Genotype and phenotype in patients with ACAN gene variants: Three cases and literature review.

OBJECTIVE: To characterize the phenotype spectrum, diagnosis, and response to growth-promoting therapy in patients with ACAN variants causing familial short stature. METHODS: Three families with ACAN variants causing short stature were reported. Similar cases in the literature were summarized, and the genotype and phenotype were analyzed. RESULTS: Three novel heterozygous variants, c.757+1G>A, (splicing), c.6229delG, p.(Asp2078Tfs*1), and c.6679C>T, p.(Gln2227*) in the ACAN gene were identified. A total of 314 individuals with heterozygous variants from 105 families and 8 individuals with homozygous variants from 4 families were confirmed to have ACAN variants from literature and our 3 cases. Including our 3 cases, the variants reported comprised 33 frameshift, 39 missense, 23 nonsense, 5 splicing, 4 deletion, and 1 translocation variants. Variation points are scattered throughout the gene, while exons 12, 15, and 10 were most common (25/105, 11/105, and 10/105, respectively). Some identical variants existing in different families could be hot variants, c.532A>T, p.(Asn178Tyr), c.1411C>T, p.(Gln471*), c.1608C>A, p.(Tyr536*), c.2026+1G>A, (splicing), and c.7276G>T, p.(Glu2426*). Short stature, early-onset osteoarthritis, brachydactyly, midfacial hypoplasia, and early growth cessation were the common phenotypic features. The 48 children who received rhGH (and GnRHa) treatment had a significant height improvement compared with before (-2.18 ± 1.06 SD vs. -2.69 ± 0.95 SD, p < 0.001). The heights of children who received rhGH (and GnRHa) treatment were significantly improved compared with those of untreated adults (-2.20 ± 1.10 SD vs. -3.24 ± 1.14 SD, p < 0.001). CONCLUSION: Our study achieves a new understanding of the phenotypic spectrum, diagnosis, and management of individuals with ACAN variants. No clear genotype-phenotype relationship of patients with ACAN variants was found. Gene sequencing is necessary to diagnose ACAN variants that cause short stature. In general, appropriate rhGH and/or GnRHa therapy can improve the adult height of affected pediatric patients caused by ACAN variants.

DeKinomics pulse-chases kinase functions in living cells.

Cellular context is crucial for understanding the complex and dynamic kinase functions in health and disease. Systematic dissection of kinase-mediated cellular processes requires rapid and precise stimulation ('pulse') of a kinase of interest, as well as global and in-depth characterization ('chase') of the perturbed proteome under living conditions. Here we developed an optogenetic 'pulse-chase' strategy, termed decaging kinase coupled proteomics (DeKinomics), for proteome-wide profiling of kinase-driven phosphorylation at second-timescale in living cells. We took advantage of the 'gain-of-function' feature of DeKinomics to identify direct kinase substrates and further portrayed the global phosphorylation of understudied receptor tyrosine kinases under native cellular settings. DeKinomics offered a general activation-based strategy to study kinase functions with high specificity and temporal resolution under living conditions.

A high-precision multi-dimensional microspectroscopic technique for morphological and properties analysis of cancer cell.

Raman and Brillouin scattering are sensitive approaches to detect chemical composition and mechanical elasticity pathology of cells in cancer development and their medical treatment researches. The application is, however, suffering from the lack of ability to synchronously acquire the scattering signals following three-dimensional (3D) cell morphology with reasonable spatial resolution and signal-to-noise ratio. Herein, we propose a divided-aperture laser differential confocal 3D Geometry-Raman-Brillouin microscopic detection technology, by which reflection, Raman, and Brillouin scattering signals are simultaneously in situ collected in real time with an axial focusing accuracy up to 1 nm, in the height range of 200 µm. The divided aperture improves the anti-noise capability of the system, and the noise influence depth of Raman detection reduces by 35.4%, and the Brillouin extinction ratio increases by 22 dB. A high-precision multichannel microspectroscopic system containing these functions is developed, which is utilized to study gastric cancer tissue. As a result, a 25% reduction of collagen concentration, 42% increase of DNA substances, 17% and 9% decrease in viscosity and elasticity are finely resolved from the 3D mappings. These findings indicate that our system can be a powerful tool to study cancer development new therapies at the sub-cell level.

