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Targeted gene disruption has rapidly become the tool of choice for the analysis of gene and protein function in routinely cultured mammalian cells. Three main technologies capable of irreversibly disrupting gene-expression exist: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. The desired outcome of the use of any of these technologies is targeted insertions and/or deletions (indels) that result in either a nonsense frame shift or splicing error that disrupts protein expression. Many excellent do-it-yourself systems for TALEN construct assembly are now available at low or no cost to academic researchers. However, for new users, screening for successful gene disruption is still a hurdle. Here, we describe efficient and cost-effective strategies for the generation of gene-disrupted cell lines. Although the focus of this chapter is on the use of TALENs, these strategies can be applied to the use of all three technologies.

This month: AI that learns patterns and facts, new protein-RNA and protein-protein relationships, engineering signaling and metabolism, and more variants of Cas9.

Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.

The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. We report that MINOS independently interacts with both preprotein translocases of the outer mitochondrial membrane and plays a role in the biogenesis of β-barrel proteins of the outer membrane., Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.

The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture. (C) 2012 Elsevier Ltd. All rights reserved.

Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.

Eukaryotic cells contain multiple organelles, which are functionally and structurally interconnected. The endoplasmic reticulum–mitochondria encounter structure (ERMES) forms a junction between mitochondria and the endoplasmic reticulum (ER). Four ERMES proteins are known in yeast, the ER-anchored protein Mmm1 and three mitochondria-associated proteins, Mdm10, Mdm12 and Mdm34, with functions related to mitochondrial morphology and protein biogenesis. We mapped the glycosylation sites of ERMES and demonstrate that three asparagine residues in the N‑terminal domain of Mmm1 are glycosylated. While the glycosylation is dispensable, the cytosolic C‑terminal domain of Mmm1 that connects to the Mdm proteins is required for Mmm1 function. To analyze the composition of ERMES, we determined the subunits by quantitative mass spectrometry. We identified the calcium-binding GTPase Gem1 as a new ERMES subunit, revealing that ERMES is composed of five genuine subunits. Taken together, ERMES represents a platform that integrates components with functions in formation of ER–mitochondria junctions, maintenance of mitochondrial morphology, protein biogenesis and calcium binding.

The sorting and assembly machinery (SAM) of mitochondria is essential for the sorting of β-barrel proteins. Different views have been presented on the role of polypeptide transport–associated (POTRA) domains in protein sorting. We show that the mitochondrial POTRA domain promotes the release of precursor proteins from the SAM complex., The mitochondrial outer membrane contains proteinaceous machineries for the translocation of precursor proteins. The sorting and assembly machinery (SAM) is required for the insertion of β‑barrel proteins into the outer membrane. Sam50 is the channel-forming core subunit of the SAM complex and belongs to the BamA/Sam50/Toc75 family of proteins that have been conserved from Gram-negative bacteria to mitochondria and chloroplasts. These proteins contain one or more N-terminal polypeptide transport-associated (POTRA) domains. POTRA domains can bind precursor proteins, however, different views exist on the role of POTRA domains in the biogenesis of β-barrel proteins. It has been suggested that the single POTRA domain of mitochondrial Sam50 plays a receptor-like function at the SAM complex. We established a system to monitor the interaction of chemical amounts of β-barrel precursor proteins with the SAM complex of wild-type and mutant yeast in organello. We report that the SAM complex lacking the POTRA domain of Sam50 efficiently binds β-barrel precursors, but is impaired in the release of the precursors. These results indicate the POTRA domain of Sam50 is not essential for recognition of β-barrel precursors but functions in a subsequent step to promote the release of precursor proteins from the SAM complex.

Although the emergence of highly pathogenic avian influenza (HPAI) of subtype H5N1 in China in 1997 had caused human fatalities and major impacts on the poultry industry, it was not until it re-eme...

