A conserved sequence in calmodulin regulated spectrin-associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth.

Calmodulin regulated spectrin-associated protein 1 (CAMSAP1) is a vertebrate microtubule-binding protein, and a representative of a family of cytoskeletal proteins that arose with animals. We reported previously that the central region of the protein, which contains no recognized functional domain, inhibited neurite outgrowth when over-expressed in PC12 cells [Baines et al., Mol. Biol. Evol. 26 (2009), p. 2005]. The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins (Baines et al. 2009). In the central region, three short well-conserved regions are characteristic of CAMSAP-family members. One of these, CAMSAP-conserved region 1 (CC1), bound to both ßIIΣ1-spectrin and Ca(2+)/calmodulin in vitro. The binding of Ca(2+)/calmodulin inhibited spectrin binding. Transient expression of CC1 in PC12 cells inhibited neurite outgrowth. siRNA knockdown of CAMSAP1 inhibited neurite outgrowth in PC12 cells or primary cerebellar granule cells: this could be rescued in PC12 cells by wild-type CAMSAP1-enhanced green fluorescent protein, but not by a CC1 mutant. We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin-binding to neurite outgrowth.

Protecting Great Barrier Reef resilience through effective management of crown-of-thorns starfish outbreaks.

Resilience-based management is essential to protect ecosystems in the Anthropocene. Unlike large-scale climate threats to Great Barrier Reef (GBR) corals, outbreaks of coral-eating crown-of-thorns starfish (COTS; Acanthaster cf. solaris) can be directly managed through targeted culling. Here, we evaluate the outcomes of a decade of strategic COTS management in suppressing outbreaks and protecting corals during the 4th COTS outbreak wave at reef and regional scales (sectors). We compare COTS density and coral cover dynamics during the 3rd and 4th outbreak waves. During the 4th outbreak wave, sectors that received limited to no culling had sustained COTS outbreaks causing significant coral losses. In contrast, in sectors that received timely and sufficient cull effort, coral cover increased substantially, and outbreaks were suppressed with COTS densities up to six-fold lower than in the 3rd outbreak wave. In the Townsville sector for example, despite exposure to comparable disturbance regimes during the 4th outbreak wave, effective outbreak suppression coincided with relative increases in sector-wide coral cover (44%), versus significant coral cover declines (37%) during the 3rd outbreak wave. Importantly, these estimated increases span entire sectors, not just reefs with active COTS control. Outbreaking reefs with higher levels of culling had net increases in coral cover, while the rate of coral loss was more than halved on reefs with lower levels of cull effort. Our results also indicate that outbreak wave progression to adjoining sectors has been delayed, probably via suppression of COTS larval supply. Our findings provide compelling evidence that proactive, targeted, and sustained COTS management can effectively suppress COTS outbreaks and deliver coral growth and recovery benefits at reef and sector-wide scales. The clear coral protection outcomes demonstrate the value of targeted manual culling as both a scalable intervention to mitigate COTS outbreaks, and a potent resilience-based management tool to "buy time" for coral reefs, protecting reef ecosystem functions and biodiversity as the climate changes.

The 3T3 neutral red uptake phototoxicity test: practical experience and implications for phototoxicity testing--the report of an ECVAM-EFPIA workshop.

