Genome Sequence of a Cellulose-Producing Bacterium,Gluconacetobacter hansenii ATCC 23769

The Gram-negative bacterium Gluconacetobacter hansenii is considered a model organism for studying cellulose synthesis. We have determined the genome sequence of strain ATCC 23769.Plants produce cellulose, an unbranched chain of β-1,4-linked glucose units, as a structural polysaccharide. It is the most abundant polymer on earth, recently receiving much interest due to its potential use as a feedstock for bioethanol. Bacteria also produce cellulose. Among these, Gluconacetobacter hansenii (previously named Acetobacter xylinus) (4) has been extensively characterized and is a model system for cellulose biosynthesis (1, 2, 7). G. hansenii produces extracellular cellulose that is devoid of lignin or hemicellulose, making it an excellent source for pure cellulose. A lack of a completely sequenced genome for this organism has been a limiting factor in identifying other key proteins involved in cellulose synthesis.The whole-genome sequencing of G. hansenii ATCC 23769 was performed using the 454 FLX-Titanium pyrosequencing technology (5). A combinatorial sequencing approach using 489,201 reads obtained from the shotgun library and 195,088 reads from an 8-kb pair end library (3) produced a total of 221,294,116 bp. These reads were assembled using the Newbler assembler, producing 88 large contigs (>500 bp) and a chromosome-sized scaffold of 3,646,142 bp with an average coverage of ×50.5. This scaffold contained exclusively chromosomal DNA and no plasmid sequences. The gaps in the large scaffold were filled by primer walking and subsequent sequencing of the PCR products. The resulting high-quality draft assembly, consisting of a large scaffold with 71 contigs, was annotated using the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) service of the National Institute of Biotechnology Information (NCBI).The chromosomal sequence of G. hansenii 23769 contains 3,547,122 bp, with a G+C content of 59%. The genome contains 3,351 genes, of which 3,308 are protein-encoding genes, accounting for 84% of the genome. There are 43 genes for tRNAs and 2 rRNA loci. The genes encoding proteins involved in cellulose synthesis are in an operon consisting of acsAB (GXY_04277), acsC (GXY_04282), and acsD (GXY_04292), as previously shown by Saxena et al. (7). Interestingly, there are two additional copies of acsAB, GXY_08864 and GXY_14452, which share 69% and 72% sequence identity, respectively, with the acsAB genes in the operon; the deduced amino acid sequences are 40% and 46% identical, respectively, with that deduced from acsAB in the operon. There are also two additional copies of acsC, GXY_08869 and GXY_014472, which share 72% and 65% DNA sequence identity, respectively, with the acsC gene in the operon; the deduced amino acid sequences share 28% and 30% amino acid identity, respectively, with that deduced from acsC. acsAB (GXY_08864) and acsC (GXY_08869) are only 17 bp apart, less than the distance (66 bp) between the acsAB and acsC genes in the operon. acsAB (GXY_14452) and acsC (GXY_14472) are separated by 3,299 bp, with three genes in between. However, acsD is present only in the operon, not duplicated elsewhere in the genome. The genome also contains three genes encoding diguanylate cyclase, as previously reported by Tal et al. (8). Diguanylate cyclase catalyzes the formation of cyclic di-GMP, a second messenger in bacteria that functions as an allosteric activator of cellulase synthase AcsAB (6).     

Synthesis and characterization of [Ru(NO)(bpp)Cl · 2H2O] [bpp = N,N′-bis(2-pyridinecarboxamide)-1,3-propane dianion] and [Ru(NO)(bpe)Cl · 2H2O] [bpe = N,N′-bis(2-pyridinecarboxamide)-1,2-ethane dianion]

Two ruthenium nitrosyl bis-pyridyl/biscarboxamido compounds, [Ru(NO)(bpp)Cl · 2H₂O] [bpp = N,N′-bis(2-pyridinecarboxamide)-1,3-propane dianion] and [Ru(NO)(bpe)Cl · 2H₂O] [bpe = N,N′-(bis-2-pyridinecarboxamide)-1,2-ethane dianion] have been characterized by ¹H NMR, ¹³C{¹H} NMR, and IR spectroscopies, electrospray ionizaton mass spectrometry, and X-ray crystallography.     

