was enhanced following simulated ischemia. Interestingly, serum deprivation was a stronger trigger of apoptosis than hypoxia. Knockdown of Sirt3 further increased apoptosis upon serum deprivation, whereas no such effect occurred upon additional hypoxia. Similarly, only upon serum deprivation but not upon simulated ischemia, silencing of Sirt3 led to a deterioration of mitochondrial function in extracellular flux analysis. In the absence of oxygen these Sirt3-dependent effects were abolished. These data indicate, that Sirt3-mediated myocardial protection is oxygen-dependent. Thus, mitochondrial respiration takes center-stage in Sirt3-dependent prevention of stress-induced myocardial damage.

     

First Encounters: Repair Sequences in Cross-Signing

Most human communication is between people who speak or sign the same languages. Nevertheless, communication is to some extent possible where there is no language in common, as every tourist knows. How this works is of some theoretical interest (Levinson, 2006 ). A nice arena to explore this capacity is when deaf signers of different languages meet for the first time and are able to use the iconic affordances of sign to begin communication. Here we focus on other-initiated repair (OIR), that is, where one signer makes clear he or she does not understand, thus initiating repair of the prior conversational turn. OIR sequences are typically of a three-turn structure (Schegloff 2007 ), including the problem source turn (T−1), the initiation of repair (T0), and the turn offering a problem solution (T+1). These sequences seem to have a universal structure (Dingemanse et al. 2013). We find that in most cases where such OIR occur, the signer of the troublesome turn (T−1) foresees potential difficulty and marks the utterance with “try markers” (Moerman, 1988 ; Sacks & Schegloff, 1979 ) which pause to invite recognition. The signers use repetition, gestural holds, prosodic lengthening, and eyegaze at the addressee as such try-markers. Moreover, when T−1 is try-marked this allows for faster response times of T+1 with respect to T0. This finding suggests that signers in these “first encounter” situations actively anticipate potential trouble and, through try-marking, mobilize and facilitate OIRs. The suggestion is that heightened meta-linguistic awareness can be utilized to deal with these problems at the limits of our communicational ability.     

Lymph node dissection for melanoma using tumescence local anaesthesia: an observational study

Background

The possibility that tumescence local anaesthesia (TLA) may lead to dissemination of tumour cells in lymph nodes is presently unclear.

Objectives

To evaluate whether infiltration by TLA influences metastatic spread and survival probability, compared to general anaesthesia (GA), based on lymph node dissection in melanoma patients.

Patients & methods

In total, 281 patients (GA: 162; TLA: 119) with cutaneous melanoma and clinically or histologically-confirmed metastases in regional lymph nodes were included. All patients underwent complete lymph node dissection.

Results

Median follow-up was 70 months. The rate of lymph node recurrence at the dissection site was 25.3% in the GA group and 17.6% in the TLA group (p = 0.082). No significant difference was found concerning 10-year melanoma-specific survival (GA: 56.2%, TLA: 67.4%; p = 0.09), disease-free survival (GA: 72.8 %, TLA: 81.1%; p = 0.095), or lymph node-free survival (GA: 72.8%, TLA: 81.1%; p = 0.095). Distant metastases-free survival appeared to be slightly reduced in the TLA group (GA: 49.9%, TLA: 64.0%; p = 0.025).

Conclusions

No differences were identified between the GA and TLA groups regarding prognostic outcome for overall survival or disease-free survival.

     

Changes in neuronal activity of cortico‐basal ganglia–thalamic networks induced by acute dopaminergic manipulations in rats

The basal ganglia are thought to be particularly sensitive to changes in dopaminergic tone, and the realization that reduced dopaminergic signaling causes pronounced motor dysfunction is the rationale behind dopamine replacement therapy in Parkinson's disease. It has, however, proven difficult to identify which neurophysiological changes that ultimately lead to motor dysfunctions. To clarify this, we have here recorded neuronal activity throughout the cortico‐basal ganglia–thalamic circuits in freely behaving rats during periods of immobility following acute dopaminergic manipulations, involving both vesicular dopamine depletion and antagonism of D1 and D2 type dopamine receptors. Synchronized and rhythmic activities were detected in the form of betaband oscillations in local field potentials and as cortical entrainment of action potentials in several basal ganglia structures. Analyses of the temporal development of synchronized oscillations revealed a spread from cortex to gradually also include deeper structures. In addition, firing rate changes involving neurons in all parts of the network were observed. These changes were typically relatively balanced within each structure, resulting in negligible net rate changes. Animals treated with D1 receptor antagonist showed a rapid onset of hypokinesia that preceded most of the neurophysiological changes, with the exception of these balanced rate changes. Parallel rate changes in functionally coupled ensembles of neurons in different structures may therefore be the first step in a cascade of neurophysiological changes underlying motor symptoms in the parkinsonian state. We suggest that balanced rate changes in distributed networks are possible mechanism of disease that should be further investigated in conditions involving dopaminergic dysfunction.     

Distinct brain representations of processed and unprocessed foods

Among all of the stimuli surrounding us, food is arguably the most rewarding for the essential role it plays in our survival. In previous visual recognition research, it has already been demonstrated that the brain not only differentiates edible stimuli from non‐edible stimuli but also is endowed with the ability to detect foods’ idiosyncratic properties such as energy content. Given the contribution of the cooked diet to human evolution, in the present study we investigated whether the brain is sensitive to the level of processing food underwent, based solely on its visual appearance. We thus recorded visual evoked potentials (VEPs) from normal‐weight healthy volunteers who viewed color images of unprocessed and processed foods equated in caloric content. Results showed that VEPs and underlying neural sources differed as early as 130 ms post‐image onset when participants viewed unprocessed versus processed foods, suggesting a within‐category early discrimination of food stimuli. Responses to unprocessed foods engaged the inferior frontal and temporal regions and the premotor cortices. In contrast, viewing processed foods led to the recruitment of occipito‐temporal cortices bilaterally, consistently with other motivationally relevant stimuli. This is the first evidence of diverging brain responses to food as a function of the transformation undergone during its preparation that provides insights on the spatiotemporal dynamics of food recognition.