Publications by authors named "Zhongqi Zhang"

73 Publications

Limited Proteolysis Coupled with Mass Spectrometry for Simultaneous Evaluation of a Large Number of Protein Variants for Their Impact on Conformational Stability.

Anal Chem 2021 Oct 12;93(42):14263-14271. Epub 2021 Oct 12.

Process Development, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320 United States.

A stable molecular structure is important in the development of a protein candidate into a therapeutic product. A therapeutic protein often contains many different variants; some of them may have an impact on the conformational stability of the protein. Conventionally, to evaluate the impact of a variant on stability, the variant must be enriched to a reasonable purity, and then its stability characterized by chromatographic or biophysical techniques. However, it is often impractical to purify and characterize each variant in a therapeutic protein. A workflow, based on limited proteolysis followed by MS detection, was established to simultaneously assess the impact of a large number of variants on conformational stability without enrichment. Because a less stable domain is more susceptible to proteolytic degradation, conformational stability of the domain can be reported from the release rate of a proteolytic peptide. A kinetic model is established to quantitatively determine the extent of domain stabilization/destabilization of different variants. The methodology is demonstrated by examining variants known to affect the stability of immunoglobulin domains, such as different -glycoforms, methionine oxidations, and sequence variants. With this methodology, near 100 variants may be evaluated within 2 days in a single experiment. Insights into the sequence-stability relationship will be obtained by monitoring the large number of low-level sequence variants, facilitating engineering of more stable molecules.
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http://dx.doi.org/10.1021/acs.analchem.1c03335DOI Listing
October 2021

Genetic Code Expansion in the Engineered Organism Vmax X2: High Yield and Exceptional Fidelity.

ACS Cent Sci 2021 Sep 31;7(9):1500-1507. Epub 2021 Aug 31.

Department of Chemistry, University of California, Berkeley, California 94720, United States.

We report that the recently introduced commercial strain of (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 noncanonical α-amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than 10-fold higher than those achieved using two different genomically recodedstrains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.ΔA.opt and C321.ΔA.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of misincorporated natural α-amino acids at the UAG-programmed position, especially in cases where the ncAA is a moderate substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that the use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve the selectivity of an aaRS toward highly desired but suboptimal ncAA substrates.
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http://dx.doi.org/10.1021/acscentsci.1c00499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8461772PMC
September 2021

Enabling development, manufacturing, and regulatory approval of biotherapeutics through advances in mass spectrometry.

Curr Opin Biotechnol 2021 Oct 8;71:206-215. Epub 2021 Sep 8.

Attribute Sciences, Process Development, Amgen Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320, United States. Electronic address:

Rapid technological advances have significantly improved the capability, versatility, and robustness of mass spectrometers which has led to them playing a central role in the development, characterization, and regulatory filings of biopharmaceuticals. Their application spans the entire continuum of drug development, starting with discovery research through product development, characterization, and marketing authorization and continues well into product life cycle management. The scope of application extends beyond traditional protein characterization and includes elements like clone selection, cell culture physiology and bioprocess optimization, investigation support, and process analytical technology. More recently, advances in the MS-based multi-attribute method are enabling the introduction of MS in a cGMP environment for routine release and stability testing. While most applications of MS to date have been for monoclonal antibodies, the successes and learnings should translate to the characterization of next-gen biotherapeutics where modalities like multispecifics could be more prevalent. In this review, we describe the most significant advances in MS and correlate them to the broad spectrum of applications to biotherapeutic development. We anticipate rapid technological improvements to continue that will further accelerate widespread deployment of MS, thereby elevating our overall understanding of product quality and enabling attribute-focused product development.
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http://dx.doi.org/10.1016/j.copbio.2021.08.001DOI Listing
October 2021

MULTI-parametric MR imaging with fLEXible design (MULTIPLEX).

Magn Reson Med 2021 Aug 31. Epub 2021 Aug 31.

UIH America, Inc., Houston, Texas, USA.

Purpose: To introduce a gradient echo (GRE) -based method, namely MULTIPLEX, for single-scan 3D multi-parametric MRI with high resolution, signal-to-noise ratio (SNR), accuracy, efficiency, and acquisition flexibility.

Theory: With a comprehensive design with dual-repetition time (TR), dual flip angle (FA), multi-echo, and optional flow modulation features, the MULTIPLEX signals contain information on radiofrequency (RF) B fields, proton density, T , susceptibility and blood flows, facilitating multiple qualitative images and parametric maps.

Methods: MULTIPLEX was evaluated on system phantom and human brains, via visual inspection for image contrasts and quality or quantitative evaluation via simulation, phantom scans and literature comparison. Region-of-interest (ROI) analysis was performed on parametric maps of the system phantom and brain scans, extracting the mean and SD of the T , , proton density (PD), and/or quantitative susceptibility mapping (QSM) values for comparison with reference values or literature.

Results: One MULTIPLEX scan offers multiple sets of images, including but not limited to: composited PDW/T W/ W, aT W, SWI, MRA (optional), B map, T map, / maps, PD map, and QSM. The quantitative error of phantom T , and PD mapping were <5%, and those in brain scans were in good agreement with literature. MULTIPLEX scan times for high resolution (0.68 × 0.68 × 2 mm ) whole brain coverage were about 7.5 min, while processing times were <1 min. With flow modulation, additional MRA images can be obtained without affecting the quality or accuracy of other images.

Conclusion: The proposed MUTLIPLEX method possesses great potential for multi-parametric MR imaging.
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http://dx.doi.org/10.1002/mrm.28999DOI Listing
August 2021

Observation and Mitigation of Leachables from Non-Product Contact Materials in Electromechanical Delivery Devices for Biotechnology Products.

J Pharm Sci 2021 Aug 12. Epub 2021 Aug 12.

