Cell size and actin architecture determine force generation in optogenetically activated cells.

Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation, and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho family. To understand better how activation of these regulators translates into cell-scale force generation in the context of different physical environments, here we combine recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that, after whole-cell RhoA activation by the CRY2/CIBN optogenetic system with a short pulse of 100 ms, single cells contract on a minute timescale in proportion to their original traction force, before returning to their original tension setpoint with near perfect precision, on a longer timescale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA response builds up quickly on a timescale of 20 s, but decays slowly on a timescale of 50 s. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA activation starts to saturate if optogenetic pulse length exceeds 50 ms, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.

Erratum to "Intratumoral Heterogeneity of MAGE-C1/CT7 and MAGE-C2/CT10 Expression in Mucosal Melanoma".

[This corrects the article DOI: 10.1155/2015/432479.].

Intratumoral Heterogeneity of MAGE-C1/CT7 and MAGE-C2/CT10 Expression in Mucosal Melanoma.

Mucosal melanoma is a rare disease, which differs from its cutaneous counterpart genetically and for its clinical behaviour. Moreover this is a heterogeneous disease based on the tissue of origin. As CT7 and CT10 are highly expressed in cutaneous melanoma and are immunogenic in this disease, we analysed their expression throughout the different subtypes of mucosal melanoma and tumor development. We detected a frequent expression of CT7 in primaries and corresponding metastases (55%) as well as for CT10 (30%). This expression resulted to be heterogeneous in the same tumor specimen and moreover influenced by the tissue of origin. Our results support the role of these antigens in immunotherapy for mucosal melanoma.

[Insights into melanoma from a pathologist's perspective]. / Lichtblicke beim schwarzen Hautkrebs aus der Sicht von Pathologen.

Malignant melanoma is the most common cause of death from skin cancer. Wide surgical excision of localized melanoma in its primary stages remains the main curative therapy. Identifying patients at an early tumour stage is therefore one of the most significant steps for treatment. In the last decades, molecular pathology rapidly established itself in melanoma research. We present new molecular methods, their significance and their application, especially focusing on BRAF( V600) mutation and BRAF-inhibiting tumor targeting therapy. Resistance to tumor targeting therapies and cell line experiments, which have evidenced a sub population with stem cell properties, illustrate melanoma heterogeneity. Efforts to develop drugs that target more than a single target gene are currently underway.

First-line temozolomide combined with bevacizumab in metastatic melanoma: a multicentre phase II trial (SAKK 50/07).

BACKGROUND: Oral temozolomide has shown similar efficacy to dacarbazine in phase III trials with median progression-free survival (PFS) of 2.1 months. Bevacizumab has an inhibitory effect on the proliferation of melanoma and sprouting endothelial cells. We evaluated the addition of bevacizumab to temozolomide to improve efficacy in stage IV melanoma. PATIENTS AND METHODS: Previously untreated metastatic melanoma patients with Eastern Cooperative Oncology Group performance status of two or more were treated with temozolomide 150 mg/m(2) days 1-7 orally and bevacizumab 10 mg/kg body weight i.v. day 1 every 2 weeks until disease progression or unacceptable toxicity. The primary end point was disease stabilisation rate [complete response (CR), partial response (PR) or stable disease (SD)] at week 12 (DSR12); secondary end points were best overall response, PFS, overall survival (OS) and adverse events. RESULTS: Sixty-two patients (median age 59 years) enrolled at nine Swiss centres. DSR12 was 52% (PR: 10 patients and SD: 22 patients). Confirmed overall response rate was 16.1% (CR: 1 patient and PR: 9 patients). Median PFS and OS were 4.2 and 9.6 months. OS (12.0 versus 9.2 months; P = 0.014) was higher in BRAF V600E wild-type patients. CONCLUSIONS: The primary end point was surpassed showing promising activity of this bevacizumab/temozolomide combination with a favourable toxicity profile. Response and OS were significantly higher in BRAF wild-type patients.

The influence of sentinel lymph node tumour burden on additional lymph node involvement and disease-free survival in cutaneous melanoma--a retrospective analysis of 392 cases.