Incidence of rhegmatogenous retinal detachments is increasing in Wenzhou, China.

AIM: To estimate and compare the incidence and characteristics of rhegmatogenous retinal detachments (RRDs) in the Wenzhou area in 2015 to 2019. METHODS: All newly developed RRD cases among residents of the Wenzhou area, from January 2015 to December 2019, were retrospectively retrieved from hospital records. Annual population data were extracted from the Wenzhou Statistical Yearbook. RESULTS: There were 3629 eligible cases. The average incidence of RRD was 7.79 cases per 100 000 population (95% confidence interval, 7.24-8.34), and the incidences were 7.99 and 7.56 for males and females, respectively. The annual incidence increased gradually from 7.26 cases per 100 000 in 2015 to 10.00 cases per 100 000 in 2019, with an overall increase of 37.74%. The highest rate of increase occurred in the age group from 60 to 69 years. Of 2750 eyes with axial length (AL) data, 1675 (60.91%) had an AL greater than 24 mm. CONCLUSION: A trend to increasing RRD incidence is observed in the Wenzhou area over the past 5-year period.

Laser transverse dual differential confocal radius measurement with high efficiency and high precision.

To meet the need for rapid, high-precision, and non-contact measurement of the radius of curvature (ROC) for large quantities of spherical optics, a radius measurement method based on transverse dual differential confocal (TDDC) detection is proposed in this study. First, a template S0 with a known ROC, R0, is axially scanned on its confocal position to obtain the fitted linear function lTDDC(z) using TDDC. Second, the template S0 is replaced by Sn, which is one of the test sample in large quantities, then the single point TDDC intensity ITDDC(Δzn) is captured without scan, which will be applied to obtain the defocus Δzn according to the linear function lTDDC(z). Finally, the ROC Rn under test is calculated using Δzn and R0. Simulation and experiments show that the measurement accuracy can achieve 8.0 ppm, and the measurement efficiency is 60 times higher than that of the traditional differential confocal scanning measurement. Measurement based on TDDC only requires scanning once and replacing Sn N times to realize the fast, high-precision, non-contact ROC detection of N pieces of spherical optics, which enables the high-efficiency and high-precision measurement of large quantities of spherical optics.

Dynamic Phosphotyrosine-Dependent Signaling Profiling in Living Cells by Two-Dimensional Proximity Proteomics.

Tyrosine phosphorylation (pTyr)-dependent signaling pathways play a vital role in various biological processes, which are spatiotemporally assembled and dynamically regulated on a minute scale by pTyr in living cells. Studying these pTyr-mediated signaling complexes is therefore challenging due to the highly dynamic nature of the protein complexes and the low abundance of pTyr. In this study, we adopted minute-resolution APEX2-based proximity labeling (PL) in living cells and Src SH2 superbinder-based pTyr peptide enrichment for simultaneously profiling these protein complexes and associated pTyr sites from the same affinity-purified sample. Upon different time courses of EGF stimulation of the living cells stably expressing APEX2-FLAG-GRB2, we constructed two-dimensional time-course curves for both interactome and tyrosine phosphoproteome. Well-annotated pTyr signaling complexes in EGFR signaling and located at the endosome were quantified with tightly correlated time-course curves for both interacting proteins and pTyr sites. Importantly, the correlated time-course curves for EGFR and endosomal HGS were well validated by targeted-parallel reaction monitoring (PRM)-MS analysis. Taking advantage of the high sensitivity of the PRM assay, the low-abundant pTyr peptide EGFR pY1110, which cannot be identified in the data-dependent acquisition (DDA) analysis, could be well quantified. Collectively, this two-dimensional proximity proteomic strategy is promising for comprehensively characterizing pTyr-mediated protein complexes with high sensitivity in living cells.