Attribute Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA. Electronic address:

Battery-powered drug delivery devices are widely used as primary containers for storing and delivering therapeutic protein products to improve patient compliance and quality of life. Compared to conventional delivery approaches such as pre-filled syringes, battery-powered devices are more complex in design requiring new materials/components for proper functionality, which could cause potential product safety and quality concerns from the extractable and leachables (E&L) of the new materials/components. In this study, E&L assessments were performed on a battery-powered delivery device during the development and qualification of the device, where novel compound 2‑hydroxy-2-methylpropiophenone (HMPP) and related compounds were observed in both E&L. The source of the HMPP and related compounds was identified to be the nonproduct contact device batteries, in which HMPP photo-initiator was used as a curing agent in the battery sealant to prevent leakage of the battery electrolytes. Toxicology assessment was performed, which showed the levels of HMPP observed in the device lots were acceptable relative to the permitted daily exposure. A drug product HMPP spike study was also performed, where no product impact was observed. Based on these assessments, an action threshold and specification limits could be established as a control strategy, if needed, to mitigate the potential risks associate with the observed leachables. As a full resolution, seven battery candidates from different suppliers were screened and one new battery was successfully qualified for the delivery devices. Overall, the holistic E&L approach was fully successful in the development and qualification of the battery-powered devices for biotherapeutic products delivery ensuring product quality and patient safety. Non-product contact materials are commonly rated as low or no risk and typically considered as out of scope of E&L activities for delivery systems following industry benchmark and regulatory agency guidance. This case study is novel as it brings into attention the materials that might not normally be in consideration during the development process. It is highly recommended to understand materials in the context of intended use on a case-by-case basis and not to generalize to ensure successful development and qualification.
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http://dx.doi.org/10.1016/j.xphs.2021.08.007DOI Listing
August 2021

Gaussian fitting algorithm with multi-geometric parameters for rotated elliptical beam profiling using pixel ion chamber.

Med Phys 2021 Sep 11;48(9):4799-4811. Epub 2021 Aug 11.

State Key Laboratory of Advanced Electromagnetic Engineering and Technology, School of Electrical and Electronic Engineering, Huazhong University of Science and Technology, Hubei, China.

Purpose: A high-precision rotated elliptical beam profiling method based on pixel ion chamber is proposed in this paper. This method aims to improve the accuracy by modeling the transverse profile of rotated beam as an ellipse with additional correlation coefficient and eliminating the fitting error due to the volume averaging effect of pixel ion chamber.

Methods: In pencil beam scanning (PBS) proton therapy systems, the transverse beam profile model is generally represented as a standard Gaussian distribution. Considering the elliptical spots, two-dimensional (2D) joint Gaussian distribution characterized with the correlation coefficient ρ is adopted in this study. Gaussian-type particle distribution with white noise was generated and processed in MATLAB to simulate the secondary particle collection in the pixel ion chamber. The simulated pixel ion chamber is a commercially available ion chamber which consists of 12 × 12 small square pixels (3.75 × 3.75 mm ) with a 0.05 mm interval. The simulated signals were preprocessed by filtering with the noise threshold and extracting the maximum simply connected domain (MSCD) of the signal. Then, five geometric parameters that identify the transverse beam profiles were fitted under different signal-to-noise ratio (SNR) conditions: the center of the beam (x , y ), the spot size (σ , σ ), and the rotation angle θ formed between the major axes of elliptical spot and the x axes of the ion chamber. First, the simulated signals were preprocessed by filtering with the noise threshold and extracting the MSCD of the signal. Second, a rectification curve of systematic error in fitted spot size versus the prescribed spot size was used to predict the systematic error due to the volume averaging effect. Finally, the effects of fitting errors on therapeutic dose were evaluated in terms of gamma index and relative dose difference.

Results: When the SNR is not less than 20 dB, the relative fitting error of spot size and the absolute fitting error of angle θ are less than 1% and 6.1°, respectively. The fitting error of beam center increases with spot size and will not exceed 0.22 mm when spot size reaches up to 12 mm. At a SNR equal to 20 dB, neither cold nor hot spots were presented in dose distribution calculated with the fitted spot parameters.

Conclusion: The improved Gaussian fitting algorithm performs well when SNR is not less than 20 dB. This method can effectively distinguish the nominal beam and rotated elliptical beam. An ideal systematic error curve can be predicted and used to correct the fitted spot size, thus eliminating the systematic error due to the volume averaging effect of the pixel ion chamber. The fitting error of spot size cannot be fully corrected, but it is negligible and shows little effect on the overall therapeutic dose.
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http://dx.doi.org/10.1002/mp.15140DOI Listing
September 2021

Study on the new dynamics and driving factors of soil pH in the red soil, hilly region of South China.

Environ Monit Assess 2021 Apr 26;193(5):304. Epub 2021 Apr 26.

School of Geography, Geomatics and Planning, Jiangsu Normal University, Xuzhou, 221116, China.

Soil acidification has always been a substantial eco-environmental problem restricting agricultural development in the red soil region of southern China. It is necessary to determine the dynamic change in soil pH in this area to formulate regional agricultural and environmental management measures. Yujiang County, a typical county with red soil acidification in southern China, was selected as the study area. Based on soil data from 1982, 2007, and 2018, the spatiotemporal variation characteristics and the latest changes in soil pH in the county were analyzed. The results show that the soil pH in Yujiang County decreased from 5.66 to 4.74 and then increased to 4.96 from 1982 to 2018, showing a trend of first decreasing and then increasing. According to the spatial distribution characteristics of soil pH, the low soil pH values in the three periods were mainly distributed in the northern mountainous areas with more forestland and dry land area and some southern hilly areas, while the paddy soil pH values in the middle low hilly areas were relatively higher. The soil pH decreased rapidly from 1982 to 2007, showing a large area of acidification. In 2007, the proportions of acidic (4.5 < pH < 5.5) and strongly acidic (pH < 4.5) soils increased by 67.37% and 10.6%, respectively, compared with that in 1982. However, from 2007 to 2018, the soil pH of the whole county increased, and the acidification trend was alleviated, which is of great significance to the regional red soil ecological environment. Through the analysis of the main factors affecting the change in soil pH, it was found that the sharp decline in soil pH in Yujiang County during 1982-2007 was mainly caused by acid rain and excessive nitrogen application. From 2007 to 2018, no significant reduction in nitrogen fertilizer in this area occurred, and although the increase in soil organic matter contributed to alleviating soil acidification, the analysis showed that the decrease in acid rain was the main reason for the rise in soil pH in Yujiang County. At the same time, notably, there is a large area of soil in the area that is still acidic, and effective control of soil acidification is still an important ecological and environmental issue in this area. In order to further improve the pH value of soil in red soil region, it is suggested that on the basis of continuous improvement of acid rain, in addition to increasing soil organic matter by returning straw to field and other measures, appropriate amount of lime or alkaline biochar can be applied to better improve the soil ecological environment in red soil hilly region.
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http://dx.doi.org/10.1007/s10661-021-09080-4DOI Listing
April 2021

GhVLN4 is involved in multiple stress responses and required for resistance to Verticillium wilt.

Plant Sci 2021 Jan 1;302:110629. Epub 2020 Aug 1.

State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China.

As structural and signaling platform in plant cell, the actin cytoskeleton is regulated by diverse actin binding proteins (ABPs). Villins are one type of major ABPs responsible for microfilament bundling, which have proved to play important roles in plant growth and development. However, the function of villins in stress tolerance is poorly understood. Here, we report the function of cotton GhVLN4 in Verticillium wilt resistance and abiotic stress tolerance. The expression of GhVLN4 was up-regulated by gibberellin, ethylene, ABA, salicylic acid, jasmonate, NaCl, PEG, and Verticillium dahliae treatment, suggesting the involvement of GhVLN4 in multiple stress and hormone responses and signaling. Virus-induced gene silencing GhVLN4 made cotton more susceptible to V. dahliae characterized by the preferential colonization and rapid growth of the fungus in both phloem and xylem of the infected stems. Arabidopsis overexpressing GhVLN4 exhibited higher resistance to V. dahliae, salt and drought than the wild-type plants. The enhanced resistance to V. dahliae is likely related to the upregulated components in SA signaling pathway; the improved tolerance to salt and drought is characterized by upregulation of the components both in ABA- related and ABA-independent signal pathways, along with altered stomatal aperture under drought. Our findings demonstrate that GhVLN4 may play important roles in regulating plant tolerance to both biotic and abiotic stresses.
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http://dx.doi.org/10.1016/j.plantsci.2020.110629DOI Listing
January 2021

Integrating hyperspectral imaging with machine learning techniques for the high-resolution mapping of soil nitrogen fractions in soil profiles.

Sci Total Environ 2021 Feb 4;754:142135. Epub 2020 Sep 4.

Jiangsu Normal University, Xuzhou 221116, China.

Soil nitrogen (N) plays a central role in soil quality and biogeochemical cycles. However, little is known about the distribution and spatial variability of the different fractions of soil N within entire soil profiles. This study aimed to investigate the potential of laboratory-based hyperspectral imaging (HSI) spectroscopy to retrieve and map total N (TN), available N (AvailN), ammonium N (NH-N), nitrate N (NO-N), and microbial biomass N (MBN) in soil profiles at a high resolution. HSI images of eleven intact soil profiles of 100 ± 5 cm depth from three typical soil types were recorded. A variety of nonlinear machine learning techniques, such as artificial neural networks (ANN), cubist regression tree (Cubist), k-nearest neighbour (KNN), support vector machine regression (SVMR) and extreme gradient boosting (XGBoost), were compared with a partial least squares regression (PLSR) to determine the most suitable model for the prediction of the various soil N fractions. Overall, the results showed that nonlinear techniques performed better than PLSR in most cases, with a high coefficient of determination (R) and low root mean square error (RMSE). Among the models, SVMR was found to be superior to the other tested models for TN (R = 0.94, RMSE = 0.17 g kg), AvailN (R = 0.94, RMSE = 13.35 mg kg), NO-N (R = 0.82, RMSE = 7.31 mg kg), and NH-N (R = 0.70, RMSE = 1.51 mg kg) based on independent validation, whereas MBN (R = 0.63, RMSE = 6.62 mg kg) was predicted best by KNN. In addition, SVMR required less computational time and was less sensitive to spectral noise. It can therefore be concluded that HSI spectroscopy combined with SVMR is suitable for the high-resolution mapping of various soil N fractions in soil profiles.
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http://dx.doi.org/10.1016/j.scitotenv.2020.142135DOI Listing
February 2021

Retraction Note to: Spinal circRNA-9119 Suppresses Nociception by Mediating the miR-26a-TLR3 Axis in a Bone Cancer Pain Mouse Model.

J Mol Neurosci 2020 Nov;70(11):1926

Department of Anesthesiology, Shunde Hospital of Southern Medical University, No. 1 Lunjiaojiazi Road, Shunde District, Foshan, 528308, Guangdong, China.

The authors have retracted this article [1] because in further experiments, they found that some experimental data cannot be verified repeatedly.
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http://dx.doi.org/10.1007/s12031-020-01693-7DOI Listing
November 2020

An evaluation of instrument types for mass spectrometry-based multi-attribute analysis of biotherapeutics.

MAbs 2020 Jan-Dec;12(1):1783062

Process Development, Amgen, Inc ., Thousand Oaks, CA, USA.