Tom40 forms the channel of the mitochondrial preprotein translocase. This beta-barrel protein assembles with alpha-helical proteins, however little is known about the mechanism of assembly. Becker et al identified a new intermediate in Tom40 assembly and show that small alpha-helical Tom proteins associate with Tom40 directly at the SAM complex., The preprotein translocase of the outer mitochondrial membrane (TOM) consists of a central β-barrel channel, Tom40, and six proteins with α-helical transmembrane segments. The precursor of Tom40 is imported from the cytosol by a pre-existing TOM complex and inserted into the outer membrane by the sorting and assembly machinery (SAM). Tom40 then assembles with α-helical Tom proteins to the mature TOM complex. The outer membrane protein Mim1 promotes membrane insertion of several α-helical Tom proteins but also affects the biogenesis of Tom40 by an unknown mechanism. We have identified a novel intermediate in the assembly pathway of Tom40, revealing a two-stage interaction of the precursor with the SAM complex. The second SAM stage represents assembly of Tom5 with the precursor of Tom40. Mim1-deficient mitochondria accumulate Tom40 at the first SAM stage like Tom5-deficient mitochondria. Tom5 promotes formation of the second SAM stage and thus suppresses the Tom40 assembly defect of mim1Δ mitochondria. We conclude that the assembly of newly imported Tom40 is directly initiated at the SAM complex by its association with Tom5. The involvement of Mim1 in Tom40 biogenesis can be largely attributed to its role in import of Tom5.

The mitochondrial outer membrane contains two translocase machineries for precursor proteins--the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of beta-barrel proteins of the outer membrane. The SAM complex is required for the assembly of precursors of the TOM complex, including not only the beta-barrel protein Tom40 but also a subset of alpha-helical subunits. While the interaction of beta-barrel proteins with the SAM complex has been studied in detail, little is known about the interaction between the SAM complex and alpha-helical precursor proteins. We report that the SAM is not static but that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of alpha-helical Tom proteins. We found that a subcomplex of TOM, Tom5-Tom40, associates with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the alpha-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the alpha-helical precursor of Tom22 and promotes its membrane integration. We suggest that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes.

Background Infections of wild birds with highly pathogenic avian influenza (AI) subtype H5N1 virus were reported for the first time in the European Union in 2006. Objectives To capture epidemiological information on H5N1 HPAI in wild bird populations through large-scale surveillance and extensive data collection. Methods Records were analysed at bird level to explore the epidemiology of AI with regard to species of wild birds involved, timing and location of infections as well as the applicability of different surveillance types for the detection of infections. Results In total, 120,706 records of birds were sent to the Community Reference Laboratory for analysis. Incidents of H5N1 HPAI in wild birds were detected in 14 EU Member States during 2006. All of these incidents occurred between February and May, with the exception of two single cases during the summer months in Germany and Spain. Conclusions For the detection of H5N1 HPAI virus, passive surveillance of dead or diseased birds appeared the most effective approach, whilst active surveillance offered better detection of low pathogenic avian influenza (LPAI) viruses. No carrier species for H5N1 HPAI virus could be identified and almost all birds infected with H5N1 HPAI virus were either dead or showed clinical signs. A very large number of Mallards (Anas platyrhynchos) were tested in 2006 and while a high proportion of LPAI infections were found in this species, H5N1 HPAI virus was rarely identified in these birds. Orders of species that appeared to be very clinically susceptible to H5N1 HPAI virus were swans, diving ducks, mergansers and grebes, supporting experimental evidence. Surveillance results indicate that H5N1 HPAI virus did not establish itself successfully in the EU wild bird population in 2006.

Scientific bird ringing was initiated to study the movements of birds, particularly annual migrants. When a ringed bird is reported to the ringing scheme we have the location of that bird at two points in time – where and when it was ringed and where and when it was found. The collation of reports of ringed birds (recoveries) allows patterns of location change with time to be described. Different methods of presenting these simple data have been devised over the years; initially, the aim was to summarise our knowledge of where birds go. Quantitative inference about population behaviour from recovery data is complicated by several factors, particularly the differences in recovery probabilities between different regions. However, the power of ringing‐data analysis is now being increased using statistical methods to correct for the expected geographical biases in recovery patterns. The quantitative interpretation of movement patterns of birds is essential for basing conservation policy on sound evidence. The...

Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2.

AimSpecies are responding to climate change by changing their distributions, creating debate about the effectiveness of existing networks of protected areas. As a contribution to this debate, we assess whether regional winter abundances and distribution of the Smew Mergellus albellus, a migratory waterbird species listed on Annex I (EU Birds Directive) that overwinters exclusively in European wetlands, changed during 1990-2011, the role of global warming in driving distributional changes and the effectiveness of the network of Special Protection Areas (SPAs, EU Birds Directive) in the context of climate change. LocationEurope. MethodsWe used site-specific counts (6,883 sites) from 16 countries covering the entire flyway to estimate annual abundance indices and trends at country, region (north-eastern, central and south-western) and flyway scales, inside and outside SPAs. We fitted autoregressive models to assess the effect of winter temperature on the annual abundance indices whilst accounting for autocorrelation. ResultsThe Smew wintering distribution shifted north-eastwards in Europe in accordance with the predictions of global warming, with increasing numbers in the north-eastern region and declines in the central region. Trends in wintering numbers were more positive in SPAs on the north-eastern and south-western part of the flyway. However, a large proportion of the wintering population remains unprotected in north-eastern areas outside of the existing SPA network. Main conclusionsSPAs accommodated climate-driven abundance changes in the north-eastern region of the wintering distribution by supporting increasing numbers of Smew in traditional and newly colonized areas. However, we highlight gaps in the current network, suggesting that urgent policy responses are needed. Given rapid changes in species distributions, we urge regular national and international assessments of the adequacy of the EU Natura 2000 network to ensure coherence in site-safeguard networks for this and other species. (Less)

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

Human mitochondrial complex I is the largest enzyme of the respiratory chain and is composed of 44 different subunits. Complex I subunits are encoded by both nuclear and mitochondrial (mt) DNA and their assembly requires a number of additional proteins. FAD-dependent oxidoreductase domain-containing protein 1 (FOXRED1) was recently identified as a putative assembly factor and FOXRED1 mutations in patients cause complex I deficiency; however, its role in assembly is unknown. Here, we demonstrate that FOXRED1 is involved in mid-late stages of complex I assembly. In a patient with FOXRED1 mutations, the levels of mature complex I were markedly decreased, and a smaller ∼475 kDa subcomplex was detected. In the absence of FOXRED1, mtDNA-encoded complex I subunits are still translated and transiently assembled into a late stage ∼815 kDa intermediate; but instead of transitioning further to the mature complex I, the intermediate breaks down to an ∼475 kDa complex. As the patient cells contained residual assembled complex I, we disrupted the FOXRED1 gene in HEK293T cells through TALEN-mediated gene editing. Cells lacking FOXRED1 had ∼10% complex I levels, reduced complex I activity, and were unable to grow on galactose media. Interestingly, overexpression of FOXRED1 containing the patient mutations was able to rescue complex I assembly. In addition, FOXRED1 was found to co-immunoprecipitate with a number of complex I subunits. Our studies reveal that FOXRED1 is a crucial component in the productive assembly of complex I and that mutations in FOXRED1 leading to partial loss of function cause defects in complex I biogenesis.

Mitochondrial F1Fo-ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear- and mitochondria-encoded subunits. Whereas chaperones for formation of the matrix-exposed hexameric F1-ATPase core domain have been identified, insight into how the nuclear-encoded F1-domain assembles with the membrane-embedded Fo-region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F1-module and peripheral stalk, but not with the assembled F1Fo-ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F1-module to the membrane embedded Fo-domain. We conclude that INAC represents a matrix-exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F1Fo-ATP synthase.

In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-xL. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family proteins.

