An overlooked phenomenon: complex interactions of potential error sources on the quality of bacterial de novo genome assemblies.

BACKGROUND: Parameters adversely affecting the contiguity and accuracy of the assemblies from Illumina next-generation sequencing (NGS) are well described. However, past studies generally focused on their additive effects, overlooking their potential interactions possibly exacerbating one another's effects in a multiplicative manner. To investigate whether or not they act interactively on de novo genome assembly quality, we simulated sequencing data for 13 bacterial reference genomes, with varying levels of error rate, sequencing depth, PCR and optical duplicate ratios. RESULTS: We assessed the quality of assemblies from the simulated sequencing data with a number of contiguity and accuracy metrics, which we used to quantify both additive and multiplicative effects of the four parameters. We found that the tested parameters are engaged in complex interactions, exerting multiplicative, rather than additive, effects on assembly quality. Also, the ratio of non-repeated regions and GC% of the original genomes can shape how the four parameters affect assembly quality. CONCLUSIONS: We provide a framework for consideration in future studies using de novo genome assembly of bacterial genomes, e.g. in choosing the optimal sequencing depth, balancing between its positive effect on contiguity and negative effect on accuracy due to its interaction with error rate. Furthermore, the properties of the genomes to be sequenced also should be taken into account, as they might influence the effects of error sources themselves.

[Regional Anaesthesia in the Prehospital Setting]. / Regionalanästhesie in der präklinischen Notfallmedizin.

Pain is often the main symptom in trauma patients. Although peripheral nerve blocks (PNB) provide fast, safe, and adequate analgesia, they are currently only rarely used outside the perioperative setting. In Germany, intravenous analgesia with non-opioid analgesics (NOPA) and strong opioids is the main treatment concept for prehospital pain. However, the use of highly potent opioids can be associated with significant side effects, especially in emergency patients. Therefore, PNBs are used in many hospitals for the treatment of perioperative pain. As with perioperative use, the advantages of early PNB in the prehospital analgesic treatment of trauma patients are obvious, especially for elderly and multimorbid patients. Early prehospital PNB can also facilitate the reduction of dislocated fractures or dislocated joints as well as the technical rescue of trauma patients. Common geriatric fractures, such as proximal femur or humerus fractures, can be treated appropriately and adequately with PNB.In this article, we show which PNB procedures can be useful in prehospital patient care and which requirements should be met for their safe use. We also present a concept for assessing whether and to what extent the prehospital use of PNB is indicated and appropriate. The aim of this article is to draw attention to PNB as a possible part of prehospital care concepts for trauma patients and to discuss its prehospital use.

Increasing Top-Down Mass Spectrometry Sequence Coverage by an Order of Magnitude through Optimized Internal Fragment Generation and Assignment.

A major limitation of intact protein fragmentation is the lack of sequence coverage within proteins' interiors. We show that collisionally activated dissociation (CAD) produces extensive internal fragmentation within proteins' interiors that fill the existing gaps in sequence coverage, including disulfide loop regions that cannot be characterized using terminal fragments. A barrier to the adoption of internal fragments is the lack of methods for their generation and assignment. To provide these, we explore the effects of protein size, mass accuracy, internal fragment size, CAD activation energy, and data preprocessing upon the production and identification of internal fragments. We also identify and mitigate the major source of ambiguity in internal fragment identification, which we term "frameshift ambiguity." Such ambiguity results from sequences containing any "middle" portion surrounded by the same composition on both termini, which upon fragmentation can produce two internal fragments of identical mass, yet out of frame by one or more amino acids (e.g., TRAIT producing TRAI or RAIT). We show that such instances permit the a priori assignment of the middle sequence portion. This insight and our optimized methods permit the unambiguous assignment of greater than 97% of internal fragments using only the accurate mass. We show that any remaining ambiguity in internal fragment assignment can be removed by consideration of fragmentation propensities or by (pseudo)-MS3. Applying these methods resulted in a 10-fold and 43-fold expanded number of identified ions, and a concomitant 7- and 16-fold increase in fragmentation sites, respectively, for native and reduced forms of a disease-associated SOD1 variant.

