Evaluation of electromagnetic navigational bronchoscopic biopsy of lung lesions performed by a thoracic surgical service.

BACKGROUND: With the increasing use of computed tomography scans for lung cancer screening and surveillance of other cancers, thoracic surgeons are being referred patients with lung lesions for biopsies. Electromagnetic navigational bronchoscopy-guided lung biopsy is a relatively new technique for bronchoscopic biopsy. Our objective was to evaluate the diagnostic yields and safety of electromagnetic navigational bronchoscopy-guided lung biopsy. METHODS: We conducted a retrospective review of patients who underwent an electromagnetic navigational bronchoscopy biopsy, performed by a thoracic surgical service, and evaluated its safety and diagnostic accuracy. RESULTS: In total, 110 patients (men 46, women 64) underwent electromagnetic navigational bronchoscopy sampling of pulmonary lesions (n = 121; median size 27 mm; interquartile range 17-37 mm). There was no procedure-related mortality. Pneumothorax requiring pigtail drainage occurred in 4 patients (3.5%). Ninety-three (76.9%) of the lesions were malignant. Eighty-seven (71.9%) of the 121 lesions had an accurate diagnosis. Accuracy increased with increased lesion size (P = .0578) with a yield of 50% for lesions <2 cm, increasing to 81% for lesions ≥2 cm. The lesions that demonstrated a positive "bronchus sign" had a yield of 87% (45/52) compared with 61% (42/69) in lesions with a negative "bronchus sign" (P = .0359). CONCLUSION: Thoracic surgeons can perform electromagnetic navigational bronchoscopy safely, with minimal morbidity and with good diagnostic yields. Accuracy increases with the presence of a bronchus sign and increasing lesion size. Patients with larger tumors and the bronchus sign may be candidates for this approach to biopsy. Further work is required to define the role of electromagnetic navigational bronchoscopy in the diagnosis of pulmonary lesions.

Exploring the Impact of Terminators on Transgene Expression in Chlamydomonas reinhardtii with a Synthetic Biology Approach.

Chlamydomonas reinhardtii has many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in the C. reinhardtii genome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design.

Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas.

Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression. Because of their modular composition, where a ligand-sensing aptamer domain is combined with an expression platform, riboswitches offer unique tools for synthetic biology applications. Here we took a mutational approach to determine functionally important nucleotide residues in the thiamine pyrophosphate (TPP) riboswitch in the THI4 gene of the model alga Chlamydomonas reinhardtii, allowing us to carry out aptamer swap using THIC aptamers from Chlamydomonas and Arabidopsis thaliana. These chimeric riboswitches displayed a distinct specificity and dynamic range of responses to different ligands. Our studies demonstrate ease of assembly as 5'UTR DNA parts, predictability of output, and utility for controlled production of a high-value compound in Chlamydomonas. The simplicity of riboswitch incorporation in current design platforms will facilitate the generation of genetic circuits to advance synthetic biology and metabolic engineering of microalgae.

Masson tumor (intravascular papillary endothelial hyperplasia) arising in a superficial temporal artery aneurysm.

Masson tumor (intravascular papillary endothelial hyperplasia) is a rare proliferation of endothelial cells within the wall of a vessel, often thought to represent an aberrant resolution of a thrombosis. We describe the unique case of a 75-year-old man who presented to the clinic with a tender, spontaneous aneurysmal dilation of his left superficial temporal artery (STA). Only 8% of all STA aneurysms are believed to be spontaneous true aneurysms, with the majority being post-traumatic pseudoaneurysms. After successful surgical resection, pathologic examination demonstrated a Masson tumor within an STA aneurysm. This paper describes a case in which both rare entities were discovered and briefly outlines the diagnostic and therapeutic modalities available.

Engineering xylose metabolism in thraustochytrid T18.

BACKGROUND: Thraustochytrids are heterotrophic, oleaginous, marine protists with a significant potential for biofuel production. High-value co-products can off-set production costs; however, the cost of raw materials, and in particular carbon, is a major challenge to developing an economical viable production process. The use of hemicellulosic carbon derived from agricultural waste, which is rich in xylose and glucose, has been proposed as a sustainable and low-cost approach. Thraustochytrid strain T18 is a commercialized environmental isolate that readily consumes glucose, attaining impressive biomass, and oil production levels. However, neither thraustochytrid growth capabilities in the presence of xylose nor a xylose metabolic pathway has been described. The aims of this study were to identify and characterize the xylose metabolism pathway of T18 and, through genetic engineering, develop a strain capable of growth on hemicellulosic sugars. RESULTS: Characterization of T18 performance in glucose/xylose media revealed diauxic growth and copious extracellular xylitol production. Furthermore, T18 did not grow in media containing xylose as the only carbon source. We identified, cloned, and functionally characterized a xylose isomerase. Transcriptomics indicated that this xylose isomerase gene is upregulated when xylose is consumed by the cells. Over-expression of the native xylose isomerase in T18, creating strain XI 16, increased xylose consumption from 5.2 to 7.6 g/L and reduced extracellular xylitol from almost 100% to 68%. Xylose utilization efficiency of this strain was further enhanced by over-expressing a heterologous xylulose kinase to reduce extracellular xylitol to 20%. Moreover, the ability to grow in media containing xylose as a sole sugar was dependent on the copy number of both xylose isomerase and xylulose kinase present. In fed-batch fermentations, the best xylose metabolizing isolate, XI-XK 7, used 137 g of xylose versus 39 g by wild type and produced more biomass and fatty acid. CONCLUSIONS: The presence of a typically prokaryotic xylose isomerase and xylitol production through a typically eukaryotic xylose reductase pathway in T18 is the first report of an organism naturally encoding enzymes from two native xylose metabolic pathways. Our newly engineered strains pave the way for the growth of T18 on waste hemicellulosic feedstocks for biofuel production.

Abnormal presentation of ascending cholangitis.

Ventriculo-peritoneal, -gallbladder, -pleural, -atrial, and -jugular shunts are all viable options in the treatment of hydrocephalus 1, 2, 3. Retrieval of these catheters can often be very difficult and may be unsuccessful or incomplete. Retained catheters can result in unforeseen and unexpected complications.

Beware of the interval cholecystectomy.

BACKGROUND: Despite limited data regarding the indications and effectiveness of percutaneous cholecystostomy (PC) in the treatment of acute cholecystitis (AC), usage has increased by over 500% since 1994. Many of these patients subsequently undergo interval cholecystectomy (IC), a procedure that has not been rigorously evaluated. This aim of this study was to quantify the morbidity and mortality associated with the IC. METHODS: We included all consecutive adult patients (>18 years old) who underwent PC and IC from January 2008 to December 2013. Conversion rate, length of operation, biliary injury, estimated blood loss, surgical site infection, length of stay, and mortality were compared with 227 patients who underwent cholecystectomy for AC during the same time interval. RESULTS: Of 18,501 patients who underwent cholecystectomy, 337 had at least one PC and 177 underwent subsequent IC. Compared with patients undergoing cholecystectomy for clinically diagnosed AC, patients undergoing IC were older (69.8 vs. 54.9 years; p < 0.001), thinner (body mass index, 28.7 vs. 31.1; p = 0.002), more complex by Tokyo grade (1.9 vs. 1.1; p < 0.001), and American Society of Anesthesia classification (3.0 vs. 2.5; p < 0.001), had longer operative times (120.7 vs. 92.5 minutes; p < 0.0001), more blood loss (30 vs. 15 mL; p = 0.01), and increased rates of conversion (26.6% vs. 12.8%; p < 0.001), surgical site infection (12.4% vs. 0.4%; p < 0.001), bowel injury (6.2% vs. 0.4%; p < 0.001), and 1-year mortality (15.3% vs. 0.4%; p < 0.01). Nonsignificant trends included significant biliary tract injury (3 vs. 0; p = 0.08) and longer length of stay (7.3 vs. 4.8 days; p = 0.39). Linear regression identified body mass index (p = 0.03), time from admission to PC (p = 0.03), and American Society of Anesthesia classification (p = 0.06) as predictors of a difficult IC. CONCLUSION: PC has been widely adopted with limited description of the subsequent IC. Our data detail the factors predicting the challenges of IC and document that it is a difficult operation associated with significant morbidity. LEVEL OF EVIDENCE: Therapeutic, level IV.

Contemporary Applications of Ultrasound in Abdominal Aortic Aneurysm Management.