Twenty per cent of sentinel lymph node (SLN)-positive melanoma patients have positive non-SLN lymph nodes in completion lymph node dissection (CLND). We investigated SLN tumour load, non-sentinel positivity and disease-free survival (DFS) to assess whether certain patients could be spared CLND. Sentinel lymph node biopsy was performed on 392 patients between 1999 and 2005. Median observation period was 38.8 months. Sentinel lymph node tumour load did not predict non-SLN positivity: 30.8% of patients with SLN macrometastases (> or =2 mm) and 16.4% with micrometastases (< or =2 mm) had non-SLN positivity (P=0.09). Tumour recurrences after positive SLNs were more than twice as frequent for SLN macrometastases (51.3%) than for micrometastases (24.6%) (P=0.005). For patients with SLN micrometastases, the DFS analysis was worse (P=0.003) when comparing those with positive non-SLNs (60% recurrences) to those without (17.6% recurrences). This difference did not translate into significant differences in DFS: patients with SLN micrometastasis, either with (P=0.022) or without additional positive non-SLNs (P<0.0001), fared worse than patients with tumour-free SLNs. The 2-mm cutoff for SLN tumour load accurately predicts differences in DFS. Non-SLN positivity in CLND, however, cannot be predicted. Therefore, contrary to other studies, no recommendations concerning discontinuation of CLND based on SLN tumour load can be deduced.

Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex.

OBJECTIVE: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na+/Ca2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes. METHODS: Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein-protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy. RESULTS: TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Co-immunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GST-pulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca2+ entry, which was dependent on TRPC3-mediated Na+ loading of myocytes. This NCX-mediated Ca2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1. CONCLUSION: These results suggest a PLC-dependent recruitment of a TRPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca2+ homeostasis.

Spitzoid malignant melanoma with lymph-node metastasis. Is a copy-number loss on chromosome 6q a marker of malignancy?

Distinction of spitzoid malignant melanomas (SMM) from Spitz nevi may be difficult or even impossible on the basis of conventional histology. In this report, a patient suffering from a primary lesion diagnosed as a Spitz nevus and a metastatic malignant melanoma approximately 4 years thereafter is described. A diagnosis of SMM was made subsequently upon review of the primary lesion. In the present analysis, we used comparative genomic hybridization (CGH) to define markers characteristic of SMM. The primary lesion revealed deletions on chromosomes 6q and 9p. In the metastasis, additional deletions on chromosomes 10p and 10q and gains of chromosome 7 were found. To our knowledge, no chromosomal aberration on chromosome 6 was hitherto demonstrated in benign melanocytic nevi. Findings reported in the literature suggest that human melanoma metastasis suppressor gene maps to 6q. In contrast, losses on chromosome 9p seem to be an early event in the development of melanoma. However, they are not only found in melanomas but are occasionally present in Spitz nevi as well as in atypical nevi. The CGH result with deletion of 6q in this difficult to diagnose primary melanocytic lesion strongly supports the diagnosis of malignant melanoma. To demonstrate the reliability of loss on chromosome 6q as a marker of SMM, a larger number of lesions must be investigated.

Primary ciliary dyskinesia: a genome-wide linkage analysis reveals extensive locus heterogeneity.

Primary ciliary dyskinesia (PCD), or immotile cilia syndrome (ICS), is an autosomal recessive disorder affecting ciliary movement with an incidence of 1 in 20000-30000. Dysmotility to complete immotility of cilia results in a multisystem disease of variable severity with recurrent respiratory tract infections leading to bronchiectasis and male subfertility. Ultrastructural defects are present in ciliated mucosa and spermatozoa. Situs inversus (SI) is found in about half of the patients (Kartagener syndrome). We have collected samples from 61 European and North American families with PCD. A genome-wide linkage search was performed in 31 multiplex families (169 individuals including 70 affecteds) using 188 evenly spaced (19cM average interval) polymorphic markers. Both parametric (recessive model) and non-parametric (identity by descent allele sharing) linkage analyses were used. No major locus for the majority of the families was identified, although the sample was powerful enough to detect linkage if 40% of the families were linked to one locus. These results strongly suggest extensive locus heterogeneity. Potential genomic regions harbouring PCD loci were localised on chromosomes 3p, 4q, 5p, 7p, 8q, 10p, 11q, 13q, 15q, 16p, 17q and 19q. Linkage analysis using PCD families with a dynein arm deficiency provided 'suggestive' evidence for linkage to chromosomal regions 8q, 16pter, while analyses using only PCD families with situs inversus resulted in 'suggestive' scores for chromosomes 8q, and 19q.