Genetically Encoded Fluorescent Proteins Enable High-Throughput Assignment of Cell Cohorts Directly from MALDI-MS Images.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) provides a unique in situ chemical profile that can include drugs, nucleic acids, metabolites, lipids, and proteins. MSI of individual cells (of a known cell type) affords a unique insight into normal and disease-related processes and is a prerequisite for combining the results of MSI and other single-cell modalities (e.g. mass cytometry and next-generation sequencing). Technological barriers have prevented the high-throughput assignment of MSI spectra from solid tissue preparations to their cell type. These barriers include obtaining a suitable cell-identifying image (e.g. immunohistochemistry) and obtaining sufficiently accurate registration of the cell-identifying and MALDI-MS images. This study introduces a technique that overcame these barriers by assigning cell type directly from mass spectra. We hypothesized that, in MSI from mice with a defined fluorescent protein expression pattern, the fluorescent protein's molecular ion could be used to identify cell cohorts. A method was developed for the purification of enhanced yellow fluorescent protein (EYFP) from mice. To determine EYFP's molecular mass for MSI studies, we performed intact mass analysis and characterized the protein's primary structure and post-translational modifications through various techniques. MALDI-MSI methods were developed to enhance the detection of EYFP in situ, and by extraction of EYFP's molecular ion from MALDI-MS images, automated, whole-image assignment of cell cohorts was achieved. This method was validated using a well-characterized mouse line that expresses EYFP in motor and sensory neurons and should be applicable to hundreds of commercially available mice (and other animal) strains comprising a multitude of cell-specific fluorescent labels.

In Vitro Liquid Extraction Surface Analysis Mass Spectrometry (ivLESA-MS) for Direct Metabolic Analysis of Adherent Cells in Culture.

Conventional metabolomic methods include extensive sample preparation steps and long analytical run times, increasing the likelihood of processing artifacts and limiting high throughput applications. We present here in vitro liquid extraction surface analysis mass spectrometry (ivLESA-MS), a variation on LESA-MS, performed directly on adherent cells grown in 96-well cell culture plates. To accomplish this, culture medium was aspirated immediately prior to analysis, and metabolites were extracted using LESA from the cell monolayer surface, followed by nano-electrospray ionization and MS analysis in negative ion mode. We applied this platform to characterize and compare lipidomic profiles of multiple breast cancer cell lines growing in culture (MCF-7, ZR-75-1, MDA-MB-453, and MDA-MB-231) and revealed distinct and reproducible lipidomic signatures between the cell lines. Additionally, we demonstrated time-dependent processing artifacts, underscoring the importance of immediate analysis. ivLESA-MS represents a rapid in vitro metabolomic method, which precludes the need for quenching, cell harvesting, sample preparation, and chromatography, significantly shortening preparation and analysis time while minimizing processing artifacts. This method could be further adapted to test drugs in vitro in a high throughput manner.

Heavy Sugar and Heavy Water Create Tunable Intact Protein Mass Increases for Quantitative Mass Spectrometry in Any Feed and Organism.

Stable isotope labeling techniques for quantitative top-down proteomics face unique challenges. These include unpredictable mass shifts following isotope labeling, which impedes analysis of unknown proteins and complex mixtures and exponentially greater susceptibility to incomplete isotope incorporation, manifesting as broadening of labeled intact protein peaks. Like popular bottom-up isotope labeling techniques, most top-down labeling methods are restricted to defined media/feed as well as amino acid auxotrophic organisms. We present a labeling method optimized for top-down proteomics that overcomes these challenges. We demonstrated this method through the spiking of 13C-sugar or 2H-water into standard laboratory feedstocks, resulting in tunable intact protein mass increases (TIPMI). After mixing of labeled and unlabeled samples, direct comparison of light and heavy peaks allowed for the relative quantitation of intact proteins in three popular model organisms, including prokaryotic and eukaryotic microorganisms and an animal. This internal standard method proved to be more accurate than label-free quantitation in our hands. Advantages over top-down SILAC include working equally well in nutrient-rich media, conceivably expanding applicability to any organism and all classes of biomolecules, not requiring high-resolving power MS for quantitation and being relatively inexpensive.