Ultrasound (US) is a well-established screening tool for detection of abdominal aortic aneurysms (AAAs) and is currently recommended not only for those with a relevant family history but also for all men and high-risk women older than 65 years of age. The advent of minimally invasive endovascular techniques in the treatment of AAAs [endovascular aneurysm repair (EVAR)] has increased the need for repeat imaging, especially in the postoperative period. Nevertheless, preoperative planning, intraoperative execution, and postoperative surveillance all mandate accurate imaging. While computed tomographic angiography and angiography have dominated the field, repeatedly exposing patients to the deleterious effects of cumulative radiation and intravenous nephrotoxic contrast, US technology has significantly evolved over the past decade. In addition to standard color duplex US, 2D, 3D, or 4D contrast-enhanced US modalities are revolutionizing AAA management and postoperative surveillance. This technology can accurately measure AAA diameter and volume, and most importantly, it can detect endoleaks post-EVAR with high sensitivity and specificity. 4D contrast-enhanced US can even provide hemodynamic information about the branch vessels following fenestrated EVARs. The need for experienced US operators and accredited vascular labs is mandatory to guarantee the reliability of the results. This review article presents a comprehensive overview of the literature on the state-of-art US imaging in AAA management, including post-EVAR follow-up, techniques, and diagnostic accuracy.

Towards developing algal synthetic biology.

The genetic, physiological and metabolic diversity of microalgae has driven fundamental research into photosynthesis, flagella structure and function, and eukaryotic evolution. Within the last 10 years these organisms have also been investigated as potential biotechnology platforms, for example to produce high value compounds such as long chain polyunsaturated fatty acids, pigments and antioxidants, and for biodiesel precursors, in particular triacylglycerols (TAGs). Transformation protocols, molecular tools and genome sequences are available for a number of model species including the green alga Chlamydomonas reinhardtii and the diatom Phaeodactylum tricornutum, although for both species there are bottlenecks to be overcome to allow rapid and predictable genetic manipulation. One approach to do this would be to apply the principles of synthetic biology to microalgae, namely the cycle of Design-Build-Test, which requires more robust, predictable and high throughput methods. In this mini-review we highlight recent progress in the areas of improving transgene expression, genome editing, identification and design of standard genetic elements (parts), and the use of microfluidics to increase throughput. We suggest that combining these approaches will provide the means to establish algal synthetic biology, and that application of standard parts and workflows will avoid parallel development and capitalize on lessons learned from other systems.

Role of riboswitches in gene regulation and their potential for algal biotechnology.

Riboswitches are regulatory elements in messenger RNA to which specific ligands can bind directly in the absence of proteins. Ligand binding alters the mRNA secondary structure, thereby affecting expression of the encoded protein. Riboswitches are widespread in prokaryotes, with over 20 different effector ligands known, including amino acids, cofactors, and Mg(2+) ions, and gene expression is generally regulated by affecting translation or termination of transcription. In plants, fungi, and microalgae, riboswitches have been found, but only those that bind thiamine pyrophosphate. These eukaryotic riboswitches operate by causing alternative splicing of the transcript. Here, we review the current status of riboswitch research with specific emphasis on microalgae. We discuss new riboswitch discoveries and insights into the underlying mechanism of action, and how next generation sequencing technology provides the motivation and opportunity to improve our understanding of these rare but important regulatory elements. We also highlight the potential of microalgal riboswitches as a tool for synthetic biology and industrial biotechnology.

Establishing Chlamydomonas reinhardtii as an industrial biotechnology host.

Microalgae constitute a diverse group of eukaryotic unicellular organisms that are of interest for pure and applied research. Owing to their natural synthesis of value-added natural products microalgae are emerging as a source of sustainable chemical compounds, proteins and metabolites, including but not limited to those that could replace compounds currently made from fossil fuels. For the model microalga, Chlamydomonas reinhardtii, this has prompted a period of rapid development so that this organism is poised for exploitation as an industrial biotechnology platform. The question now is how best to achieve this? Highly advanced industrial biotechnology systems using bacteria and yeasts were established in a classical metabolic engineering manner over several decades. However, the advent of advanced molecular tools and the rise of synthetic biology provide an opportunity to expedite the development of C. reinhardtii as an industrial biotechnology platform, avoiding the process of incremental improvement. In this review we describe the current status of genetic manipulation of C. reinhardtii for metabolic engineering. We then introduce several concepts that underpin synthetic biology, and show how generic parts are identified and used in a standard manner to achieve predictable outputs. Based on this we suggest that the development of C. reinhardtii as an industrial biotechnology platform can be achieved more efficiently through adoption of a synthetic biology approach.

