Lytic to temperate switching of viral communities.

Microbial viruses can control host abundances via density-dependent lytic predator-prey dynamics. Less clear is how temperate viruses, which coexist and replicate with their host, influence microbial communities. Here we show that virus-like particles are relatively less abundant at high host densities. This suggests suppressed lysis where established models predict lytic dynamics are favoured. Meta-analysis of published viral and microbial densities showed that this trend was widespread in diverse ecosystems ranging from soil to freshwater to human lungs. Experimental manipulations showed viral densities more consistent with temperate than lytic life cycles at increasing microbial abundance. An analysis of 24 coral reef viromes showed a relative increase in the abundance of hallmark genes encoded by temperate viruses with increased microbial abundance. Based on these four lines of evidence, we propose the Piggyback-the-Winner model wherein temperate dynamics become increasingly important in ecosystems with high microbial densities; thus 'more microbes, fewer viruses'.

Optimizing and evaluating the reconstruction of Metagenome-assembled microbial genomes.

BACKGROUND: Microbiome/host interactions describe characteristics that affect the host's health. Shotgun metagenomics includes sequencing a random subset of the microbiome to analyze its taxonomic and metabolic potential. Reconstruction of DNA fragments into genomes from metagenomes (called metagenome-assembled genomes) assigns unknown fragments to taxa/function and facilitates discovery of novel organisms. Genome reconstruction incorporates sequence assembly and sorting of assembled sequences into bins, characteristic of a genome. However, the microbial community composition, including taxonomic and phylogenetic diversity may influence genome reconstruction. We determine the optimal reconstruction method for four microbiome projects that had variable sequencing platforms (IonTorrent and Illumina), diversity (high or low), and environment (coral reefs and kelp forests), using a set of parameters to select for optimal assembly and binning tools. METHODS: We tested the effects of the assembly and binning processes on population genome reconstruction using 105 marine metagenomes from 4 projects. Reconstructed genomes were obtained from each project using 3 assemblers (IDBA, MetaVelvet, and SPAdes) and 2 binning tools (GroopM and MetaBat). We assessed the efficiency of assemblers using statistics that including contig continuity and contig chimerism and the effectiveness of binning tools using genome completeness and taxonomic identification. RESULTS: We concluded that SPAdes, assembled more contigs (143,718 ± 124 contigs) of longer length (N50 = 1632 ± 108 bp), and incorporated the most sequences (sequences-assembled = 19.65%). The microbial richness and evenness were maintained across the assembly, suggesting low contig chimeras. SPAdes assembly was responsive to the biological and technological variations within the project, compared with other assemblers. Among binning tools, we conclude that MetaBat produced bins with less variation in GC content (average standard deviation: 1.49), low species richness (4.91 ± 0.66), and higher genome completeness (40.92 ± 1.75) across all projects. MetaBat extracted 115 bins from the 4 projects of which 66 bins were identified as reconstructed metagenome-assembled genomes with sequences belonging to a specific genus. We identified 13 novel genomes, some of which were 100% complete, but show low similarity to genomes within databases. CONCLUSIONS: In conclusion, we present a set of biologically relevant parameters for evaluation to select for optimal assembly and binning tools. For the tools we tested, SPAdes assembler and MetaBat binning tools reconstructed quality metagenome-assembled genomes for the four projects. We also conclude that metagenomes from microbial communities that have high coverage of phylogenetically distinct, and low taxonomic diversity results in highest quality metagenome-assembled genomes.

Oxygen minimum zones harbour novel viral communities with low diversity.