Multi-attribute methods (MAM), based on proteolytic digestion followed by liquid chromatography-mass spectrometry analysis of proteolytic peptides, have gained substantial attention in the biopharmaceutical industry for quantifying a variety of quality attributes for therapeutic proteins. Most MAM developed so far have been based on high-resolution mass spectrometers, due to their superb resolving power to distinguish analyte signals from interferences. Lower-resolution instruments, if demonstrated suitable, may further promote the adoption of the technology due to their low cost, small footprint, and ease of use. In this work, we compared the performance of a high-resolution instrument with a few low-resolution quadrupole-type instruments in quantifying a diverse set of quality attributes in a monoclonal antibody product. Different modes of operation for the quadrupole instruments, including scan mode, selected-ion monitoring and multiple-reaction monitoring, were evaluated. The high-resolution instrument has superb performance, with a quantitation limit of 0.002%. Single-quadrupole instruments in scan mode, on the other hand, provide a quantitation limit of about 1%, which may be fit-for-purpose for many routine MAM applications.
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http://dx.doi.org/10.1080/19420862.2020.1783062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531562PMC
July 2021

circStrn3 is involved in bone cancer pain regulation in a rat model.

Acta Biochim Biophys Sin (Shanghai) 2020 May;52(5):495-505

Department of Anesthesiology, The First Affiliated Hospital of Jinan University, Guangzhou 510632, China.

Bone cancer pain (BCP) is a common chronic pain that is caused by a primary or metastatic bone tumor. More detailed molecular mechanisms of BCP are warranted. In this study, we established a BCP rat model. The von Frey hair test, body weight, and hematoxylin and eosin staining were employed. We screened differentially expressed circRNAs (DECs) between the BCP group and sham group. The results revealed that 850 DECs were significantly up-regulated and 644 DECs were significantly down-regulated in the BCP group. Furthermore, we identified 1177 differentially expressed genes (DEGs) significantly up-regulated and 565 DEGs significantly down-regulated in the BCP group. Gene Ontology annotation of all 1742 DEGs revealed that biological regulation of metabolic processes, cellular processes, and binding were the top enriched terms. For Kyoto Encyclopedia of Genes and Genomes analysis, phagosome, HTLV-I infection, proteoglycans in cancer, and herpes simplex infection were significantly enriched in this study. In addition, we identified four selected circRNAs, chr6:72418120|72430205, chr20:7561057|7573740, chr18:69943105|69944476, and chr5:167516581|167558250, by quantitative real time PCR. chr6:72418120|72430205 (circStrn3) was selected for further study based on expression level and the circRNA-miRNA-mRNA network table. Western blot analysis suggested that knockdown of circStrn3 could effectively induce Walker 256 cell apoptosis. In summary, our study provided a more in-depth understanding of the molecular mechanisms of BCP.
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http://dx.doi.org/10.1093/abbs/gmaa018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270972PMC
May 2020

Complete Extraction of Protein Dynamics Information in Hydrogen/Deuterium Exchange Mass Spectrometry Data.

Authors:
Zhongqi Zhang

Anal Chem 2020 05 26;92(9):6486-6494. Epub 2020 Apr 26.

Process Development, Amgen Incorporated, One Amgen Center Drive, Thousand Oaks, California 91320, United States.

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has been used to study protein conformation and conformational dynamics. A continuous labeling experiment, followed by proteolytic digestion and MS analysis, generates a large amount of data, containing information on protein conformation and conformational dynamics. Lacking appropriate computational methods, information hidden inside the isotope distribution is often omitted and not extracted. In this work, a computational model is described to simulate the determined isotope pattern for each proteolytic peptide at each labeling time. Optimizing the model with experimental data yields conformational protection as well as protein unfolding/folding kinetics. With this method, complete extraction of protein dynamics information in the HDX-MS data is achieved. Information derived from the method is reliable as the model is mostly based on first-principles with very few assumptions. It is demonstrated that the protein dynamics information extracted from one or two labeling time points approaches or exceeds the information derived from an entire deuterium uptake time course by the traditional method. Application of the computational method to an IgG1 antibody under mild denaturing conditions indicates that the unfolding of each immunoglobulin domain can be explained by a simple two-state unfolding process, with different unfolding rate for each domain.
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http://dx.doi.org/10.1021/acs.analchem.9b05724DOI Listing
May 2020

Quantitative structure-chromatographic retention relationship of synthesized peptides (HGRFG, NPNPT) and their derivatives.

Anal Biochem 2020 05 27;597:113653. Epub 2020 Feb 27.

College of Life Science, Northwest University, Xi'an, 710069, PR China. Electronic address:

Carapax Trionycis extract peptides (HGRFG, NPNPT) are able to protect against CCl-induced liver fibrosis. Therefore, this study applies to deal with chromatographic lipophilicity determination of synthesized peptides (HGRFG, NPNPT) and their derivatives using reversed-phase high performance liquid chromatography (RP-HPLC) combined with methanol-water mobile phase and two reversed-phase chromatographic columns (COSMOISL 5C18-MS-II and SHIMADZU-C18). The chromatographic lipophilicity of the analyzed compounds was expressed as logk constant and correlated with lipophilicity descriptors. Quantitative structure-retention relationships (QSRR) analysis was performed to imitate chromatographic lipophilicity behavior using molecular descriptors. Modeling was performed using linear regression (LR) and multiple linear regression (MLR) methods with the help of principal component analysis (PCA) and hierarchical cluster analysis (HCA). The most influential molecular descriptors were lipophilicity descriptors, which are important for molecules ability to pass through biological membranes. All established QSRR models were statistically validated by standards, cross- and external validation parameters. According to these statistical validation parameters, MLR models (R > 0.856) were better for chromatographic lipophilicity prediction of peptide compounds. It can be concluded that chromatographic systems with COSMOISL 5C18-MS-II column were better for modeling of logk than systems with SHIMADZU-C18 column. Modeling was performed in order to obtain lipophilicity profiles of investigated compounds as future drug candidates.
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http://dx.doi.org/10.1016/j.ab.2020.113653DOI Listing
May 2020

Ketamine Regulates Phosphorylation of CRMP2 To Mediate Dendritic Spine Plasticity.

J Mol Neurosci 2020 Mar 5;70(3):353-364. Epub 2019 Dec 5.

