Pressure cycling technology for challenging proteomic sample processing: application to barnacle adhesive.

AbstractSuccessful proteomic characterization of biological material depends on the development of robust sample processing methods. The acorn barnacle Amphibalanus amphitrite is a biofouling model for adhesive processes, but the identification of causative proteins involved has been hindered by their insoluble nature. Although effective, existing sample processing methods are labor and time intensive, slowing progress in this field. Here, a more efficient sample processing method is described which exploits pressure cycling technology (PCT) in combination with protein solvents. PCT aids in protein extraction and digestion for proteomics analysis. Barnacle adhesive proteins can be extracted and digested in the same tube using PCT, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. PCT methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than highly polar hexafluoroisopropanol, leading to the identification and description of >40 novel proteins at the interface. Some of these have homology to proteins with elastomeric properties or domains involved with protein-protein interactions, while many have no sequence similarity to proteins in publicly available databases, highlighting the unique adherent processes evolved by barnacles. The methods described here can not only be used to further characterize barnacle adhesive to combat fouling, but may also be applied to other recalcitrant biological samples, including aggregative or fibrillar protein matrices produced during disease, where a lack of efficient sample processing methods has impeded advancement. Data are available via ProteomeXchange with identifier PXD012730

Molt-dependent transcriptomic analysis of cement proteins in the barnacle Amphibalanus amphitrite

Abstract Background A complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. Barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. Little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion. Methods The main body and sub-mantle tissue of the barnacle Amphibalanus amphitrite (basionym Balanus amphitrite) were collected in pre- and post-molt stages. RNA-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze the protein content of barnacle secretions. Results We report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. While no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue (where the cement glands are located) compared to the main barnacle body. We report the discovery of a novel 114kD cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. Further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity. Conclusions The expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. Our results reveal a novel and potentially prominent protein to the mix of cement-related components in A. amphitrite. Despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. Our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate.http://deepblue.lib.umich.edu/bitstream/2027.42/115487/1/12864_2015_Article_2076.pd

Heterozygous COL4A3 Variants in Histologically Diagnosed Focal Segmental Glomerulosclerosis

Introduction: Steroid-resistant nephrotic syndrome (SRNS) is one of the most frequent causes for chronic kidney disease in childhood. In ~30% of these cases a genetic cause can be identified. The histological finding in SRNS is often focal segmental glomerulosclerosis (FSGS). In rare cases, however, pathogenic variants in genes associated with Alport syndrome can be identified in patients with the histological finding of FSGS. Materials and Methods: Clinical information was collected out of clinical reports and medical history. Focused molecular genetic analysis included sequencing of COL4A5 and COL4A3 in the index patient. Segregation analysis of identified variants was performed in the parents and children of the index patient. Results: The female index patient developed mild proteinuria and microscopic hematuria in childhood (12 years of age). The histological examination of the kidney biopsies performed at the age of 21, 28, and 32 years showed findings partly compatible with FSGS. However, immunosuppressive treatment of the index patient did not lead to a sufficient reduction of in part nephrotic-range proteinuria. After the patient developed hearing impairment at the age of 34 years and her daughter was diagnosed with microscopic hematuria at the age of 6 years, re-examination of the index's kidney biopsies by electron microscopy revealed textural changes of glomerular basement membrane compatible with Alport syndrome. Molecular genetic analysis identified two missense variants in COL4A3 in a compound heterozygous state with maternal and paternal inheritance. One of them is a novel variant that was also found in the 6 year old daughter of the index patient who presented with microscopic hematuria. Discussion: We were able to show that a novel variant combined with a previously described variant in compound heterozygous state resulted in a phenotype that was histologically associated with FSGS. Molecular genetic analysis therefore can be essential to solve difficult cases that show an unusual appearance and therefore improve diagnostic accuracy. Additionally, unnecessary and inefficient treatment with multiple side effects can be avoided

PEDIA: prioritization of exome data by image analysis.

PURPOSE: Phenotype information is crucial for the interpretation of genomic variants. So far it has only been accessible for bioinformatics workflows after encoding into clinical terms by expert dysmorphologists. METHODS: Here, we introduce an approach driven by artificial intelligence that uses portrait photographs for the interpretation of clinical exome data. We measured the value added by computer-assisted image analysis to the diagnostic yield on a cohort consisting of 679 individuals with 105 different monogenic disorders. For each case in the cohort we compiled frontal photos, clinical features, and the disease-causing variants, and simulated multiple exomes of different ethnic backgrounds. RESULTS: The additional use of similarity scores from computer-assisted analysis of frontal photos improved the top 1 accuracy rate by more than 20-89% and the top 10 accuracy rate by more than 5-99% for the disease-causing gene. CONCLUSION: Image analysis by deep-learning algorithms can be used to quantify the phenotypic similarity (PP4 criterion of the American College of Medical Genetics and Genomics guidelines) and to advance the performance of bioinformatics pipelines for exome analysis

Self-Assembly of Protein Nanofibrils Orchestrates Calcite Step Movement through Selective Nonchiral Interactions

The recognition of atomically distinct surface features by adsorbed biomolecules is central to the formation of surface-templated peptide or protein nanostructures. On mineral surfaces such as calcite, biomolecular recognition of, and self-assembly on, distinct atomic kinks and steps could additionally orchestrate changes to the overall shape and symmetry of a bulk crystal. In this work, we show through <i>in situ</i> atomic force microscopy (AFM) experiments that an acidic 20 kDa cement protein from the barnacle <i>Megabalanus rosa</i> (MRCP20) binds specifically to step edge atoms on {101̅4} calcite surfaces, remains bound and further assembles over time to form one-dimensional nanofibrils. Protein nanofibrils are continuous and organized at the nanoscale, exhibiting striations with a period of ca. 45 nm. These fibrils, templated by surface steps of a preferred geometry, in turn selectively dissolve underlying calcite features displaying the same atomic arrangement. To demonstrate this, we expose the protein solution to bare and fibril-associated rhombohedral etch pits to reveal that nanofibrils accelerate only the movement of fibril-forming steps when compared to undecorated steps exposed to the same solution conditions. Calcite mineralized in the presence of MRCP20 results in asymmetric crystals defined by frustrated faces with shared mirror symmetry, suggesting a similar step-selective behavior by MRCP20 in crystal growth. As shown here, selective surface interactions with step edge atoms lead to a cooperative regime of calcite modification, where templated long-range protein nanostructures shape crystals

Copy-Number Variations Measured by Single-Nucleotide–Polymorphism Oligonucleotide Arrays in Patients with Mental Retardation

Whole-genome analysis using high-density single-nucleotide–polymorphism oligonucleotide arrays allows identification of microdeletions, microduplications, and uniparental disomies. We studied 67 children with unexplained mental retardation with normal karyotypes, as assessed by G-banded chromosome analyses. Their DNAs were analyzed with Affymetrix 100K arrays. We detected 11 copy-number variations that most likely are causative of mental retardation, because they either arose de novo (9 cases) and/or overlapped with known microdeletions (2 cases). The eight deletions and three duplications varied in size from 200 kb to 7.5 Mb. Of the 11 copy-number variations, 5 were flanked by low-copy repeats. Two of those, on chromosomes 15q25.2 and Xp22.31, have not been described before and have a high probability of being causative of new deletion and duplication syndromes, respectively. In one patient, we found a deletion affecting only a single gene, MBD5, which codes for the methyl-CpG-binding domain protein 5. In addition to the 67 children, we investigated 4 mentally retarded children with apparent balanced translocations and detected four deletions at breakpoint regions ranging in size from 1.1 to 14 Mb