[Maternal morbidity after abdominal cesarean section in relation to bacterial amniotic fluid colonization and premature rupture of fetal membranes]. / Mütterliche Morbidität nach abdominaler Schnittentbindung in Abhängigkeit vom Keimbefall des Fruchtwassers und vom vorzeitigen Blasensprung.

At the time of Caesarean section, amniotic fluid was collected transabdominally from 1025 patients. In 990 patients cultures were performed on the amniotic fluid. The incidence of Caesarean section of all patients was 16.1%, the perinatal mortality in all patients 0.61%, in cases of Caesarean section 1.93%. The influence of bacterial colonisation in the amniotic cavity was analyzed with respect to premature ruptured membranes and frequency of vaginal examinations on the maternal post-operative morbidity (wound-seroma and abscess 2.4%, febrile morbidity 17%, urinary tract infection 19%). Bacterial contamination was present in 33.3% of the amniotic fluid or uterine cavity, after ruptured membranes in 44.3%, without ruptured membranes in 23.35%. There is a significant influence of the positive cultured amniotic fluid on the wound healing and on the febrile morbidity, while the urinary tract infection has been not influenced. The bacterial contamination of the amniotic fluid is correlated to the frequency of vaginal examination and the presence of ruptured membranes. The following microbiologic isolates were found in the amniotic fluids: Staphylococcus epidermidis 36.38%, Streptococcus B 12.23%, Streptococcus D 10.3%, Escherichia coli 8.42%, Staphylococcus aureus 7.88%, Peptococci 3.26%, Streptococcus salivarius 2.99%, Bacteroides 2.4%. According to these data, patients with ruptured membranes of an interval of more than 12 hrs and with more than 7 vaginal examinations could be selected for antibiotic prophylaxis to achieve a reduction in febrile morbidity and in wound infection.

[Hemodynamic effects of the induction of general anesthesia after low thoracic epidural anesthesia]. / Effets hémodynamiques de l'induction d'une anesthésie générale après installation d'une péridurale thoracique basse.

Sixteen ASA 1 or 2 patients scheduled for abdominal surgery were included in the study after they had given their informed consent. Thirty minutes after starting a low-thoracic epidural anaesthesia (median level of sensitivity loss: T5), the patients were randomly given an intravenous bolus injection of either thiopentone (4 mg.kg-1, n = 8) or etomidate (0.5 mg.-1, n = 8), associated with succinylcholine 1 mg.kg-1. One minute after induction of general anaesthesia, the patients were intubated and mechanically ventilated (V(T) 8 ml.kg-1, rate 12 c.min-1). Mean arterial blood pressure (MAP) (oscillometric method), cardiac output (CO) (transthoracic bioimpedance) and heart rate were recorded semi-continuously. Total peripheral resistances (TPR) were calculated using the formula TPR = (MA/CO)*80. There were no differences between the groups in patient age, height, weight, and cardiovascular consequences of epidural anaesthesia. After anaesthetic induction and before endotracheal intubation, there was a slight decrease in CO in both groups, without any change in MAP. After intubation, MAP increased in both groups through peripheral vasoconstriction, whereas CO did not increase further. A significant tachycardia was occurred only seen in the thiopentone group, before and after tracheal intubation. This study showed that thiopentone and etomidate were suitable drugs for anaesthetic induction in a patient under epidural blockade. However, the absence of tachycardia following etomidate may be beneficial in cardiac patients. The monitoring of cardiac output determinants during thiopentone and etomidate anaesthesia require further invasive investigations.

[Effects of lidocaine on arterial and venous circulation of limbs in man]. / Effets de la lidocaïne sur la circulation artérielle et veineuse des membres chez l'homme.

