Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Relationship between postabsorptive respiratory exchange ratio and plasma free fatty acid concentrations

The relationship between overnight postabsorptive (fasting) respiratory exchange ratio (RER) and plasma FFA concentrations was addressed using data from three separate protocols, each of which involved careful control of the antecedent diet. Protocol 1 examined the relationship between fasting RER and the previous daytime RER. In Protocol 2 fasting, RER and plasma palmitate concentrations were measured in 29 women and 31 men (body mass index <30 kg·m⁻²). Protocol 3 analyzed data from Nielsen et al. (Nielsen, S., Z. K. Guo, J. B. Albu, S. Klein, P. C. O''Brien, M. D. Jensen. 2003. Energy expenditure, sex and endogenous fuel availability in humans. J. Clin. Invest. 111: 981-988.) to understand how fasting RER and palmitate concentrations relate within individuals during four consecutive measurements. The results were as follows: 1) Fasting RER was correlated (r = 0.74, P < 0.001) with the previous day''s average RER, and less so with RER variability. 2) Fasting RER was correlated (r = −0.39, P = 0.007) with fasting plasma palmitate concentrations. 3) The pattern of the RER/palmitate relationship was similar within individuals and between individuals; a negative slope was observed significantly more often than a positive slope (χ² test; P < 0.001). Our findings suggest that, despite a fixed food quotient, the slight departures from energy equilibrium in a controlled General Clinical Research Center environment can effect plasma FFA concentrations. We suggest that including indirect calorimetry as part of FFA metabolism studies may aid in data interpretation.     

Primate stem cells: bridge the translation from basic research to clinic application

A growing body of literature has shown that stem cells are very effective for the treatment of degenerative diseases in rodents but these exciting results have not translated to clinical practice. The difference results from the divergence in genetic, metabolic, and physiological phenotypes between rodents and humans. The high degree of similarity between non-human primates(NHPs) and humans provides the most accurate models for preclinical studies of stem cell therapy. Using a NHP model to understand the following key issues, which cannot be addressed in humans or rodents, will be helpful for extending stem cell applications in the basic science and the clinic. These issues include pluripotency of primate stem cells, the safety and efficiency of stem cell therapy, and transplantation procedures of stem cells suitable for clinical translation. Here we review studies of the above issues in NHPs and current challenges of stem cell applications in both basic science and clinical therapies. We propose that the use of NHP models, in particular combining the serial production and transplantation procedures of stem cells is the most useful for preclinical studies designed to overcome these challenges.     

Composition and peptide maps of cross-linked human choriogonadotropin-receptor complexes on porcine granulosa cells

Radioiodinated human choriogonadotropin was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to the hormone receptor on porcine granulosa cells and the solubilized sample was electrophoresed. Cross-linked samples revealed four additional bands of slower electrophoretic mobility in addition to the hormone alpha, beta, and alpha beta dimer bands. The four bands corresponded to masses of 68, 74, 102, and 136 kDa whereas the alpha beta dimer band corresponded to 50 kDa. Formation of the four bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding can be prevented by excess of native hormone but not by follitropin. A monofunctional analog of the cross-linking reagent failed to produce the four bands. They were also produced by cross-linking Triton X-100-solubilized hormone-receptor complexes. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of the cross-linked sample were treated with reagents that cleave covalent cross-links and then electrophoresed in a second dimension gel, 18-, 24-, 28-, and 34-kDa components were released, in addition to the alpha and beta subunits of the native hormone. Simultaneous peptide mapping of the cross-linked complexes in the gel matrix with Staphylococcus V8 protease or papain revealed progressive proteolysis to generate terminal fragments of 30 or 27 kDa, respectively. These fragments were unique to and commonly present in the 74-, 102-, and 136-kDa hormone-receptor complexes but were not produced by proteolysis of the cross-linked human choriogonadotropin (hCG) alpha beta dimer or the hCG alpha subunit. Apparently, the radioactively labeled segment(s) of the alpha subunit of 125I-hCG was cross-linked to the 24-kDa component. The results demonstrate the protein nature of the receptor and suggest that 125I-hCG was initially cross-linked to the 24-kDa component to generate the 74-kDa complex, then the 28- and 34-kDa components were sequentially cross-linked to the 24-kDa component in the 74-kDa complex to generate the 102- and 134-kDa complexes.     

Contrasting patterns in elevational diversity between microorganisms and macroorganisms

Aim Data and analyses of elevational gradients in diversity have been central to the development and evaluation of a range of general theories of biodiversity. Elevational diversity patterns have, however, been severely understudied for microbes, which often represent decomposer subsystems. Consequently, generalities in the patterns of elevational diversity across different trophic levels remain poorly understood. Our aim was to examine elevational gradients in the diversity of macroinvertebrates, diatoms and bacteria along a stony stream that covered a large elevational gradient. Location Laojun Mountain, Yunnan province, China. Methods The sampling scheme included 26 sites spaced at elevational intervals of 89 m from 1820 to 4050 m elevation along a stony stream. Macroinvertebrate and diatom richness were determined based on the morphology of the specimens. Taxonomic richness for bacteria was quantified using a molecular fingerprinting method. Over 50 environmental variables were measured at each site to quantify environmental variables that could correlate with the patterns of diversity. We used eigenvector‐based spatial filters with multiple regressions to account for spatial autocorrelation. Results The bacterial richness followed an unexpected monotonic increase with elevation. Diatoms decreased monotonically, and macroinvertebrate richness showed a clear unimodal pattern with elevation. The unimodal richness pattern for macroinvertebrates was best explained by the mid‐domain effect (r² = 0.72). The diatom richness was best explained by the variation in nutrient supply, and the increase in bacterial richness with elevation may be related to an increased carbon supply. Main conclusions We found contrasting patterns in elevational diversity among the three studied multi‐trophic groups comprising unicellular and multicellular aquatic taxa. We also found that there may be fundamental differences in the mechanisms underlying these species diversity patterns.     

Culture of newborn monkey liver epithelial progenitor cells in chemical defined serum-free medium

Studies with hepatic progenitor cells from non-human primates would allow better understanding of their human counterparts. In this study, rhesus monkey liver epithelial progenitor cells (mLEPCs) were derived from a small piece of newborn livers in chemical defined serum-free medium. Digested hepatic cells were treated in Ca²⁺-containing medium to form cell aggregates. Two types of cell aggregates were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 d in our cultures. The remaining type consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f, and E-cad, the markers of hepatic stem cells, but were negative for α-fetoprotein, albumin, and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as α-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake, and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19-positive cholangiocytes with formation of doughnut-like structure. Thus, mLEPCs with bipotency were derived from newborn monkey liver and may serve as a preclinical model for assessment of cell therapy in humans.     

Morphological and Molecular Identification of Spirometra Tapeworms (Cestoda: Diphyllobothriidae) from Carnivorous Mammals in the Serengeti and Selous Ecosystems of Tanzania

Spirometra tapeworms (Cestoda: Diphyllobothriidae) collected from carnivorous mammals in Tanzania were identified by the DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and internal transcribed spacer 1 (ITS1), and by morphological characteristics. A total of 15 adult worms were collected from stool samples and carcasses of Panthera leo, Panthera pardus, and Crocuta crocuta in the Serengeti and Selous ecosystems of Tanzania. Three Spirometra species: S. theileri, S. ranarum and S. erinaceieuropaei were identified based on morphological features. Partial cox1 sequences (400 bp) of 10 specimens were revealed. Eight specimens showed 99.5% similarity with Spirometra theileri ({"type":"entrez-nucleotide","attrs":{"text":"MK955901","term_id":"1796114604","term_text":"MK955901"}}MK955901), 1 specimen showed 99.5% similarity with the Korean S. erinaceieuropaei and 1 specimen had 99.5% similarity with Myanmar S. ranarum. Sequence homology estimates for the ITS1 region of S. theileri were 89.8% with S. erinaceieuropaei, 82.5% with S. decipiens, and 78.3% with S. ranarum; and 94.4% homology was observed between S. decipiens and S. ranarum. Phylogenetic analyses were performed with 4 species of Spirometra and 2 species of Dibothriocephalus (=Diphyllobothrium). By both ML and BI methods, cox1 and ITS1 gave well supported, congruent trees topology of S. erinaceieuropaei and S. theileri with S. decipiens and S. ranarum forming a clade. The Dibothriocephalus species were sisters of each other and collectively forming successive outgroups. Our findings confirmed that 3 Spirometra species (S. theileri, S. ranarum, and S. erinaceieuropaei) are distributed in the Serengeti and Selous ecosystems of Tanzania.