This is the report from the "ECVAM-EFPIA workshop on 3T3 NRU Phototoxicity Test: Practical Experience and Implications for Phototoxicity Testing", jointly organized by ECVAM and EFPIA and held on the 25-27 October 2010 in Somma Lombardo, Italy. The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 within the European Commission Joint Research, based on a Communication from the European Commission (1991). The main objective of ECVAM is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine and replace the use of laboratory animals. The European Federation of Pharmaceuticals Industries and Association (EFPIA) represent the pharmaceutical industry operating in Europe. Through its direct membership of 31 national associations and 40 leading pharmaceutical companies, EFPIA is the voice on the EU scene of 2200 companies committed to researching, developing and bringing to patients new medicines that improve health and the quality of life around the world. The workshop, co-chaired by Joachim Kreysa (ECVAM) and Phil Wilcox (GSK, EFPIA) involved thirty-five experts from academia, regulatory authorities and industry, invited to contribute with their experiences in the field of phototoxicology. The main objectives of the workshop were: -to present 'in use' experience of the pharmaceutical industry with the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU-PT), -to discuss why it differs from the results in the original validation exercise, -to discuss technical issues and consider ways to improve the usability of the 3T3 NRU-PT for (non-topical) pharmaceuticals, e.g., by modifying the threshold of chemical light absorption to trigger photo-toxicological testing, and by modifying technical aspects of the assay, or adjusting the criteria used to classify a positive response. During the workshop, the assay methodology was reviewed by comparing the OECD Test Guideline (TG 432) with the protocols used in testing laboratories, data from EFPIA and JPMA 'surveys' were presented and possible reasons for the outcomes were discussed. Experts from cosmetics and pharmaceutical industries reported on their experience with the 3T3 NRU-PT and evidence was presented for phototoxic clinical symptoms that could be linked to certain relevant molecules. Brainstorming sessions discussed if the 3T3 NRU-PT needed to be improved and whether alternatives to the 3T3 NRU-PT exist. Finally, the viewpoint from EU and US regulators was presented. In the final session, the conclusions of the meeting were summarized, with action points. It was concluded that the 3T3 NRU-PT identifies phototoxicological hazards with a 100% sensitivity, and thus is accepted as the tier one test that correctly identifies the absence of phototoxic potential. Consequently, positive results in the 3T3 NRU-PT often do not translate into a clinical phototoxicity risk. Possible ways to improve the practical use of this assay include: (i) adaptation of changed UV/vis-absorption criteria as a means to reduce the number of materials tested, (ii) reduction of the highest concentration to be tested, and (iii) consideration of modifying the threshold criteria for the prediction of a positive call in the test.

The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins.

We describe a structural domain common to proteins related to human calmodulin-regulated spectrin-associated protein1 (CAMSAP1). Analysis of the sequence of CAMSAP1 identified a domain near the C-terminus common to CAMSAP1 and two other mammalian proteins KIAA1078 and KIAA1543, which we term a CKK domain. This domain was also present in invertebrate CAMSAP1 homologues and was found in all available eumetazoan genomes (including cnidaria), but not in the placozoan Trichoplax adherens, nor in any nonmetazoan organism. Analysis of codon alignments by the sitewise likelihood ratio method gave evidence for strong purifying selection on all codons of mammalian CKK domains, potentially indicating conserved function. Interestingly, the Drosophila homologue of the CAMSAP family is encoded by the ssp4 gene, which is required for normal formation of mitotic spindles. To investigate function of the CKK domain, human CAMSAP1-enhanced green fluorescent protein (EGFP) and fragments including the CKK domain were expressed in HeLa cells. Both whole CAMSAP1 and the CKK domain showed localization coincident with microtubules. In vitro, both whole CAMSAP1-glutathione-s-transferase (GST) and CKK-GST bound to microtubules. Immunofluorescence using anti-CAMSAP1 antibodies on cerebellar granule neurons revealed a microtubule pattern. Overexpression of the CKK domain in PC12 cells blocked production of neurites, a process that requires microtubule function. We conclude that the CKK domain binds microtubules and represents a domain that evolved with the metazoa.

A current practice for predicting ocular toxicity of systemically delivered drugs.

The ability to predict ocular side effects of systemically delivered drugs is an important issue for pharmaceutical companies. Although animal models involving standard clinical ophthalmic examinations and postmortem microscopic examinations of eyes are still used to identify ocular issues, these methods are being supplemented with additional in silico, in vitro, and in vivo techniques to identify potential safety issues and assess risk. The addition of these tests to a development plan for a potential new drug provides the opportunity to save time and money by detecting ocular issues earlier in the program. This review summarizes a current practice for minimizing the potential for systemically administered, new medicines to cause adverse effects in the eye.

Let's just hold a real vote.

The planned RCN consultation to gauge how nurses feel about balloting on industrial action is a step in the right direction, but for heaven's sake why don't we just hold a real vote? No one is interested in a test vote - not the media, not members. A real vote would be headline news and RCN members would be enthused and motivated. As it is, we are just kicking the ball into the long grass yet again. There will be a low turnout and we will end up shrugging our shoulders and saying 'There's nothing to be done'. Jeremy Hunt must be laughing his socks off.

Nurses are best placed to ensure the ethical application of DNRs.

Last week the BBC reported that doctors in England have placed 'do not resuscitate' orders on patients without discussion with their families.

College listened to our concerns about governance planning.

As RCN steward for Gwynedd, I was one of the small group of members who met RCN council chair Kath McCourt and senior staff to discuss concerns that a review of legal and governance structures at the college could weaken the influence of its regional boards and branches (news May 23).

Cardiac muscle cell cytoskeletal protein 4.1: analysis of transcripts and subcellular location--relevance to membrane integrity, microstructure, and possible role in heart failure.

The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies.