Bacterial genome chimaerism and the origin of mitochondria

Many studies have sought to determine the origin and evolution of mitochondria. Although the Alphaproteobacteria are thought to be the closest relatives of the mitochondrial progenitor, there is dispute as to what its particular sister group is. Some have argued that mitochondria originated from ancestors of the order Rickettsiales, or more specifically of the Rickettsiaceae family, while others believe that ancestors of the family Rhodospirillaceae are also equally likely the progenitors. To resolve some of these disputes, sequence similarity searches and phylogenetic analyses were performed against mitochondria-related proteins in Saccharomyces cerevisiae. The 86 common matches of 5 Alphaproteobacteria (Rickettsia prowazekii, Rhodospirillum rubrum, Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Ochrobactrum anthropi) to yeast mitochondrial proteins were distributed fairly evenly among the 5 species when sorted by highest identity or score. Moreover, exploratory phylogenetic analyses revealed that among these common matches, 44.19% (38) had branched most closely with O. anthropi, while only 34.88% (30) corresponded with Rickettsia prowazekii. More detailed phylogenetic analyses with additional Alphaproteobacteria and including genes from the mitochondria of Reclinomonas americana found matches of mitochondrial genes to those of members of the Rickettsiaceae, Anaplasmataceae, and Rhodospirillaceae families. The results support the idea that notable bacterial genome chimaerism has occurred en route to the formation of mitochondria.     

Purpose-driven biomaterials research in liver-tissue engineering

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications.     

Diatom community structure on in-service cruise ship hulls

Diatoms are an important component of marine biofilms found on ship hulls. However, there are only a few published studies that describe the presence and abundance of diatoms on ships, and none that relate to modern ship hull coatings. This study investigated the diatom community structure on two in-service cruise ships with the same cruise cycles, one coated with an antifouling (AF) system (copper self-polishing copolymer) and the other coated with a silicone fouling-release (FR) system. Biofilm samples were collected during dry docking from representative areas of the ship and these provided information on the horizontal and vertical zonation of the hull, and intact and damaged coating and niche areas. Diatoms from the genera Achnanthes, Amphora and Navicula were the most common, regardless of horizontal ship zonation and coating type. Other genera were abundant, but their presence was more dependent on the ship zonation and coating type. Samples collected from damaged areas of the hull coating had a similar community composition to undamaged areas, but with higher diatom abundance. Diatom fouling on the niche areas differed from that of the surrounding ship hull and paralleled previous studies that investigated differences in diatom community structure on static and dynamically exposed coatings; niche areas were similar to static immersion and the hull to dynamic immersion. Additionally, diatom richness was greater on the ship with the FR coating, including the identification of several new genera to the biofouling literature, viz. Lampriscus and Thalassiophysa. These results are the first to describe diatom community composition on in-service ship hulls coated with a FR system. This class of coatings appears to have a larger diatom community compared to copper-based AF systems, with new diatom genera that have the ability to stick to ship hulls and withstand hydrodynamic forces, thus creating the potential for new problematic species in the biofilm.     

In vivo co-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner

Co-localization of biochemical processes plays a key role in the directional control of metabolic fluxes toward specific products in cells. Here, we employ in vivo scaffolds made of RNA that can bind engineered proteins fused to specific RNA binding domains. This allows proteins to be co-localized on RNA scaffolds inside living Escherichia coli. We assembled a library of eight aptamers and corresponding RNA binding domains fused to partial fragments of fluorescent proteins. New scaffold designs could co-localize split green fluorescent protein fragments to produce activity as measured by cell-based fluorescence. The scaffolds consisted of either single bivalent RNAs or RNAs designed to polymerize in one or two dimensions. The new scaffolds were used to increase metabolic output from a two-enzyme pentadecane production pathway that contains a fatty aldehyde intermediate, as well as three and four enzymes in the succinate production pathway. Pentadecane synthesis depended on the geometry of enzymes on the scaffold, as determined through systematic reorientation of the acyl-ACP reductase fusion by rotation via addition of base pairs to its cognate RNA aptamer. Together, these data suggest that intra-cellular scaffolding of enzymatic reactions may enhance the direct channeling of a variety of substrates.     

The Influence of Nanocrystal Aggregates on Photovoltaic Performance in Nanocrystal–Polymer Bulk Heterojunction Solar Cells

CdSe nanocrystals (NCs) can be used as an electron acceptor in solar cells, employing organic ligands to passivate their surface and make them processable from solution. The nature and abundance of impurities present after NC ligand exchange from oleic acid to n‐butylamine are identified. A further purification step using hexane as a selective solvent is described, which excludes NC aggregates from solution. The influence of NC aggregates on photovoltaic device performance is studied in a CdSe:poly[2‐methoxy‐5‐(3′,7′‐dimethyloctyloxy)‐1,4‐phenylene vinylene] (MDMO‐PPV) bulk heterojunction solar cell. The exclusion of NC aggregates leads to a four‐fold increase in device power conversion efficiency (PCE) in optimized devices. A superior blend morphology leading to improved charge generation and a better NC percolation network is identified as the main causes of this increased solar cell performance.     

Epidemiology of Acute Pancreatitis in Hospitalized Children in the United States from 2000–2009

Background

Single-center studies suggest an increasing incidence of acute pancreatitis (AP) in children. Our specific aims were to (i) estimate the recent secular trends, (ii) assess the disease burden, and (iii) define the demographics and comorbid conditions of AP in hospitalized children within the United States.

Methods

We used the Healthcare Cost and Utilization Project Kids’ Inpatient Database, Agency for Healthcare Research and Quality for the years 2000 to 2009. Extracted data were weighted to generate national-level estimates. We used the Cochrane-Armitage test to analyze trends; cohort-matching to evaluate the association of AP and in-hospital mortality, length of stay, and charges; and multivariable logistic regression to test the association of AP and demographics and comorbid conditions.

Results

We identified 55,012 cases of AP in hospitalized children (1–20 years of age). The incidence of AP increased from 23.1 to 34.9 (cases per 10,000 hospitalizations per year; P<0.001) and for all-diagnoses 38.7 to 61.1 (P<0.001). There was an increasing trend in the incidence of both primary and all-diagnoses of AP (P<0.001). In-hospital mortality decreased (13.1 to 7.6 per 1,000 cases, P<0.001), median length of stay decreased (5 to 4 days, P<0.001), and median charges increased ($14,956 to $22,663, P<0.001). Children with AP compared to those without the disease had lower in-hospital mortality (adjusted odds ratio, aOR 0.86, 95% CI, 0.78–0.95), longer lengths of stay (aOR 2.42, 95% CI, 2.40–2.46), and higher charges (aOR 1.62, 95% CI, 1.59–1.65). AP was more likely to occur in children older than 5 years of age (aORs 2.81 to 5.25 for each 5-year age interval). Hepatobiliary disease was the comorbid condition with the greatest association with AP.

Conclusions

These results demonstrate a rising incidence of AP in hospitalized children. Despite improvements in mortality and length of stay, hospitalized children with AP have significant morbidity.     

Metabolomics Analysis Identifies Intestinal Microbiota-Derived Biomarkers of Colonization Resistance in Clindamycin-Treated Mice

Background

The intestinal microbiota protect the host against enteric pathogens through a defense mechanism termed colonization resistance. Antibiotics excreted into the intestinal tract may disrupt colonization resistance and alter normal metabolic functions of the microbiota. We used a mouse model to test the hypothesis that alterations in levels of bacterial metabolites in fecal specimens could provide useful biomarkers indicating disrupted or intact colonization resistance after antibiotic treatment.

Methods

To assess in vivo colonization resistance, mice were challenged with oral vancomycin-resistant Enterococcus or Clostridium difficile spores at varying time points after treatment with the lincosamide antibiotic clindamycin. For concurrent groups of antibiotic-treated mice, stool samples were analyzed using quantitative real-time polymerase chain reaction to assess changes in the microbiota and using non-targeted metabolic profiling. To assess whether the findings were applicable to another antibiotic class that suppresses intestinal anaerobes, similar experiments were conducted with piperacillin/tazobactam.

Results

Colonization resistance began to recover within 5 days and was intact by 12 days after clindamycin treatment, coinciding with the recovery bacteria from the families Lachnospiraceae and Ruminococcaceae, both part of the phylum Firmicutes. Clindamycin treatment caused marked changes in metabolites present in fecal specimens. Of 484 compounds analyzed, 146 (30%) exhibited a significant increase or decrease in concentration during clindamycin treatment followed by recovery to baseline that coincided with restoration of in vivo colonization resistance. Identified as potential biomarkers of colonization resistance, these compounds included intermediates in carbohydrate or protein metabolism that increased (pentitols, gamma-glutamyl amino acids and inositol metabolites) or decreased (pentoses, dipeptides) with clindamycin treatment. Piperacillin/tazobactam treatment caused similar alterations in the intestinal microbiota and fecal metabolites.

Conclusions

Recovery of colonization resistance after antibiotic treatment coincided with restoration of several fecal bacterial metabolites. These metabolites could provide useful biomarkers indicating intact or disrupted colonization resistance during and after antibiotic treatment.     

Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily

4-Amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase catalyzes a complex rearrangement of 5-aminoimidazole ribonucleotide (AIR) to form HMP-P, the pyrimidine moiety of thiamine phosphate. We determined the three-dimensional structures of HMP-P synthase and its complexes with the product HMP-P and a substrate analog imidazole ribotide. The structure of HMP-P synthase reveals a homodimer in which each protomer comprises three domains: an N-terminal domain with a novel fold, a central (betaalpha)(8) barrel and a disordered C-terminal domain that contains a conserved CX(2)CX(4)C motif, which is suggestive of a [4Fe-4S] cluster. Biochemical studies have confirmed that HMP-P synthase is iron sulfur cluster-dependent, that it is a new member of the radical SAM superfamily and that HMP-P and 5'-deoxyadenosine are products of the reaction. M?ssbauer and EPR spectroscopy confirm the presence of one [4Fe-4S] cluster. Structural comparisons reveal that HMP-P synthase is homologous to a group of adenosylcobalamin radical enzymes. This similarity supports an evolutionary relationship between these two superfamilies.     

Reconfiguration of a Multi-oscillator Network by Light in the Drosophila Circadian Clock

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Rapid and Robust PCR-Based All-Recombinant Cloning Methodology

We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology.