Monomer Levels Research Articles

Analysis of Alkylphenol ethoxylates (APEOs) from tannery sediments using LC–MS and their environmental risks

Alkyl phenol polyethoxylates (APEOs) are a major group of high production volume chemicals, extensively used as nonionic surfactants in industrial, agricultural and domestic sectors. These surfactants and its respective metabolites are found to be persistent and toxic. Mainly, they act as endocrine disruptors by mimicking the natural hormones. India being a developing country, witnesses pollution due to various industries including tannery. In the present study, sediment samples were collected from the Vellore district of Tamil Nadu, India to investigate the occurrence of APEOs. The sediments were extracted by ultrasonication and analyzed in a liquid chromatography mass spectrometer. Octyl phenol ethoxylates (OPEOs) were not observed in any of the samples. Nonyl phenol ethoxylates (NPEOs) were detected in the range of ND – 36 mg/kg with 85 % detection frequency. The occurrence of NPEOs in sediment indicates its wide usage in tannery and its partitioning behavior in environment. The levels of NPEOs in the study were found to be unsafe according to the sediment guidelines of various studies. Since NPEOs were observed in sediment samples, possibilities of occurrence of their monomers and metabolites cannot be ruled out. Therefore, further studies are warranted for understanding the levels of monomers and metabolites in order to ascertain the environmental risks more appropriately.

Quantification of the HIV-1 virus-like particle production process by super-resolution imaging: From VLP budding to nanoparticle analysis.

Virus-like particles (VLPs) offer great promise in the field of nanomedicine. Enveloped VLPs are a class of these nanoparticles and their production process occurs by a budding process, which is known to be the most critical step at intracellular level. In this study, we developed a novel imaging method based on super-resolution fluorescence microscopy (SRFM) to assess the generation of VLPs in living cells. This methodology was applied to study the production of Gag VLPs in three animal cell platforms of reference: HEK 293-transient gene expression (TGE), High Five-baculovirus expression vector system (BEVS) and Sf9-BEVS. Quantification of the number of VLP assembly sites per cell ranged from 500 to 3,000 in the different systems evaluated. Although the BEVS was superior in terms of Gag polyprotein expression, the HEK 293-TGE platform was more efficient regarding the assembly of Gag as VLPs. This was translated into higher levels of non-assembled Gag monomer in BEVS harvested supernatants. Furthermore, the presence of contaminating nanoparticles was evidenced in all three systems, specifically in High Five cells. The SRFM-based method here developed was also successfully applied to measure the concentration of VLPs in crude supernatants. The lipid membrane of VLPs and the presence of nucleic acids alongside these nanoparticles could also be detected using common staining procedures. Overall, a complete picture of the VLP production process was achieved in these three production platforms. The robustness and sensitivity of this new approach broaden the applicability of SRFM toward the development of new detection, diagnosis and quantification methods based on confocal microscopy in living systems.

Nuclear actin regulates cell proliferation and migration via inhibition of SRF and TEAD

Actin dynamics regulate cell behaviour in response to physiological signals. Here we demonstrate a novel role for nuclear actin in inhibiting cell proliferation and migration. We demonstrate that physiological signals that elevate cAMP, which is anti-mitogenic in vascular smooth muscle cells, increases nuclear actin monomer levels. Expression of a nuclear-targeted polymerisation-defective actin mutant (NLS-ActinR62D) inhibited proliferation and migration. Preventing nuclear actin monomer accumulation by enhancing its nuclear export or polymerisation reversed the anti-mitogenic and anti-migratory effects of cAMP. Transcriptomic analysis identified repression of proliferation and migration associated genes regulated by serum response factor (SRF) and TEA Domain (TEAD) transcription factors. Accordingly, NLS-ActinR62D inhibited SRF and TEAD activity and target gene expression, and these effects were reversed by constitutively-active mutants of the TEAD and SRF co-factors YAP, TAZ and MKL1. In summary, intranuclear actin inhibits proliferation and migration by inhibiting YAP-TEAD and MKL-SRF activity. This mechanism explains the anti-mitogenic and anti-migratory properties of physiological signals that elevate cAMP. SummaryMcNeill et al show that increased levels of intranuclear actin monomer inhibit cell proliferation and migration by inhibiting MKL1-SRF and YAP/TAZ-TEAD-dependent gene expression. This mechanism mediates the anti-mitogenic and anti-migratory effects of physiological signals that elevate cyclic-AMP.

