Crambescin C1 Acts as A Possible Substrate of iNOS and eNOS Increasing Nitric Oxide Production and Inducing In Vivo Hypotensive Effect.

Crambescins are guanidine alkaloids from the sponge Crambe crambe. Crambescin C1 (CC) induces metallothionein genes and nitric oxide (NO) is one of the triggers. We studied and compared the in vitro, in vivo, and in silico effects of some crambescine A and C analogs. HepG2 gene expression was analyzed using microarrays. Vasodilation was studied in rat aortic rings. In vivo hypotensive effect was directly measured in anesthetized rats. The targets of crambescines were studied in silico. CC and homo-crambescine C1 (HCC), but not crambescine A1 (CA), induced metallothioneins transcripts. CC increased NO production in HepG2 cells. In isolated rat aortic rings, CC and HCC induced an endothelium-dependent relaxation related to eNOS activation and an endothelium-independent relaxation related to iNOS activation, hence both compounds increase NO and reduce vascular tone. In silico analysis also points to eNOS and iNOS as targets of Crambescin C1 and source of NO increment. CC effect is mediated through crambescin binding to the active site of eNOS and iNOS. CC docking studies in iNOS and eNOS active site revealed hydrogen bonding of the hydroxylated chain with residues Glu377 and Glu361, involved in the substrate recognition, and explains its higher binding affinity than CA. The later interaction and the extra polar contacts with its pyrimidine moiety, absent in the endogenous substrate, explain its role as exogenous substrate of NOSs and NO production. Our results suggest that CC serve as a basis to develop new useful drugs when bioavailability of NO is perturbed.

Outcomes of surgical resection after neoadjuvant chemoimmunotherapy in locally advanced stage IIIA non-small-cell lung cancer.

OBJECTIVES: This analysis aimed to evaluate perioperative outcomes of surgical resection following neoadjuvant treatment with chemotherapy plus nivolumab in resectable stage IIIA non-small-cell lung cancer. METHODS: Eligible patients received neoadjuvant chemotherapy (paclitaxel + carboplatin) plus nivolumab for 3 cycles. Reassessment of the tumour was carried out after treatment and patients with at least stable disease as best response underwent pulmonary resection. After surgery, patients received adjuvant treatment with nivolumab for 1 year. Surgical data were collected from the NADIM database and patient charts were reviewed for additional surgical details. RESULTS: Among 46 patients who received neoadjuvant treatment, 41 (89.1%) underwent surgery. Two patients rejected surgery and 3 did not fulfil resectability criteria. There were 35 lobectomies (85.3%), 3 of which were sleeve lobectomies (9.4%), 3 bilobectomies (7.3%) and 3 pneumonectomies (7.3%). Video-assisted thoracoscopy was the initial approach in 51.2% of cases, with a conversion rate of 19% (n = 4). There was no operative mortality at either 30 or 90 days. The most common complications were prolonged air leak (n = 8), pneumonia (n = 5) and arrhythmia (n = 4). Complete resection (R0) was achieved in all patients who underwent surgery, downstaging was observed in 37 patients (90.2%) and major pathological response in 34 patients (82.9%). CONCLUSIONS: Surgical resection following induction therapy with chemotherapy plus nivolumab appears to be safe and offers appropriate oncological outcomes. Perioperative morbidity and mortality rates in our study were no higher than previously reported in this setting. A minimally invasive approach is, therefore, feasible.

Prevalence of Clinical COPD Phenotypes in Patients Who Are Candidates for Lung Transplantation, Complications and Post-Transplant Survival.

Introduction: The prevalence of COPD phenotypes that are referred for assessment for lung transplantation is unknown, as well as whether specific phenotype influences post-transplant evolution in those patients who receive it. Material and methods: Ambispective observational study without intervention. The main objective was to know the prevalence of the different COPD phenotypes of the patients referred for the evaluation of a lung transplant. Secondary objective were to compare their clinical characteristics, to perform an analysis of post-transplant survival or complications according to their phenotype. Results: 502 patients were evaluated for lung transplantation, of which 173 met the study criteria. 31.21% of the patients were discarded for transplantation on a first visit. The final cohort of potential transplant candidates who completed the pre-transplant study was 119 (69%) and 47 finally received a lung transplant (39.5%). The most frequent COPD phenotype evaluated for lung transplantation was the exacerbator (59%), followed by the non-exacerbator (38%) and the Asthma COPD Overlap [ACO] (3%). 59.8% of the exacerbator-phenotype patients assessed did not complete the pre-transplant study. Exacerbator-phenotype patients have a lower post-transplant survival (1115.1 days [standard deviation-DE-587]) vs. ACO: 1432 days [DE 507.5] and Non-exacerbators: 1317.8 days [DE 544.7] p = 0.16), although this difference has not been statistically significant. Conclusions: The most frequent COPD phenotype assessed for lung transplantation is the exacerbator, although more than half of these patients fail to complete the pre-transplant study.

Introducción: Se desconoce la prevalencia de los fenotipos de EPOC que son remitidos para valorar el trasplante pulmonar, así como si un fenotipo específico tiene influencia en la evolución postrasplante en aquellos pacientes que lo reciben. Materiales y métodos: Estudio observacional ambispectivo, sin intervención. El objetivo principal fue conocer la prevalencia de los diferentes fenotipos de EPOC de los pacientes que fueron remitidos para valorar un trasplante de pulmón. Los objetivos secundarios fueron comparar sus características clínicas para realizar un análisis de la supervivencia postrasplante o de las complicaciones de acuerdo con el fenotipo. Resultados: Se valoró a 502 pacientes para la realización de un trasplante de pulmón, de los cuales 173 cumplían los criterios del estudio. El 31,21% de los pacientes fue descartado para el trasplante en la primera visita. La cohorte final de candidatos potenciales a trasplante que completaron el estudio pretrasplante fue de 119 pacientes (69%) y, finalmente, 47 recibieron el trasplante de pulmón (39,5%). El fenotipo de EPOC que se evaluó con mayor frecuencia para el trasplante de pulmón fue el agudizador (59%), seguido del no agudizador (38%) y el mixto EPOC-asma (3%). El 59,8% de los pacientes con fenotipo agudizador valorados no completó el estudio pretrasplante. Los pacientes con fenotipo agudizador presentan una supervivencia postrasplante más baja (1.115,1 días; desviación estándar [DE]: 587) frente a los mixto EPOC-asma (1.432 días; DE: 507,5) y los no agudizadores (1.317,8 días; DE: 544,7; p = 0,16), aunque esta diferencia no fue estadísticamente significativa. Conclusiones: El fenotipo más frecuentemente estudiado para el trasplante de pulmón es el agudizador, aunque más de la mitad de estos pacientes no completa el estudio pretrasplante.

Improving zebrafish embryo xenotransplantation conditions by increasing incubation temperature and establishing a proliferation index with ZFtool.

BACKGROUND: Zebrafish (Danio rerio) is a model organism that has emerged as a tool for cancer research, cancer being the second most common cause of death after cardiovascular disease for humans in the developed world. Zebrafish is a useful model for xenotransplantation of human cancer cells and toxicity studies of different chemotherapeutic compounds in vivo. Compared to the murine model, the zebrafish model is faster, can be screened using high-throughput methods and has a lower maintenance cost, making it possible and affordable to create personalized therapies. While several methods for cell proliferation determination based on image acquisition and quantification have been developed, some drawbacks still remain. In the xenotransplantation technique, quantification of cellular proliferation in vivo is critical to standardize the process for future preclinical applications of the model. METHODS: This study improved the conditions of the xenotransplantation technique - quantification of cellular proliferation in vivo was performed through image processing with our ZFtool software and optimization of temperature in order to standardize the process for a future preclinical applications. ZFtool was developed to establish a base threshold that eliminates embryo auto-fluorescence and measures the area of marked cells (GFP) and the intensity of those cells to define a 'proliferation index'. RESULTS: The analysis of tumor cell proliferation at different temperatures (34 °C and 36 °C) in comparison to in vitro cell proliferation provides of a better proliferation rate, achieved as expected at 36°, a maintenance temperature not demonstrated up to now. The mortality of the embryos remained between 5% and 15%. 5- Fluorouracil was tested for 2 days, dissolved in the incubation medium, in order to quantify the reduction of the tumor mass injected. In almost all of the embryos incubated at 36 °C and incubated with 5-Fluorouracil, there was a significant tumor cell reduction compared with the control group. This was not the case at 34 °C. CONCLUSIONS: Our results demonstrate that the proliferation of the injected cells is better at 36 °C and that this temperature is the most suitable for testing chemotherapeutic drugs like the 5-Fluorouracil.

The association of bacterial C9-based TTX-like compounds with Prorocentrum minimum opens new uncertainties about shellfish seafood safety.

In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INa.

Marine guanidine alkaloids crambescidins inhibit tumor growth and activate intrinsic apoptotic signaling inducing tumor regression in a colorectal carcinoma zebrafish xenograft model.

The marine environment constitutes an extraordinary resource for the discovery of new therapeutic agents. In the present manuscript we studied the effect of 3 different sponge derived guanidine alkaloids, crambescidine-816, -830, and -800. We show that these compounds strongly inhibit tumor cell proliferation by down-regulating cyclin-dependent kinases 2/6 and cyclins D/A expression while up-regulating the cell cyclin-dependent kinase inhibitors -2A, -2D and -1A. We also show that these guanidine compounds disrupt tumor cell adhesion and cytoskeletal integrity promoting the activation of the intrinsic apoptotic signaling, resulting in loss of mitochondrial membrane potential and concomitant caspase-3 cleavage and activation. The crambescidin 816 anti-tumor effect was fnally assayed in a zebrafish xenotransplantation model confirming its potent antitumor activity against colorectal carcinoma in vivo.Considering these results crambescidins could represent promising natural anticancer agents and therapeutic tools.

