Observation of Silicone Oil Within the Vitreous and Sclera Following Intravitreal Administration of Biotherapeutics Using Insulin Syringes in Cynomolgus Monkeys.

Silicone oil droplets have been reported in the eyes of human patients following intravitreous (IVT) injections with several marketed biotherapeutic products. Intravitreous administration of a novel biotherapeutic in a 14-week cynomolgus monkey study using insulin syringes was associated with 2, non-test-article-related phenomena: "vitreous floater/clear sphere" on indirect ophthalmoscopy and intrascleral "foreign material near injection track" on histopathology. Retrospective analysis of 81 other preclinical studies of IVT administration of novel biotherapeutics found a greater frequency of clear spheres in monkey IVT studies using insulin syringes and formulations containing polysorbate.

Quantitation of Cyclosporin A in Cell Culture Media by Differential Mobility Mass Spectrometry (DMS-MS/MS).

Cell permeability is an important factor in determining the bioavailability of therapeutics that is usually measured by cell culture testing. The concentration of pharmaceutical in a medium such as Hank's Balanced Salt Solution with HEPES organic buffer (HBSS-HEPES) is measured at a series of time points, making simplicity and high throughput of the analytical method important characteristics. We report an electrospray differential mobility spectrometry mass spectrometry method (nanoESI-DMS-MS) for the rapid determination of cyclosporin A (CsA, cyclosporine) concentration in such a buffer.

The expanding role of mass spectrometry in the field of vaccine development.

Biological mass spectrometry has evolved as a core analytical technology in the last decade mainly because of its unparalleled ability to perform qualitative as well as quantitative profiling of enormously complex biological samples with high mass accuracy, sensitivity, selectivity and specificity. Mass spectrometry-based techniques are also routinely used to assess glycosylation and other post-translational modifications, disulfide bond linkage, and scrambling as well as for the detection of host cell protein contaminants in the field of biopharmaceuticals. The role of mass spectrometry in vaccine development has been very limited but is now expanding as the landscape of global vaccine development is shifting towards the development of recombinant vaccines.

Tracking matrix effects in the analysis of DNA adducts of polycyclic aromatic hydrocarbons.

LC-MS using electrospray ionization is currently the method of choice in bio-organic analysis covering a wide range of applications in a broad spectrum of biological media. The technique is noted for its high sensitivity but one major limitation that hinders achievement of its optimal sensitivity is the signal suppression due to matrix inferences introduced by the presence of co-extracted compounds during the sample preparation procedure. The analysis of DNA adducts of common environmental carcinogens is particularly sensitive to such matrix effects as sample preparation is a multistep process which involves "contamination" of the sample due to the addition of enzymes and other reagents for digestion of the DNA in order to isolate the analyte(s).

Translational assessment of cardiac contractility by echocardiography in the telemetered rat.

Introduction: Cardiac contractility was evaluated using standard inotropic agents in rats. We compared indices of cardiac contractility, i.e.

Determination of site selectivity of different carcinogens for preferential mutational hot spots in oligonucleotide fragments by ion-pair reversed-phase nano liquid chromatography tandem mass spectrometry.

Ion-pair reversed-phase nano liquid chromatography coupled with nanospray ion trap mass spectrometry was used to investigate site selectivity of the known carcinogens N-acetoxy-2-acetylaminofluorene, N-hydroxy-4-aminobiphenyl and (+/-)-anti-benzo[a]pyrene diol epoxide with the synthetic double-strand 14-mer long oligonucleotide fragment of the p53 gene containing two mutational hot-spot codons (5'-P-ACC155 CGC156 GTC157 CGC158 GC/5'-GCG CGG ACG CGG GT). The investigation was performed using a monolithic polystyrene divinylbenzene capillary column and triethylammonium bicarbonate as an ion-pair reagent. The exact location of the carcinogen on the modified oligonucleotide backbone was determined using characteristic collision-induced dissociation fragmentation patterns obtained under negative-ion mode ionization.

Hip arthroscopy: from the beginning to the future--an innovator's perspective.

Hip arthroscopy is one of the fastest-growing areas of orthopaedic surgery. There are many reasons for this, including a better understanding of the pathophysiology of damage to the hip joint, improvements in imaging and technology advancements in arthroscopic instrumentation. This manuscript documents the historical development of hip arthroscopy, in general, as well as advances and ideas that have led to common techniques with regard to portal placement, traction and instrumentation.

Understanding gas phase modifier interactions in rapid analysis by differential mobility-tandem mass spectrometry.

A systematic study involving the use and optimization of gas-phase modifiers in quantitative differential mobility-mass spectrometry (DMS-MS) analysis is presented using nucleoside-adduct biomarkers of DNA damage as an important reference point for analysis in complex matrices. Commonly used polar protic and polar aprotic modifiers have been screened for use against two deoxyguanosine adducts of DNA: N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP) and N-(deoxyguanosin-8-y1)-2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP). Particular attention was paid to compensation voltage (CoV) shifts, peak shapes, and product ion signal intensities while optimizing the DMS-MS conditions.

Extending the dynamic range of the ion trap by differential mobility filtration.

A miniature, planar, differential ion mobility spectrometer (DMS) was interfaced to an LCQ classic ion trap to conduct selective ion filtration prior to mass analysis in order to extend the dynamic range of the trap. Space charge effects are known to limit the functional ion storage capacity of ion trap mass analyzers and this, in turn, can affect the quality of the mass spectral data generated. This problem is further exacerbated in the analysis of mixtures where the indiscriminate introduction of matrix ions results in premature trap saturation with non-targeted species, thereby reducing the number of parent ions that may be used to conduct MS/MS experiments for quantitation or other diagnostic studies.

A differential mobility spectrometry/mass spectrometry platform for the rapid detection and quantitation of DNA adduct dG-ABP.

Rationale: There is continued interest in exploring new analytical technologies for the detection and quantitation of DNA adducts, biomarkers which provide direct evidence of exposure and genetic damage in cells. With the goal of reducing clean-up steps and improving sample throughput, a Differential Mobility Spectrometry/Mass Spectrometry (DMS/MS) platform has been introduced for adduct analysis.

Methods: A DMS/MS platform has been utilized for the analysis of dG-ABP, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl (4-ABP).

Risk of sciatic nerve traction injury during hip arthroscopy—is it the amount or duration? An intraoperative nerve monitoring study.

Background: Using intraoperative nerve monitoring we prospectively studied the prevalence, pattern, and predisposing factors for sciatic nerve traction injury during hip arthroscopy.

Methods: The transcranial motor (tcMEP) and/or somatosensory (SSEP) evoked potentials of seventy-six patients undergoing hip arthroscopy in the lateral position were recorded. Changes in the posterior tibial and common peroneal nerves were evaluated to assess the effects of the amount and duration of traction on nerve function.

The analysis of DNA adducts: the transition from (32)P-postlabeling to mass spectrometry.

The technique of (32)P-postlabeling, which was introduced in 1982 for the analysis of DNA adducts, has long been the method of choice for in vivo studies because of its high sensitivity as it requires only <10μg DNA to achieve the detection of 1 adduct in 10(10) normal bases. (32)P-postlabeling has therefore been utilized in numerous human and animal studies of DNA adduct formation. Like all techniques (32)P-postlabeling does have several disadvantages including the use of radioactive phosphorus, lack of internal standards, and perhaps most significantly does not provide any structural information for positive identification of unknown adducts, a shortcoming that could significantly hamper progress in the field.

Sialic acid speciation using capillary electrophoresis: optimization of analyte derivatization and separation.

Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) was employed for rapid sialic acid speciation, facilitating the quantitative determination of N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) on glycoproteins. Derivatization of the sialic acids with 2-aminoacridone (2-AMAC), using classical reductive amination in a nonaqueous solvent, led to the spontaneous decarboxylation of the sialic acid residues as determined by CE-LIF and offline mass spectrometric analysis. Modification of both the labeling conditions, to drive the decarboxylation reaction to completion and the CE-LIF parameters to separate the neutral species by complexation with a neutral coated capillary and borate reversed polarity, led to a robust platform for the rapid, sensitive, and quantitative speciation of sialic acids.

GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization and sequencing of oligonucleotide adducts.

The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in-house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids.

Reversed-phase ion-pair liquid chromatography electrospray ionization tandem mass spectrometry for separation, sequencing and mapping of sites of base modification of isomeric oligonucleotide adducts using monolithic column.

In this manuscript, an efficient high resolution reversed phase-ion pairing-liquid chromatography electrospray ionization tandem mass spectrometry (RP-IP-LC-MS/MS) method for separation of isomeric modified oligonucleotides using a polymeric (styrene-divinylbenzene) monolithic capillary column is presented. The effects of different ion pairing reagents (IPR), their concentration, mobile phase additives and conditions were evaluated towards achieving the highest possible resolution and chromatographic separation of isomeric oligonucleotides. Ion pairing reagents and mobile phase conditions were evaluated using as model N-acetylaminofluorene [AAF] adducted ss-oligonucleotides (CCC CGA GCA ATC TCA AT).

Mass spectrometric based analysis, characterization and applications of circulating cell free DNA isolated from human body fluids.

In the past decade, cell free DNA, or circulating cell free DNA, or cell free circulating DNA, isolated from body fluids such as plasma/serum/urine has emerged as an important tool for clinical diagnostics. The molecular biology of circulating cell free DNA is poorly understood but there is currently an increased effort to understand the origin, mechanism of its circulation, and sensitive characterization for the development of diagnostic applications. There has been considerable progress towards these goals using real time polymerase chain reaction technique (rt-PCR).

The vanishing zero revisited: thresholds in the age of genomics.

The concept of the vanishing zero, which was first discussed 50 years ago in relation to pesticide residues in foods and food crops, focused on the unintended regulatory consequences created by ever-increasing sensitivity and selectivity of analytical methods, in conjunction with the ambiguous wording of legislation meant to protect public health. In the interim, the ability to detect xenobiotics in most substrates has increased from tens of parts per million to parts per trillion or less, challenging our ability to interpret the biological significance of exposures at the lowest detectable levels. As a result the focus of risk assessment, especially for potential carcinogens, has shifted from defining an acceptable level, to extrapolating from the best available analytical results.

Assessing the limitations to terpenoid indole alkaloid biosynthesis in Catharanthus roseus hairy root cultures through gene expression profiling and precursor feeding.

The production of pharmaceutically important terpenoid indole alkaloids (TIAs) from Catharanthus roseus is partly regulated at the transcriptional level. In this study, limitations in TIA biosynthesis from C. roseus hairy root cultures were assessed through gene expression profiling and precursor feeding.

The influence of cytosine methylation on the chemoselectivity of benzo[a]pyrene diol epoxide-oligonucleotide adducts determined using nanoLC/MS/MS.

Benzo[a]pyrene is a major carcinogen implicated in human lung cancer. Almost 60% of human lung cancers have a mutation in the p53 tumor suppressor gene at several specific codons. An on-line nanoLC/MS/MS method using a monolithic nanocolumn was applied to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, (+/-)-(7R,8S,9S,10R)-benzo[a]pyrene 7,8-diol 9,10-epoxide [(+/-)-anti-benzo[a]pyrene diol epoxide (BPDE)], which was reacted in vitro with a synthesized 14-mer double stranded oligonucleotide (5'-ACCCG5CG7TCCG11CG13C-3'/5'-GCGCGGGCGCGGGT-3') derived from the p53 gene.

Surgical correction of "cam-type" femoroacetabular impingement: a cadaveric comparison of open versus arthroscopic debridement.

Purpose: The goal of this study was to compare open and arthroscopic surgical techniques for "cam-type" femoroacetabular impingement in terms of feasibility and reliability.

Methods: We used 5 fresh-frozen cadaver specimens (10 hips). Anteroposterior and cross-table radiographs were taken for each.

Separation and sequencing of isomeric oligonucleotide adducts using monolithic columns by ion-pair reversed-phase nano-HPLC coupled to ion trap mass spectrometry.

An ion-pair reversed-phase nano-high-performance liquid chromatography (IP-RP-nano-HPLC) method using a monolithic poly(styrene-divinylbenzene) (PS-DVB) column coupled to nanoelectrospray ionization mass spectrometry (nano-ESI-MS) was evaluated to separate and identify isomeric oligonucleotide adducts derived from the covalent binding of (+/-)-anti-7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] to double-stranded (ds) 5'-PO4--ACCCGCGTCCGCGC-3'/5'-GCGCGGGCGCGGGT-3' oligonucleotide. The influence of three different nanospray emitters on electrospray signal was evaluated in terms of analyte ion sensitivity. The best nanoelectrospray performance for the oligonucleotides was observed with the distal metal-coated emitter.

Hip arthroscopy by the lateral approach.

Performing hip arthroscopy with the patient in the lateral decubitus position is advantageous in aiding in visualization of the hip joint, in maneuvering instruments in obese patients, and in facilitating entry to the hip joint in patients with spurs on the anterolateral aspect of the acetabulum. The patient is placed in the lateral decubitus position with the hip on which the surgery is being performed on the top. The leg is placed in traction and a well-padded perineal post is applied for countertraction.