Alpha-synuclein differentially reduces surface expression of N-methyl-d-aspartate receptors in the aging human brain

The aging brain is associated with reduced cell surface expression of N-methyl-d-aspartate receptors (NMDARs), but the mechanism remains poorly understood. In the present study, we showed that in the striatum and hippocampus but not the cerebellum and parietal cortex, levels of α-synuclein monomers and oligomers increased with age, which correlated negatively with the expression of GluN1, and positively with the expression of total Rab5B. The oligomer-α-synuclein exhibited a stronger correlation with the expression of surface GluN1 and total Rab5B. In MES23.5 cells, the monomer- or oligomer-α-synuclein were shown to increase in a manner dependent on the concentrations of the added monomers and oligomers. Again, the oligomer-α-synuclein showed more potent effects than the monomer-α-synuclein on surface GluN1 and total Rab5B expression. Accordingly, the oligomer-treated cells showed a greater reduction in NMDA-evoked Ca2+ influx than the monomer-treated cells, which was largely inhibited by pistop2, a clathrin inhibitor. These results suggest that the age-dependent accumulation of α-synuclein monomers and oligomers differentially contributes to the reduction in surface NMDAR expression in selective brain regions.

Electromagnetic control of spin ordered Mn3 qubits: a density functional study.

[Mn3O(O2CMe)(dpd3/2)]2 is composed of two monomers each of which contain three Mn atoms at the vertices of an equilateral triangle. A full analysis of the electronic and magnetic structure of the dimer shows that each Mn atom carries a local spin of S = 2 while other spin states are energetically much higher. This result suggests application for conventional as well as quantum tasks. A detailed analysis of the electronic and magnetic structure of the monomer, on the other hand, suggests that there are three spin states of S = 1, S = 3/2 and S = 2 per monomer which are energetically competitive. We found that while monomer-monomer interactions are very weak, the coupling of monomers via covalent linkers affects both the magnetization and electronic energy levels of monomers. In particular, the isolated monomers prefer a ground state with local spin of S = 1 on Mn atoms and an antiferromagnetically ordered structure while the dimers possess a ground state with local spin of S = 2 on Mn atoms and a ferromagnetically ordered structure. The investigation of the polarizability of both monomer and dimer is examined for antiferromagnetically ordered structures which induces a high dipole moment of 0.08 (a.u.) and 0.16 (a.u.) for monomer and dimer, respectively. The energy of the antiferromagnetic structure is also high compared to other spin-electric molecules.

PERSISTENT TAPETAL CELL2 Is Required for Normal Tapetal Programmed Cell Death and Pollen Wall Patterning.

The timely programmed cell death (PCD) of the tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development, including the deposition and patterning of the pollen wall. Although several genes involved in tapetal PCD and pollen wall development have been characterized, the underlying regulatory mechanism remains elusive. Here we report that PERSISTENT TAPETAL CELL2 (PTC2), which encodes an AT-hook nuclear localized protein in rice (Oryza sativa), is required for normal tapetal PCD and pollen wall development. The mutant ptc2 showed persistent tapetal cells and abnormal pollen wall patterning including absent nexine, collapsed bacula, and disordered tectum. The defective tapetal PCD phenotype of ptc2 was similar to that of a PCD delayed mutant, ptc1, in rice, while the abnormal pollen wall patterning resembled that of a pollen wall defective mutant, Transposable Element Silencing Via AT-Hook, in Arabidopsis (Arabidopsis thaliana). Levels of anther cutin monomers in ptc2 anthers were significantly reduced, as was expression of a series of lipid biosynthetic genes. PTC2 transcript and protein were shown to be present in the anther after meiosis, consistent with the observed phenotype. Based on these data, we propose a model explaining how PTC2 affects anther and pollen development. The characterization of PTC2 in tapetal PCD and pollen wall patterning expands our understanding of the regulatory network of male reproductive development in rice and will aid future breeding approaches.

Abnormal Biomarkers at Rest in Individuals with Sickle Cell Trait

Abstract Individuals with sickle cell trait (SCT) participate in competitive athletics and/or military training. Some have vigorously trained or competed without incident, while others have encountered serious medical complications. Universal precautions are recommended to reduce the risk of exercise collapse associated with sickle cell trait (ECAST) and exertion related death (ERD). However, there remain deaths in sport and military training attributed to SCT. ECAST and/or ERD are relatively unpredictable and are accompanied by rhabdomyolysis, thrombosis, hemolysis, renal dysfunction, and inflammation. Abnormal thrombotic markers have been reported in subjects with SCT at rest. This pilot study measured total protein (myolysis/hemolysis), creatine kinase (myolysis), haptoglobin (hemolysis), D-dimer and fibrin monomers (thrombosis) at rest in four subjects with SCT and four healthy controls at four time intervals at least two weeks apart. AS subjects demonstrated lower haptoglobin, higher total protein, and higher creatine kinase levels compared to AA subjects, suggesting subclinical hemolysis and myocyte destruction at rest. D-dimer and fibrin monomer were positive in 56.3% and 12.5% respectively of SCT subjects and negative for all AA subjects. The SCT subjects demonstrated day-to-day variability of D-dimer and fibrin monomer levels with positive tests on some, but not all of the four testing occasions. These data support previous findings that SCT subjects exhibit thrombotic activity at rest and suggest the potential for hemolysis and myolysis. These abnormal biomarkers may prove useful to predict risk (baseline values), identify responders to exercise, or identify early stage onset of ECAST (during exercise) to reduce exertion-related adverse events.

Mechanical and adhesive properties of hybrid epoxy-polyhydroxyurethane network polymers

Epoxies and polyurethanes are both important classes of materials with wide-ranging applications. The merging of both chemistries presents new opportunities to investigate materials with potentially unique or improved performance. In this work, a series of formulations with varying levels of epoxy and cyclic carbonate monomers were reacted with a multifunctional amine to produce network polymers with hybrid functionality. The spectroscopic, swelling, thermal-mechanical, tensile, and adhesive behaviors were evaluated. The materials performed as expected based on the proposed network structure as determined through small-molecule model studies. In particular, the hybrid network polymers performed admirably as adhesives with significantly improved performance over an epoxy–amine control.

Experimental and Modeling Investigation of Radical Homopolymerization of 2‐(Methacryloyloxyethyl) Trimethylammonium Chloride in Aqueous Solution

AbstractThe radical homopolymerization kinetics of 2‐(methacryloyloxyethyl) trimethylammonium chloride (TMAEMC) in aqueous solution is investigated across a wide range of initial monomer contents (5–35 wt%), ionic strengths, and pH levels using an in‐situ NMR technique to track monomer consumption over the complete conversion range. Molar mass distributions (MMD) of the final homopolymers are also examined, with additional batch and semi‐batch experiments conducted in a stirred vessel. The rates of monomer conversion and polymer MMDs are dependent on initial monomer content but almost entirely independent of pH and the presence of salts, with some acceleration of rate observed for low monomer levels at very high salt concentration. To aid with the interpretation of these results, the conductivity and counterion activity of monomer and polymer mixtures are measured to determine the extent of electrostatic interactions at various levels of conversion. These results are combined with recently reported measurements of TMAEMC homopropagation kinetics to develop a TMAEMC homopolymerization model that captures the systematic decrease in rates of monomer conversion observed with increased initial monomer content during batch polymerization as well as provides a good representation of semi‐batch polymerization.

S-Glutathionylated Serine Proteinase Inhibitors as Biomarkers for Radiation Exposure in Prostate Cancer Patients

In biological tissues, radiation causes the formation of reactive oxygen species (ROS), some of which lead to sequential oxidation of certain protein cysteine residues. Resultant cysteinyl radicals are subject to post-translational modification through S-glutathionylation. The present clinical trial was designed to determine if S-glutathionylated serine protease inhibitors (serpins) in blood could be used as biomarkers of exposure to radiation. 56 male prostate cancer patients treated with radiotherapy were enrolled in the trial and levels of S-glutathionylated serpins A1 and A3 were assessed by immunoblotting. Patients were classified into three groups: (1) external beam radiation therapy (EBRT); (2) brachytherapy (BT); (3) both EBRT and BT. Prior to treatment, baseline plasma levels of both unmodified and S-glutathionylated serpins were similar in each group. We identified elevated plasma levels of S-glutathionylated serpin A1 monomer, trimer and serpin A3 monomer in patient blood following radiation. Maximal increased levels of these S-glutathionylated serpins were correlated with increased duration of radiotherapy treatments. We conclude that it is practical to quantify patient plasma S-glutathionylated serpins and that these post-translationally modified proteins are candidate biomarkers for measuring radiation exposure. This provides a platform for use of such biomarkers in trials with the range of drugs that, like radiation, produce ROS.

Co-administration of iloprost and eptifibatide in septic shock (CO-ILEPSS)\u2014a randomised, controlled, double-blind investigator-initiated trial investigating safety and efficacy

BackgroundPart of the pathophysiology in septic shock is a progressive activation of the endothelium and platelets leading to widespread microvascular injury with capillary leakage, microthrombi and consumption coagulopathy. Modulating the inflammatory response of endothelium and thrombocytes might attenuate this vicious cycle and improve outcome.MethodThe CO-ILEPSS trial was a randomised, placebo-controlled, double-blind, pilot trial. Patients admitted to the intensive care unit with septic shock were randomised and allocated in a 2:1 ratio to active treatment with dual therapy of iloprost 1 ng/kg/min and eptifibatide 0.5 μg/kg/min for 48 h or placebo. The primary outcomes were changes in biomarkers reflecting endothelial activation and disruption, platelet consumption and fibrinolysis. We compared groups with mixed models, post hoc Wilcoxon signed-rank test and Mann-Whitney U test.ResultsWe included 24 patients of which 18 (12 active, 6 placebo) completed the full 7-day trial period and were included in the per-protocol analyses of the primary outcomes. Direct comparison between groups showed no differences in the primary outcomes. Analyses of within-group delta values revealed that biomarkers of endothelial activation and disruption changed differently between groups with increasing levels of thrombomodulin (p = 0.03) and nucleosomes (p = 0.02) in the placebo group and decreasing levels of sE-Selectin (p = 0.007) and sVEGFR1 (p = 0.005) in the active treatment group. Platelet count decreased the first 48 h in the placebo group (p = 0.049) and increased from baseline to day 7 in the active treatment group (p = 0.023). Levels of fibrin monomers declined in the active treatment group within the first 48 h (p = 0.048) and onwards (p = 0.03). Furthermore, there was a significant reduction in SOFA score from 48 h (p = 0.024) and onwards in the active treatment group.Intention-to-treat analyses of all included patients showed no differences in serious adverse events including bleeding, use of blood products or mortality.ConclusionOur results could indicate benefit from the experimental treatment with reduced endothelial injury, reduced platelet consumption and ensuing reduction in fibrinolytic biomarkers along with improved SOFA score. The results of the CO-ILEPSS trial are exploratory and hypothesis generating and warrant further investigation in a large-scale trial.Trial registrationClinicaltrials.com, NCT02204852 (July 30, 2014); EudraCT no. 2014-002440-41

Biochemical characterization and expression analysis of lignification in two pear (Pyrus ussuriensis Maxim.) varieties with contrasting stone cell content.

As lignified stone cells reduce fruit quality, we investigated lignin deposition, phenolic metabolites, and expression of lignin biosynthetic genes during fruit development to elucidate the molecular mechanism of stone cell lignification using histological, biochemical, and transcriptional data from two Ussurian pear varieties (Jianba and Nanguo) with contrasting stone cell content. Lignin content and distribution coincided with stone cell accumulation. As per LC-MS analysis, Jianba exhibited higher levels of lignin monomers and hydroxycinnamates than Nanguo, consistently with lignin amount in each case. However, flavonoid content was much higher in Nanguo. Transcriptional data showed that most monolignol biosynthesis-related genes were particularly upregulated in Jianba during lignin accumulation; especially CCR and LAC, two monolignol biosynthesis-specific genes, were substantially upregulated in Jianba fruits at critical stages. Therefore, differences in stone cell content between "Jianba" and "Nanguo" may result from differential expression of lignin synthase genes located downstream of the lignin biosynthesis pathway. Taken together, our data may provide a deeper understanding of the molecular mechanism for stone cell lignification in pear fruit.

Identifying Nonaffine Softening Modes in Glassy Polymer Networks: A Pathway to Chemical Design.

Using molecular simulations and theory, we develop an explicit mapping of the contribution of molecular relaxation modes in glassy thermosets to the shear modulus, where the relaxations were tuned by altering the polarity of side groups. Specifically, motions at the domain, segmental, monomer, and atomic levels are taken from molecular dynamics snapshots and directly linked with the viscoelasticity through a framework based in the lattice dynamics of amorphous solids. This unique approach provides direct insight into the roles of chemical groups in the stress response, including the time scale and spatial extent of relaxations during mechanics. Two thermoset networks with differing concentrations of polar side groups were examined, dicyclopentadiene (DCPD) and 5-norbornene-2-methanol (NBOH). A machine learning method is found to be effective for quantifying large-scale correlated motions, while more local chemical relaxations are readily identified by direct inspection. The approach is broadly applicable and enables rapid predictions of the frequency-dependent modulus for any glass.

Perfusion CHO cell culture applied to lower aggregation and increase volumetric productivity for a bispecific recombinant protein

Secreted recombinant proteins can aggregate during cell culture. We studied a poorly-behaved bispecific scaffold that increasingly aggregated (up to 62% high molecular weight species, HMW) as a function of culture time in a fed-batch and intensified cell culture processes. We identified that protein aggregates increased with accumulated protein concentration inside the bioreactor. Furthermore, results indicated that a maximum product concentration was reached beyond which no additional soluble protein accumulated in culture even when doubling the integrated viable cell density with the intensified process, suggesting additional secreted protein was precipitating. To overcome this limitation and maintain the cell-specific productivity (qp) in culture, we explored a perfusion process where recombinant protein was continuously removed from the bioreactor to maintain low product concentration and consequently, minimize protein aggregation. We studied different viable cell densities (VCDs) inside the bioreactor (one to five-fold) and found a corresponding two-fold modulation of monomer levels. In all VCD conditions, qp was maintained. On the contrary, the previous intensified process showed an “apparent” 2.5-fold decrease in qp at the end of culture because of the presumed limited protein solubility at higher concentrations. The combination of lower aggregate levels and constant qp resulted in up to four to five-fold increase in recoverable product (i.e., monomer) with the improved perfusion process.

Transcriptomic and metabolomic profiling of Camellia sinensis L. cv. 'Suchazao' exposed to temperature stresses reveals modification in protein synthesis and photosynthetic and anthocyanin biosynthetic pathways.

To determine the mechanisms in tea plants responding to temperature stresses (heat and cold), we examined the global transcriptomic and metabolomic profiles of the tea plant cultivar 'Suchazao' under moderately low temperature stress (ML), severely low temperature stress (SL), moderately high temperature stress (MH) and severely high temperature stress (SH) using RNA-seq and high performance liquid chromatography tandem mass spectrometry/mass spectrometry (HPLC-MS/MS), respectively. The identified differentially expressed genes indicated that the synthesis of stress-resistance protein might be redirected to cope with the temperature stresses. We found that heat shock protein genes Hsp90 and Hsp70 played more critical roles in tea plants in adapting to thermal stress than cold, while late embryogenesis abundant protein genes (LEA) played a greater role under cold than heat stress, more types of zinc finger genes were induced under cold stress as well. In addition, energy metabolisms were inhibited by SH, SL and ML. Furthermore, the mechanisms of anthocyanin synthesis were different under the cold and heat stresses. Indeed, the CsUGT75C1 gene, encoding UDP-glucose:anthocyanin 5-O-glucosyl transferase, was up-regulated in the SL-treated leaves but down-regulated in SH. Metabolomics analysis also showed that anthocyanin monomer levels increased under SL. These results indicate that the tea plants share certain foundational mechanisms to adjust to both cold and heat stresses. They also developed some specific mechanisms for surviving the cold or heat stresses. Our study provides effective information about the different mechanisms tea plants employ in surviving cold and heat stresses, as well as the different mechanisms of anthocyanin synthesis, which could speed up the genetic breeding of heat- and cold-tolerant tea varieties.

Abnormal Biomarkers at Rest in Individuals with Sickle Cell Trait

<h3>ABSTRACT</h3> Individuals with sickle cell trait (SCT) participate in competitive athletics and/or military training. Some have vigorously trained or competed without incident; whereas, others have encountered serious medical complications. Universal precautions are recommended to reduce the risk of exercise collapse associated with sickle cell trait (ECAST) and exercise-related death (ERD). However, there remain deaths in sport and military training attributed to SCT. ECAST and/or ERD are relatively unpredictable and are accompanied by rhabdomyolysis, thrombosis, hemolysis, renal dysfunction, and inflammation. Abnormal thrombotic markers have been reported in patients with SCT at rest. This pilot study measured total protein (TP; myolysis/hemolysis), creatine kinase (CK; myolysis), haptoglobin (hemolysis), dimerized plasmin fragment D (D-dimer), and fibrin monomers (thrombosis) at rest in 4 patients with SCT and 4 healthy patients at 4-time intervals at least 2 weeks apart. <i>AS</i> patients demonstrated lower haptoglobin, higher TP, and higher CK levels compared with <i>AA</i> patients, suggesting subclinical hemolysis and myocyte destruction at rest. Findings for the D-dimer and fibrin monomer tests were positive in 56.3% and 12.5%, respectively, of SCT patients and negative for all <i>AA</i> patients. The SCT patients demonstrated day-to-day variability of D-dimer and fibrin monomer levels with positive test results on some, but not all, of the 4 testing occasions. These data support previous findings that SCT patients exhibit thrombotic activity at rest and suggest the potential for hemolysis and myolysis. These abnormal biomarkers may prove useful to predict risk (baseline values), identify responders to exercise, or identify early-onset ECAST (during exercise) to reduce exertion-related adverse events.

A critical appraisal of amyloid-β-targeting therapies for Alzheimerdisease.

Brain accumulation of the amyloid-β (Aβ) peptide is believed to be the initial event inthe Alzheimer disease (AD) process. Aβ accumulation begins 15-20 years before clinical symptoms occur, mainly owing to defective brain clearance of the peptide. Over the past 20years, we have seen intensive efforts to decrease the levels of Aβ monomers, oligomers, aggregates and plaques using compounds that decrease production, antagonize aggregation orincrease brain clearance of Aβ. Unfortunately, these approaches have failed to show clinical benefit in large clinical trials involving patients with mild to moderate AD. Clinical trials in patients at earlier stages of the disease are ongoing, but the initial results have not been clinically impressive. Efforts are now being directed against Aβ oligomers, the most neurotoxic molecular species, and monoclonal antibodies directed against these oligomers are producing encouraging results. However, Aβ oligomers are in equilibrium with both monomeric and aggregated species; thus, previous drugs that efficiently removed monomeric Aβ or Aβ plaques should have produced clinical benefits. In patients with sporadic AD, Aβ accumulation could be a reactive compensatory response to neuronal damage of unknown cause, and alternative strategies, including interference with modifiable risk factors, might be needed to defeat this devastating disease.

ChemGenome2.1: An Ab Initio Gene Prediction Software.

Gene prediction, also known as gene identification, gene finding, gene recognition, or gene discovery, is among one of the important problems of molecular biology and is receiving increasing attention due to the advent of large-scale genome sequencing projects. We designed an ab initio model (called ChemGenome) for gene prediction in prokaryotic genomes based on physicochemical characteristics of codons. In this chapter, we present the methodology of the latest version of this model ChemGenome2.1 (CG2.1). The first module of the protocol builds a three-dimensional vector from three calculated quantities for each codon-the double-helical trinucleotide base pairing energy, the base pair stacking energy, and an index of the propensity of a codon for protein-nucleic acid interactions. As this three-dimensional vector moves along any genome, the net orientation of the resultant vector should differ significantly for gene and non-genic regions to make a distinction feasible. The predicted putative protein-coding genes from above parameters are passed through a second module of the protocol which reduces the number of false positives by utilizing a filter based on stereochemical properties of protein sequences. The chemical properties of amino acid side chains taken into consideration are the presence of sp3 hybridized γ carbon atom, hydrogen bond donor ability, short/absence of δ carbon and linearity of the side chains/non-occurrence of bi-dentate forks with terminal hydrogen atoms in the side chain. The final prediction of the potential protein-coding genes is based on the frequency of occurrence of amino acids in the predicted protein sequences and their deviation from the frequency values of Swissprot protein sequences, both at monomer and tripeptide levels. The final screening is based on Z-score. Though CG2.1 is a gene finding tool for prokaryotes, considering the underlying similarity in the chemical and physical properties of DNA among prokaryotes and eukaryotes, we attempted to evaluate its applicability for gene finding in the lower eukaryotes. The results give a hope that the concept of gene finding based on physicochemical model of codons is a viable idea for eukaryotes as well, though, undoubtedly, improvements are needed.

