Comprehensive biodiversity analysis via ultra-deep patterned flow cell technology: a case study of eDNA metabarcoding seawater.

The characterization of biodiversity is a crucial element of ecological investigations as well as environmental assessment and monitoring activities. Increasingly, amplicon-based environmental DNA metabarcoding (alternatively, marker gene metagenomics) is used for such studies given its ability to provide biodiversity data from various groups of organisms simply from analysis of bulk environmental samples such as water, soil or sediments. The Illumina MiSeq is currently the most popular tool for carrying out this work, but we set out to determine whether typical studies were reading enough DNA to detect rare organisms (i.e., those that may be of greatest interest such as endangered or invasive species) present in the environment. We collected sea water samples along two transects in Conception Bay, Newfoundland and analyzed them on the MiSeq with a sequencing depth of 100,000 reads per sample (exceeding the 60,000 per sample that is typical of similar studies). We then analyzed these same samples on Illumina's newest high-capacity platform, the NovaSeq, at a depth of 7 million reads per sample. Not surprisingly, the NovaSeq detected many more taxa than the MiSeq thanks to its much greater sequencing depth. However, contrary to our expectations this pattern was true even in depth-for-depth comparisons. In other words, the NovaSeq can detect more DNA sequence diversity within samples than the MiSeq, even at the exact same sequencing depth. Even when samples were reanalyzed on the MiSeq with a sequencing depth of 1 million reads each, the MiSeq's ability to detect new sequences plateaued while the NovaSeq continued to detect new sequence variants. These results have important biological implications. The NovaSeq found 40% more metazoan families in this environment than the MiSeq, including some of interest such as marine mammals and bony fish so the real-world implications of these findings are significant. These results are most likely associated to the advances incorporated in the NovaSeq, especially a patterned flow cell, which prevents similar sequences that are neighbours on the flow cell (common in metabarcoding studies) from being erroneously merged into single spots by the sequencing instrument. This study sets the stage for incorporating eDNA metabarcoding in comprehensive analysis of oceanic samples in a wide range of ecological and environmental investigations.

Limited hepatitis B immunoglobulin with potent nucleos(t)ide analogue is a cost-effective prophylaxis against hepatitis B virus after liver transplantation.

BACKGROUND: Prophylaxis against hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT) includes lifelong hepatitis B immunoglobulin (HBIG) and oral antiviral agent(s). In the presence of high-genetic-barrier nucleos(t)ide analogues, the need for lifelong HBIG is questioned. We evaluated the safety and cost-effectiveness of a limited HBIG course. METHODS: OLT from 2006 to 2013 were reviewed. Patients with pre-OLT hepatitis B virus surface antigen who received HBV prophylaxis with 2 HBIG doses (anhepatic and first post-operative day; 10,000 units/dose) and potent nucleos(t)ide analogues were included. The primary end point was HBV recurrence (HBV-DNA detection). RESULTS: Thirteen patients (primary transplants) were included, median Model for End-Stage Liver Disease score was 18, and there was no fulminant failure; HBV-DNA was detected in 4 patients at OLT. After OLT, 10 patients received entecavir and/or tenofovir. Median follow-up was 23 months. One recurrence occurred (7.7%) at month 13 (HBV-DNA: 14 IU/mL); the graft maintained excellent function. This minimal viremic expression is related to hepatocellular carcinoma recurrence with neoplastic replication carrying integrated HBV-DNA; thus, there is no defined HBV viral recurrence. No graft loss or patient death was related to HBV recurrence. The 1-year patient and graft survival rate was 84.6%. Cost-savings in the first year was $178,100 per patient when compared with Food and Drug Administration-approved HBIG dosing. CONCLUSIONS: In the era of potent oral nucleos(t)ide analogues, a limited HBIG course appears to be cost-effective in preventing HBV recurrence.

Nucleotide bias causes a genomewide bias in the amino acid composition of proteins.

We analyzed the nucleotide contents of several completely sequenced genomes, and we show that nucleotide bias can have a dramatic effect on the amino acid composition of the encoded proteins. By surveying the genes in 21 completely sequenced eubacterial and archaeal genomes, along with the entire Saccharomyces cerevisiae genome and two Plasmodium falciparum chromosomes, we show that biased DNA encodes biased proteins on a genomewide scale. The predicted bias affects virtually all genes within the genome, and it could be clearly seen even when we limited the analysis to sets of homologous gene sequences. Parallel patterns of compositional bias were found within the archaea and the eubacteria. We also found a positive correlation between the degree of amino acid bias and the magnitude of protein sequence divergence. We conclude that mutational bias can have a major effect on the molecular evolution of proteins. These results could have important implications for the interpretation of protein-based molecular phylogenies and for the inference of functional protein adaptation from comparative sequence data.

Transient lectin binding by white matter tract border zone microglia in the foetal rabbit brain.

Axonal growth cones of developing white matter tracts are guided through the cerebrum by interactions with cell surface and extracellular matrix molecules expressed by glial cells that mediate cell adhesion and contact-dependent inhibition. Specific carbohydrates are considered essential for the proper functioning of these molecular complexes. We studied developmental aspects of complex carbohydrate expression by white matter glia in the foetal rabbit brain using the tomato lectin Lycopersicon esculentum, which has affinity for components of the extracellular matrix proteins and cell surface proteins (N-acetylglucosamine) and activated lysosomal membrane glycoproteins (N-acetyllactosamine). Concentrations of the lectin-positive glia were transiently found immediately adjacent to developing white matter tracts of the foetal rabbit brain from 22 to 32 days' gestation. The number of positive cells markedly diminished by the fourth post-natal day and in the adult brain. The lectin-positive glia did not react with antibody to glial fibrillary acidic protein. However, they did express the macrophage surface antigen, Mac-1, indicating that the lectin binding reflected the presence of microglial activated lysosomal membranes. These data suggest that, in addition to their role as central nervous system scavengers, microglia are involved in a specifically timed function in the neurodevelopmental programme of white matter tract formation.

Flow cytometric detection and binding studies of human endometrial stromal cell epidermal growth factor receptor in monolayer culture: influence of progesterone.

Epidermal growth factor (EGF) has been shown to modulate endometrial differentiation in vivo and in vitro. Therefore, endometrial stromal cell EGF receptors were characterized in intact endometrial stromal cells, cultured in vitro. The methods used for characterization were flow cytometry, binding and displacement studies, and gel electrophoresis followed by autoradiography. In flow cytometry the histogram of labelled stromal cells was identical to Caski cells, which served as a positive control. EGF binding revealed the typical binding hierarchy for EGF receptors. The Scatchard analysis showed a curvilinear plot with a calculated dissociation constant of 0.36 nM for high affinity binding sites. In autoradiography a band of approximately 170 kDa was visualized corresponding to the known size of the EGF receptor. The intensity of this band was increased by pretreatment of stromal cells with 10 nM progesterone for 4 days. Furthermore, stimulation with progesterone led to an increase in specific EGF binding activity of stromal cells by 21% compared to control. These data indicate that intact stromal cells in monolayer culture maintain specific EGF receptors, which are up-regulated by progesterone.

Characterization of receptors for insulin-like growth factor type I on cultured human endometrial stromal cells: downregulation by progesterone.

Because insulin-like growth factor type I (IGF-I) is reputed to be involved in the endometrial decidualization, we analyzed the expression of IGF-I receptors in an in vitro system of human endometrial stromal cells. Competitive binding studies of both intact stromal cells and membrane preparation indicated the presence of specific components with high affinity for binding IGF-I. Half-maximum displacement was obtained with 2.3 nmol/l native IGF-I, whereas insulin was unable to achieve half-maximum displacement even at higher concentrations. This IGF-I binding component was found to be a saturable protein in respect of the radioligand [125I]IGF-I, with a dissociation constant of 0.16 nmol/l. Affinity cross-linking studies revealed a labelled band of approximate relative molecular mass 135000, corresponding to the known alpha-subunit of IGF-I receptor. This band was significantly inhibited dose-dependently by the IGF-I receptor monoclonal antibody alpha-IR3 or native IGF-I, suggesting that the IGF-I binding component in the membrane of stromal cells has the identity of the alpha-subunit of IGF-I receptor. Cell proliferation in vitro was stimulated by progesterone. Furthermore, progesterone downregulated the [125I]IGF-I binding activity by downregulation of the IGF-I membrane receptor of human endometrial stromal cells. These data show that the IGF-I receptor is a functionally integral component of the stromal cell membrane structure, and its expression might be directly modulated by progesterone and, therefore, might play an important role in the preparation of the stroma for successful embryo implantation.

Characterization and identification of EGF-receptors as an integral component of membrane structure of human endometrial stromal cells in vitro.