1. This strategy aims to increase the contribution which ringing makes to conservation science and ornithological research, recognising that the primary outputs of the scheme are scientific publications based on data gathered by volunteer ringers. The strategy envisages a greater involvement of ringers in planned projects that are designed to provide specific types of information. Clear advice on priority species and categories of birds (eg age classes, seasons of ringing, breeding vs roosting) to be ringed and recaptured will be given to those ringing outside centrally organised projects. 2. The research programme will focus on two main topics, population dynamics and migration and movements. In addition research on life‐histories, moult and condition should be conducted when opportunities allow. 3. The Ringing Scheme makes a vital contribution to studies of population dynamics by providing data on survival rates and productivity, and sometimes also on numbers. Popidation dynamics models based on such da...

Disorders of mitochondrial oxidative phosphorylation (OXPHOS) present with almost any symptom, at any age with any mode of inheritance. OXPHOS disorders are the most common group of inherited metabolic diseases and occur in ~1/5000 births. There is no ‘gold standard’ diagnostic test and most paediatric diagnoses rely on finding an enzyme defect while molecular investigations are typically restricted to testing for common mutations or sequencing of specific candidate genes. While next generation sequencing (NGS) approaches have yielded great insights into the identification of pathogenic mutations, these generally focus on sequencing of known mitochondrial and disease related genes, yielding a molecular diagnosis in ~60% of cases. Recent advances in proteomics have improved such that much of the human cellular proteome can now be determined in a matter of hours. In addition, with the use of quantitaive approaches including stable isotope labeling of cells in culture (SILAC), differences between the proteomes of diseased and controls cells can be measured. We are now investigating the use of quantitative proteomics as a complementery tool to NGS in the molecular diagnosis of OXPHOS disease, and to ultimately identify novel candidate genes that cause disease.

The feeding ecology of Greenland white-fronted geese Anser albifrons flavirostris was studied during .spring staging in Iceland 1997. Geese feeding on Poa pratense dominated hayfields (> 80% cover) were highly selective, selecting for Deschampsia caespitosa which comprised only 10% of the sward. Geese fed most on the south-facing fringes of Deschampsia tussocks. Subsequent analysis showed that the southern fringes of Deschampsia tussocks supported significantly greater biomass (27% greater mass of green material) and that leaves growing on the southern faces had significantly higher protein content than those on the northern faces (33.9% vs 30.5%)- It appears that the geese maximise their nutritional intake in spring by selecting the grass species of highest quality and taking the most nutritious parts of the plants.

The effects of simulated goose grazing on Phleum pratense plants were tested in an Iceland hayfield during the spring goose staging period (19 April-11 May 1997). Plants in an area exclosed from the influence of grazing and the nutrient effects of goose faeces were subject to the removal of the youngest lamina once, three and four times during this period. Clipping three and four times resulted in 25-41% increases in cumulative elongation of youngest laminae compared with unclipped plants. Total cumulative lamina growth of entire plants showed no significant difference between unclipped plants and those clipped three and four times, hence no overcompensation occurred. Sequential clipping elevated the protein content of the youngest laminae from 20% to 27-33%, whereas there was no change amongst shoots clipped only once. Because geese only consume the youngest lamina of each Phleum plant, measurements from this experiment showed that regular physical removal of growing biomass doubled the biomass of preferred tissue available to geese and increased the potential protein intake 3.5 times at experimental clipping frequencies similar to levels of sequential harvesting observed amongst staging geese compared to less frequent harvesting. These increases were achieved without any fertilising effects of goose faeces implicated in such effects in previous studies.

