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PubMed2025

Emerging protein sequencing technologies: proteomics without mass spectrometry?

INTRODUCTION: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been a leading method for proteomics for 30 years. Advantages provided by LC-MS/MS are offset by significant disadvantages, including cost. Recently, several non-mass spectrometric methods have emerged, but little information is available about their capacity to analyze the complex mixtures routine for mass spectrometry. AREAS COVERED: We review recent non-mass-spectrometric methods for sequencing proteins and peptides, including those using nanopores, sequencing by degradation, reverse translation, and short-epitope mapping, with comments on bioinformatics challenges, fundamental limitations, and areas where new technologies will be more or less competitive with LC-MS/MS. In addition to conventional literature searches, instrument vendor websites, patents, webinars, and preprints were also consulted to give a more up-to-date picture. EXPERT OPINION: Many new technologies are promising. However, demonstrations that they outperform mass spectrometry in terms of peptides and proteins identified have not yet been published, and astute observers note important disadvantages, especially relating to the dynamic range of single-molecule measurements of complex mixtures. Still, even if the performance of emerging methods proves inferior to LC-MS/MS, their low cost could create a different kind of revolution: a dramatic increase in the number of biology laboratories engaging in new forms of proteomics research.

Proteomics↗

PubMed1993

Specific induction of uncoiling and recombination by azacytidine in classical satellite-containing constitutive heterochromatin.

Azacytidine (ACR) is known to induce uncoiling and somatic association involving the constitutive heterochromatin of human chromosomes 1, 9, 15, and 16 and the Y. These regions are composed of alphoid and classical satellite DNA sequences. Using specific probes for chromosomes 1 and 16, we have performed two-color fluorescence in situ hybridization on human lymphocytes cultured in the presence of ACR. We demonstrate that for these two chromosomes (1) uncoiling and association specifically occur in classical satellite-containing regions at the first cell generation, (2) breakages also affect these regions, and (3) somatic recombinations occur between these regions and lead to translocations at the next cell generation. These results suggest that changes in methylation of repetitive DNA sequences are related to chromosomal instability occurring during cell transformation and tumorigenesis.

Azacitidine↗

PubMed2000

Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene.

A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cell-mediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1(SF2) p55(Gag) were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55(Gag) protein expression to levels that ranged from 322- to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55(Gag) and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gag protease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8(+) cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.

AIDS Vaccines↗

PubMed2004

Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome.

As genomic sequencing of Leishmania nears completion, functional analyses that provide a global genetic perspective on biological processes are important. Despite polycistronic transcription, RNA transcript abundance can be measured using microarrays. To provide a resource to evaluate cDNA arrays, we undertook 5' expressed sequence tag analysis of 2183 full-length randomly selected cDNAs from Leishmania major promastigote (days 3, 7, 10 of culture in vitro), and lesion-derived amastigote libraries. PCR-amplified inserts from 1830 of these cDNA representing 1001 unique genes were spotted onto microarrays, and compared internally with PCR-amplified open reading frames (ORFs) from 904 genes representing 842 unique genes annotated in the L. major genome. Microarrays were screened with RNA from procyclic, metacyclic and amastigote populations of L. major. Redundant clones on the array gave highly reproducible results, providing confidence in identification of stage-specific gene expression. Four hundred and thirty unique (i.e. non-redundant) stage-specific genes were identified. A higher percentage of stage-specific gene expression was observed in amastigotes ( approximately 35%) compared to metacyclics ( approximately 12%) for both cDNAs and ORFs, but cDNAs provided a richer source of regulated genes than currently annotated ORFs from the Leishmania genome. In mapping cDNAs onto the Leishmania genome, we noted that approximately 42% aligned to regions not recognised as genes using current predictive annotation tools. These genes are highly represented in our stage-specific genes, and therefore represent important drug targets and vaccine candidates. Careful annotation of cDNAs onto the Leishmania genome will be important before producing the next generation of oligonucleotide arrays based on annotated genes of the genomic sequencing project.

Animals↗

PubMed2005

Transposable element insertion location bias and the dynamics of gene drive in mosquito populations.

Some vector-borne disease control strategies using transgenic mosquitoes require transgene spread to high frequency in populations. Transposable elements (TEs) are DNA sequences that replicate and transpose within the genomes of other organisms and may therefore be represented in the next generation in higher frequencies than predicted by Mendelian segregation. This over-representation has allowed some TEs to spread through natural populations. Transgenes incorporated within a TE sequence are expected to be driven into populations as long as there is a positive balance between fitness costs and over-representation. Models have been used to examine parameters that affect this balance but did not take into account biased insertion of TEs to linked sites in the genome. A simulation model was created to examine the impact of insertion bias on TE spread in mosquito populations. TEs that induce no fitness costs are predicted to increase in frequency over a wide range of parameter values but spread is slower for lower levels of transposition and non-local movement. If TEs are costly, high proportions of local movement can slow or halt spread. To function as a robust transgene drive mechanism a TE should replicate and transpose > 10%/insert/generation, induce < 1% fitness cost/insert, and move preferentially to unlinked sites in the genome.

Animals↗

PubMed1991

Cytogenesis in the monkey retina.

Time of cell origin in the retina of the rhesus monkey (Macaca mulatta) was studied by plotting the number of heavily radiolabeled nuclei in autoradiograms prepared from 2- to 6-month-old animals, each of which was exposed to a pulse of 3H-thymidine (3H-TdR) on a single embryonic (E) or postnatal (P) day. Cell birth in the monkey retina begins just after E27, and approximately 96% of cells are generated by E120. The remaining cells are produced during the last (approximately 45) prenatal days and into the first several weeks after birth. Cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in the foveal and perifoveal regions by E56. Despite extensive overlap, a class-specific sequence of cell birth was observed. Ganglion and horizontal cells, which are born first, have largely congruent periods of cell genesis with the peak between E38 and E43, and termination around E70. The first labeled cones were apparent by E33, and their highest density was achieved between E43 and E56, tapering to low values at E70, although some cones are generated in the far periphery as late as E110. Amacrine cells are next in the cell birth sequence and begin genesis at E43, reach a peak production between E56 and E85, and cease by E110. Bipolar cell birth begins at the same time as amacrines, but appears to be separate from them temporally since their production reaches a peak between E56 and E102, and persists beyond the day of birth. Müller cells and rod photoreceptors, which begin to be generated at E45, achieve a peak, and decrease in density at the same time as bipolar cells, but continue genesis at low density on the day of birth. Thus, bipolar, Müller, and rod cells have a similar time of origin. The maximal temporal separation of cell birth is between cones and amacrine cells so that cell generation exhibits two relatively distinct phases: the first phase gives rise to ganglion, horizontal, and cone cells, and the second phase to amacrine, bipolar, rod, and Müller cells. In addition, cells of the first phase are generated faster than the second phase cells, and there are differences in the topography of spread of labeled cells between the two phases. Each cell class displays a central-to-peripheral gradient in genesis, although the spatiotemporal characteristics of the gradients differ between the classes.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed1989

[Automated measurement of distance vision based on the DIN strategy].

A method for automated measurement of far vision is described which meets the test requirements laid down in the new DIN standards. The subject sits 5 m from a high-resolution monitor on which either Landolt rings or Snellen's types are generated by a computer. By moving a joystick the subject indicates to the computer whether he can see the critical detail (e.g., the direction of opening of the Landolt ring). Depending on the subject's input and the course of the test so far, the computer generates the next test symbol until the threshold criterion is reached. The sequence of presentation of the symbols and the threshold criterion are also in accordance with the DIN standard. Initial measurements of far vision using this automated system produced similar results to those obtained by conventional methods.

Distance Perception↗

PubMed2026

Dysregulated Sheddase Signalling as a Molecular Driver of Plaque Instability Revealed by Integrative Transcriptomics.

Atherosclerosis is a major cause of mortality due to chronic and progressive low-grade inflammation and fibroproliferative remodelling of the intima of arteries. Comprehensive understanding of the interplay between plaque biology and the mechanisms underlying plaque vulnerability and rupture is essential. Here, we aimed to investigate the transcriptomic profiles of stable and unstable atherosclerotic plaques using RNA sequencing data from human carotid atherosclerotic plaque samples based on next-generation knowledge discovery (NGKD) methods. High-throughput RNA-seq data from plaques dissected in stable and unstable regions of four patients were obtained from the Gene Expression Omnibus (GEO) database. GEO RNA-seq Experiments Interactive Navigator (GREIN) software was used to obtain raw gene-level counts and filtered metadata for this dataset. The data were further filtered and normalized using Express analyst to derive differentially expressed genes (DEGs) in unstable plaques compared to stable plaques. The DEGs were further analysed using WebGestalt, STRING DB, preranked gene set enrichment analysis (GSEA), and Ingenuity Pathway Analysis (IPA) software. We identified 4792 DEGs in unstable plaques based on a p-value cutoff of < 0.05. NGKD analysis revealed that the sheddase pathway, collagen degradation, activation of matrix metalloproteinases (MMPs), and extracellular matrix (ECM) degradation ranked among the top five upregulated pathways, whereas the inhibition of MMPs and smooth muscle contraction pathways were identified as the most prominent downregulated pathways in unstable plaques. We found that the sheddase pathway was one of the most significantly upregulated canonical pathways in unstable plaques and this finding opens new avenues for potential therapeutic interventions in patients with atherosclerosis.

Humans↗

PubMed2006

Anticipating the 1,000 dollar genome.

A new generation of DNA-sequencing platforms will become commercially available over the next few years. These instruments will enable re-sequencing of human genomes at a previously unimagined throughput and low cost. Here, I examine why the 1,000 dollar human genome is an important goal for research and clinical diagnostics, and what will be required to achieve it.

Equipment and Supplies↗

PubMed1998

DNA synthesis, mismatch repair and cancer.

The entire nucleotide sequence of the genome must be transmitted from one generation to the next with no or few errors. Preservation of this integrity requires multiple genes whose alteration can lead to an early event in tumorigenesis by increasing the mutation rate. This mutator phenotype would provide a continuing pool of mutants upon which selection could act to promote a tumor. Recent evidence consistant with this hypothesis is the mutator phenotype of tumor cells of patients with a hereditary form of colon cancer (HNPCC) which exhibit a several hundred-fold increase in spontaneous mutations in addition to a high degree of microsatellite instability. The multiple genomic alterations increasingly reported as associated with most cancers may therefore be linked to a variety of DNA metabolic processes guardians of the genome, including fidelity of the DNA synthesis and mismatch repair. The connection between cancer and deregulation of nucleotide synthesis, imbalance of the pools of nucleotides, deficiency of DNA polymerases, and mismatch repair is the subject of this review. We consider how perturbation in these DNA transactions results in instability of the genome and cancer.

Base Pair Mismatch↗

PubMed2003

Discriminant snakes for 3D reconstruction of anatomical organs.

In this work a new statistic deformable model for 3D segmentation of anatomical organs in medical images is proposed. A statistic discriminant snake performs a supervised learning of the object boundary in an image slice to segment the next slice of the image sequence. Each part of the object boundary is projected in a feature space generated by a bank of Gaussian filters. Then, clusters corresponding to different boundary pieces are constructed by means of linear discriminant analysis. Finally, a parametric classifier is generated from each contour in the image slice and embodied into the snake energy-minimization process to guide the snake deformation in the next image slice. The discriminant snake selects and classifies image features by the parametric classifier and deforms to minimize the dissimilarity between the learned and found image features. The new approach is of particular interest for segmenting 3D images with anisotropic spatial resolution, and for tracking temporal image sequences. In particular, several anatomical organs from different imaging modalities are segmented and the results compared to expert tracings.

Algorithms↗

PubMed1994

A deductive database system for analyzing human nucleotide sequence data.