The epidermal growth factor EGF is a well known mitogenic agent, elicits significant biological responses in intact animals, organs and cell cultures, and might be involved in the endometrial receptivity for successful embryo implantation. Although immunohistological and binding studies have detected EGF-binding activity in endometrial tissues, the nature of the EGF- binding site has not been fully investigated. Therefore, this study was performed to characterize and identify the nature of the endometrial stromal cell EGF-binding activity and to demonstrate EGF effects on stromal cell growth under in vitro conditions. Stimulation of cellular proliferation with EGF in various concentrations for 6 days revealed a significant increase of the proliferation rate up to 150% of that found in control subjects in a dose-dependent fashion, reaching a maximum stimulation at 10 nM EGF. Binding studies with labelled 125I-EGF confirmed the localization of the EGF-binding site in the membrane fraction of stromal cells. Furthermore, this binding activity was found to be a ligand-saturable molecular species with a dissociation constant of about 1.75 nM, and with a high specificity to EGF ligands. Affinity cross-linking studies revealed that the stromal cell membrane EGF-binding component is a protein molecule of 170,000 daltons, and has no subunit structure bound with disulphide bridges. The electrophoretic migration pattern of this molecule corresponds to EGF-receptor species already identified in other cells and tissues. This was further documented by specific reactivity with EGF-receptor antibodies and EGF/alpha-TGF peptides as well. In conclusion, the EGF-binding activity of stromal cell membranes has the nature and identity of EGF-receptor species, and is a functionally integral component of the stromal cell membrane structure.

The primary structure of the hemoglobin from the lobe-lipped bat (Chalinolobus morio, Microchiroptera).

The hemoglobin of the lobe-lipped bat (Chalinolobus morio, Vespertilionidae) is composed of 45% HbI and 55% HbII. Both components show identical alpha-chains but differ at the following three positions of their beta-chains: beta I/beta II 21: Glu/Asp, 70: Ser/Ala, and 135: Gln/Leu. High performance liquid chromatography revealed pure alpha-chains and a mixture of only partly separated beta-chains. Based on this material, the primary structures of all three globin chains could be achieved by automatic Edman degradation of the whole chains and peptides obtained by trypsin hydrolysis. Compared to human hemoglobin, Chalinolobus shows 17 replacements in the alpha-chains and 24/22 in the beta-chains. A sequence comparison of the globin chains from the three vespertilionid bats Chalinolobus morio and Myotis velifer (Vespertilioninae) as well as Antrozous pallidus (Nyctophilinae) supports a close relationship of the former only for the beta-chains. Molecular modeling showed that the replacements involved in three alpha 1/beta 1 and one alpha 1/beta 2 subunit interface contacts do not cause any interruption. All phosphate binding sites and amino acid residues responsible for the Bohr effect are unchanged. Thus normal physiological properties should be expected for Chalinolobus morio hemoglobin.

The primary structure of the hemoglobin from the tomb bat (Taphozous georgianus, Microchiroptera).

The primary structures of the alpha- and beta-chains of the single hemoglobin component from the tomb bat (Taphozous georgianus, Microchiroptera) are presented. After chain separation by reversed-phase HPLC the sequences could be determined by automatic gas and liquid phase Edman degradation of the chains and their tryptic peptides. The alpha- and beta-chains differ from human hemoglobin by 14 and 18 replacements, respectively. Compared to the total number of amino-acid exchanges, the exchange rate in the interhelical regions of the alpha-chains is surprisingly high (25%). It seems unlikely that substitutions at contact positions affect the oxygen binding properties of the hemoglobin.

The primary structure of the hemoglobin from the Australian ghost bat (Macroderma gigas, Microchiroptera).

The Australian ghost bat (Macroderma gigas, Microchiroptera) has two hemoglobin components in the ratio 3:2. They share identical beta-chains and differ by three replacements in the alpha-chains. The primary structures of all three chains are presented. They could be separated by high-performance liquid chromatography. The sequences were determined by automatic liquid and gas phase Edman degradation of the chains and their tryptic peptides. The two alpha-chains show 18 and 19 and the beta-chains 15 exchanges compared to human alpha- and beta-chains, respectively. The divergent evolution of Macroderma gigas and Megaderma lyra, two representatives of the family Megadermatidae, is discussed. An influence of replacements at functionally important positions on the hemoglobin oxygen affinity seems unlikely.

Transitional cell carcinoma of the renal pelvis with ovarian metastasis.

This article describes the first reported case of a primary transitional cell carcinoma of the renal pelvis metastatic to the ovary. The clinical presentation in our patient was similar to that of a primary ovarian carcinoma. The differential diagnosis of a primary or metastatic transitional cell carcinoma in the ovary is important and has therapeutic as well as prognostic implications.