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Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in the cell bodies of dopaminergic neurons in the substantia nigra (SN) pars compacta. These neurons are lost in neurodegenerative diseases, such as Parkinson’s disease and dementia with Lewy bodies. Although it is known that lipids, proteins, and environmental toxins accumulate in NMGs, the function of NMGs has not yet been finally clarified as well as their origin and the synthesis of neuromelanin. We, therefore, isolated NMGs and surrounding SN tissue from control patients by laser microdissection and analyzed the proteomic profile by tandem mass spectrometry. With our improved workflow, we were able to (1) strengthen the regularly reported link between NMGs and lysosomes, (2) detect tyrosine hydroxylase to be highly abundant in NMGs, which may be related to neuromelanin synthesis and (3) indicate a yet undescribed link between stress granules (SGs) and NMGs. Based on our findings, we cautiously hypothesize, that SGs may be the origin of NMGs or form in close proximity to them, potentially due to the oxidative stress caused by neuromelanin-bound metals.

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.

Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra pars compacta. In addition to neuromelanin, NMGs contain proteins, lipids and metals. As NMG-containing dopaminergic neurons are preferentially lost in Parkinson’s disease and dementia with Lewy bodies (DLB), it is assumed that NMGs may play a role in neurodegenerative processes. Until now, this role is not completely understood and needs further investigation. We therefore set up an exploratory proteomic study to identify differences in the proteomic profile of NMGs from DLB patients (n = 5) compared to healthy controls (CTRL, n = 5). We applied a laser microdissection and mass-spectrometry-based approach, in which we used targeted mass spectrometric experiments for validation. In NMG-surrounding (SNSurr.) tissue of DLB patients, we found evidence for ongoing oxidative damage and an impairment of protein degradation. As a potentially disease-related mechanism, we found α-synuclein and protein S100A9 to be enriched in NMGs of DLB cases, while the abundance of several ribosomal proteins was significantly decreased. As S100A9 is known to be able to enhance the formation of toxic α-synuclein fibrils, this finding points towards an involvement of NMGs in pathogenesis, however the exact role of NMGs as either neuroprotective or neurotoxic needs to be further investigated. Nevertheless, our study provides evidence for an impairment of protein degradation, ongoing oxidative damage and accumulation of potentially neurotoxic protein aggregates to be central mechanisms of neurodegeneration in DLB.

Target skeletal muscle fibres - defined by different concentric areas in oxidative enzyme staining - can occur in patients with neurogenic muscular atrophy. Here, we used our established hypothesis-free proteomic approach with the aim of deciphering the protein composition of targets. We also searched for potential novel interactions between target proteins.Targets and control areas were laser microdissected from skeletal muscle sections of 20 patients with neurogenic muscular atrophy. Samples were analysed by a highly sensitive mass spectrometry approach, enabling relative protein quantification. The results were validated by immunofluorescence studies. Protein interactions were investigated by yeast two-hybrid assays, coimmunoprecipitation experiments and bimolecular fluorescence complementation.More than 1000 proteins were identified. Among these, 55 proteins were significantly over-represented and 40 proteins were significantly under-represented in targets compared to intraindividual control samples. The majority of over-represented proteins were associated with the myofibrillar Z-disc and actin dynamics, followed by myosin and myosin-associated proteins, proteins involved in protein biosynthesis and chaperones. Under-represented proteins were mainly mitochondrial proteins. Functional studies revealed that the LIM domain of the over-represented protein LIMCH1 interacts with isoform A of Xin actin-binding repeat-containing protein 1 (XinA).In particular, proteins involved in myofibrillogenesis are over-represented in target structures, which indicate an ongoing process of sarcomere assembly and/or remodelling within this specific area of the muscle fibres. We speculate that target structures are the result of reinnervation processes in which filamin C-associated myofibrillogenesis is tightly regulated by the BAG3-associated protein quality system.