Department of Anatomy, Neuroscience Laboratory for Cognitive and Developmental Disorders, Medical College of Jinan University, Guangzhou, 510630, Guangdong, China.

Ketamine is widely used in infants and young children for anesthesia, and subanesthetic doses of ketamine make neurons form new protrusions and promote synapse formation. However, the precise pathological mechanisms remain to be elucidated. In this study, we demonstrated that ketamine administration significantly increased dendritic spine density and maturity in rat cortical neurons in vivo and in vitro. Western blot analysis showed that CRMP2 protein expression was significantly increased in cerebral cortex of ketamine group, and phosphorylation levels of CRMP at Thr514 and Ser522 were significantly reduced. Furthermore, overexpression of CRMP2 promoted the growth of cortical neuron processes and dendritic spines. Although the dendritic field was more complex after adding ketamine and the density of dendritic spines increased, there was no statistical difference and no obvious superposition effect was observed. Moreover, both Ser522 mutant construction of CRMP2, GFP-CRMP2-522D, and mcherry-CDK5 showed similar inhibitory effects on neurite outgrowth, which could be rescued by ketamine. The frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were significantly inhibited when GFP-CRMP2-522D and mCherry-CDK5 were transfected into cortical neurons and this trend could also be rescued by ketamine. In general, this study reveals a new mechanism by which ketamine promotes the growth and development of dendritic spines in developing cortical neurons.
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http://dx.doi.org/10.1007/s12031-019-01419-4DOI Listing
March 2020

Effects of urbanization on productivity of terrestrial ecological systems based on linear fitting: a case study in Jiangsu, eastern China.

Sci Rep 2019 11 20;9(1):17140. Epub 2019 Nov 20.

School of Geography, Geomatics, and Planning, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, China.

The terrestrial ecosystem productivity and foundation of regional ecosystem services have been significantly influenced by recent urbanization processes. This study assesses the changes in terrestrial ecosystem productivity in Jiangsu from the years of 2000 to 2015 in response to the urbanization. A linear model that incorporates the traditional equalization method is proposed to improve the estimation accuracy of net primary productivity (NPP) loss. Results revealed that the land area of urban construction expanded rapidly during the research period to encompass an area of 8672.8 km. The rate of expansion was highest during 2005-2010. Additionally, the expansion rate of urban construction land was considerably higher in southern Jiangsu compared to the northern areas. The NPP exhibited a rising tendency from the year of 2000 to 2015, and varied from 33.30 to 40.23 Tg C/y. It was higher in the central parts, which include the cities of Yancheng and Nantong. The increase in urban construction land has resulted in a significant reduction in the terrestrial ecosystem productivity, i.e. a cumulative NPP loss of 2.55-2.88 Tg C during the research period. The NPP losses due to the conversion from cropland to constrction land were the highest, followed by the wetland. The work in this paper indicates that the rate of future productivity losses in terrestrial ecosystem in northern Jiangsu would be faster than the southern areas.
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http://dx.doi.org/10.1038/s41598-019-53789-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868215PMC
November 2019

Correction to: Spinal circRNA-9119 Suppresses Nociception by Mediating the miR-26a-TLR3 Axis in a Bone Cancer Pain Mouse Model.

J Mol Neurosci 2020 01;70(1):19-20

Department of Anesthesiology, Shunde Hospital of Southern Medical University, No. 1 Lunjiaojiazi Road, Shunde District, Foshan, 528308, Guangdong, China.

The original version of this article unfortunately contained a mistake in Fig. 2.
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http://dx.doi.org/10.1007/s12031-019-01407-8DOI Listing
January 2020

Impact of Fc N-glycan sialylation on IgG structure.

MAbs 2019 Nov-Dec;11(8):1381-1390. Epub 2019 Sep 2.

Department of Attribute Sciences, Process Development, Amgen, Inc , Thousand Oaks, California , USA.

Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.
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http://dx.doi.org/10.1080/19420862.2019.1655377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816437PMC
June 2020

Spinal circRNA-9119 Suppresses Nociception by Mediating the miR-26a-TLR3 Axis in a Bone Cancer Pain Mouse Model.

J Mol Neurosci 2020 Jan 31;70(1):9-18. Epub 2019 Jul 31.

Department of Anesthesiology, Shunde Hospital of Southern Medical University, No. 1 Lunjiaojiazi Road, Shunde District, Foshan, 528308, Guangdong, China.

Altered expression of circular RNA (circRNA) is recognized as a contributor to malignant pain where microRNA (miRNA) exerts an essential effect. We generated a murine model for bone malignancy pain in which 2472 osteolytic sarcoma cells were injected into the femurs of mice. CircRNA microarray and quantitative PCR (qPCR) and revealed that circ9119 expression was repressed in the spinal cord of bone malignancy pain model mice, which is the first relay site involved in the transmission of nociceptive information to the cerebrum of mice that receive spinal analgesics for malignancy pain. Overexpression of circ9119 by plasmid injection in the model mice reduced progressive thermal hyperalgesia and mechanical hyperalgesia. Bioinformatics prediction and dual-luciferase reporter assay showed that circ9119 functions as a sponge of miR-26a, which targets the TLR3 3'-untranslated region. Furthermore, expression of miR-26a was elevated and TLR3 level was repressed in bone malignancy pain model mice, which were counteracted by circ9119 in the spinal cord of tumor-bearing mice. Moreover, excessive expression of miR-26a was involved in the recovery of mice from progressive thermal hyperalgesia and mechanical hyperalgesia triggered via circ9119. TLR3 knockdown in bone malignancy pain model mice thoroughly impaired pain in the initial stages and reduced the effects of circ9119 on hyperalgesia. Our research findings indicate that targeting the circ9119-miR-26a-TLR3 axis may be a promising analgesic strategy to manage malignancy pain.
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http://dx.doi.org/10.1007/s12031-019-01378-wDOI Listing
January 2020

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Nat Methods 2019 07 27;16(7):595-602. Epub 2019 Jun 27.

Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
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http://dx.doi.org/10.1038/s41592-019-0459-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614034PMC
July 2019

Efficacy and Safety of Tedizolid Phosphate versus Linezolid in a Randomized Phase 3 Trial in Patients with Acute Bacterial Skin and Skin Structure Infection.

Antimicrob Agents Chemother 2019 07 24;63(7). Epub 2019 Jun 24.

Leeds Teaching Hospitals and University of Leeds, Leeds, United Kingdom.

Tedizolid phosphate is approved for the treatment of acute bacterial skin and skin structure infection (ABSSSI) caused by Gram-positive bacteria in the United States, Europe, and other countries. In this multicenter, double-blind, phase 3 study, 598 adult ABSSSI patients in China, Taiwan, the Philippines, and the United States were randomized to receive 200 mg of tedizolid, intravenously (i.v.)/orally (p.o.), once daily for 6 days or 600 mg of linezolid, i.v./p.o. twice daily for 10 days. The primary endpoint was early clinical response rate at 48 to 72 h. Secondary endpoints included programmatic and investigator-assessed outcomes at end-of-therapy (EOT) and posttherapy evaluation (PTE) visits. Safety was also evaluated. In the intent-to-treat (ITT) population, 75.3% of tedizolid-treated patients and 79.9% of linezolid-treated patients were early responders (treatment difference, -4.6%; 95% confidence interval [CI], -11.2, 2.2). After exclusion of patients who never received the study drug (tedizolid,  = 8; linezolid,  = 1; modified ITT), comparable early response rates were observed (tedizolid, 77.4%; linezolid, 80.1%; treatment difference, -2.7%; 95% CI, -9.4, 3.9). Secondary endpoints showed high and similar clinical success rates in the ITT and clinically evaluable (CE) populations at EOT and PTE visits (e.g., CE-PTE for tedizolid, 90.4%; for linezolid, 93.5%). Both drugs were well tolerated, and no death occurred. Eight patients experienced phlebitis with tedizolid while none did with linezolid; hence, drug-related treatment-emergent adverse events were reported in a slightly higher proportion in the tedizolid (20.9%) arm than in the linezolid arm (15.8%). The study demonstrated that tedizolid in a primarily Asian population was an efficacious and well-tolerated treatment option for ABSSSI patients. (This study has been registered at ClinicalTrials.gov under registration no. NCT02066402.).
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http://dx.doi.org/10.1128/AAC.02252-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591607PMC
July 2019

Reliable LC-MS Multiattribute Method for Biotherapeutics by Run-Time Response Calibration.

Anal Chem 2019 04 4;91(8):5252-5260. Epub 2019 Apr 4.

Process Development , Amgen Inc. , One Amgen Center Drive , Thousand Oaks , California 91320 , United States.

A major challenge of a mass-spectrometry-based quantitative multiattribute method (MAM) for biotherapeutics is its high variability between instruments. For reproducible attribute measurements, not only is a similar instrument model required, but the instruments must also be tuned to the same condition. This poses great long-term challenges, considering the rapid development of new instrumentations. In addition, differences in digestion efficiency, peptide recovery, and artificial modifications during sample preparation also contribute to variability between laboratories. To overcome these challenges, new mathematical methods are developed to calculate the attribute abundance in the sample, using the reference standard (RS) material as calibrant. Most quality attributes in the RS remain constant throughout the life of the standard, and therefore, the RS can serve as a calibrant to correct for the difference between instruments or sample preparation procedures. Because RS data are usually collected in a MAM assay, no additional work is required from the analyst. Data from a large number of attributes demonstrated that these methodologies greatly reduced instrument-to-instrument and sample preparation variabilities. With these methodologies, a consistent instrument model and sample preparation procedure is no longer a requirement. As a result, changes in digestion procedure and advances in instrumentations will not significantly affect the assay result.
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http://dx.doi.org/10.1021/acs.analchem.9b00027DOI Listing
April 2019

Efficacy and Safety of Intravitreal Aflibercept for Polypoidal Choroidal Vasculopathy: Two-Year Results of the Aflibercept in Polypoidal Choroidal Vasculopathy Study.

Am J Ophthalmol 2019 08 6;204:80-89. Epub 2019 Mar 6.

Department of Ophthalmology, Kyushu University Hospital, Fukuoka, Japan.

Purpose: We sought to evaluate longer-term efficacy and safety of intravitreal aflibercept monotherapy (IAI) vs IAI plus rescue photodynamic therapy (rPDT) in patients with polypoidal choroidal vasculopathy (PCV).

Design: This was a prospective multicenter, double-masked, sham-controlled randomized clinical study across 62 centers.

Methods: In this phase 3b/4 study, patients with PCV with best-corrected visual acuity of 73-24 Early Treatment Diabetic Retinopathy Study letters (20/40-20/320 Snellen equivalent) received IAI 2 mg every 4 weeks until week 12, when they were randomized 1:1 to receive IAI or IAI plus rPDT if rescue criteria were met. Patients not requiring rescue received IAI every 8 weeks; those requiring rescue received IAI every 4 weeks plus sham/active PDT. At week 52 (the primary endpoint), IAI was noninferior to IAI plus rPDT. After week 52, treatment intervals could be extended beyond 8 weeks at the investigators' discretion. Noninferiority of IAI vs IAI plus rPDT for mean best-corrected visual acuity change from baseline to week 96 was evaluated.

Results: Over 96 weeks, 54 patients (17.0%) met rescue criteria. At week 96, IAI was noninferior to IAI plus rPDT in terms of Early Treatment Diabetic Retinopathy Study letters gained (+10.7 vs +9.1, P = .48). Proportions of patients with complete polyp regression (33.1% vs 29.1%) or without active polyps (82.1% vs 85.6%) were similar. In year 2, the mean number of injections was 4.6 in both arms. No new safety signals were observed.