The effects of intravenous lidocaine on limb arteries and veins were investigated in a placebo-controlled study. Seven young healthy volunteers, 23 to 28-years-old, were included. Electrocardiogram, arterial pressure and arm and leg blood flows were recorded continuously. Systolic and diastolic blood pressures were measured in the left arm by finger photoplethysmography. Limb blood flow and the limb venous system were studied by venous occlusive plethysmography. The venous parameters studied were venous tone, lowest closing pressure, venous volume at 30 mmHg, and venous distensibility. After an initial bolus of 1.5 mg.kg-1 lidocaine had been given, 30, and then 60, micrograms.kg-1.min-1 were given for one hour each. Plasma noradrenaline and serum lidocaine titres were measured before giving the lidocaine, and at the end of each one hour period. Placebo consisted in a two hour infusion of 0.25 ml.min-1 normal saline. Lidocaine titres were 1.64 +/- 0.40 microgram.ml-1 after one hour, and 2.55 +/- 0.69 microgram.ml-1 after two hours. Lidocaine increased vascular resistances in both the forearm (+81% to +93%) and the calf (+38% to +57%). There was a concomitant increase in mean arterial blood pressure (+21% to +28%) without any change in heart rate. There was a significant dose-dependent increase in plasma noradrenaline levels during the second period of the lidocaine infusion with respect to the preinfusion period and the same period during the placebo infusion. Venous capacitance measured before any infusion had been started was greater in the leg than in the arm.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of housing on the development of calluses and subcutaneous mucus cysts on the limbs of swine]. / Zum Einfluss der Haltung auf die Anbildung von Schwielen und subkutanen Schleimbeuteln an den Gliedmassen der Schweine.

Extremities of pigs often show swelling of various size and consistency. The aim of the present investigation was to determine the cause of the swelling with special emphasis on housing conditions. A total of 168 animals kept on slatted and/or partly slatted floors and 55 pigs kept on straw were examined. The swelling was partially caused by connective tissue proliferation only, but more often consisted of non inflamed subcutaneous bursae. Skeletal changes near the periost could never be seen. In pigs kept on straw weals and subcutaneous bursae practically never developed, whereas in animals kept on concrete, these changes did occur more frequently and were more pronounced with time.

Ultrastructure of somatotrophs of rats with median eminence lesions: studies in basal conditions and after thyrotropin-releasing hormone stimulation.

Anterior pituitaries from female Sprague-Dawley rats (160-200 g b.w) bearing electrolytic lesions of the median eminence were qualitatively and quantitatively investigated by electron microscopy and the findings compared with those from intact animals or sham-operated controls. This study was performed in basal conditions and after stimulation with thyrotropin-releasing hormone. Animals with lesions showed a reduction of both the number of somatotrophic cells and growth hormone granules. After injection of thyrotropin-releasing hormone exocytosis of growth hormone granules from somatotrophs was frequent in rats with lesions of the median eminence but rare in sham-operated animals. The differences were statistically significant.

Light and electron microscopic studies of thyrotrophin releasing hormone-induced growth hormone and prolactin release in hypophysectomized rats bearing an ectopic pituitary gland.

A light microscopic and ultrastructural study of anterior pituitary glands transplanted under the kidney capsule of hypophysectomized rats was performed, in basal conditions and after stimulation with thyrotrophin releasing hormone, at various intervals (24 h-2 months) after transplantation. Confirming previous biochemical findings, the results suggest that with time, somatotrophs acquire sensitivity to thyrotrophin releasing hormone and respond with increased exocytosis at doses that were found ineffective in pituitary glands in situ. Thyrotrophin releasing hormone stimulation did not seem to influence the morphology of prolactin cells, which were already highly active under basal conditions at all time intervals.

Ultrastructural study of the adenohypophysis of the Chinese hamster.

The adenohypophysis of normal Chinese hamsters of both sexes was examined ultrastructurally. Organs were fixed by intravascular perfusion with S-collidine-buffered glutaraldehyde solution. Seven types of cells were differentiated and, according to morphological characteristics, classified as (1) mammotropes, with very large (400-800 nm) polymorphous secretory granules; (2) somatotropes, either in the storage phase with numerous large, dense granules (average 300 nm), or in the hormone synthesis phase, with abundant endoplasmic reticulum and large Golgi apparatus; (3) corticotropes, with irregular cell shape, and granules (average 160 nm) arranged in lines parallel to the cell membrane; (4) FSH gonadotropes, with abundant and dilated endoplasmic reticulum, and granules (190-320 nm) uniformly distributed in the cytoplasm; (5) LH gonadotropes, with granules (120-220 nm) of varied density; (6) thyrotropes, with irregular cell shape and very small granules (120-160 nm), and (7) agranulated cells. The ultrastructure of the adenohypophysis of the Chinese hamster corresponds closely with observations reported in rats, mice and Syrian hamsters.