CYTOCHROME P450 3A13 AND ENDOTHELIN JOINTLY MEDIATE DUCTUS ARTERIOSUS CONSTRICTION TO OXYGEN IN MICE

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3Anull (Cyp3a(-/-)) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a(-/-) DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a(-/-) DA. However, responses of RA-treated WT and Cyp3a(-/-) mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a(-/-) newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally

Dendrimer-Based Fluorescent Indicators: In Vitro and In Vivo Applications

BACKGROUND: The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. PRINCIPAL FINDINGS: We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. CONCLUSION: We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups

ERK Is Involved in the Reorganization of Somatosensory Cortical Maps in Adult Rats Submitted to Hindlimb Unloading

Sensorimotor restriction by a 14-day period of hindlimb unloading (HU) in the adult rat induces a reorganization of topographic maps and receptive fields. However, the underlying mechanisms are still unclear. Interest was turned towards a possible implication of intracellular MAPK signaling pathway since Extracellular-signal-Regulated Kinase 1/2 (ERK1/2) is known to play a significant role in the control of synaptic plasticity. In order to better understand the mechanisms underlying cortical plasticity in adult rats submitted to a sensorimotor restriction, we analyzed the time-course of ERK1/2 activation by immunoblot and of cortical reorganization by electrophysiological recordings, on rats submitted to hindlimb unloading over four weeks. Immunohistochemistry analysis provided evidence that ERK1/2 phosphorylation was increased in layer III neurons of the somatosensory cortex. This increase was transient, and parallel to the changes in hindpaw cortical map area (layer IV). By contrast, receptive fields were progressively enlarged from 7 to 28 days of hindlimb unloading. To determine whether ERK1/2 was involved in cortical remapping, we administered a specific ERK1/2 inhibitor (PD-98059) through osmotic mini-pump in rats hindlimb unloaded for 14 days. Results demonstrate that focal inhibition of ERK1/2 pathway prevents cortical reorganization, but had no effect on receptive fields. These results suggest that ERK1/2 plays a role in the induction of cortical plasticity during hindlimb unloading

Evaluation of lymph node numbers for adequate staging of Stage II and III colon cancer

<p>Abstract</p> <p>Background</p> <p>Although evaluation of at least 12 lymph nodes (LNs) is recommended as the minimum number of nodes required for accurate staging of colon cancer patients, there is disagreement on what constitutes an adequate identification of such LNs.</p> <p>Methods</p> <p>To evaluate the minimum number of LNs for adequate staging of Stage II and III colon cancer, 490 patients were categorized into groups based on 1-6, 7-11, 12-19, and ≥ 20 LNs collected.</p> <p>Results</p> <p>For patients with Stage II or III disease, examination of 12 LNs was not significantly associated with recurrence or mortality. For Stage II (HR = 0.33; 95% CI, 0.12-0.91), but not for Stage III patients (HR = 1.59; 95% CI, 0.54-4.64), examination of ≥20 LNs was associated with a reduced risk of recurrence within 2 years. However, examination of ≥20 LNs had a 55% (Stage II, HR = 0.45; 95% CI, 0.23-0.87) and a 31% (Stage III, HR = 0.69; 95% CI, 0.38-1.26) decreased risk of mortality, respectively. For each six additional LNs examined from Stage III patients, there was a 19% increased probability of finding a positive LN (parameter estimate = 0.18510, p < 0.0001). For Stage II and III colon cancers, there was improved survival and a decreased risk of recurrence with an increased number of LNs examined, regardless of the cutoff-points. Examination of ≥7 or ≥12 LNs had similar outcomes, but there were significant outcome benefits at the ≥20 cutoff-point only for Stage II patients. For Stage III patients, examination of 6 additional LNs detected one additional positive LN.</p> <p>Conclusions</p> <p>Thus, the 12 LN cut-off point cannot be supported as requisite in determining adequate staging of colon cancer based on current data. However, a minimum of 6 LNs should be examined for adequate staging of Stage II and III colon cancer patients.</p

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Mathematical modeling reveals the functional implications of the different nuclear shuttling rates of Erk1 and Erk2.