Conclusion: IAI monotherapy was noninferior to IAI with rescue PDT up to 96 weeks, and functional and anatomical improvements achieved at 52 weeks were maintained. Few patients required rescue PDT, which provided no additional visual benefit.
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http://dx.doi.org/10.1016/j.ajo.2019.02.027DOI Listing
August 2019

Amino Acid Misincorporation Propensities Revealed through Systematic Amino Acid Starvation.

Biochemistry 2018 12 28;57(49):6767-6779. Epub 2018 Nov 28.

Process Development , Amgen, Inc. , 1 Amgen Center Drive , Thousand Oaks , California 91320 , United States.

Elevated amino acid misincorporation levels during protein translation can cause disease and adversely impact biopharmaceutical product quality. Our previous work, along with that of others, identified numerous low-level unintended sequence variants. However, because of the limited analytical detection efficiency, we believed that these observations represented only a fraction of biologically relevant outcomes. Because amino acid misincorporation can be exacerbated by amino acid starvation, we believed that a more comprehensive set of sequence variants could be derived through systematic starvation. Our goals for this study were therefore (1) to systematically characterize misincorporation patterns under amino acid starvation and (2) to elucidate the major misincorporation mechanisms and propensities for cultured mammalian cells. To the best of our knowledge, this is the first study to use controlled systematic starvation to maximize the observation of unique sequence variants to provide a more holistic perspective of amino acid misincorporation. Our findings bridge the two prevailing lines of research and propose that both base mismatches during codon recognition (especially G/U and wobble mismatches) and misacylation are common and major amino acid misincorporation mechanisms. This proposal is also supported by the observation of mechanistic additivity between the base mismatch and misacylation mechanisms. In addition, we observed significant overlap in misincorporation mechanisms and propensities among cell lines and organisms. Lastly, we explored factors that can lead to codon-associated misincorporation behavior.
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http://dx.doi.org/10.1021/acs.biochem.8b00976DOI Listing
December 2018

Spatial distribution characteristics of soil organic carbon in subtropical forests of mountain Lushan, China.

Environ Monit Assess 2018 Aug 23;190(9):545. Epub 2018 Aug 23.

School of Geography, Geomatics and Planning, Jiangsu Normal University, Xuzhou, 221116, China.

The study on the spatial distribution of forest soil organic carbon (SOC) is of great significance for accurate assessment of carbon storage in forest ecosystems. In the present study, by taking eight kinds of forest soils of Mountain Lushan in the subtropical area as the research object, we studied the spatial distribution characteristics of SOC in this mountainous area. The results showed that the SOC content and SOC density (SOCD) of main forest types in the Mountain Lushan were lower than the national and the world average. The soil layer of Lushan forest was thinner, and the SOC and active SOC (ASOC) contents of different forest types and SOCDs are the highest in the surface soil. SOCD of the topsoil accounts for 32.64-54.03% of the total SOCD in the whole soil profile. Surface litter is an important source of SOC, and the different vegetation types are the important reason for the different spatial distribution of SOC in this area. Soil SOC contents in the high-altitude forest (bamboo forest, deciduous broadleaf forest, Pinus taiwanensis forest, evergreen-deciduous forest, and coniferous-broadleaved mixed forest) were higher than those in the low-altitude forest (evergreen broadleaf forest, shrub, and Pinus massoniana forest). However, the difference in SOC content exhibited at the altitude gradient is significantly lower than that in SOC in the soil profile. This indicates that both soil depth and elevation are the important factors that affected SOC distribution. However, the influence of soil depth on spatial distribution of SOC may be more complex than that of altitude. Vegetation types and soil properties are the main reasons for the large differences of reduction rate in the contents of SOC and ASOC.
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http://dx.doi.org/10.1007/s10661-018-6906-xDOI Listing
August 2018

A systematic dissection of sequence elements determining β-Klotho and FGF interaction and signaling.

Sci Rep 2018 07 23;8(1):11045. Epub 2018 Jul 23.

Department of Cardiometabolic Disorders, Amgen Discovery Research, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA, 94080, USA.

Endocrine fibroblast growth factors (FGFs) require Klotho transmembrane proteins as necessary co-receptors to activate FGF receptor (FGFR) signaling. In particular, FGF19 and FGF21 function through β-Klotho to regulate glucose and lipid metabolism. Recent research has focused on elucidating how these two FGFs interact with β-Klotho and FGFRs to activate downstream signaling. In this study, using hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS), we identified regions on the β-Klotho protein that likely participate in ligand interaction, and vice versa. Alanine and arginine mutagenesis were carried out to further probe the contributions of individual residues to receptor/ligand interactions. Using biochemical and cell-based signaling assays with full-length proteins, we show that both the KL1 and KL2 domains of β-Klotho participate in ligand interaction, and these binding sites on β-Klotho are shared by FGF19 and FGF21. In addition, we show that two highly conserved regions in the C-terminal tail of FGF19 and FGF21 are responsible for interaction with the co-receptor. Our results are consistent with recent publications on the crystal structures of the Klotho proteins and provide insight into how endocrine FGFs interact with co-receptors for signal transduction.
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http://dx.doi.org/10.1038/s41598-018-29396-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056499PMC
July 2018

Contraceptive efficacy and safety of estradiol valerate/dienogest in a healthy female population: a multicenter, open-label, uncontrolled Phase III study.

Int J Womens Health 2018 7;10:257-266. Epub 2018 Jun 7.

Bayer Pharmaceuticals, AG, Berlin, Germany.

Background: To investigate the efficacy and safety of a combined oral contraceptive containing estradiol valerate and dienogest (EV/DNG) in healthy Asian women.

Methods: In this multicenter Phase III study, women received oral EV/DNG in a 28-day regimen for 13 cycles. The primary efficacy endpoint was the number of unintended pregnancies, measured by the Pearl Index (PI); secondary efficacy endpoints included bleeding pattern and cycle control parameters. Adverse events were monitored during the study and overall satisfaction with treatment was determined on completion of the study.