An oversupply of nutrients with a loss of metabolic flexibility and subsequent cardiac dysfunction are hallmarks of diabetic cardiomyopathy. Even if excess substrate is offered, the heart suffers energy depletion as metabolic fluxes are diminished. To study the effects of a high glucose supply, a stably glucose transporter type 4 (GLUT4)-overexpressing cell line presenting an onset of diabetic cardiomyopathy-like phenotype was established. Long-term hyperglycaemia effects were analysed. Rat cardiomyoblasts overexpressing GLUT4 (H9C2KE2) were cultured under normo- and hyperglycaemic conditions for long-term. Expression profiles of several proteins were compared to non-transfected H9C2 cells (H9C2) using RT-qPCR, proteomics-based analysis, or Western blotting. GLUT4 surface analysis, glucose uptake, and cell morphology changes as well as apoptosis/necrosis measurements were performed using flow cytometry. Additionally, brain natriuretic peptide (BNP) levels, reactive oxygen species (ROS) formation, glucose consumption, and lactate production were quantified. Long-term hyperglycaemia in H9C2KE2 cells induced increased GLUT4 presence on the cell surface and was associated with exaggerated glucose influx and lactate production. On the metabolic level, hyperglycaemia affected the tricarboxylic acid (TCA) cycle with accumulation of fumarate. This was associated with increased BNP-levels, oxidative stress, and lower antioxidant response, resulting in pronounced apoptosis and necrosis. Chronic glucose overload in cardiomyoblasts induced by GLUT4 overexpression and hyperglycaemia resulted in metabolically stimulated proteome profile changes and metabolic alterations on the TCA level.

A detailed description of pathophysiological effects that viruses exert on their host is still challenging. For the first time, we report a highly controllable viral expression model based on an iPS-cell line from a healthy human donor. The established viral model system enables a dose-dependent and highly localized RNA-virus expression in a fully controllable environment, giving rise for new applications for the scientific community.

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson's disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.

Aims We investigated N471D WASH complex subunit strumpellin (Washc5) knock‐in and Washc5 knock‐out mice as models for hereditary spastic paraplegia type 8 (SPG8). Methods We generated heterozygous and homozygous N471D Washc5 knock‐in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock‐out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. Results Homozygous N471D Washc5 knock‐in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock‐in mice. WASHC5‐related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock‐out mice showing normal WASHC5 levels could not be bred to homozygosity. Conclusions While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.

Cerebrospinal fluid (CSF) diagnostics has emerged as a valid tool for a variety of neurological diseases. However, CSF diagnostics has been playing a subordinate role in the diagnosis of many neurological conditions. Thus, in the multitude of neuromuscular diseases in which motor neurons are affected, a CSF sample is rarely taken routinely. However, CSF diagnostics has the potential to specify the diagnosis and monitor the treatment of neuromuscular disorders. In this review, we therefore focused on a variety of neuromuscular diseases, among them amyotrophic lateral sclerosis (ALS), peripheral neuropathies, and spinal muscular atrophy (SMA), for which CSF diagnostics has emerged as a promising option for determining the disease itself and its progression. We focus on potentially valuable biomarkers among different disorders, such as neurofilaments, cytokines, other proteins, and lipids to determine their suitability, differentiating between different neurological disorders and their potential to determine early disease onset, disease progression, and treatment outcome. We further recommend novel approaches, e.g., the use of mass spectrometry as a promising alternative techniques to standard ELISA assays, potentially enhancing biomarker significance in clinical applications.

Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. All data are available via ProteomeXchange with the identifier PXD025359. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach, identified a discriminative peptide panel, and confirmed our panel using a public data set.

For the quantification of certain proteins of interest within a complex sample, Western blot analysis is the most widely used method. It enables detection of a target protein based on the use of specific antibodies. However, the whole procedure is often very time-consuming. Nevertheless, with the development of fast blotting systems and further development of immunostaining methods, a reduction of the processing time can be achieved. Major challenges for the reliable protein quantification by Western blotting are adequate data normalization and stable protein detection. Usually, normalization of the target protein signal is performed based on housekeeping proteins (e.g., glyceraldehyde 3-phosphate dehydrogenase, ß-actin) with the assumption that those proteins are expressed constitutively at the same level across experiments. However, several studies have already shown that this is not always the case making this approach suboptimal. Another strategy uses total protein normalization where the abundance of the target protein is related to the total protein amount in each lane. This approach is independent of a single loading control, and precision of quantification and reliability is increased. For Western blotting several detection methods are available, e.g., colorimetric, chemiluminescent, radioactive, fluorescent detection. Conventional colorimetric staining tends to suffer from low sensitivity, limited dynamic range, and low reproducibility. Chemiluminescence-based methods are straightforward, but the detected signal does not linearly correlate to protein abundance (from protein amounts5μg) and have a relatively narrow dynamic range. Radioactivity is harmful to health. To overcome these limitations, stain-free methods were developed allowing the combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer to the membrane. Here, we present a rapid Western blot protocol, which combines fast blotting using the iBlot system and fast immunostaining utilizing ReadyTector

