Impact of training sets on classification of high-throughput bacterial 16s rRNA gene surveys

Taxonomic classification of the thousands–millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naïve Bayesian classifier (for example, the Ribosomal Database Project II (RDP) classifier), due to favorable trade-offs among automation, speed and accuracy. The resulting classification depends on the reference sequences and taxonomic hierarchy used to train the model; although the influence of primer sets and classification algorithms have been explored in detail, the influence of training set has not been characterized. We compared classification results obtained using three different publicly available databases as training sets, applied to five different bacterial 16S rRNA gene pyrosequencing data sets generated (from human body, mouse gut, python gut, soil and anaerobic digester samples). We observed numerous advantages to using the largest, most diverse training set available, that we constructed from the Greengenes (GG) bacterial/archaeal 16S rRNA gene sequence database and the latest GG taxonomy. Phylogenetic clusters of previously unclassified experimental sequences were identified with notable improvements (for example, 50% reduction in reads unclassified at the phylum level in mouse gut, soil and anaerobic digester samples), especially for phylotypes belonging to specific phyla (Tenericutes, Chloroflexi, Synergistetes and Candidate phyla TM6, TM7). Trimming the reference sequences to the primer region resulted in systematic improvements in classification depth, and greatest gains at higher confidence thresholds. Phylotypes unclassified at the genus level represented a greater proportion of the total community variation than classified operational taxonomic units in mouse gut and anaerobic digester samples, underscoring the need for greater diversity in existing reference databases.     

Proteomic analyses of transgenic LQT1 and LQT2 rabbit hearts elucidate an increase in expression and activity of energy producing enzymes

Various biochemical and genomic mechanisms are considered to be a hallmark of metabolic remodeling in the stressed heart, including the hypertrophied and failing heart. In this study, we used quantitative proteomic 2-D Fluorescence Difference In-Gel Electrophoresis (2-D DIGE) in conjunction with mass spectrometry to demonstrate differential protein expression in the hearts of transgenic rabbit models of Long QT Syndrome 1 (LQT1) and Long QT Syndrome 2 (LQT2) as compared to littermate controls (LMC). The results of our proteomic analysis revealed upregulation of key metabolic enzymes involved in all pathways associated with ATP generation, including creatine kinase in both LQT1 and LQT2 rabbit hearts. Additionally, the expression of lamin-A protein was increased in both LQT1 and LQT2 rabbit hearts as was the expression of mitochondrial aldehyde dehydrogenase and desmoplakin in LQT1 and LQT 2 rabbit hearts, respectively. Results of the proteomic analysis also demonstrated down regulation in the expression of protein disulfide-isomerase A3 precuorsor and dynamin-like 120 kDa protein (mitochondrial) in LQT1, and of alpha-actinin 2 in LQT2 rabbit hearts. Up regulation of the expression of the enzymes associated with ATP generation was substantiated by the results of selective enzyme assays in LQT1 and LQT2 hearts, as compared to LMC, which revealed increases in the activities of glycogen phosphorylase (+50%, +65%, respectively), lactate dehydrogenase (+25%, +25%) pyruvate dehydrogenase (+31%, +22%), and succinate dehydrogenase (+32%, +60%). The activity of cytochrome c-oxidase, a marker for the mitochondrial function was also found to be significantly elevated (+80%) in LQT1 rabbit hearts as compared with LMC. Western blot analysis in LQT1 and LQT2 hearts compared to LMC revealed an increase in the expression of very-long chain-specific acyl-CoA dehydrogenase (+35%, +33%), a rate-limiting enzymes in β-oxidation of fatty acids. Collectively, our results demonstrate similar increases in the expression and activities of key ATP-generating enzymes in LQT1 and LQT2 rabbit hearts, suggesting an increased demand, and in turn, increased energy supply across the entire metabolic pathway by virtue of the upregulation of enzymes involved in energy generation.     