Potential pitfalls in analyzing structural uncoupling of eNOS: aging is not associated with increased enzyme monomerization.

Homodimer formation is essential for the normal activity of endothelial nitric oxide synthase (eNOS). Structural uncoupling of eNOS, with generation of enzyme monomers, is thought to contribute to endothelial dysfunction in several vascular disorders, including aging. However, low-temperature SDS-PAGE of healthy arteries has revealed considerable variation between studies in the relative expression of eNOS dimers and monomers. While assessing structural uncoupling of eNOS in aging arteries, we identified methodological pitfalls that might contribute to such variation. Therefore, using human cultured aortic endothelial cells and aortas from young and aged Fischer-344 rats, we investigated optimal approaches for analyzing the expression of eNOS monomers and dimers. The results demonstrated that published differences in treatment of cell lysates can significantly impact the relative expression of several eNOS species, including denatured monomers, partially folded monomers, dimers, and higher-order oligomers. In aortas, experiments initially confirmed a large increase in eNOS monomers in aging arteries, consistent with structural uncoupling. However, these monomers were actually endogenous IgG, which, under these conditions, has mobility similar to eNOS monomers. Increased IgG levels in aged aortas likely reflect the aging-induced disruption of endothelial junctions and increased arterial penetration of IgG. After removal of the IgG signal, there were low levels of eNOS monomers in young arteries, which were not significantly different in aged arteries. Therefore, structural uncoupling of eNOS is not a prominent feature in young healthy arteries, and the process is not increased by aging. The study also identifies optimal approaches to analyze eNOS dimers and monomers. NEW & NOTEWORTHY Structural uncoupling of endothelial nitric oxide synthase (eNOS) is considered central to endothelial dysfunction. However, reported levels of eNOS dimers and monomers vary widely, even in healthy arteries. We demonstrate that sample processing can alter relative levels of eNOS species. Moreover, endothelial dysfunction in aging aortas results in IgG accumulation, which, because of similar mobility to eNOS monomers, could be misinterpreted as structural uncoupling. Indeed, enzyme monomerization is not prominent in young or aging arteries.

CONTAMINATION OF ANTI-VEGF DRUGS FOR INTRAVITREAL INJECTION: How Do Repackaging and Newly Developed Syringes Affect the Amount of Silicone Oil Droplets and Protein Aggregates?

The particle counts and the nature of particles of three different antivascular endothelial growth factor agents (VEGF) in different containers in a laboratory setting were compared. Original prefilled ranibizumab glass syringes, original vials with aflibercept, and repacked ready-to-use plastic syringes with bevacizumab from a compounding pharmacy and a compounding company (CC) were analyzed. Particle counts and size distributions were quantified by different particle characterization methods (nephelometry, light obscuration, Micro-Flow Imaging, nanotracking analysis, resonant mass measurement). Using high-performance size-exclusion chromatography (HP-SEC), levels of protein drug monomer and soluble aggregates were determined. Nearly all samples showed similar product quality. Light obscuration and Micro-Flow Imaging showed a 4-fold to 9-fold higher total particle count in compounding company bevacizumab (other samples up to 42,000 particles/mL). Nanotracking analysis revealed highest values for compounding company bevacizumab (6,375 million particles/mL). All containers showed similar amounts of silicone oil microdroplets. Ranibizumab showed lowest particle count of all tested agents with only one monomer peak in HP-SEC. Repackaged bevacizumab from different suppliers showed varying product quality. All three tested agents are available in similar quality regarding particulate purity and silicone oil microdroplet count. Repackaging can have a major impact on the quality.