Data-independent acquisition (DIA) has recently developed as a powerful tool to enhance the quantification of peptides and proteins within a variety of sample types, by overcoming the stochastic nature of classical data-dependent approaches, as well as by enabling the identification of all peptides detected in a mass spectrometric event. Here, we describe a workflow for the establishment of a sample-fitting DIA method using Spectronaut Pulsar X (Biognosys, Switzerland).

Data-independent acquisition (DIA) has recently developed as a powerful tool to enhance the quantification of peptides and proteins within a variety of sample types, by overcoming the stochastic nature of classical data-dependent approaches, as well as by enabling the identification of all peptides detected in a mass spectrometric event. Here, we describe a workflow for the establishment of a sample-fitting DIA method using Spectronaut Pulsar X (Biognosys, Switzerland).

For the quantification of certain proteins of interest within a complex sample, Western blot analysis is the most widely used method. It enables detection of a target protein based on the use of specific antibodies. However, the whole procedure is often very time-consuming. Nevertheless, with the development of fast blotting systems and further development of immunostaining methods, a reduction of the processing time can be achieved. Major challenges for the reliable protein quantification by Western blotting are adequate data normalization and stable protein detection. Usually, normalization of the target protein signal is performed based on housekeeping proteins (e.g., glyceraldehyde 3-phosphate dehydrogenase, s-actin) with the assumption that those proteins are expressed constitutively at the same level across experiments. However, several studies have already shown that this is not always the case making this approach suboptimal. Another strategy uses total protein normalization where the abundance of the target protein is related to the total protein amount in each lane. This approach is independent of a single loading control, and precision of quantification and reliability is increased. For Western blotting several detection methods are available, e.g., colorimetric, chemiluminescent, radioactive, fluorescent detection. Conventional colorimetric staining tends to suffer from low sensitivity, limited dynamic range, and low reproducibility. Chemiluminescence-based methods are straightforward, but the detected signal does not linearly correlate to protein abundance (from protein amounts >5μg) and have a relatively narrow dynamic range. Radioactivity is harmful to health. To overcome these limitations, stain-free methods were developed allowing the combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer to the membrane. Here, we present a rapid Western blot protocol, which combines fast blotting using the iBlot system and fast immunostaining utilizing ReadyTector® all-in-one solution with the Smart Protein Layers (SPL) approach.

The pathological hallmark of Parkinson&rsquo, s disease (PD) is the loss of neuromelanin-containing dopaminergic neurons within the substantia nigra pars compacta (SNpc). Additionally, numerous studies indicate an altered synaptic function during disease progression. To gain new insights into the molecular processes underlying the alteration of synaptic function in PD, a proteomic study was performed. Therefore, synaptosomes were isolated by density gradient centrifugation from SNpc tissue of individuals at advanced PD stages (N = 5) as well as control subjects free of pathology (N = 5) followed by mass spectrometry-based analysis. In total, 362 proteins were identified and assigned to the synaptosomal core proteome. This core proteome comprised all proteins expressed within the synapses without regard to data analysis software, gender, age, or disease. The differential analysis between control subjects and PD cases revealed that CD9 antigen was overrepresented and fourteen proteins, among them Thymidine kinase 2 (TK2), mitochondrial, 39S ribosomal protein L37, neurolysin, and Methionine-tRNA ligase (MARS2) were underrepresented in PD suggesting an alteration in mitochondrial translation within synaptosomes.