Dysregulation of very-long-chain fatty acid metabolism causes membrane saturation and induction of the unfolded protein response.

The unfolded protein response (UPR) senses defects in the endoplasmic reticulum (ER) and orchestrates a complex program of adaptive cellular remodeling. Increasing evidence suggests an important relationship between lipid homeostasis and the UPR. Defects in the ER membrane induce the UPR, and the UPR in turn controls the expression of some lipid metabolic genes. Among lipid species, the very-long-chain fatty acids (VLCFAs) are relatively rare and poorly understood. Here, we show that loss of the VLCFA-coenzyme A synthetase Fat1, which is essential for VLCFA utilization, results in ER stress with compensatory UPR induction. Comprehensive lipidomic analyses revealed a dramatic increase in membrane saturation in the fat1Δ mutant, likely accounting for UPR induction. In principle, this increased membrane saturation could reflect adaptive membrane remodeling or an adverse effect of VLCFA dysfunction. We provide evidence supporting the latter, as the fat1Δ mutant showed defects in the function of Ole1, the sole fatty acyl desaturase in yeast. These results indicate that VLCFAs play essential roles in protein quality control and membrane homeostasis and suggest an unexpected requirement for VLCFAs in Ole1 function.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Minimal n-noids in hyperbolic and anti-de Sitter 3-space.

We construct minimal surfaces in hyperbolic and anti-de Sitter 3-space with the topology of a n-punctured sphere by loop group factorization methods. The end behaviour of the surfaces is based on the asymptotics of Delaunay-type surfaces, i.e. rotational symmetric minimal cylinders. The minimal surfaces in H3 extend to Willmore surfaces in the conformal 3-sphere S 3 = H3∪S2∪H3.

Extraskeletal Osteosarcoma: A Rare Case Arising in Phthisis Bulbi with a Review of the Literature.

A 60-year-old male presented with an increasingly painful and swollen phthisical eye. Enucleation revealed a large mass obliterating the eye with extension into the adjacent extraocular muscle. Histologic examination showed high-grade osteosarcoma. Systemic work-up showed no disease elsewhere, and a diagnosis of orbital extraskeletal osteosarcoma was rendered. Complete resection was not possible, and neoadjuvant radiation was given. The patient subsequently developed pulmonary metastasis and died of disease 5 months after initial diagnosis. Given the highly aggressive nature of this malignancy, raising awareness that extraskeletal osteosarcoma may arise in the background of phthisis bulbi will facilitate timely and accurate diagnosis and treatment.

Tributylphosphate extraction behavior of bismuthate-oxidized americium.

Higher oxidation states of americium have long been known; however, options for their preparation in acidic solution are limited. The conventional choice, silver-catalyzed peroxydisulfate, is not useful at nitric acid concentrations above about 0.3 M. We investigated the use of sodium bismuthate as an oxidant for Am (3+) in acidic solution. Room-temperature oxidation produced AmO 2 (2+) quantitatively, whereas oxidation at 80 degrees C produced AmO 2 (+) quantitatively. The efficacy of the method for the production of oxidized americium was verified by fluoride precipitation and by spectroscopic absorbance measurements. We performed absorbance measurements using a conventional 1 cm cell for high americium concentrations and a 100 cm liquid waveguide capillary cell for low americium concentrations. Extinction coefficients for the absorbance of Am (3+) at 503 nm, AmO 2 (+) at 514 nm, and AmO 2 (2+) at 666 nm in 0.1 M nitric acid are reported. We also performed solvent extraction experiments with the hexavalent americium using the common actinide extraction ligand tributyl phosphate (TBP) for comparison to the other hexavalent actinides. Contact with 30% tributyl phosphate in dodecane reduced americium; it was nevertheless extracted using short contact times. The TBP extraction of AmO 2 (2+) over a range of nitric acid concentrations is shown for the first time and was found to be analogous to that of uranyl, neptunyl, and plutonyl ions.