Green genes: bioinformatics and systems-biology innovations drive algal biotechnology.

Many species of microalgae produce hydrocarbons, polysaccharides, and other valuable products in significant amounts. However, large-scale production of algal products is not yet competitive against non-renewable alternatives from fossil fuel. Metabolic engineering approaches will help to improve productivity, but the exact metabolic pathways and the identities of the majority of the genes involved remain unknown. Recent advances in bioinformatics and systems-biology modeling coupled with increasing numbers of algal genome-sequencing projects are providing the means to address this. A multidisciplinary integration of methods will provide synergy for a systems-level understanding of microalgae, and thereby accelerate the improvement of industrially valuable strains. In this review we highlight recent advances and challenges to microalgal research and discuss future potential.

Unraveling vitamin B12-responsive gene regulation in algae.

Photosynthetic microalgae play a vital role in primary productivity and biogeochemical cycling in both marine and freshwater systems across the globe. However, the growth of these cosmopolitan organisms depends on the bioavailability of nutrients such as vitamins. Approximately one-half of all microalgal species requires vitamin B12 as a growth supplement. The major determinant of algal B12 requirements is defined by the isoform of methionine synthase possessed by an alga, such that the presence of the B12-independent methionine synthase (METE) enables growth without this vitamin. Moreover, the widespread but phylogenetically unrelated distribution of B12 auxotrophy across the algal lineages suggests that the METE gene has been lost multiple times in evolution. Given that METE expression is repressed by the presence of B12, prolonged repression by a reliable source of the vitamin could lead to the accumulation of mutations and eventually gene loss. Here, we probe METE gene regulation by B12 and methionine/folate cycle metabolites in both marine and freshwater microalgal species. In addition, we identify a B12-responsive element of Chlamydomonas reinhardtii METE using a reporter gene approach. We show that complete repression of the reporter occurs via a region spanning -574 to -90 bp upstream of the METE start codon. A proteomics study reveals that two other genes (S-Adenosylhomocysteine hydrolase and Serine hydroxymethyltransferase2) involved in the methionine-folate cycle are also repressed by B12 in C. reinhardtii. The strong repressible nature and high sensitivity of the B12-responsive element has promising biotechnological applications as a cost-effective regulatory gene expression tool.

Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity.

Thiamin (vitamin B1) is an essential micronutrient needed as a cofactor for many central metabolic enzymes. Animals must have thiamin in their diet, whereas bacteria, fungi, and plants can biosynthesize it de novo from the condensation of a thiazole and a pyrimidine moiety. Although the routes to biosynthesize these two heterocycles are not conserved in different organisms, in all cases exogenous thiamin represses expression of one or more of the biosynthetic pathway genes. One important mechanism for this control is via thiamin-pyrophosphate (TPP) riboswitches, regions of the mRNA to which TPP can bind directly, thus facilitating fine-tuning to maintain homeostasis. However, there is little information on how modulation of riboswitches affects thiamin metabolism in vivo. Here we use the green alga, Chlamydomonas reinhardtii, which regulates both thiazole and pyrimidine biosynthesis with riboswitches in the THI4 (Thiamin 4) and THIC (Thiamin C) genes, respectively, to investigate this question. Our study reveals that regulation of thiamin metabolism is not the simple dogma of negative feedback control. Specifically, balancing the provision of both of the heterocycles of TPP appears to be an important requirement. Furthermore, we show that the Chlamydomonas THIC riboswitch is controlled by hydroxymethylpyrimidine pyrophosphate, as well as TPP, but with an identical alternative splicing mechanism. Similarly, the THI4 gene is responsive to thiazole. The study not only provides insight into the plasticity of the TPP riboswitches but also shows that their maintenance is likely to be a consequence of evolutionary need as a function of the organisms' environment and the particular pathway used.

Comparative analysis of ß-carotene hydroxylase genes for astaxanthin biosynthesis.