Oxygen minimum zones (OMZs) are oceanographic features that affect ocean productivity and biodiversity, and contribute to ocean nitrogen loss and greenhouse gas emissions. Here we describe the viral communities associated with the Eastern Tropical South Pacific (ETSP) OMZ off Iquique, Chile for the first time through abundance estimates and viral metagenomic analysis. The viral-to-microbial ratio (VMR) in the ETSP OMZ fluctuated in the oxycline and declined in the anoxic core to below one on several occasions. The number of viral genotypes (unique genomes as defined by sequence assembly) ranged from 2040 at the surface to 98 in the oxycline, which is the lowest viral diversity recorded to date in the ocean. Within the ETSP OMZ viromes, only 4.95% of genotypes were shared between surface and anoxic core viromes using reciprocal BLASTn sequence comparison. ETSP virome comparison with surface marine viromes (Sargasso Sea, Gulf of Mexico, Kingman Reef, Chesapeake Bay) revealed a dissimilarity of ETSP OMZ viruses to those from other oceanic regions. From the 1.4 million non-redundant DNA sequences sampled within the altered oxygen conditions of the ETSP OMZ, more than 97.8% were novel. Of the average 3.2% of sequences that showed similarity to the SEED non-redundant database, phage sequences dominated the surface viromes, eukaryotic virus sequences dominated the oxycline viromes, and phage sequences dominated the anoxic core viromes. The viral community of the ETSP OMZ was characterized by fluctuations in abundance, taxa and diversity across the oxygen gradient. The ecological significance of these changes was difficult to predict; however, it appears that the reduction in oxygen coincides with an increased shedding of eukaryotic viruses in the oxycline, and a shift to unique viral genotypes in the anoxic core.

Mild proteolytic digestion restores exocytotic activity to N-ethylmaleimide-inactivated cell surface complex from sea urchin eggs.

The Ca2+-stimulated release of cortical vesicle (cortical granule) contents from the cell surface complex (CSC) of the sea urchin egg is an in vitro model for exocytosis. To gain insight into the molecular mechanism of exocytosis we investigated the sensitivity of this model to sulfhydryl modification and proteolytic digestion. Our findings include the following: (a) Proteolytic treatment with trypsin or pronase of CSC prepared from the eggs of Strongylocentrotus purpuratus increased the free Ca2+ concentration required to elicit exocytosis. Although a small increase in the Ca2+ threshold was detected after mild proteolysis, high concentrations of trypsin (0.5 mg/ml) and prolonged incubation (3 h) were required to render the CSC unresponsive to high concentrations of Ca2+ (0.5 mM). Despite the severity of the proteolytic digestions required to inactivate the CSC, the individual cortical vesicles remained intact, as gauged by the latency of ovoperoxidase, a cortical vesicle enzyme. (b) As previously shown (Haggerty, J. C., and R. C. Jackson, 1983, J. Biol. Chem. 258:1819-1825), cortical exocytosis can be blocked by sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). In this report we demonstrate that NEM inhibits by increasing the Ca2+ threshold required for exocytosis. When CSC that had been completely inactivated by NEM modification was briefly digested, on ice, with a low concentration of trypsin (or several other proteases), exocytotic activity was restored. Although the Ca2+ threshold of the reactivated CSC was slightly higher than that of untreated CSC, it was nearly identical to that of control CSC, which was trypsinized but not treated with NEM. We discuss the significance of these results with regard to the molecular mechanism of exocytosis.

Subcellular localization of transporters along the rat blood-brain barrier and blood-cerebral-spinal fluid barrier by in vivo biotinylation.

Nutrient transporters and ABC efflux pumps at the blood-brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel luminal surface. Using this approach, we analyzed the luminal/abluminal localization of a number of blood-brain barrier transporters identified by quantitative PCR profiling as being highly expressed and enriched in rat brain endothelial cells compared with whole brain. We also examined the apical/basal-lateral distribution of transporters in the choroid plexus, a secondary site for transport of nutrients between the blood and CNS. We detected P-glycoprotein (Pgp) (Abcb1), ATP-binding cassette (Abc) g2, multidrug resistance protein (Mrp) 4 (Abcc4), glucose transporter 1 (Glut1) (Slc2a1), Lat1 (Slc7a5), and monocarboxylate transporter-1 (Mct1) (Slc16a1) on the luminal surface of rat cerebral microvessels by both immunofluorescence staining and Western blotting of in vivo biotinylated proteins. Mrp1 (Abcc1) appeared primarily abluminal by immunofluorescence staining, and was barely detectable in the biotinylated protein fraction. Organic anion transporter (Oat) 3 (Slc22a8), organic anion transporter polypeptide (Oatp) 2b1 (Slco2b1, Oatpb), and Mrp5 (Abcc5) were not detected on the luminal surface using either method, while Oatp1a4 (Slco1a4, Oatp2) appeared to partially localize to the microvessel lumen by immunofluorescence staining, but was not detected in the biotinylated protein fraction by Western blotting. Lat1, Mrp1 and Mrp4 were detected on the basal-lateral surface of lateral ventricle choroid plexus epithelial cells. Mrp5, Oct3 and Oatp2b1 (Oatpb) were detected in the ependymal cells lining the ventricle. We did not detect Pgp expression in choroid plexus by immunofluorescence staining. In vivo biotinylation provides a method for domain-specific labeling of luminal surface proteins within the capillaries of the blood-brain barrier, allowing for biochemical analysis of protein localization and facilitating optical discrimination of the luminal and abluminal endothelial surfaces.

Aura-biomes are present in the water layer above coral reef benthic macro-organisms.

As coral reef habitats decline worldwide, some reefs are transitioning from coral- to algal-dominated benthos with the exact cause for this shift remaining elusive. Increases in the abundance of microbes in the water column has been correlated with an increase in coral disease and reduction in coral cover. Here we investigated how multiple reef organisms influence microbial communities in the surrounding water column. Our study consisted of a field assessment of microbial communities above replicate patches dominated by a single macro-organism. Metagenomes were constructed from 20 L of water above distinct macro-organisms, including (1) the coral Mussismilia braziliensis, (2) fleshy macroalgae (Stypopodium, Dictota and Canistrocarpus), (3) turf algae, and (4) the zoanthid Palythoa caribaeorum and were compared to the water microbes collected 3 m above the reef. Microbial genera and functional potential were annotated using MG-RAST and showed that the dominant benthic macro-organisms influence the taxa and functions of microbes in the water column surrounding them, developing a specific "aura-biome". The coral aura-biome reflected the open water column, and was associated with Synechococcus and functions suggesting oligotrophic growth, while the fleshy macroalgae aura-biome was associated with Ruegeria, Pseudomonas, and microbial functions suggesting low oxygen conditions. The turf algae aura-biome was associated with Vibrio, Flavobacterium, and functions suggesting pathogenic activity, while zoanthids were associated with Alteromonas and functions suggesting a stressful environment. Because each benthic organism has a distinct aura-biome, a change in benthic cover will change the microbial community of the water, which may lead to either the stimulation or suppression of the recruitment of benthic organisms.

Identification and characterization of a cell surface proteoglycan on keratinocytes.

Proteoglycans fill the intercellular space between keratinocytes but their structure and function are not well understood. We have identified and partially characterized one intercellular proteoglycan on human keratinocytes, for which we propose the name epican (epidermal intercellular proteoglycan). Monoclonal antibodies (MoAb) were generated from a mixture of keratinocyte proteoglycans. One, designated MoAb17, identified the core protein of an intercellular proteoglycan that had an apparent mobility of greater than 250 kDa on Western blots. The core protein itself had an apparent mobility of 180 kDa following deglycosylation with trifluoromethanesulfonic acid. Enzymatic deglycosylation revealed that most core protein molecules were substituted with heparan sulfate but that some carried chondroitin sulfate instead. Smaller forms of the core protein were more abundant in tissue-culture medium than in cell extracts. This proteoglycan was localized by immunofluorescence to the intercellular space of the epidermis and the surface of keratinocytes in vitro, particularly at cell-cell contacts. MoAb17 did not react with protoglycans extracted from other skin cells, nor did it bind to basement membranes or connective tissue. Comparison of Western immunoblots using MoAb17 and antibodies to core proteins of other proteoglycans suggested that epican is not related to syndecan but is a member of the CD44 family.

CD44 expression on epidermal melanocytes.

We examined CD44 expression on melanocytes to begin to understand what role CD44 might have in the normal behavior of melanocytes and to provide a basis for comparing CD44 expression in melanoma cells. CD44 was expressed on the entire surface of melanocytes and accentuated at the tips of dendritic processes. Two predominant forms of CD44 are expressed on cultured human foreskin melanocytes. One form has the covalent addition of chondroitin sulfate, whereas the other form has no chondroitin sulfate. Both use the hematopoietic, or CD44H, core protein. Using polymerase chain reaction primers that span the site where alternative splicing of CD44 occurs, we found only the cDNA coding CD44H. 12-O-Tetradecanoylphorbol 13-acetate increases the size of the chondroitin sulfate chain(s) attached to CD44 but not the proportion of CD44 molecules that carry chondroitin sulfate. Ninety percent of proteoglycans on melanocytes are chondroitin sulfate proteoglycans, and the CD44 chondroitin sulfate proteoglycan represented 10% of that total. These data show that CD44H is expressed as a "part-time" chondroitin sulfate proteoglycan on normal cultured melanocytes.

The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44.

Epican, a heparan sulfate proteoglycan, was recently identified on the surface of keratinocytes with the aid of a monoclonal antibody to its core protein. Using that antibody to screen a human keratinocyte cDNA library, a clone encoding the entire epican core protein was selected and sequenced. The core protein of epican is a form of CD44. The deduced protein sequence of 699 amino acids has a novel 339 amino acid domain inserted into the proximal extracellular domain of the standard, leukocyte form of CD44. The additional domain adds a number of potential N- and O-linked glycosylation sites and two proteolysis sites to this form of CD44.

The presence of antithyroid antibodies in patients with affective and nonaffective psychiatric disorders.

We determined the frequency of antithyroglobulin and antimicrosomal antibodies in 173 consecutively admitted psychiatric inpatients. (We found antithyroid antibodies in 8% (5/65) of patients with DSM-III major depression, 13% (4/31) with biploar disorder, and in 0% (0/4) of those with schizoaffective disorder.) The rate of antibody occurrence was unrelated to lithium exposure either within individual diagnostic categories or for the sample as a whole. The overall frequency of positive antithyroid antibody titers in patients with DSM-III affective disorder, 9% (9/99), did not differ from that in patients with nonaffective disorders, 10% (7/68). However, patients with bipolar affective disorder-mixed or bipolar affective disorder-depressed had a higher rate of positive antithyroid antibody titers than other patients. Our findings confirm earlier reports that thyroid disorders may be particularly common in patients with bipolar affective disorder, even in the absence of lithium exposure. However, as antithyroid antibodies also occurred at a relatively high rate in nonaffective disorders, the possible psychiatric effects of autoimmune thyroiditis do not appear to be limited to affective dysregulation.

Organic brain syndrome associated with marginal hypothyroidism.

The authors report organic brain dysfunction in two psychiatric patients with grade 2 hypothyroidism; one was depressed and one had a paranoid psychosis with depressive features. The depression and psychosis responded to psychotropic medication and L-thyroxine, but the cognitive dysfunction improved only partially.

Relationship of serum TSH concentration and antithyroid antibodies to diagnosis and DST response in psychiatric inpatients.

Basal serum TSH concentration, antithyroid antibody titers, and DST response were evaluated in 124 psychiatric patients with affective symptoms. DST nonsuppressors were more likely than DST suppressors to have thyroid abnormalities.

Antithyroid antibodies in depressed patients.

The presence of antithyroid (antimicrosomal and antithyroglobulin) antibodies was assessed in 45 psychiatric inpatients with prominent depressive symptoms (28 with DSM-III major depression). Nine patients (20%) had detectable titers of antithyroid antibodies, a rate considerably higher than the 5%-10% observed in the normal population. Each of these nine patients with symptomless autoimmune thyroiditis had normal baseline serum thyrotropin concentrations and normal thyroid function (as assessed by T4, T3 uptake, and free thyroxine index). These findings support the hypothesis of subtle thyroid dysfunction in a sizable sample of psychiatric inpatients with prominent depressive symptoms.

Subclinical hypothyroidism: a modifiable risk factor for depression?

The authors assessed the lifetime history of major depression in 16 subjects with subclinical hypothyroidism and 15 subjects whose thyroid function was completely normal. The lifetime frequency of depression was significantly higher in the subjects who met the criteria for subclinical hypothyroidism (56%) than in those who did not (20%), suggesting that subclinical hypothyroidism may lower the threshold for the occurrence of depression.

Neuroendocrine effects of intravenous clomipramine in depressed patients and healthy subjects.