Acute inflammatory demyelinating optic neuritis: current concepts in diagnosis and management.

BACKGROUND: Optic neuritis (ON), defined as an inflammatory demyelinating optic neuropathy, is a frequent cause of visual loss owing to optic nerve dysfunction in young or middle-aged patients. ON can be seen in isolation or in association with multiple sclerosis (MS). Highlighting the importance of this association is the fact that approximately 20% of patients with MS will present with ON. METHODS: Review was conducted of the literature and pertinent clinical trials. CONCLUSION: Although the vision prognosis of patients with ON is excellent, with or without the use of corticosteroids, a minority of patients will suffer from significantly poor vision. ON may be the heralding manifestation of MS; the risk stratification for the future development of MS in patients presenting with ON can be determined by the number of white matter lesions on the baseline cerebral magnetic resonance imaging study. To date, 2 randomized, placebo-controlled studies have found that patients with a clinically isolated demyelinating syndrome, such as ON, at risk for MS, may benefit from the early institution of a disease-modifying drug.

The Eger Macular Stressometer: pilot study.

PURPOSE: To evaluate the sensitivity of the Eger Macular Stressometer (EMS) for early screening of age-related macular degeneration (AMD) in a clinical practice. We examined the null hypothesis that AMD eyes have EMS recovery times (RTs) that do not differ from eyes with cataract, diabetic retinopathy, or glaucoma. DESIGN: The design of this study was a nonrandomized clinical trial. METHODS: Ninety-two eyes from 92 patients with vision 20/80 or better, age 50 and older, of either gender, and any ethnic origin, were recruited into one of four groups: AMD (30 eyes), normal or mild cataract (30 eyes), diabetic retinopathy (16 eyes), and glaucoma (16 eyes). Recovery times were obtained with the EMS, according to manufacturer's instructions. RESULTS: The mean (SD) [median] RT for the AMD group was 11.8 (7.6) [9] seconds, the normal/cataract group 10.0 (4.3) [9] seconds, the diabetic retinopathy group 8.4 (3.0) [8] seconds, and glaucoma group 8.6 (2.4) [8] seconds. Recovery time did not appear to be related to group (P =.58), age (P =.50), visual acuity (P =.52), or sex (P =.23). CONCLUSIONS: We found EMS RT distributions did not differ between AMD, cataract, diabetic retinopathy, and glaucoma groups. The EMS in its current form is not a sensitive screening tool for AMD. Further testing is needed to examine EMS sensitivity with other macular diseases such as central serous choroidopathy and diabetic macular edema.

Superior ophthalmic vein thrombosis in a patient with dacryocystitis-induced orbital cellulitis.

A 71-year-old-man presented with chronic left-sided epiphora and a 5-day history of progressive left orbital swelling that had started with a "bump" on the left side of his nose. Orbital CT revealed left-sided preseptal and postseptal inflammation, along with marked thickening of the left superior ophthalmic vein. Orbital MRI with gadolinium enhancement and fat suppression revealed a low-intensity signal in the left superior ophthalmic vein, consistent with a superior ophthalmic vein thrombosis. There was no cavernous sinus involvement. A diagnosis was made of left-sided dacryocystitis-induced orbital cellulitis and superior ophthalmic vein thrombosis. Treatment consisted of intravenous vancomycin, followed by early dacryocystorhinostomy and postoperative intravenous dexamethasone. Anticoagulation was not used. Within 1 week after surgery, the orbital congestion had dramatically improved. Though rare, isolated superior ophthalmic vein thrombosis can be a harbinger of cavernous sinus thrombosis; therefore, early detection is the key to avoiding cavernous sinus thrombosis.