New insights into the first oxidative phenol coupling reaction during vancomycin biosynthesis

OxyB catalyzes the first oxidative phenol coupling reaction in vancomycin biosynthesis. OxyB is a P450 hemoprotein whose activity is strictly dependent upon the presence of molecular oxygen. Here, it was shown that label from (18)O(2) is not incorporated into the monocyclic product during catalysis by OxyB. In addition, it was shown that OxyB can convert a model hexapeptide substrate containing (R)-Tyr6, instead of (S)-Tyr6, covalently linked as a C-terminal thioester to a peptidyl carrier protein (PCP-7S) derived from the vancomycin non-ribosomal peptide synthetase (NRPS), into the corresponding epimeric monocyclic product. The binding of this epimeric hexapeptide-PCP conjugate to the Fe(III) form of OxyB, as monitored by UV-vis spectroscopy, revealed a K(d)=35+/-5 microM. Thus, the enzyme reveals a surprising lack of stereospecificity in the binding and transformation of these epimeric substrates.     

Flat Feet,Happy Feet? Comparison of the Dynamic Plantar Pressure Distribution and Static Medial Foot Geometry between Malawian and Dutch Adults

In contrast to western countries, foot complaints are rare in Africa. This is remarkable, as many African adults walk many hours each day, often barefoot or with worn-out shoes. The reason why Africans can withstand such loading without developing foot complaints might be related to the way the foot is loaded. Therefore, static foot geometry and dynamic plantar pressure distribution of 77 adults from Malawi were compared to 77 adults from the Netherlands. None of the subjects had a history of foot complaints. The plantar pressure pattern as well as the Arch Index (AI) and the trajectory of the center of pressure during the stance phase were calculated and compared between both groups. Standardized pictures were taken from the feet to assess the height of the Medial Longitudinal Arch (MLA). We found that Malawian adults: (1) loaded the midfoot for a longer and the forefoot for a shorter period during roll off, (2) had significantly lower plantar pressures under the heel and a part of the forefoot, and (3) had a larger AI and a lower MLA compared to the Dutch. These findings demonstrate that differences in static foot geometry, foot loading, and roll off technique exist between the two groups. The advantage of the foot loading pattern as shown by the Malawian group is that the plantar pressure is distributed more equally over the foot. This might prevent foot complaints.     

DIETARY LUFENURON REDUCES EGG HATCH AND INFLUENCES PROTEIN EXPRESSION IN THE FRUIT FLY Bactrocera latifrons (HENDEL)

Lufenuron (LFN), a chitin synthase inhibitor, impacts the fertility of Ceratitis capitata, Bactrocera dorsalis, B. cucurbitae, and B. latifrons. We posed the hypothesis that LFN curtails egg hatch in the solanaceous fruit fly, B. latifrons. In this study, newly emerged virgin adults were sexed and fed for 12 days with varying concentrations of LFN‐laced agar diets until sexual maturation. Eggs were collected from 12‐d‐old adults and the egg hatch was assessed. Egg hatch decreased in adults reared on LFN‐treated diets. LFN‐treated media did not influence fertility after one gender was reared on experimental and the other on control media before mating. Exposure to LFN‐treated medium after mating led to reduced egg hatch. We infer that LFN is not a permanent sterilant, and reduced egg hatch depends on continuous exposure to dietary LFN after mating. Proteomic analysis identified two differentially expressed proteins, a pheromone binding protein and a chitin binding protein, between adults maintained on LFN‐treated and control diets. Expression of two genes encoding chitin synthase 2, and chitin binding protein, was altered in adults exposed to dietary LFN. LFN treatments also led to increased expression of two odorant binding proteins one in females and one in males. We surmise these data support our hypothesis and provide insight into LFN actions.     

A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.     

The relationship of aerobic capacity,anaerobic peak power and experience to performance in CrossFit exercise

CrossFit is becoming increasingly popular as a method to increase fitness and as a competitive sport in both the Unites States and Europe. However, little research on this mode of exercise has been performed to date. The purpose of the present investigation involving experienced CrossFit athletes and naïve healthy young men was to investigate the relationship of aerobic capacity and anaerobic power to performance in two representative CrossFit workouts: the first workout was 12 minutes in duration, and the second was based on the total time to complete the prescribed exercise. The participants were 32 healthy adult males, who were either naïve to CrossFit exercise or had competed in CrossFit competitions. Linear regression was undertaken to predict performance on the first workout (time) with age, group (naïve or CrossFit athlete), VO₂max and anaerobic power, which were all significant predictors (p < 0.05) in the model. The second workout (repetitions), when examined similarly using regression, only resulted in CrossFit experience as a significant predictor (p < 0.05). The results of the study suggest that a history of participation in CrossFit competition is a key component of performance in CrossFit workouts which are representative of those performed in CrossFit, and that, in at least one these workouts, aerobic capacity and anaerobic power are associated with success.