Protein sequence identification by tandem mass spectroscopy (LC‐MS/MS) identifies thousands of protein sequences even in complex mixtures, and provides valuable insight into the biological functions ...of different cells. For non‐model organisms, transcriptomes are generally used to allow peptide identification, an important addition to their use as a gene catalog allowing the potential metabolic activities of cells to be determined. We used LC‐MS/MS data to identify which of the six possible reading frames in the transcriptome was actually used by the cell to make protein, and asked whether this would have an impact on downstream analyses using the dataset. We combined results from several LC‐MS/MS experiments designed to identify peptide sequences in extracts from the dinoflagellate Lingulodinium polyedra using a 74 655‐sequence transcriptome. We compiled a list of 6628 translated nucleic acid sequences that contained the ensemble of peptide matches (termed MS‐validated sequences) and assessed the similarity in downstream analyses between this data set and the 6628 nucleic acid sequences from which they were derived. When compared with BLASTx analyses of the DNA sequences, the MS‐validated protein‐sequences‐analyzed using BLASTp showed differences in gene ontology, had more identified BLAST hits, and contained more KEGG pathway enzymes. The MS‐validated protein sequences also differ from datasets containing longest open reading frame (ORF) protein sequences. We also note a poor correlation between the levels of protein and mRNA abundance, a comparison not previously performed for dinoflagellates. The differences observed between analyses of MS‐validated protein sequence and nucleic acid sequence datasets suggest use of the former may provide a more accurate representation of cellular capacity than the latter. Developing MS‐validated protein sequence datasets may also speed interpretation of MS‐MS spectra in bottom up proteomics experiments.
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•Nightly bioluminescence requires increased luciferase and substrate binding protein.•Nightly DNA replication involves increased proliferating cell nuclear antigen (PCNA)•Daily ...increases in enzymes generating UV protection can act as sunscreen.•Daily increases in enzymes modifying cellular pH may compensate for CO2 fixation.
Protein levels were assessed in the dinoflagellate Lingulodinium polyedra over the course of a diurnal cycle using a label-free LC-MS/MS approach. Roughly 1700 proteins were quantitated in a triplicate dataset over a daily period, and 13 were found to show significant rhythmic changes. Included among the proteins found to be most abundant at night were the two bioluminescence proteins, luciferase and luciferin binding protein, as well as a proliferating cell nuclear protein involved in the nightly DNA replication. Aconitase and a pyrophosphate fructose-6-phosphate-1-phosphotransferase were also found to be more abundant at night, suggestive of an increased ability to generate ATP by glucose catabolism when photosynthesis does not occur. Among the proteins more abundant during the day were found a 2-epi-5-epi-valiolone synthase, potentially involved in synthesis of mycosporin-like amino acids that can act as a “microbial sunscreen”, and an enzyme synthesizing vitamin B6 which is known to protect against oxidative stress. A lactate oxidoreductase was also found to be more abundant during the day, perhaps to counteract the pH changes due to carbon fixation by facilitating conversion of pyruvate to lactate. This unbiased proteomic approach reveals novel insights into the daily metabolic changes of this dinoflagellate. Furthermore, the observation that only a limited number of proteins vary support a model where metabolic flux through pathways can be controlled by variations in a select few, possibly rate limiting, steps. Data are available via ProteomeXchange with identifier PXD006994.
Brief exposure of skin to near-infrared (NIR) laser light has been shown to augment the immune response to intradermal vaccination and thus act as an immunologic adjuvant. Although evidence indicates ...that the NIR laser adjuvant has the capacity to activate innate subsets including dendritic cells (DCs) in skin as conventional adjuvants do, the precise immunological mechanism by which the NIR laser adjuvant acts is largely unknown. In this study we sought to identify the cellular target of the NIR laser adjuvant by using an established mouse model of intradermal influenza vaccination and examining the alteration of responses resulting from genetic ablation of specific DC populations. We found that a continuous wave (CW) NIR laser adjuvant broadly modulates migratory DC (migDC) populations, specifically increasing and activating the Lang
and CD11b
Lang
subsets in skin, and that the Ab responses augmented by the CW NIR laser are dependent on DC subsets expressing CCR2 and Langerin. In comparison, a pulsed wave NIR laser adjuvant showed limited effects on the migDC subsets. Our vaccination study demonstrated that the efficacy of the CW NIR laser is significantly better than that of the pulsed wave laser, indicating that the CW NIR laser offers a desirable immunostimulatory microenvironment for migDCs. These results demonstrate the unique ability of the NIR laser adjuvant to selectively target specific migDC populations in skin depending on its parameters, and highlight the importance of optimization of laser parameters for desirable immune protection induced by an NIR laser-adjuvanted vaccine.