OBJECTIVE: Neuroendocrine challenge paradigms have been used to asses serotonergic systems in depression, but limitations in the specificity of many of these tests have been noted. In this study, the neuroendocrine responses to acute intravenous administration of the serotonin (5-HT) reuptake inhibitor clomipramine were assessed in depressed patients and matched control subjects. METHODS: Thirty hospitalized patients who met DSM-III-R criteria for major depression, and 30 healthy control subjects who were matched for age, sex, and season of year for the time of study, received 12.5 mg of intravenously administered clomipramine. RESULTS: The depressed patients demonstrated significant blunting of prolactin responses to clomipramine, as well as trends toward blunted ACTH and cortisol responses. There was no difference between the patient and control groups in growth hormone responses, plasma clomipramine levels, or self-reports of side effects. CONCLUSIONS: These data support the hypothesis that depressed patients have abnormal neuroendocrine responses to the intravenous administration of the 5-HT reuptake inhibitor clomipramine. Further study is required to delineate the mechanisms responsible for the abnormal response to intravenously administered clomipramine in depression.

Depression in women treated for gynecological cancer: clinical and neuroendocrine assessment.

To determine the prevalence of major depression in cancer patients and assess the usefulness of the dexamethasone suppression test (DST) and the thyrotropin-releasing hormone (TRH) stimulation test for diagnosing major depression in these patients, the authors studied 83 women hospitalized for gynecological cancer. Nineteen (23%) had major depression according to DSM-III criteria. The sensitivity and specificity of the DST were 40% and 88%, respectively. No relationship between DST and TRH test results was found. These findings indicate a high prevalence of depression in cancer patients, but further research on these tests in cancer patients is needed; their routine use with cancer patients is premature at this time.

Life adaptation after percutaneous transluminal coronary angioplasty and coronary artery bypass grafting.

Life adaptation of 32 patients who had undergone percutaneous transluminal coronary angioplasty (PTCA) for coronary stenosis was compared with that of 15 patients who had coronary artery bypass grafting (CABG). Patients were matched for psychosocial, anatomic and cardiac functions. Life adaptation was measured at 6 and 15 months after PTCA or CABG by the Psychosocial Adjustment to Illness Scale (PAIS), a multidimensional instrument that evaluates change in 7 primary life domains. The overall PAIS scores for patients who had undergone PTCA were significantly better (p less than 0.04) than the scores for those who had undergone CABG after 6 months, and this superior functioning continued after 15 months (p less than 0.05). After 6 months patients who had undergone PTCA functioned better at work (p less than 0.005), in sexual performance (p less than 0.0001) and with their families (p less than 0.002). The improvement in work functioning continued at 15 months (p less than 0.04), but the differences in sexual and family domains became nonsignificant.

Use of the dexamethasone suppression test with DSM-III criteria in psychiatrically hospitalized adolescents.

Although developmental factors may complicate the assessment of major affective disorder in the adolescent patient, studies suggest that the same descriptive criteria used in the assessment of the adult patient can be utilized to diagnose major depression in the adolescent patient. In order to determine if the dexamethasone suppression test (DST) has a similar sensitivity and specificity for DSM-III major depression in adolescents compared to that found in adult patients, we administered the DST to 55 adolescents admitted to an inpatient psychiatric unit. Each of the adolescents exhibited depressive symptoms; 23 fulfilled DSM-III criteria for major depressive syndromes (20 major depression; 3 schizoaffective), and 32 fulfilled the criteria for other DSM-III diagnoses. Of the 23 patients with major affective disorder, 10 (43%) exhibited nonsuppression of serum cortisol after dexamethasone. Of the 32 patients with depressive symptoms and other diagnoses, five (16%) exhibited nonsuppression; on follow up, three of the five nonsuppressors fulfilled criteria for major depression. These findings suggest that DSM-III criteria can be used to diagnose major depression in the adolescent patient and that the DST may play an important role in detecting or confirming the diagnosis of major depressive disorders in adolescent patients.

Strongyloidiasis presenting as depression: a case report.

A case is described in which an occult parasitic infection. Strongyloidiasis, presented with symptoms suggestive of hypochondriacal depression. The diagnosis had been missed in several extensive medical evaluation and was made belatedly by stool specimen following unsuccessful treatment with antidepressant medication. Clinical features of this common infection are reviewed.