Astaxanthin (3,3'-dihydroxy-4,4'-diketo-ß-carotene) (1) is a carotenoid of significant commercial value due to its superior antioxidant potential, application as a component of animal feeds, and ongoing research that links its application to the treatment and prevention of human pathologies. The high commercial cost of 1 is also based upon its complex synthesis. Chemical synthesis has been demonstrated, but produces a mixture of stereoisomers with limited applications. Production from biological sources is limited to natural producers with complex culture requirements. The biosynthetic pathway for 1 is well studied; however, questions remain that prevent optimized production in heterologous systems. Presented is a direct comparison of 12 ß-carotene (2) hydroxylases derived from archaea, bacteria, cyanobacteria, and plants. Expression in Escherichia coli enables a comparison of catalytic activity with respect to zeaxanthin (3) and 1 biosynthesis. The most suitable ß-carotene hydroxylases were subsequently expressed from an efficient dual expression vector, enabling 1 biosynthesis at levels up to 84% of total carotenoids. This supports efficient 1 biosynthesis by balanced expression of ß-carotene ketolase and ß-carotene hydroxylase genes. Moreover, our work suggests that the most efficient route for astaxanthin biosynthesis proceeds by hydroxylation of ß-carotene to zeaxanthin, followed by ketolation.

A high-throughput screen for the identification of improved catalytic activity: ß-carotene hydroxylase.

Astaxanthin is a natural product of immense value. Its biosynthesis has been investigated extensively and typically requires the independent activity of two proteins, a ß-carotene ketolase and ß-carotene hydroxylase. Rational engineering of this pathway has produced limited success with respect to the biological production of astaxanthin. Random mutagenesis of the ß-carotene ketolase has also been pursued. However, to date, no suitable method has been developed for the investigation of the ß-carotene hydroxylase because ß-carotene and zeaxanthin cannot be differentiated visually, unlike ß-carotene and canthaxanthin. Thus, random mutagenesis and efficient selection of improved ß-carotene hydroxylase clones is not feasible. Presented here are the steps required for the efficient generation of a ß-carotene hydroxylase random mutagenesis library in Escherichia coli. Subsequently presented is a novel high-throughput screening method for the rapid identification of clones with enhanced ß-carotene hydroxylase activity. The validity of the presented method is confirmed by functional expression of the mutated proteins, combined with accurate quantification of produced carotenoids. The developed method has potential applications in the development of biological systems for improved carotenoid biosynthesis, as well as robust astaxanthin production.

Efficient extraction of canthaxanthin from Escherichia coli by a 2-step process with organic solvents.

Canthaxanthin has a substantial commercial market in aquaculture, poultry production, and cosmetic and nutraceutical industries. Commercial production is dominated by chemical synthesis; however, changing consumer demands fuel research into the development of biotechnology processes. Highly productive microbial systems to produce carotenoids can be limited by the efficiency of extraction methods. Extraction with hexane, acetone, methanol, 2-propanol, ethanol, 1-butanol, tetrahydrofuran and ethyl acetate was carried out with each solvent separately, and subsequently the most efficient solvents were tested in combination, both as mixtures and sequentially. Sequential application of methanol followed by acetone proved most efficient. Extraction efficiency remained stable over a solvent to biomass range of 100:1 to 55:1, but declined significantly at a ratio of 25:1. Application of this method to a canthaxanthin-producing Escherichia coli production system enabled efficient canthaxanthin extraction of up to 8.5 mg g(-1) dry biomass.

Characterization of cyanobacterial beta-carotene ketolase and hydroxylase genes in Escherichia coli, and their application for astaxanthin biosynthesis.

Carotenoid biosynthesis is highly conserved and well characterized up to the synthesis of beta-carotene. Conversely, the synthesis of astaxanthin from beta-carotene is less well characterized. Regardless, astaxanthin is a highly sought natural product, due to its various industrial applications and elevated antioxidant capacity. In this article, 12 beta-carotene ketolase and 4 beta-carotene hydroxylase genes, isolated from 5 cyanobacterial species, are investigated for their function, and potential for microbial astaxanthin synthesis. Further, this in vivo comparison identifies and applies the most promising genetic elements within a dual expression vector, which is maintained in Escherichia coli. Here, combined overexpression of individual beta-carotene ketolase and beta-carotene hydroxylase genes, within a beta-carotene accumulating host, enables a 23.5-fold improvement in total carotenoid yield (1.99 mg g(-1)), over the parental strain, with >90% astaxanthin.