After protection from hunting on the wintering range in 1982/83, complete surveys of Greenland white-fronted geese at all known Irish and British wintering resorts have been carried out annually. These showed that this population increased by 5.0% per annum from 16, 541 in spring 1983 to 30, 459 in spring 1995, characterised by a 6.6% annual increase during 1982/83-1991/92, followed by a less rapid increase in subsequent years. In addition, regular counts of at least eight wintering flocks also exist prior to 1982/83. Five of these (including the two most important, Islay in Scotland and Wexford in Ireland) showed no trend before protection, but significant increases after legislation. Two other flocks at protected sites showed increasing numbers prior to changes in legislation, followed by stable numbers afterwards and the eighth flock increased in number before and after protection. On Islay, a significant increase in crude adult annual survival rate (based on census data) occurred after the hunting ban. Numbers on Islay continue to show linear increase. At Wexford, there was no significant difference between crude adult survival before and after the hunting ban where, after a short period of increase, numbers stabilised at 8, 000-10, 000 after 1990. There were no significant differences in the proportions of young birds before and after protection in these two flocks. Despite overall population increase, seven flocks have become extinct during 1982-1995 and a further five are close to extinction. Eighteen flocks have declined since protection, 35 showed no significant trends and 20 showed increases. Multivariate analysis suggests size, number and quality of feeding areas, levels of disturbance, flock size and latitude influence flock status-smallest most southerly flocks on fewest, poor quality limited feeding ranges showing most serious declines. The consequences of increasing concentration of the population at a few wintering areas need urgent attention and mechanisms should be sought to maintain current range, particularly on traditional semi-natural or low intensity agricultural land.

Summary Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity of RCS in vitro and in vivo, independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization. We demonstrate that, accordingly, the efficiency of mitochondria-dependent cell growth depends on cristae shape. Thus, RCS assembly emerges as a link between membrane morphology and function., Graphical Abstract, Highlights • Dissociation of cristae remodeling from OMM permeabilization • Cristae shape determines assembly of respiratory chain supercomplexes • Efficiency of mitochondrial respiration and cellular growth depends on cristae shape, The ability to perturb cristae shape without affecting other key aspects of mitochondrial physiology reveals that membrane shape influences supercomplex assembly and stability to regulate mitochondrial respiration and cellular respiratory growth. Quaternary structures such as supercomplexes therefore emerge as a link between membrane morphology and function.

Greenland White-fronted Geese wintering in Ireland and Britain stage for 3 weeks in Iceland in spring before migrating onwards to breeding areas in west Greenland. The geese now depart their wintering quarters 12–15 days earlier than in 1973 because they attain necessary fat stores earlier than in previous years. Icelandic temperatures at critical midway staging areas have shown no significant change since 1973, creating a potential mismatch in food availability along the migratory route. Greenland White-fronted Geese have shifted from consuming below-ground plant storage organs in Iceland in spring to grazing managed hayfields created since the 1950s where fresh grass shoot growth occurs despite sub-zero temperatures, when traditional natural foods are physically inaccessible to staging geese due to frozen substrates. Rates of fat accumulation (measured by field scores of abdominal profiles) and mass change (measured in captured geese) were the same in the springs of 1997, 1998 and 1999 as in that of 2007 when the migration episode was 10 days earlier. Hence, earlier arrival in Iceland in 2007 did not occur at cost to refuelling rates there. The shift to acquiring energy from artificial grasslands has enabled Greenland White-fronted Geese to arrive in Iceland earlier, but has apparently not impaired their ability to accumulate fat reserves required for onwards migration, which occurs at the same rate, only earlier in the spring.

Biogenesis of the translocase of the outer mitochondrial membrane (TOM complex) involves the assembly of the central β-barrel forming protein Tom40 with six different subunits that are embedded in the membrane via α-helical transmembrane segments. The sorting and assembly machinery (SAM complex) of the outer membrane plays a central role in this process. The SAM complex mediates the membrane integration of β-barrel precursor proteins including Tom40. The small Tom proteins Tom5 and Tom6 associate with the precursor of Tom40 at the SAM complex at an early stage of the assembly process and play a stimulatory role in the formation of the mature TOM complex. A fraction of the SAM components interacts with the outer membrane protein mitochondrial distribution and morphology protein 10 (Mdm10) to form the SAM-Mdm10 machinery; however, different views exist on the function of the SAM-Mdm10 complex. We report here that the third small Tom protein, Tom7, plays an inhibitory role at two distinct steps in the biogenesis of the TOM complex. First, Tom7 plays an antagonistic role to Tom5 and Tom6 at the early stage of Tom40 assembly at the SAM complex. Second, Tom7 interacts with Mdm10 that is not bound to the SAM complex, and thus promotes dissociation of the SAM-Mdm10 complex. Since the SAM-Mdm10 complex is required for the biogenesis of Tom22, Tom7 delays the assembly of Tom22 with Tom40 at a late stage of assembly of the TOM complex. Thus, Tom7 modulates the biogenesis of topologically different proteins, the β-barrel forming protein Tom40 and Tom22 that contains a transmembrane α-helix.

β-barrel membrane proteins in Gram-negative bacteria, mitochondria, and chloroplasts are assembled by highly conserved multi-protein complexes. The mechanism by which these molecular machines fold and insert their substrates is poorly understood. It has not been possible to dissect the folding and insertion pathway because the process has not been reproduced in a biochemical system. We purified the components that fold and insert E. coli outer membrane proteins and reconstituted β-barrel protein assembly in proteoliposomes using the enzymatic activity of a protein substrate to report on its folding state. The assembly of this protein occurred without an energy source but required a soluble chaperone in addition to the multi-protein assembly complex.

SummaryThe biogenesis of mitochondria requires the import of a large number of proteins from the cytosol [1, 2]. Although numerous studies have defined the proteinaceous machineries that mediate mitochondrial protein sorting, little is known about the role of lipids in mitochondrial protein import. Cardiolipin, the signature phospholipid of the mitochondrial inner membrane [3–5], affects the stability of many inner-membrane protein complexes [6–12]. Perturbation of cardiolipin metabolism leads to the X-linked cardioskeletal myopathy Barth syndrome [13–18]. We report that cardiolipin affects the preprotein translocases of the mitochondrial outer membrane. Cardiolipin mutants genetically interact with mutants of outer-membrane translocases. Mitochondria from cardiolipin yeast mutants, as well as Barth syndrome patients, are impaired in the biogenesis of outer-membrane proteins. Our findings reveal a new role for cardiolipin in protein sorting at the mitochondrial outer membrane and bear implications for the pathogenesis of Barth syndrome.

Long-distance migrations are among the wonders of the natural world, but this multi-taxon review shows that the characteristics of species that undertake such movements appear to make them particularly vulnerable to detrimental impacts of climate change. Migrants are key components of biological systems in high latitude regions, where the speed and magnitude of climate change impacts are greatest. They also rely on highly productive seasonal habitats, including wetlands and ocean upwellings that, with climate change, may become less food-rich and predictable in space and time. While migrants are adapted to adjust their behaviour with annual changes in the weather, the decoupling of climatic variables between geographically separate breeding and non-breeding grounds is beginning to result in mistimed migration. Furthermore, human land-use and activity patterns will constrain the ability of many species to modify their migratory routes and may increase the stress induced by climate change. Adapting conservation strategies for migrants in the light of climate change will require substantial shifts in site designation policies, flexibility of management strategies and the integration of forward planning for both people and wildlife. While adaptation to changes may be feasible for some terrestrial systems, wildlife in the marine ecosystem may be more dependent on the degree of climate change mitigation that is achievable.

Mitochondria and the nucleus are key features that distinguish eukaryotic cells from prokaryotic cells. Mitochondria originated from a bacterium that was endosymbiotically taken up by another cell more than a billion years ago. Subsequently, most mitochondrial genes were transferred and integrated into the host cell's genome, making the evolution of pathways for specific import of mitochondrial proteins necessary. The mitochondrial protein translocation machineries are composed of numerous subunits. Interestingly, many of these subunits are at least in part derived from bacterial proteins, although only few of them functioned in bacterial protein translocation. We propose that the primitive alpha-proteobacterium, which was once taken up by the eukaryote ancestor cell, contained a number of components that were utilized for the generation of mitochondrial import machineries. Many bacterial components of seemingly unrelated pathways were integrated to form the modern cooperative mitochondria-specific protein translocation system.

The Scottish Raptor Monitoring Scheme (SRMS) comprises 7 partner organizations and was established in 2002 after i) the publication of the UK Government's Raptor Working Group Report that made recommendations for enhanced monitoring, ii) increased applied data needs (e.g., for site designation), and iii) concerns for the status of some species. The SRMS has 3 major objectives: i) to facilitate cooperation between parties; ii) to provide robust information on Scottish raptor populations by determining trends in numbers, range, survival, and productivity and understanding the causes of change; and iii) to maintain high and uniform standards for the collection, collation, auditing, and analysis of data and reporting of information. Data are collected for 19 species: 14 diurnal raptors, 4 owls, and 1 corvid, the Common Raven. Here we describe the development of the scheme, challenges, and achievements during its first 4 y, the nature and value of the data collected, and plans for the future.

To manage and conserve wildlife populations effectively it is necessary to use methods that identify the often non-linear trends in populations, have an inbuilt assessment of trend quality and can analyse count data from a range of spatial scales. We present a method of trend analysis using generalised additive models. These produce smoothed indices of abundance that can be used to assess population change from one or more sites or time periods, with any number of estimates of abundance per index period. We apply this method to count data collected under the Wetland Bird Survey, a national scheme that monitors waterbirds in the United Kingdom. To highlight declining populations, ‘alerts’ were raised if the population decline was equal to or greater than 50%. Significance was determined using bootstrapped confidence intervals for analyses that included many sites, or a novel Monte-Carlo method for single site analyses. The impact of missing data, species count variability and the number of months used to calculate the population change was greater at individual sites than for national datasets, which were relatively insensitive to changes in the above parameters. For single sites it is essential that three or more counts be made per index period if reliable estimates of population change are required. We propose that the method presented could be applied to a wide range of national or other monitoring schemes for a variety of taxa.

BACKGROUND: Activating mutations of the FLT3 gene are common in acute myeloid leukemia (AML), with approximately 25-30% of cases containing an internal tandem duplication (ITD) in the juxtamembrane domain. The presence of a FLT3-ITD mutation is associated with a poor prognosis, the severity of which, can be modulated by the combination of co-occuring mutations. In animal models, expression of Flt3-ITD by transgenesis, bone marrow transplantation or gene knock-in does not lead to an acute leukemia but a myeloproliferative disease, resembling CMML, suggesting a requirement for additional co-operating mutations (Lee et al, Cancer Cell. 2007, 12: 367). This is supported by in vivo models which demonstrate that the combination of the Flt3-ITD mutation with other genetic lesions leads to the development of an acute leukemia in mice (Chen et al, Genes Dev. 2013, 27: 1974). AIMS: To identify and characterise novel genes that alter Flt3-ITD induced MPN by using an N-ethyl-N-Nitroso-urea (ENU) mutagenesis strategy in mice with an Flt3-ITD homozygous knock-in background. METHODS: An autosomal dominant screen for Flt3-ENU co-operating mutations was carried out at the Australian Phenomics Facility by mating ENU-mutagenised male mice homozygous for the Flt3-ITD knock-in allele to homozygous Flt3-ITD females. G1 mice were screened for changes in blood cell parameters indicative of an altered disease state (compared to that induced by Flt3-ITD alone). Mice with blood cell parameters outside two standard deviations of the relevant G1 mean were identified as potential mutation carriers and bred to Flt3-ITD homozygous knock-in mice to test for heritability of the phenotype. Where pedigrees were generated demonstrating heritable phenotypes, multiple affected and unaffected littermates were subject to exome sequencing and analysis to identify a list of candidate gene mutations segregating with the disease phenotype. RESULTS: 150 G1 mice were screened, leading to the identification of four pedigrees with heritable phenotypes marked by an exacerbated MPN. Exome sequencing has identified a short list of 3 genes for one pedigree (pedigree 37) that includes a mutation in Neurofibromatosis 1 (Nf1), a gene known to be involved in the induction of juvenile myelomonocytic leukemia and frequently lost in AML (Parkin et al,Clin Cancer Res 2010, 16:4135). In another pedigree (pedigree 24) we identified a mutation in Ndufa10 as the single candidate segregating with the phenotype (Figure 1A-B). Ndufa10 encodes a subunit of the mitochondrial respiratory complex I, which is the first and largest complex in the mitochondrial electron transport chain. Importantly, germline mutations in this gene lead to a complex I deficiency syndrome in humans, indicating that it is a critical subunit of this complex. We hypothesise that mutation of Ndufa10 leads to altered cellular metabolism in hematopoietic stem and progenitor cells which contributes to exacerbation of the MPN, possibly through an alteration in production of reactive oxygen species and a shift in the balance between glycolysis and oxidative phosphorylation. Breeding of the Ndufa10 mutation onto a non Flt3-ITD background shows that action of the mutation is not dependent on the presence of Flt3-ITD, as these mice also have altered blood counts, including increased WBC (Figure 1C). CONCLUSIONS: It is possible to identify mutations that exacerbate Flt3-ITD induced MPN through mutagenesis and an efficient blood screening strategy. In addition, using this strategy, we have identified novel mutations that act independently of Flt3-ITD to induce changes in the haematological compartment. Translation of these findings to human AML may indicate pathways that will be targets for new and complementary treatments in AML. Figure 1. A. Flt3-ITD Pedigree 24 indicating affected mice and genotyping for the Ndufa10 mutation. +/+=Ndufa10 wt, m/+=Ndufa10 heterozygous mutant. B. WBC counts for male mice from Pedigree 24 at 15-17 weeks (+/+, n=3; m/+, n=8). WBC from ENU G1 mice are shown as a comparison (G1, n=75). C. WBC counts for male mice on a wildtype C57 background at 16-18 weeks. +/+=Ndufa10 wt (n=7), m/+=Ndufa10 heterozygous mutant (n=8), m/m=Ndufa10 homozygous mutant (7). Figure 1. A. Flt3-ITD Pedigree 24 indicating affected mice and genotyping for the Ndufa10 mutation. +/+=Ndufa10 wt, m/+=Ndufa10 heterozygous mutant. B. WBC counts for male mice from Pedigree 24 at 15-17 weeks (+/+, n=3; m/+, n=8). WBC from ENU G1 mice are shown as a comparison (G1, n=75). C. WBC counts for male mice on a wildtype C57 background at 16-18 weeks. +/+=Ndufa10 wt (n=7), m/+=Ndufa10 heterozygous mutant (n=8), m/m=Ndufa10 homozygous mutant (7). Disclosures No relevant conflicts of interest to declare.

