The role of L-type voltage-gated calcium channels Cav1.2 and Cav1.3 in normal and pathological brain function

The use of specific activators and inhibitors that penetrate the central nervous system has suggested an essential functional role of L-type calcium channels (LTCC) in several important physiological processes of the brain, including the modulation of the mesoaccumbal dopamine signalling pathway, synaptic transmission of auditory stimuli and synaptic plasticity of neutral and aversive learning and memory processes. However, the lack of selectivity of available pharmacological agents towards the most prominent LTCC isoforms in the brain, namely Ca_v1.2 and Ca_v1.3, has hampered the elucidation of the precise contribution made by each specific channel isoform within these specific physiological processes. Modern genetic approaches, both in rodents and in human, have recently enhanced our understanding of the selective functional roles of Ca_v1.2 and Ca_v1.3 channels. In rodents, the characterisation of global and conditional isoform-specific knockouts suggests a contribution of Ca_v1.2 channels in spatial memory formation, whereas Ca_v1.3 channels seem to be involved in the consolidation of fear memories and in neurodegenerative mechanisms associated with the development of Parkinson’s disease. With regard to the molecular mechanisms underlying drug addiction, Ca_v1.3 channels are necessary for the development and Ca_v1.2 channels for the expression of cocaine and amphetamine behavioural sensitisation. In humans, both the identification of naturally occurring LTCC variants (“channelopathies”) and unbiased genome-wide association studies have linked LTCCs to working memory performance in healthy individuals and schizophrenic patients. Individually, CACNA1C polymorphisms and CACNA1D variants have been linked to a variety of psychiatric diseases and to congenital deafness, respectively. However, the contribution of individual LTCCs and their polymorphisms to human brain function and diseases remains unclear, necessitating the use of isoform-specific pharmacological agents.     

Apoptosis of resident and inflammatory macrophages before and during the inflammatory response of the virgin bovine mammary gland

Background  

Macrophages may play a prominent role in defense of the bovine mammary gland, and their functionality is necessary for successful eradication of bacterial pathogens. In contrast to necrosis, however, apoptosis has not yet been studied in macrophages from bovine mammary glands. Therefore, the aim of this study was to confirm the occurrence of apoptosis in macrophages from resting heifer mammary glands and during the inflammatory response.     

Circulating tumor DNA dynamics and recurrence risk in patients undergoing curative intent resection of colorectal cancer liver metastases: A prospective cohort study

BackgroundIn patients with resectable colorectal liver metastases (CRLM), the role of pre- and postoperative systemic therapy continues to be debated. Previous studies have shown that circulating tumor DNA (ctDNA) analysis, as a marker of minimal residual disease, is a powerful prognostic factor in patients with nonmetastatic colorectal cancer (CRC). Serial analysis of ctDNA in patients with resectable CRLM could inform the optimal use of perioperative chemotherapy. Here, we performed a validation study to confirm the prognostic impact of postoperative ctDNA in resectable CRLM observed in a previous discovery study.Methods and findingsWe prospectively collected plasma samples from patients with resectable CRLM, including presurgical and postsurgical samples, serial samples during any pre- or postoperative chemotherapy, and serial samples in follow-up. Via targeted sequencing of 15 genes commonly mutated in CRC, we identified at least 1 somatic mutation in each patient’s tumor. We then designed a personalized assay to assess 1 mutation in plasma samples using the Safe-SeqS assay. A total of 380 plasma samples from 54 patients recruited from July 2011 to Dec 2014 were included in our analysis. Twenty-three (43%) patients received neoadjuvant chemotherapy, and 42 patients (78%) received adjuvant chemotherapy after surgery. Median follow-up was 51 months (interquartile range, 31 to 60 months). At least 1 somatic mutation was identified in all patients’ tumor tissue. ctDNA was detectable in 46/54 (85%) patients prior to any treatment and 12/49 (24%) patients after surgery. There was a median 40.93-fold (19.10 to 87.73, P < 0.001) decrease in ctDNA mutant allele fraction with neoadjuvant chemotherapy, but ctDNA clearance during neoadjuvant chemotherapy was not associated with a better recurrence-free survival (RFS). Patients with detectable postoperative ctDNA experienced a significantly lower RFS (HR 6.3; 95% CI 2.58 to 15.2; P < 0.001) and overall survival (HR 4.2; 95% CI 1.5 to 11.8; P < 0.001) compared to patients with undetectable ctDNA. For the 11 patients with detectable postoperative ctDNA who had serial ctDNA sampling during adjuvant chemotherapy, ctDNA clearance was observed in 3 patients, 2 of whom remained disease-free. All 8 patients with persistently detectable ctDNA after adjuvant chemotherapy have recurred. End-of-treatment (surgery +/− adjuvant chemotherapy) ctDNA detection was associated with a 5-year RFS of 0% compared to 75.6% for patients with an undetectable end-of-treatment ctDNA (HR 14.9; 95% CI 4.94 to 44.7; P < 0.001). Key limitations of the study include the small sample size and the potential for false-positive findings with multiple hypothesis testing.ConclusionsWe confirmed the prognostic impact of postsurgery and posttreatment ctDNA in patients with resected CRLM. The potential utility of serial ctDNA analysis during adjuvant chemotherapy as an early marker of treatment efficacy was also demonstrated. Further studies are required to define how to optimally integrate ctDNA analyses into decision-making regarding the use and timing of adjuvant therapy for resectable CRLM.Trial registrationACTRN12612000345886.     

Nanoscale organization of β2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize β₂-adrenergic receptors (β₂AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the β₂AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the β₂AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of β₂AR are observed in the membrane of the HEK293 cells that stably overexpress β₂AR-GFP and β₂AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use β₂AR-Venus fusion proteins as models for β₂AR function. These observations are critical for designing future cell models and assays based on β₂AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.     

Effects of arm and leg loading on sprint performance

The effects of loading on sprint kinematics were examined in 24 male students. The moment of inertia of either the arms or legs was increased by up to 50% of their unloaded values and the time for distances of 0.5–15 m and 15–30 m from a sprint start was measured. An increase in leg loading was associated with a gradual decrease in velocity of both sprint phases, while the change associated with arm loading was modest and significant only in the second phase. The decrease in sprint velocity was predominantly due to a reduction in stride rate, while the stride length remained almost unchanged. It was concluded that leg loading affected sprint velocity more than arm loading, and also that the velocity was reduced due to a decrease in the stride rate rather than in the stride length. Accepted: 10 November 1997