Results: A total of 954 Asian women (97.7% of subjects assigned to study medication; mean age 33.4 years) were treated. Five pregnancies were reported during EV/DNG treatment over 796.34 relevant woman-years of exposure, giving an unadjusted PI of 0.63 and a cumulative failure rate of 0.0049; 3 pregnancies during EV/DNG treatment over 760.35 relevant woman-years of exposure gave an adjusted PI of 0.39. The bleeding pattern improved during the reporting periods within the study. The proportion of women who experienced withdrawal bleeding decreased with treatment (84.9% of women during Cycle 1 vs 79.3% in Cycle 13), and the mean length of withdrawal bleeding decreased with treatment (4.2 vs 3.4 days). The number and maximum length of intracyclic bleeding/spotting episodes also decreased with EV/DNG. EV/DNG was well tolerated, and 92% of women included in the study were very satisfied or somewhat satisfied with EV/DNG.

Conclusion: EV/DNG showed high contraceptive efficacy, was well tolerated in Asian women, and may be effectively used in this population.

Clinical Trials Registry: ClinicalTrials.gov identifier: NCT01638910.
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http://dx.doi.org/10.2147/IJWH.S157056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995293PMC
June 2018

Efficacy and Safety of Intravitreal Aflibercept for Polypoidal Choroidal Vasculopathy in the PLANET Study: A Randomized Clinical Trial.

JAMA Ophthalmol 2018 07;136(7):786-793

Department of Ophthalmology, Kyushu University Hospital, Fukuoka, Japan.

Importance: Polypoidal choroidal vasculopathy (PCV) is common in Asian populations, but an optimal treatment approach remains to be confirmed.

Objective: To evaluate intravitreal aflibercept injection (IAI) in participants with PCV and compare IAI monotherapy with IAI plus rescue photodynamic therapy (PDT).

Design, Setting, And Participants: This 96-week, double-masked, sham-controlled phase 3b/4 randomized clinical trial was conducted at multiple centers in Australia, Germany, Hong Kong, Hungary, Japan, Singapore, South Korea, and Taiwan from May 2014 to August 2016, and included adults 50 years or older with symptomatic macular PCV and a best-corrected visual acuity of 73 to 24 Early Treatment Diabetic Retinopathy Study letters (20/40-20/320 Snellen equivalent).

Interventions: Participants received 2 mg of IAI at weeks 0, 4, and 8. At week 12, participants with a suboptimal response were randomized 1:1 to receive IAI plus sham PDT (IAI monotherapy) or a "rescue" of IAI plus rescue PDT (IAI/PDT). Participants who did not qualify for rescue received IAI every 8 weeks; those qualifying for rescue received IAI every 4 weeks plus sham/active PDT. When the rescue criteria were no longer met, injection intervals were gradually extended to 8 weeks.

Main Outcomes And Measures: Noninferiority of IAI monotherapy to IAI/PDT for mean change in best-corrected visual acuity from baseline to week 52 (95% CI of the difference entirely above -5 letters).

Results: Of the 318 participants, the mean (SD) age was 70.6 (8.2) years, 96 (30.2%) were women, and 152 (47.8%) were Japanese. Monotherapy with IAI was noninferior to IAI/PDT for the primary end point (+10.7 vs +10.8 letters, respectively; 95% CI, -2.9 to 1.6; P = .55), with few participants requiring rescue therapy (19 [12.1%] vs 23 [14.3%], respectively). Participants in both treatment groups had similar reductions in central subfield thickness from baseline to week 52 (-137.7 [IAI monotherapy] vs -143.5 μm [IAI/PDT]). At week 52, 49 (38.9%) and 60 participants (44.8%) had no polypoidal lesions observed on indocyanine green angiography in the IAI monotherapy and IAI/PDT groups, respectively. Furthermore, 116 (81.7%) and 136 (88.9%), respectively, had no polypoidal lesions with leakage. The most frequent ocular adverse events were conjunctival hemorrhage (IAI monotherapy, 8 [5.1%]) and dry eye (IAI/PDT, 9 [5.6%]).

Conclusions And Relevance: Improvement in visual and/or functional outcomes was achieved in more than 85% of participants who were treated with IAI monotherapy, with no signs of leakage from polypoidal lesions in more than 80%. As fewer than 15% met the criteria of a suboptimal response to receive PDT, the potential benefit of adding PDT cannot be determined.

Trial Registration: ClinicalTrials.gov Identifier: NCT02120950.
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http://dx.doi.org/10.1001/jamaophthalmol.2018.1804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136040PMC
July 2018

Molecular basis for the loss-of-function effects of the Alzheimer's disease-associated R47H variant of the immune receptor TREM2.

J Biol Chem 2018 08 24;293(32):12634-12646. Epub 2018 May 24.

From Amgen Discovery Research, Amgen Inc., San Francisco, California 94080,

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with and characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased and stability of TREM2 contribute to loss of function in disease.
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http://dx.doi.org/10.1074/jbc.RA118.002352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093241PMC
August 2018

MS-based conformation analysis of recombinant proteins in design, optimization and development of biopharmaceuticals.

Methods 2018 07 18;144:134-151. Epub 2018 Apr 18.

Process Development, Amgen, 1 Amgen Center Drive, Thousand Oaks, CA 91320, United States. Electronic address:

Mass spectrometry (MS)-based methods for analyzing protein higher order structures have gained increasing application in the field of biopharmaceutical development. The predominant methods used in this area include native MS, hydrogen deuterium exchange-MS, covalent labeling, cross-linking and limited proteolysis. These MS-based methods will be briefly described in this article, followed by a discussion on how these methods contribute at different stages of discovery and development of protein therapeutics.
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http://dx.doi.org/10.1016/j.ymeth.2018.04.011DOI Listing
July 2018
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