Mutations of bone morphogenetic protein receptor type II (BMPR-II) have been associated with familial and idiopathic pulmonary arterial hypertension (PAH). BMPR-II is a member of the transforming ...growth factor-beta receptor superfamily. It consists of extracellular, transmembrane, and kinase domains, and a unique C-terminus with mostly unknown function. However, a number of PAH-causing mutations are predicted to truncate the C-terminus, suggesting that this domain plays an important role in the homeostasis of pulmonary vessels. In this study, we sought to elucidate the functional role of this C-terminus by seeking its interacting partners. Using yeast two-hybrid screening, we identified c-Src tyrosine kinase as a binding partner of this C-terminus. In vitro co-immunoprecipitation confirmed their interaction. Mutations truncating the C-terminus disrupted their interaction, while missense mutation within kinase domain reduced their interaction. In addition, BMPR-II and c-Src tyrosine kinase colocalized within intracellular aggregates when overexpressed in HEK293 cells. Moreover, mutations truncating the C-terminus disrupted their colocalization, whereas missense mutation within kinase domain had no effect on their colocalization. Furthermore, BMP ligand stimulation decreased c-Src-activating phosphorylation at Tyrosine 418 in pulmonary smooth muscle cells in both time- and concentration-dependent manners. Mutations that truncated the C-terminus abolished this response. Taken together, these results suggest a model in which proliferative effect of c-Src by vasoactive molecules is balanced by opposing effect of BMP signaling in basal state, and the loss of this balance due to BMPR2 mutations leads to increased c-Src activity and subsequently cell growth.
Interferon consensus sequence binding protein (ICSBP) is a transcription factor of the interferon (IFN) regulatory factor (IRF) family. Mice with a null mutation of ICSBP exhibit two prominent ...phenotypes related to previously described activities of the IRF family. The first is enhanced susceptibility to virus infections associated with impaired production of IFNγ. The second is deregulated hematopoiesis in both ICSBP−/− and ICSBP+/− mice that manifests as a syndrome similar to human chronic myelogenous leukemia. The chronic period of the disease progresses to a fatal blast crisis characterized by a clonal expansion of undifferentiated cells. Normal mice injected with cells from mice in blast crisis developed acute leukemia within 6 weeks of transfer. These results suggest a novel role for ICSBP in regulating the proliferation and differentiation of hematopoietic progenitor cells.
Dinoflagellates are notorious for their ability to form the harmful algal blooms known as “red tides,” yet the mechanisms underlying bloom formation remain poorly understood. Despite recent advances ...in nucleic acid sequencing, which have generated transcriptomes from a wide range of species exposed to a variety of different conditions, measuring changes in RNA levels have not generally produced great insight into dinoflagellate cell biology or environmental physiology, nor do we have a thorough grasp on the molecular events underpinning bloom formation. Not only is the transcriptomic response of dinoflagellates to environmental change generally muted, but there is a markedly low degree of congruency between mRNA expression and protein expression in dinoflagellates. Herein we discuss the application of high-throughput proteomics to the study of dinoflagellate biology. By profiling the cellular protein complement (the proteome) instead of mRNA (the transcriptome), the biomolecular events that underlie the changes of phenotypes can be more readily evaluated, as proteins directly determine the structure and the function of the cell. Recent advances in proteomics have seen this technique become a high-throughput method that is now able to provide a perspective different from the more commonly employed nucleic acid sequencing. We suggest that the time is ripe to exploit these new technologies in addressing the many mysteries of dinoflagellate biology, such as how the symbiotic dinoflagellate inhabiting reef corals acclimate to increases in temperature, as well as how harmful algal blooms are initiated at the sub-cellular level. Furthermore, as dinoflagellates are not the only eukaryotes that demonstrate muted transcriptional responses, the techniques addressed within this review are amenable to a wide array of organisms.
Abstract A brief exposure of skin to a low-power, non-tissue damaging laser light has been demonstrated to augment immune responses to intradermal vaccination. Both preclinical and clinical studies ...show that this approach is simple, effective, safe and well tolerated compared to standard chemical or biological adjuvants. Until now, these laser exposures have been performed using a diode-pumped solid-state laser (DPSSL) devices, which are expensive and require labor-intensive maintenance and special training. Development of an inexpensive, easy-to-use and small device would form an important step in translating this technology toward clinical application. Here we report that we have established a handheld, near-infrared (NIR) laser device using semiconductor diodes emitting either 1061, 1258, or 1301 nm light that costs less than $4000, and that this device replicates the adjuvant effect of a DPSSL system in a mouse model of influenza vaccination. Our results also indicate that a broader range of NIR laser wavelengths possess the ability to enhance vaccine immune responses, allowing engineering options for the device design. This small, low-cost device establishes the feasibility of using a laser adjuvant approach for mass-vaccination programs in a clinical setting, opens the door for broader testing of this technology with a variety of vaccines and forms the foundation for development of devices ready for use in the clinic.
A specialized extracellular matrix of proteins and polysaccharides controls the morphology and packing of calcium carbonate crystals and becomes occluded within the mineralized composite during ...formation of the molluscan shell and pearl. We have cloned and characterized the cDNA coding for Lustrin A, a newly described matrix protein from the nacreous layer of the shell and pearl produced by the abalone, Haliotis rufescens, a marine gastropod mollusc. The full-length cDNA is 4,439 base pairs (bp) long and contains an open reading frame coding for 1,428 amino acids. The deduced amino acid sequence reveals a highly modular structure with a high proportion of Ser (16%), Pro (14%), Gly (13%), and Cys (9%). The protein contains ten highly conserved cysteine-rich domains interspersed by eight proline-rich domains; a glycine- and serine-rich domain lies between the two cysteine-rich domains nearest the C terminus, and these are followed by a basic domain and a C-terminal domain that is highly similar to known protease inhibitors. The glycine- and serine-rich domain and at least one of the proline-rich domains show sequence similarity to proteins of two extracellular matrix superfamilies (one of which also is involved in the mineralized matrixes of bone, dentin, and avian eggshell). The arrangement of alternating cysteine-rich domains and proline-rich domains is strikingly similar to that found in frustulins, the proteins that are integral to the silicified cell wall of diatoms. Its modular structure suggests that Lustrin A is a multifunctional protein, whereas the occurrence of related sequences suggest it is a member of a multiprotein family.
Recent work has shown that conventional surfactants form ordered aggregates of well-defined shape and size at solid−liquid interfaces. , Here we report interfacial aggregate structures as a function ...of surfactant geometry by using gemini surfactants with varying tail and spacer lengths. On the anionic cleavage plane of mica, aggregates tend to favor a lower curvature than in solution but follow the same general variation with surfactant geometry (i.e., with larger headgroup areas resulting in greater curvature). These morphologies on mica correlate well with those observed in surfactant−silicate mesophases, where electrostatic binding of headgroups also plays a dominant role. In addition, interfacial sphere-to-rod transitions are induced on mica (as in free solution) by binding with a headgroup-specific counterion. In contrast to mica, the hydrophobic cleavage plane of graphite interacts with surfactant tailgroups, giving rise to interfacial aggregates that are surface-controlled and relatively independent of surfactant geometry. This interaction is used to heterogeneously nucleate a surfactant−silicate mesophase which is interfacially controlled and differs from the bulk phase.
Bone mineral density or bone mass alone cannot reliably predict fracture risk in patients. It is generally accepted that bone quality, including the properties of the organic matrix of bone, should ...also be considered. Collagen type I accounts for about 90% of this organic matrix. The other 10% are accounted for by various proteins and proteoglycans usually summarized by the term noncollagenous proteins (NCPs). These NCPs have a large influence on the nanoscale organization of bone. In addition, some NCPs have intriguing properties that could strongly influence bone matrix material properties; they can form self-healing networks based on ion-mediated bonds. Such behavior was also reported for trabecular bone fracture surfaces, rejoined after cleavage. To obtain proof that this behavior of bone is due to NCPs, an immunohistochemical approach was chosen for the work presented in this communication. Antibodies for phosphoserine, which is abundant in many NCPs but not in collagen type I, as well as antibodies for osteopontin and bone sialoprotein, were used on human trabecular bone fracture surfaces and microfractured trabeculae. Signals were detected using secondary gold-labeled antibodies and backscattered scanning electron microscopy. We found homogenous NCP coverage of fracture surfaces and elevated signals on bridging ligaments. Osteopontin and bone sialoprotein were detected in localized patches. Overall, this work suggests that the self-healing effect of trabecluar bone fracture surfaces, rejoined after cleavage, can be explained by the presence of NCPs. In addition, we conclude that NCPs also constitute the interface that is disrupted when bone fails, attributing them high importance for bone matrix material properties and fracture risk.
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