Background: Processing of post-mortem human brain tissue samples like Neuromelanin-containing substantia nigra is a challenging task in proteomics research in the context of neurodegenerative diseases such as Parkinson’s disease, due to the availability of only limited sample amounts. Therefore, a highly efficient lysis and digestion approach is needed to guarantee high reproducibility and a good quantification performance. We conducted a systematic study in order to compare qualitatively and quantitatively six different sample preparation protocols for substantia nigra brain tissue collected by laser microdissection.Results: LC-MS/MS analysis revealed that the combination of formic acid and in-solution digestion with trypsin leads to an enhanced peptide and protein identification as well as a robust and reliable protein quantification with a very good reproducibility.Conclusion: We recommend this protocol for future studies of human substantia nigra tissue as well as NM granules in the context of neurodegenerative disorders like Parkinson syndrome.

ObjectiveTo determine whether a new indel mutation in the dimerization domain of filamin C (FLNc) causes a hereditary myopathy with protein aggregation in muscle fibers, we clinically and molecularly studied a German family with autosomal dominant myofibrillar myopathy (MFM).MethodsWe performed mutational analysis in 3 generations, muscle histopathology, and proteomic studies of IM protein aggregates. Functional consequences of the FLNC mutation were investigated with interaction and transfection studies and biophysics molecular analysis.ResultsEight patients revealed clinical features of slowly progressive proximal weakness associated with a heterozygous c.8025_8030delCAAGACinsA (p.K2676Pfs*3) mutation in FLNC. Two patients exhibited a mild cardiomyopathy. MRI of skeletal muscle revealed lipomatous changes typical for MFM with FLNC mutations. Muscle biopsies showed characteristic MFM findings with protein aggregation and lesion formation. The proteomic profile of aggregates was specific for MFM-filaminopathy and indicated activation of the ubiquitin-proteasome system (UPS) and autophagic pathways. Functional studies revealed that mutant FLNc is misfolded, unstable, and incapable of forming homodimers and heterodimers with wild-type FLNc.ConclusionsThis new MFM-filaminopathy family confirms that expression of mutant FLNC leads to an adult-onset muscle phenotype with intracellular protein accumulation. Mutant FLNc protein is biochemically compromised and leads to dysregulation of protein quality control mechanisms. Proteomic analysis of MFM protein aggregates is a potent method to identify disease-relevant proteins, differentiate MFM subtypes, evaluate the relevance of gene variants, and identify novel MFM candidate genes.

Introduction: Diabetic cardiomyopathy is characterized by oversupply of nutrients with concurrent loss of metabolic flexibility. Even in excess substrate availability the heart suffers energy depletion as metabolic fluxes are diminished. We established a stably GLUT4 overexpressing cell line derived from H9C2 that presents a diabetic cardiomyopathy like phenotype. Protein expression patterns are analyzed by proteomic approaches. Material and Methods: Rat cardiomyoblasts stably overexpressing GLUT4 (H9C2-GLUT4) were cultured under hyperglycemic conditions (30mM glucose) either short time (3 days) or long time (more than 3 months). Expression profile was compared to non-transfected H9C2 cells (H9C2), using same culture conditions. Cell culture samples were prepared for mass spectrometric analysis by sample lysis followed by tryptic digestion. Peptides were analyzed on an Orbitrap Elite (ThermoFisher, Scientific) and resulting files were taken for the identification and quantification of proteins using MaxQuant and Perseus. DAVID Bioinformatics Resources 6.8 functioned as Pathway and GO term enrichment tool for further in depth analysis of the data set. Results: We were able to quantify 2,841 proteins of whose 881 were found to be significantly regulated (5% FDR; ANOVA corrected p-value). Further analysis of long time and short time exposure of either WT or GLUT4 overexpressing cells lead to the identification of 135 significantly expressed proteins in the WT and 112 significantly expressed proteins in the H9C2-GLUT4 model (5% FDR; ANOVA corrected p-value). In depth pathway and GO term enrichment analysis support effects on metabolism, protein biosynthesis and handling, and ECM composition. Discussion: Chronic glucose overload in cardiomyoblasts induced by GLUT4 overexpression and hyperglycemia resulted in a metabolic induced proteome profile change. These models may thus increase our knowledge of biological effects of chronic hyperglycemia on the diabetic heart. Disclosure B. Stratmann: Speaker’s Bureau; Self; AstraZeneca, Neoplas Tools GmbH. B. Eggers: None. Y. Mattern: None. K. Marcus: None. D. Tschoepe: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH. Research Support; Self; AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Neoplas tools GmbH.