Social status and cortisol levels in singing rock hyraxes

Many mammals use acoustic signals to communicate with conspecifics. Rock hyraxes (Procavia capensis) are social mammals whose vocal communication is usually restricted to quiet sounds used between nearby individuals. Loud repetitive warning trills are an exception. In our study site, a third of the adult male hyraxes also produces a rich, complex and loud vocalization we term 'singing'. In this study, we examine whether singers, which are more conspicuous by the act of singing, have higher cortisol (i.e. basal stress; C) levels than non-singers, and whether there is an association between social status and stress hormones in male hyraxes. We show that 'singing' males are different from the general adult male population in that their C levels are higher than those of silent males. Only in singers, C levels are associated with social rank, with dominants showing the highest levels. Singers are also on average older and more dominant than most other sexually mature non-singing males. Further, they copulate more than non-singers, suggesting that singing males may have higher reproductive success. Our results support the ‘stress of domination’ hypothesis and indicate that in the rock hyrax singing may reflect high competitive ability, designating singers as a distinct class of males, unique in their personal attributes and behavior.     

The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses

Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants.     

Bacteria associated with mucus and tissues of the coral Oculina patagonica in summer and winter

The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 x 10(7) and 8.3 x 10(8)/cm2 of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different alpha-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (> 99.5% identity). Of these, 295 were novel (< 99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.     

Effectiveness of sucrose analgesia in newborns undergoing painful medical procedures

Background

Sucrose is widely used to manage procedural pain in term newborns despite a lack of evidence of its effectiveness for different procedures and infant populations. Our objectives were to evaluate the effectiveness and safety of sucrose in newborns undergoing various medical procedures within 2 days of birth.

Methods

We performed a double-blind, randomized controlled trial. We included newborns (≥ 36 weeks gestation) of diabetic mothers and nondiabetic mothers. Each newborn received 2 mL of a 24%-sucrose or placebo solution before all procedures. We used the Premature Infant Pain Profile to assess pain during intramuscular injection of vitamin K, venipuncture for the newborn screening test and the first 3 heel lances for glucose monitoring (newborns of diabetic mothers only). Scores ranged from from 0 (no pain) to 18 (maximum pain).

Results

We included 240 newborns (120 from diabetic mothers, 120 from nondiabetic mothers). The overall mean pain score was lower among newborns who received sucrose than among those who received a placebo (mean difference –1.3, 95% confidence interval [CI] –2.0 to –0.6). We found that pain scores during intramuscular injection did not differ significantly between the sucrose and placebo groups for newborns of diabetic or nondiabetic mothers (newborns of nondiabetic mothers: mean difference –1.1, 95% CI –2.4 to 0.2; newborns of diabetic mothers: mean difference –1.0, 95% CI –2.4 to 0.4). During venipuncture, newborns who received sucrose had lower pain scores compared with those who received a placebo (newborns of nondiabetic mothers: mean difference –3.2, 95% CI –4.6 to –1.8; newborns of diabetic mothers: mean difference –2.4, 95% CI –3.8 to –1.0). Among newborns of diabetic mothers, there was no difference in pain during the first 3 heel lances or mean glucose levels between the sucrose and placebo groups (p = 0.94 and p = 0.29 respectively).

Interpretation

We found a modest reduction of pain in newborns of both diabetic and nondiabetic mothers when sucrose was used for all medical procedures performed in the first 2 days after birth. However, when each procedure was analyzed separately, we found that the effectiveness of sucrose was limited to venipuncture for the newborn screening test. (http://Clinicaltrials.gov trial register no. {"type":"clinical-trial","attrs":{"text":"NCT00213213","term_id":"NCT00213213"}}NCT00213213.)All newborns experience pain from medical procedures in the first days after birth. At the very least, newborns receive an intramuscular injection of vitamin K and venipuncture or heel-lance for newborn screening tests. Newborns of diabetic mothers, about 4% of newborns, undergo additional heel-lances for monitoring glucose levels.^1,2 Untreated pain from these procedures can have long-lasting effects.^3,4 We previously reported that newborns of diabetic mothers who received an average of 10 heel-lances within 2 days of birth exhibited a heightened pain response during subsequent venipunctures compared with newborns from nondiabetic mothers.²Studies aimed at reducing pain and its consequences in newborns are clinically important.^5–7 Sucrose (table sugar) has great potential for routine use.⁶ Sucrose is a naturally occurring sweetener with analgesic effects in newborns. Although not fully understood, the mechanism of action is thought to involve activation of the endogenous opioid system through taste.⁸ This is supported by the presence of opioid receptors on the tongue and animal studies showing that analgesia can be reversed by opioid antagonists during noxious stimulation.⁹ Moreover, chronic consumption of sweeteners has been shown to negate the analgesic effects of opioids.¹⁰In systematic reviews, single doses of sucrose have been reported to reduce pain in newborns undergoing commonly performed medical procedures, including heel-lancing and venipuncture.^11,12 Based on these data, national and international pain management guidelines promote the widespread use of sucrose.^13–16 However, sucrose has not been evaluated for all commonly performed procedures in healthy term newborns, and there are concerns that sucrose may raise blood glucose levels in newborns of diabetic mothers. It is necessary to demonstrate that sucrose is effective and safe for varied procedures, repeated use and diverse infant populations.We sought to evaluate the effectiveness and safety of sucrose in newborns of diabetic mothers and nondiabetic mothers undergoing routine painful medical procedures during the first 2 days after birth. Our hypothesis was that sucrose reduces pain during intramuscular injection of vitamin K, venipuncture for the newborn screening test and heel lance for blood glucose monitoring without serious adverse effects.     