The mitogen-activated protein kinase (MAPK) family of proteins is involved in regulating cellular fates such as proliferation, differentiation and apoptosis. In particular, the dynamics of the Erk/Mek system, which has become the canonical example for MAPK signaling systems, have attracted considerable attention. Erk is encoded by two genes, Erk1 and Erk2, that until recently had been considered equivalent as they differ only subtly at the sequence level. However, these proteins exhibit radically different trafficking between cytoplasm and nucleus and this fact may have functional implications. Here we use spatially resolved data on Erk1/2 to develop and analyze spatio-temporal models of these cascades, and we discuss how sensitivity analysis can be used to discriminate between mechanisms. Our models elucidate some of the factors governing the interplay between signaling processes and the Erk1/2 localization in different cellular compartments, including competition between Erk1 and Erk2. Our approach is applicable to a wide range of signaling systems, such as activation cascades, where translocation of molecules occurs. Our study provides a first model of Erk1 and Erk2 activation and their nuclear shuttling dynamics, revealing a role in the regulation of the efficiency of nuclear signaling

At least 2 distinct pathways generating reactive oxygen species mediate vascular cell adhesion molecule-1 induction by advanced glycation end products

Objective—The interaction of advanced glycation end products (AGEs) with their main receptor RAGE in endothelial cells induces intracellular generation of reactive oxygen species (ROS) and the expression of vascular cell adhesion molecule (VCAM)-1. We investigated the role of distinct sources of ROS, including the mitochondrial electron transport chain, NAD(P)H oxidase, xanthine oxidase, and arachidonic acid metabolism, in AGE-induced VCAM-1 expression. Methods and Results—The induction of ROS and VCAM-1 by AGEs in cultured human umbilical vein endothelial cells was specifically blocked by an anti-RAGE antibody. The inhibition of NAD(P)H oxidase by apocynin and diphenylene iodonium, and of the mitochondrial electron transport system at complex II by thenoyltrifluoroacetone (TTFA), significantly inhibited both AGE-induced ROS production and VCAM-1 expression, whereas these effects were potentiated by rotenone and antimycin A, specific inhibitors of mitochondrial complex I and III, respectively. The inhibition of Cu/Zn superoxide dismutase inhibited both ROS and VCAM-1 induction, indicating that H2O2 by this source is involved as a mediator of VCAM-1 expression by AGEs. Conclusions—Altogether, these results demonstrate that ROS generated by both NAD(P)H-oxidase and the mitochondrial electron transport system are involved in AGE signaling through RAGE, and indicate potential targets for the inhibition of the atherogenic signals triggered by AGE-RAGE interaction

Spatio-temporal dynamics and localization of MeCP2 and pathological mutants in living cells

MECP2 is an X-linked gene coding for a protein functioning as a transcriptional repressor. The protein MeCP2 (Methyl CpG-binding protein) is an abundant component of pericentric heterochromatin and its mutations or duplications are present in around 80% of patients with a neurological disorder known as Rett Syndrome. Although MeCP2 action depends critically on its binding to chromatin, very little is known about the dynamics of this process. Using fluorescence recovery after photobleaching in controlled conditions concentration, we demonstrated that most GFP-MeCP2 fusion protein associates strongly and reversibly to pericentric heterocromatin whereas the remaining fraction is bound irreversibly. The mobility of the methyl-binding protein is influenced by the differentiation state of the host cells. Furthermore, residues downstream of the methyl-binding domain are critical for the interaction with chromatin. Whereas the binding is stabilised by the central region it is likely modulated by the most C-terminal region. Importantly, these residues are missing in several disease causing mutations. Our data suggest that alterations in the affinity of MeCP2 for chromatin might contribute to the pathological effects of mutations causing Rett Syndrom