In a common proteomics analysis today, the origins of our sample in the vial are known and therefore a database dependent approach to identify the containing peptides can be used. The first YPIC challenge though provided us with 19 synthetic peptides, which together formed an English sentence. For the identification of these peptides, a de-novo approach was used, which brought us together with an internet search engine to the hidden sentence. But only having the sentence was not sufficient for us, we also wanted to identify as many as possible of the spectra in our data. Therefore, we created and refined a database approach from the de-novo method and finally could identify the peptide-sentence with a good overlap.

Protein aggregation is a pathological feature of neurodegenerative disorders. We previously demonstrated that protein inclusions in the brain are composed of supersaturated proteins, which are abundant and aggregation-prone, and form a metastable subproteome. It is not yet clear, however, whether this phenomenon is also associated with non-neuronal protein conformational disorders. To respond to this question, we analyzed proteomic datasets from biopsies of patients with genetic and acquired protein aggregate myopathy (PAM) by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that a metastable subproteome is present in skeletal muscle from healthy patients. The expression of this subproteome escalate as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion-containing fibers, proteins within the metastable subproteome rise in abundance, suggesting that they escape regulation. Taken together, our results show in the context of a human conformational disorder that the supersaturation of a metastable subproteome underlies widespread aggregation and correlates with the histopathological state of the tissue.

Secondary mitochondrial dysfunction is a feature in a wide variety of human protein aggregate diseases caused by mutations in different proteins, both in the central nervous system and in striated muscle. The functional relationship between the expression of a mutated protein and mitochondrial dysfunction is largely unknown. In particular, the mechanism how this dysfunction drives the disease process is still elusive. To address this issue for protein aggregate myopathies, we performed a comprehensive, multi-level analysis of mitochondrial pathology in skeletal muscles of human patients with mutations in the intermediate filament protein desmin and in muscles of hetero- and homozygous knock-in mice carrying the R349P desmin mutation. We demonstrate that the expression of mutant desmin causes disruption of the extrasarcomeric desmin cytoskeleton and extensive mitochondrial abnormalities regarding subcellular distribution, number and shape. At the molecular level, we uncovered changes in the abundancy and assembly of the respiratory chain complexes and supercomplexes. In addition, we revealed a marked reduction of mtDNA- and nuclear DNA-encoded mitochondrial proteins in parallel with large-scale deletions in mtDNA and reduced mtDNA copy numbers. Hence, our data demonstrate that the expression of mutant desmin causes multi-level damage of mitochondria already in early stages of desminopathies. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1592-7) contains supplementary material, which is available to authorized users.

Proteomics using LC-MS/MS has become one of the main methods to analyze the proteins in biological samples in high-throughput. But the existing mass spectrometry instruments are still limited with respect to resolution and measurable mass ranges, which is one of the main reasons why shotgun proteomics is the major approach. Here, proteins are digested, which leads to the identification and quantification of peptides instead. While often neglected, the important step of protein inference needs to be conducted to infer from the identified peptides to the actual proteins in the original sample.In this work, we highlight some of the previously published and newly added features of the tool PIA – Protein Inference Algorithms, which helps the user with the protein inference of measured samples. We also highlight the importance of the usage of PSI standard file formats, as PIA is the only current software supporting all available standards used for spectrum identification and protein inference. Additionally, we briefly describe the benefits of working with workflow environments for proteomics analyses and show the new features of the PIA nodes for the KNIME Analytics Platform. Finally, we benchmark PIA against a recently published dataset for isoform detection.PIA is open source and available for download on GitHub (https://github.com/mpc-bioinformatics/pia) or directly via the community extensions inside the KNIME analytics platform.

Species distribution models have recently become important tools in ecological research. Prediction of suitable habitats for threatened and endangered species is essential for the conservation and management of their native habitats. A landscape scale approach is relevant for biodiversity conservation since landscape planning and management are generally conducted at wide spatial scales, focusing on areas with complex landscape configuration as a consequence of human activities. The aims of this study were to test a maximum entropy approach (Maxent) to the development of a niche-based model for species of conservation interest and to relate this model to landscape structure metrics. The results obtained here showed a good predictive power of Maxent for the three target species and highlighted the importance of landscape structure analysis for the detection of patterns of habitat suitability. Moreover, this work stressed that combining classical environmental information with landscape structure in...