The analysis of the human genome is one of the most significant topics in both biology and medical science. There is a growing need for a well-designed database system for searching and analyzing the human genome data. We developed a deductive database system to search and analyze nucleotide sequence data derived from the GenBank primates data. A deductive database system is a next generation one and it contains an inference mechanism that can handle problems beyond the capabilities of classical database systems. Database queries are described in logical rules. These rules are simple even for molecular biologists who are not experts in computer programs because they are declarative and do not require the procedural commands that are usually used in computer programs. Furthermore, queries based on logical rules are powerful enough to express complicated biological problems. Particularly, recursive rules are suitable for examining secondary structures of nucleotide sequences. In our analysis of TfR's IRE, we noted five stem-and-loop structures.

Artificial Intelligence↗

PubMed2025

Ischemic Injury Drives Nascent Tumor Growth Via Accelerated Hematopoietic Aging.

BACKGROUND: Patients with peripheral artery disease have an increased risk of cancer development. Aging-associated changes in hematopoietic stem and progenitor cells (HSPCs), including inflammation and increased myelopoiesis, are implicated in both cardiovascular disease and cancer, but their contributions to cardiovascular disease-driven tumor progression are unclear. OBJECTIVES: This study sought to study tumor growth after peripheral ischemia and consequent changes within the HSPC bone marrow compartment to uncover mechanisms through which altered hematopoiesis promotes cancer. METHODS: Mammary cancer (E0771) growth was monitored in C57BL/6J mice after hind limb ischemia (HLI) or sham surgery. The tumor immune microenvironment, circulatory immune cells, and HSPC compartment were assessed by flow cytometry. Next-generation single-cell RNA and assay for transposase-accessible chromatin sequencing of bone marrow progenitors was performed to assess the distinct and synergistic transcriptomic and epigenetic changes of cancer and peripheral ischemia. The functional impact on tumor progression and persistence of ischemia-induced epigenetic reprogramming of HSPCs and their myeloid progeny was examined by bone marrow transplantation. RESULTS: Peripheral ischemia increased monocyte and neutrophil output at the expense of lymphocytes, driven by a shift toward CD150hi myeloid-biased hematopoietic stem cells. This was associated with accelerated cancer growth and enrichment of tumors with myeloid cells (monocytes, macrophages, neutrophils) and regulatory T cells. Increased myelopoiesis was also supported by sequencing analyses showing HLI and tumor-induced transcriptional and epigenetic enrichment for inflammatory (NLRP3 inflammasome) and aging-associated neogenin-1, thrombospondin-1) signatures in subsets of monocyte/dendritic progenitors. HLI-accelerated tumor growth and myeloid-skewing was transmissible via bone marrow transplantation, indicating long-term reprogramming of innate immune responses. CONCLUSIONS: Peripheral ischemia enhances inflammaging of hematopoietic stem cells and long-lasting alterations to antitumoral immunity, accelerating breast tumor growth.

bone marrow transplantation↗

PubMed1996

Ectopic expression of the maize homeobox genes ZmHox1a or ZmHox1b causes pleiotropic alterations in the vegetative and floral development of transgenic tobacco.

The ZmHox1a and ZmHox1b (for Zea mays homeobox) genes map on chromosomes 8 and 6, respectively. Both homeobox genes encode proteins that show 91% similarity and are transcribed simultaneously in meristematic and proliferating cells of the maize plant. To gain insight into the biological function of these genes, both open reading frames were expressed in tobacco, under the control of the cauliflower mosaic virus 35S promoter. The resulting transgenic ZmHox1a or ZmHox1b plants showed identical phenotypic alterations that fall into three classes: size reduction, formation of adventitious shoots, and homeotic floral transformations. Approximately 30% of the ZmHox1-expressing plants grew to only one-third of the wild-type size, and most axillary buds gave rise to lateral shoots. Flower abnormalities included formation of petaloid stamens and development of secondary flowers within the primary gynoecium. Therefore, the ectopic expression of the maize ZmHox1 homeobox gene products affects the vegetative as well as the reproductive phase of tobacco plants. All phenotypic alterations were transmitted to the next generation.

Amino Acid Sequence↗

PubMed2001

Analysis of transcriptional regulation of human breast aromatase by in vitro and in vivo studies.

Aromatase mRNA in normal breast tissues is mainly transcribed from exon 1b (I.4) of the gene. However, in breast cancer tissues, it is often transcribed from exon 1c/1d (I.3/PII). Such a switching from exon 1b to exon 1c/1d has often found concomitantly with elevated levels of aromatase mRNA. To elucidate the molecular mechanism of a switching of multiple exons 1 and enhanced expression of aromatase in the breast tissues, we identified essential elements responsible for transcription from exon 1b. Gel shift assays indicated that there are two essential elements for transcription from exon 1b between -300 and -500 bp in the promoter region. The two unique elements have homologous consensus DNA sequences, and competed for binding of the specific nuclear protein with each other. We next generated transgenic mouse expressing a reporter gene controlled by multiple promoters in the human aromatase gene. The mouse gave tissue-specific expression of the reporter gene and tissue-specific utilization of the alternative promoter regions. These results show that this transgenic mouse is a good model animal for the study of tissue-specific regulation of human aromatase gene.

Alternative Splicing↗

PubMed2000

An associational model of birdsong sensorimotor learning II. Temporal hierarchies and the learning of song sequence.

Understanding the neural mechanisms underlying serially ordered behavior is a fundamental problem in motor learning. We present a computational model of sensorimotor learning in songbirds that is constrained by the known functional anatomy of the song circuit. The model subsumes our companion model for learning individual song "syllables" and relies on the same underlying assumptions. The extended model addresses the problem of learning to produce syllables in the correct sequence. Central to our approach is the hypothesis that the Anterior Forebrain Pathway (AFP) produces signals related to the comparison of the bird's own vocalizations and a previously memorized "template." This "AFP comparison hypothesis" is challenged by the lack of a direct projection from the AFP to the song nucleus HVc, a candidate site for the generator of song sequence. We propose that sequence generation in HVc results from an associative chain of motor and sensory representations (motor --> sensory --> next motor. ) encoded within the two known populations of HVc projection neurons. The sensory link in the chain is provided, not by auditory feedback, but by a centrally generated efference copy that serves as an internal prediction of this feedback. The use of efference copy as a substitute for the sensory signal explains the ability of adult birds to produce normal song immediately after deafening. We also predict that the AFP guides sequence learning by biasing motor activity in nucleus RA, the premotor nucleus downstream of HVc. Associative learning then remaps the output of the HVc sequence generator. By altering the motor pathway in RA, the AFP alters the correspondence between HVc motor commands and the resulting sensory feedback and triggers renewed efference copy learning in HVc. Thus, auditory feedback-mediated efference copy learning provides an indirect pathway by which the AFP can influence sequence generation in HVc. The model makes predictions concerning the role played by specific neural populations during the sensorimotor phase of song learning and demonstrates how simple rules of associational plasticity can contribute to the learning of a complex behavior on multiple time scales.

Animals↗

PubMed1993

Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are now feasible and may form the basis of the next generation of automated microbial identification systems. However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA. Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents. The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured.

Bacteria↗

PubMed2000

Ladder sequencing.

Ladder sequencing of polypeptides involves progressive N- or C-terminal amino acid truncation via chemical or enzymatic treatments. Peptide ladders are generated in which each component differs from the next by one residue. The ladder components are analyzed by mass spectrometry, and the amino acid sequence is deduced from the mass differences between consecutive fragments. Chemical procedures are common in N-terminal degradation, whereas proteolytic digestion is often used in C-terminal sequence analysis. Matrix-assisted laser desorption/ionization mass spectrometry is widespread for one-step readout of the peptide ladders and provides high sensitivity in combination with robustness and ease of use. The particular advantage of ladder sequencing in relation to other techniques for sequence analysis is the high data acquisition rate and the very good sample throughput that can be achieved. Multiple determinations are carried out within minutes at high sensitivity and low sample consumption. Several reports demonstrate analysis at the low picomole to femtomole level.

Peptide Hydrolases↗