Troubleshooting of single port video-assisted thoracoscopic lung resection.

By now single port video-assisted thoracoscopic surgery is in full swing all over the world. Series of papers have been proved its safety and feasibility. There are still some tricks which could help beginner to overcome the obstacle of operation. In this article, we tried to focus on how to retrieve the specimen from chest wall cavity simply and how to deal with the bleeding episode during operation.

Crambescin C1 Exerts a Cytoprotective Effect on HepG2 Cells through Metallothionein Induction.

The Mediterranean marine sponge Crambe crambe is the source of two families of guanidine alkaloids known as crambescins and crambescidins. Some of the biological effects of crambescidins have been previously reported while crambescins have undergone little study. Taking this into account, we performed comparative transcriptome analysis to examine the effect of crambescin-C1 (CC1) on human tumor hepatocarcinoma cells HepG2 followed by validation experiments to confirm its predicted biological activities. We report herein that, while crambescin-A1 has a minor effect on these cells, CC1 protects them against oxidative injury by means of metallothionein induction even at low concentrations. Additionally, at high doses, CC1 arrests the HepG2 cell cycle in G0/G1 and thus inhibits tumor cell proliferation. The findings presented here provide the first detailed approach regarding the different effects of crambescins on tumor cells and provide a basis for future studies on other possible cellular mechanisms related to these bioactivities.

Chronic ciguatoxin treatment induces synaptic scaling through voltage gated sodium channels in cortical neurons.

Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin. These findings identify an unanticipated role for ciguatoxin in the regulation of homeostatic plasticity in central neurons involving NaV channels and raise the possibility that some of the neurological symptoms of ciguatera might be explained by these compensatory mechanisms.

Technique of uniportal VATS major pulmonary resections.

The surgical approach to lung resections is evolving constantly. Since the video-assisted thoracoscopic surgery (VATS) anatomic lobectomy for lung cancer was described two decades ago, many units have successfully adopted this technique. The VATS lobectomy can be defined as the individual dissection of veins, arteries and bronchus, with a mediastinal lymphadenectomy, using a videothoracoscopic approach visualized on screen and involving 2 to 4 incisions or ports, with no rib spreading. However, the surgery can be performed by only one incision with similar outcomes. Since 2010, when the uniportal approach was introduced for major pulmonary resections, the technique has been spreading worldwide. This technique provides a direct view of the target tissue. The parallel instrumentation achieved during the single-port approach mimics the maneuvers performed during open surgery. It represents a less invasive approach than the multiport technique, and minimizes the compression of the intercostal nerve. As the surgeon's experience with the uniportal VATS lobectomy grows, more complex cases can be performed by using this approach, thus expanding the indications for single-incision thoracoscopic lobectomy.

Uniportal video-assisted thoracoscopic lymph node dissection.

Lymphadenectomy is an important part of lung cancer surgery. At the moment, video-assisted thoracoscopic (VATS) is the most common approach to remove these tumors, when it is technically possible. With our current experience in VATS in major resections we have obtained a radical videothoracoscopic mediastinal lymphadenectomy, and single-port provides us with the best anatomic instrumentation and a direct view.

Management of complications by uniportal video-assisted thoracoscopic surgery.

Since the video-assisted thoracoscopic surgery (VATS) anatomic lobectomy for lung cancer was described two decades ago, many units have successfully adopted this technique. VATS lobectomy is a safe and effective approach for the treatment not only of early stage lung cancer but also for more advanced disease. It represents a technical challenge. As the surgeon's experience grows, more complex or advanced cases are approached using the VATS approach. However, as VATS lobectomy has been applied to more advanced cases, the rate of conversion to open thoracotomy has increased, particularly early in the surgeon's learning curve, mostly due to the occurrence of complications. The best strategy for facing complications of VATS lobectomy is to prevent them from happening. Avoiding complications is subject to an appropriate preoperative workup and patient selection. Planning for a VATS resection as safely as possible involves the consideration of the patient´s characteristics and the anticipated technical aspects of the case. Awareness of the possibility of intraoperative complications of VATS lobectomy is mandatory to avoid them, and the development of management strategies is necessary to limit morbidity if they occur.

Indication for VATS sublobar resections in early lung cancer.

When dealing with early non-small cell lung cancer (NSCLC) sublobar resections still remain part of the surgical armamentarium. In selected patients with lung cancer, the combination of the potential benefits of parenchyma sparing procedures to the limited trauma provided by Video Assisted Thoracic Surgery (VATS) techniques can become very appealing. Two main groups are included: non-anatomical (wedges) and anatomical (segmentectomies) excisions. We describe the techniques, results and potential indications of both of these techniques.