Petroleum Pollution Bioremediation Using Water-Insoluble Uric Acid as the Nitrogen Source

The biodegradation of hydrocarbon pollutants in open systems is limited by the availability of a utilizable nitrogen source. This limitation can be overcome by using uric acid. Enrichment cultures grown on crude oil-uric acid media yielded mixed and pure cultures that degraded petroleum. In a simulated open system, uric acid bound to crude oil and was available for bacterial growth and petroleum biodegradation.     

The Hunneberg Stage (Ordovician) in the area east of St. Petersburg, north-western Russia

The bio- and lithostratigraphy of the poorly known early Ordovician (Arenig) Hunneberg Stage is described from the area east of St. Petersburg (Putilovo Quarry and Lava river), where the Lakity Beds (Leetse Fm.) consist of glauconitic sands and silty clays. The described fauna includes graptolites, conodonts, brachiopods, acritarchs, foraminifers, and several problematic groups. The Lakity Beds are unique within the lower Ordovician sequence of the East Baltic in the abundance of graptolites, co-occurring with conodonts. The conodonts from the lower part of the Lakity Beds belong to the uppermost subzone (Oelandodus elongatus — Acodus deltatus deltatus) of theParoistodus proteus Zone, coinciding with the lower part of theTetragraptus phyllograptoides Zone. This suggests that there is a hiatus between the Lakity Beds and the underlying Nazya Formation. The lower Lakity Beds also contain the brachiopodsLeptembolon lingulaeformis andEosiphonotreta cf.acrotretomorpha, which suggest that they may be correlated with the Joa Member of the Leetse Formation in North Estonia. In the upper part of the Lakity Beds, conodonts of thePrioniodus elegans Zone co-occur with graptolites indicating the upper subzone of theTetragraptus phyllograptoides Zone in the Scandinavian sections. There are still problems of defining and correlating the base of the Billingen Regional Stage, which are outside the scope of this paper.     

Lipopolysaccharide exposure during purification of human monocytes by adherence increases their recovery and cytolytic activity

Exposure of monocytes to lipopolysaccharide (LPS) during, but not after, adherence purification increased their cytolytic activity in short-term 51Cr-release assays against K562 target cells. In the absence of LPS only a minority of monocytes could be recovered by adherence. With 1 ng/ml to 10 micrograms/ml LPS present during the 1-hr adherence procedure, however, monocytes spread more extensively on serum-coated plastic and glass surfaces and virtually all of the monocytes in a mononuclear leukocyte preparation were recovered in the adherent fraction. While increasing the recovery of monocytes threefold, LPS exposure during adherence also increased monocyte purity as assessed by peroxidase staining, morphology, and indirect immunofluorescence with monoclonal Mo2. The proportion of Leu-11-positive NK cells in the adherent fraction did not change. Depletion of NK cells by treatment with anti-Leu-11b and complement eliminated cytolytic activity from the nonadherent, but not from the adherent, fraction isolated with LPS. Thus, addition of LPS during adherence produced a monocyte preparation with enhanced cytolytic activity not attributable to NK contaminants. To test whether LPS caused production of lymphokines that activate monocytes, we tested supernatants of unseparated mononuclear leukocytes for the capacity to stimulate purified monocytes for cytolysis. Such supernatants stimulated monocytes more effectively than LPS alone. We conclude that LPS stimulates monocytes for cytolysis most effectively during adherence purification because LPS allows the recovery of weakly adherent monocytes with high cytolytic capacity; also, LPS may stimulate production of lymphokines that further augment monocyte cytolytic activity.