ABSTRACT
The ability to preserve microbial communities in faecal samples is essential as increasing numbers of studies seek to use the gut microbiome to identify biomarkers of disease. Here we use shotgun metagenomics to rigorously evaluate the technical and compositional reproducibility of five room temperature (RT) microbial stabilisation methods compared to the best practice of flash-freezing. These methods included RNALater, OMNIGene-GUT, a dry BBL swab, LifeGuard, and a novel method for preserving faecal samples, a Copan FLOQSwab in an active drying tube (FLOQSwab-ADT). Each method was assessed using six replicate faecal samples from five participants, totalling 180 samples. The FLOQSwab-ADT performed best for both technical and compositional reproducibility, followed by RNAlater and OMNIgene-GUT. LifeGuard and the BBL swab had unpredictable outgrowth of Escherichia species in at least one replicate from each participant. We further evaluated the FLOQSwab-ADT in an additional 239 samples across 10 individuals after storage at -20 °C, RT, and 50 °C for four weeks compared to fresh controls. The FLOQSwab-ADT maintained its performance across all temperatures, indicating this method is an excellent alternative to existing RT stabilisation methods.
ABSTRACT
Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with approximately 50% protein identity to adenosylhomocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-l-homocysteine, the by-product of S-adenosyl-l-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs (zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.
Subject(s)
Adenosylhomocysteinase/chemistry , Gene Expression Regulation, Developmental , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , Phosphatidylinositols/metabolism , Phospholipids/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/physiology , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Calcium Channels/metabolism , Cloning, Molecular , Humans , Inositol 1,4,5-Trisphosphate Receptors , Molecular Sequence Data , Phylogeny , Receptors, Cytoplasmic and Nuclear/metabolism , Sequence Homology, Amino Acid , ZebrafishABSTRACT
Classic Hodgkin's lymphoma (HL) tissue contains a small population of morphologically distinct malignant cells called Hodgkin and Reed-Sternberg (HRS) cells, associated with the development of HL. Using 3'-rapid amplification of cDNA ends (RACE) we identified an alternative mRNA for the DEC-205 multilectin receptor in the HRS cell line L428. Sequence analysis revealed that the mRNA encodes a fusion protein between DEC-205 and a novel C-type lectin DCL-1. Although the 7.5-kb DEC-205 and 4.2-kb DCL-1 mRNA were expressed independently in myeloid and B lymphoid cell lines, the DEC-205/DCL-1 fusion mRNA (9.5 kb) predominated in the HRS cell lines (L428, KM-H2, and HDLM-2). The DEC-205 and DCL-1 genes comprising 35 and 6 exons, respectively, are juxtaposed on chromosome band 2q24 and separated by only 5.4 kb. We determined the DCL-1 transcription initiation site within the intervening sequence by 5'-RACE, confirming that DCL-1 is an independent gene. Two DEC-205/DCL-1 fusion mRNA variants may result from cotranscription of DEC-205 and DCL-1, followed by splicing DEC-205 exon 35 or 34-35 along with DCL-1 exon 1. The resulting reading frames encode the DEC-205 ectodomain plus the DCL-1 ectodomain, the transmembrane, and the cytoplasmic domain. Using DCL-1 cytoplasmic domain-specific polyclonal and DEC-205 monoclonal antibodies for immunoprecipitation/Western blot analysis, we showed that the fusion mRNA is translated into a DEC-205/DCL-1 fusion protein, expressed in the HRS cell lines. These results imply an unusual transcriptional control mechanism in HRS cells, which cotranscribe an mRNA containing DEC-205 and DCL-1 prior to generating the intergenically spliced mRNA to produce a DEC-205/DCL-1 fusion protein.
Subject(s)
Antigens, CD/chemistry , Hodgkin Disease/metabolism , Lectins, C-Type/chemistry , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Alternative Splicing , Amino Acid Sequence , Base Sequence , Blotting, Northern , Blotting, Western , Cell Line , Chromosomes/metabolism , Cytoplasm/metabolism , DNA, Complementary/metabolism , Enzyme-Linked Immunosorbent Assay , Exons , HL-60 Cells , Humans , Jurkat Cells , Lectins/metabolism , Minor Histocompatibility Antigens , Models, Genetic , Molecular Sequence Data , Precipitin Tests , Protein Structure, Tertiary , RNA Splicing , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
Dendritic cells (DC) are the professional antigen-presenting cells that initiate immune responses. While DC take up antigen, migrate to lymph nodes and present processed antigen to T lymphocytes, little is known of the intracellular biochemical pathways controlling these events. Using the differential display technique, employing the activated blood DC-like cell line L428, we isolated a cDNA induced during DC differentiation likely to have a regulatory function. This cDNA encoded a putative 530-amino-acid (aa) protein consisting of a unique hydrophilic domain (106 aa) and a domain (424 aa) similar to the methylation pathway enzyme S-adenosylhomocysteine hydrolase (AHCY). Therefore, this molecule was termed DC-expressed AHCY-like molecule (DCAL). DCAL mRNA was expressed moderately in fresh blood DC, but was not detectable in other peripheral blood mononuclear cells. DCAL mRNA increased markedly during activation of blood and skin DC (Langerhans cells), but was diminished in terminally differentiated tonsil DC. Cultured monocytes expressed little DCAL mRNA, but levels increased markedly when differentiated into DC by cytokines GM-CSF and IL-4. The DCAL gene [Chromosome (Chr) 1] and another previously identified DCAL-like molecule KIAA0828 (Chr 7) differed from the AHCY gene (Chr 20) in gene organization. Thus, DCAL may have a role in controlling critical events in DC differentiation and belong to a novel family of AHCY-like molecules.