To counteract habitat fragmentation, the connectivity of a landscape should be enhanced. Corridors are thought to facilitate movement between disconnected patches of habitat, and linear strips of habitat connecting isolated patches are a popular type of corridor. On the other hand, the creation of new corridors can lead to fragmentation of the surrounding habitat. For example, heathland corridors connect patches of heathland and alternatively hedgerows connect patches of woodland. Nevertheless, these corridors themselves also break up previously connected patches of their surrounding habitat and in so doing fragment another type of habitat (heathland corridors fragment woodlands and woodland strips or hedgerows fragment heath-lands). To overcome this challenge we propose the use of semi-open habitats (a mixture of heathland and woodland vegetation) as conservation corridors to enable dispersal of both stenotopic heathland and woodland species. We used two semi-open corridors with a mosaic of heathland and woody vegetation to investigate the efficiency of semi-open corridors for species dispersal and to assess whether these corridors might be a suitable approach for nature conservation. We conducted a mark-recapture study on three stenotopic flightless carabid beetles of heathlands and woodlands and took an inventory of all the carabid species in two semi-open corridors. Both methodological approaches showed simultaneous immigration of woodland and heathland species in the semi-open corridor. Detrended correspondence analysis showed a clear separation of the given habitats and affirmed that semi-open corridors are a good strategy for connecting woodlands and heathlands. The best means of creating and preserving semi-open corridors is probably through extensive grazing. To counteract habitat fragmentation, the connectivity of a landscape should be enhanced. Corridors are thought to facilitate movement between disconnected patches of habitat, and linear strips of habitat connecting isolated patches are a popular type of corridor. On the other hand, the creation of new corridors can lead to fragmentation of the surrounding habitat. For example, heathland corridors connect patches of heathland and alternatively hedgerows connect patches of woodland. Nevertheless, these corridors themselves also break up previously connected patches of their surrounding habitat and in so doing fragment another type of habitat (heathland corridors fragment woodlands and woodland strips or hedgerows fragment heathlands). To overcome this challenge we propose the use of semi-open habitats (a mixture of heathland and woodland vegetation) as conservation corridors to enable dispersal of both stenotopic heathland and woodland species. We used two semi-open corridors with a mosaic of heathland and woody vegetation to investigate the efficiency of semi-open corridors for species dispersal and to assess whether these corridors might be a suitable approach for nature conservation. We conducted a mark-recapture study on three stenotopic flightless carabid beetles of heathlands and woodlands and took an inventory of all the carabid species in two semi-open corridors. Both methodological approaches showed simultaneous immigration of woodland and heathland species in the semi-open corridor. Detrended correspondence analysis showed a clear separation of the given habitats and affirmed that semi-open corridors are a good strategy for connecting woodlands and heathlands. The best means of creating and preserving semi-open corridors is probably through extensive grazing.

Habitat fragmentation can be defined as the modification of once natural habitats into smaller, isolated subareas surrounded by other types of habitat (more or less hostile; the matrix; e. g. Valladares et al., 2006). Fragmentation includes both, the separation of habitats and habitat loss, but the most dramatic and consistently negative effects on biodiversity can be attributed to habitat loss (Fahrig, 2003). In our central European man-made landscape more and more habitats become destroyed or fragmented because of the increasing anthropogenic need of available land and despite the growing knowledge about the problem. Roads, residential, and industrial areas separate formerly connected habitats into small remnants and thus create small subpopulations. Accordingly, habitat fragmentation and the associated effects like (1) the biodiversity decline of stenotopic species in smaller habitats (Desender et al., 1999; Magura et al., 2001), (2) the loss of genetic diversity and variability (Debinski & Holt, 2000; Keller & Largiader, 2003), and (3) a higher probability of inbreeding in smaller populations, belong to the main reasons for extinction of species (Groom et al., 2006). Especially stenotopic species with low dispersal power are endangered because the exchange of specimen between different habitat patches is reduced or entirely inhibited (Hanski et al., 1995). Thus, standardised, comprehensible quantification methods of fragmentation are greatly important for the development of management and conservation plans for habitat networks.