Transmembrane proteins with β-barrel structure are characteristic of the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts ( 1 ). β-Barrel membrane proteins typically include a central pore that is lined by multiple antiparallel β strands. The functions of these proteins range from protein transport to metabolite flux. On page 890 of this issue, Hagan et al. ( 2 ) report a major step toward a molecular understanding of β-barrel membrane insertion and folding.

The purpose of this study was to determine if high-frequency (HF) ultrasound (20–80 MHz) is sensitive to the change of average human body in temperature (approximately 37 °C) to average room temperatures of approximately 20 °C to 25 °C. When temperatures decrease, water molecules within organic tissue expand, decreasing their density. Changes in temperatures can alter the number of molecules found within a given area, altering tissue density. Two parameters in high-frequency ultrasound (20–80 MHz) have been found to be sensitive to a range of pathologies in resected margins from breast conservation surgery: The number of peaks (the peak density) in the waveform spectrum and the slope of the Fourier transform of the waveform spectrum. Changes in temperatures may affect the accuracy of these two parameters. To test this hypothesis, through-transmission and pulse-echo measurements were acquired fresh (within one hour of slaughter from local butchers) aorta samples from porcine hearts. Results will be present...

The outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria contain abundant β-barrel proteins that are essential for the transport of proteins and metabolites. Identification of the mitochondrial sorting and assembly machinery (SAM complex) revealed a new protein import pathway and sparked interest in mitochondrial β-barrel biogenesis. A central SAM component, Sam50, is conserved from bacteria to humans and a related protein is also found in chloroplasts, implying a conserved mechanism of β-barrel sorting in eukaryotes and prokaryotes (Pfanner et al., 2004; Paschen et al., 2005; Dolezal et al., 2006; Bos et al., 2007).