Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. Download video file.^{(65M, mp4)}     

The role of waves in the colonisation of terrestrial sediments deposited in the marine environment

Elevated rates of sediment run-off, as a result of changes in land-use and climate, are a significant threat to marine coastal communities, with a potential to cause broad-scale, long-term alteration of habitats. Individual sedimentation events can smother estuarine flats with terrigenous sediments, creating a significant disturbance to local benthic communities. Variations in the degree to which a habitat is altered, the rate at which mixing occurs, and species-specific dispersal and responses to the altered habitat, suggest that colonisation of terrestrial sediment depositions will vary with location, both between and within estuaries. This study was designed to explore the effect that variations in wave-induced hydrodynamics would have on long-term colonisation of terrestrial sediment depositions on intertidal flats. Sites for the experimental deposition of terrestrial sediment were located along a gradient in wave exposure, with only limited variation in immersion times (30 min) and ambient sediment particle sizes (predominantly fine sand). Over 20 months, periodic measurements were made of factors predicted to affect colonisation: the sediment characteristics of the deposited sediment; local-scale wave climate; bioturbation of the deposited sediment; and local populations of benthic invertebrates. Neither opportunistic use of the new resource, progressive recovery or facilitation by colonising macrofauna was observed. Little vertical mixing of the deposited and existing sediment by either waves or bioturbators occurred; instead bedload transport was the dominant process. Local differences in hydrodynamic conditions and macrobenthic communities resulted in site-specific colonisation of the experimental plots. The strength and duration of the macrofaunal response to deposited sediment observed in this study suggest that chronic small-scale (m's) patchy deposition of terrestrial sediment in the intertidal marine environment has a strong potential to alter both habitats and communities.     

Low molecular mass protein patterns in mycobacterial culture filtrates and purified protein derivatives

Mycobacterial polypeptides from 2 kDa to 14 kDa may be involved in host response to infection and be useful for new diagnostics and vaccines. Tris-tricine SDS-polyacrylamide gel electrophoresis separation of proteins in tuberculin purified protein derivative (PPD) and in 6-8-week culture filtrates of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and other Mycobacterium species demonstrated as many as 10 low molecular mass bands. Common and distinct bands were observed among different species and PPD. These low molecular mass culture filtrate proteins may represent potential diagnostic reagents and vaccines for Mycobacterium tuberculosis.     

Citrullination Was Introduced into Animals by Horizontal Gene Transfer from Cyanobacteria

Protein posttranslational modifications add great sophistication to biological systems. Citrullination, a key regulatory mechanism in human physiology and pathophysiology, is enigmatic from an evolutionary perspective. Although the citrullinating enzymes peptidylarginine deiminases (PADIs) are ubiquitous across vertebrates, they are absent from yeast, worms, and flies. Based on this distribution PADIs were proposed to have been horizontally transferred, but this has been contested. Here, we map the evolutionary trajectory of PADIs into the animal lineage. We present strong phylogenetic support for a clade encompassing animal and cyanobacterial PADIs that excludes fungal and other bacterial homologs. The animal and cyanobacterial PADI proteins share functionally relevant primary and tertiary synapomorphic sequences that are distinct from a second PADI type present in fungi and actinobacteria. Molecular clock calculations and sequence divergence analyses using the fossil record estimate the last common ancestor of the cyanobacterial and animal PADIs to be less than 1 billion years old. Additionally, under an assumption of vertical descent, PADI sequence change during this evolutionary time frame is anachronistically low, even when compared with products of likely endosymbiont gene transfer, mitochondrial proteins, and some of the most highly conserved sequences in life. The consilience of evidence indicates that PADIs were introduced from cyanobacteria into animals by horizontal gene transfer (HGT). The ancestral cyanobacterial PADI is enzymatically active and can citrullinate eukaryotic proteins, suggesting that the PADI HGT event introduced a new catalytic capability into the regulatory repertoire of animals. This study reveals the unusual evolution of a pleiotropic protein modification.     

Sequence analysis of mitochondrial DNA from Podospora anserina. Pervasiveness of a class I intron in three separate genes

A 48 kb region of the 95 kb mitochondrial genome of Podospora anserina has been mapped and sequenced (1 kb = 10(3) base-pairs). The DNA sequence of the genes for ND2, 3, 4, ATPase 6 and URFC are presented here. As in Neurospora crassa, the ND2 and 3 genes consist of a unit separated by one TAA stop codon. ND3, 4 and ATPase 6 are interrupted by class I introns. All three introns are remarkably similar in the C-domain of their secondary structure, sufficient enough to designate them as new subgroup, class IC introns. The open reading frames of the ND3 and 4 introns bear a high sequence similarity to the open reading frame of the class IB introns of ATPase 6 from N. crassa and ND1 from Neurospora intermedia Varkud. We also show that the tRNA Met-2 gene is duplicated and is involved in a recombinational event. The 5' region of URFC is also duplicated but no involvement of this gene with recombination or formation of plasmids is known. The evolutionary significance of the similarities of intron secondary structures and open reading frames of the ND3, 4 and ATPase 6 genes is discussed, including the possible separate evolution of structural and coding sequences.     

Comparison of fermentation reactions in different regions of the human colon

Colonic contents were obtained from two human sudden-death victims within 3 h of death. One of the subjects (1) was methanogenic, the other (2) was a non-CH, producer. Measurements of bacterial fermentation products showed that in both individuals short-chain fatty acids, lactate and ethanol concentrations were highest in the caecum and ascending colon. In contrast, products of protein fermentation, such as ammonia, branched chain fatty acids and phenolic compounds, progressively increased from the right to the left colon, as did the pH of gut contents. In Subject 1, cell population densities of methanogenic bacteria (MB) increased distally through the gut and methanogenic activity was lower in the right (0.78–1–18 μmol CH₄ produced/h/g dry wt contents) than in the left colon (1.34 μmol CH₄ produced/h/g dry wt contents). Methane production rates did not correlate with MB numbers.
Sulphate-reducing bacteria (SRB) were not found and dissimilatory sulphate reduction was not detected in any region of the colon. Methanogenic bacteria did not occur in subject 2, but high numbers of SRB were present throughout the gut ( ca 10⁹/g dry wt contents). Sulphate reduction rates were maximal in the ascending and transverse colons (0.24 and 0.22 μmol ³⁵SO^2–₄ reduced/h/g dry wt contents, respectively). Short-chain fatty acid production by caecal contents was up to eight-fold higher than contents from the sigmoid/rectum. These findings demonstrate significant differences in fermentation reactions in different regions of the large gut.     

Long noncoding RNA complementarity and target transcripts abundance

Eukaryotic mRNA metabolism regulates its stability, localization, and translation using complementarity with counter-part RNAs. To modulate their stability, small and long noncoding RNAs can establish complementarity with their target mRNAs. Although complementarity of small interfering RNAs and microRNAs with target mRNAs has been studied thoroughly, partial complementarity of long noncoding RNAs (lncRNAs) with their target mRNAs has not been investigated clearly. To address that research gap, our lab investigated whether the sequence complementarity of two lncRNAs, lincRNA-p21 and OIP5-AS1, influenced the quantity of target RNA expression. We predicted a positive correlation between lncRNA complementarity and target mRNA quantity. We confirmed this prediction using RNA affinity pull down, microarray, and RNA-sequencing analysis. In addition, we utilized the information from this analysis to compare the quantity of target mRNAs when two lncRNAs, lincRNA-p21 and OIP5-AS1, are depleted by siRNAs. We observed that human and mouse lincRNA-p21 regulated target mRNA abundance in complementarity-dependent and independent manners. In contrast, affinity pull down of OIP5-AS1 revealed that changes in OIP5-AS1 expression influenced the amount of some OIP5-AS1 target mRNAs and miRNAs, as we predicted from our sequence complementarity assay. Altogether, the current study demonstrates that partial complementarity of lncRNAs and mRNAs (even miRNAs) assist in determining target RNA expression and quantity.     

Abnormal erythrocyte choline and influx in Alzheimer's disease

Choline transport and levels were studied in erythrocytes from patients with Alzheimer's disease and age-matched controls using stable isotopic tracer techniques. The mean erythrocyte choline in the Alzheimer group was 50.1 nmol ml-1 compared to 15.5 nmol ml-1 in the controls. This was significant using a Student's t test at a P of less than 0.0005. Influx of choline into the erythrocyte correlated inversely with erythrocyte choline with high significance. This study suggests that erythrocyte choline is elevated in a subset of patients with Alzheimer's disease and that the low affinity transport system is also abnormal in these patients. This abnormality of choline transport may play a role in the pathogenesis of Alzheimer's disease in some patients.