Inner limiting membrane peeling prevents secondary epiretinal membrane after vitrectomy for proliferative diabetic retinopathy.

AIM: To evaluate the role of internal limiting membrane (ILM) peeling in preventing secondary epiretinal membrane (ERM) formation in pars plana vitrectomy (PPV) for proliferative diabetic retinopathy (PDR). METHODS: This retrospective study analyzed the medical records of patients who underwent PPV for PDR and were followed up for minimum 3mo. ILM peeling was performed based on the intraoperative surgeons' judgments. ERM was assessed by optical coherence tomography photography. The relationship between ILM peeling and postoperative ERM was analyzed. RESULTS: In total, 212 eyes from 197 patients were included in this study. The incidence of secondary ERM in the ILM non-peeling group was significantly higher than that in the ILM peeling group (37.0% vs 14.0%; P<0.001). Multivariate logistical regression revealed that ILM peeling was highly associated with the prevention of secondary ERM development [odds ratio 0.38; 95% confidence interval 0.17-0.86; P<0.05]. CONCLUSION: ILM peeling during PPV for PDRs can effectively reduce the incidence of secondary ERM development and is worth consideration by vitreoretinal surgeons.

Exosomes From Cancer-Associated Mesenchymal Stem Cells Transmit TMBIM6 to Promote the Malignant Behavior of Hepatocellular Carcinoma via Activating PI3K/AKT Pathway.

Objective: Cancer-associated mesenchymal stem cells (MSCs) regulate the progression of cancers through exosome-delivered components, while few studies are conducted on hepatocellular carcinoma (HCC). This study aimed to evaluate the effect of exosomes from HCC-associated MSCs (HCC-MSCs) on HCC cellular functions and the potential regulatory mechanism. Methods: HCC cells (Huh7 and PLC) were cultured normally or co-cultured with HCC-MSCs, HCC-MSCs plus GW4869, or HCC-MSC-derived exosomes; then mRNA sequencing and RT-qPCR validation were conducted. Subsequently, candidate genes were sorted out and modified in HCC cells. Next, TMBIM6-modified HCC-MSCs were used to treat HCC cells. Results: Both HCC-MSCs and their derived exosomes promoted proliferation, invasion, sphere formation ability but suppressed apoptosis in HCC cells (all p < 0.05); however, the effect of HCC-MSCs on these cellular functions was repressed by exosome inhibitor (GW4869). Subsequently, TMBIM6, EEF2, and PRDX1 were sorted out by mRNA sequencing and RT-qPCR validation as candidate genes implicated in the regulation of HCC cellular functions by HCC-MSC-derived exosomes. Among them, TMBIM6 had a potent effect (all p < 0.05), while EEF2 and PRDX1 had less effect on regulating HCC cell viability and invasion. Next, direct silencing TMBIM6 repressed viability, sphere formation, invasion, epithelial-mesenchymal transition (EMT), and PI3K/AKT pathway but promoted apoptosis in HCC cells; however, overexpressing TMBIM6 showed the opposite effect. Furthermore, incubating with exosomes from TMBIM6-modified HCC-MSCs presented a similar effect as direct TMBIM6 modification in HCC cells. Conclusion: HCC-MSC-derived exosomes transmit TMBIM6 to promote malignant behavior via PI3K/AKT pathway in HCC.

Generic Plug-and-Play Strategy for High-Throughput Analysis of PTM-Mediated Protein Complexes.

Protein complexes mediated by various post-translational modifications (PTMs) play important roles in almost every aspect of biological processes. PTM-mediated protein complexes often have weak and transient binding properties, which limit their unbiased profiling especially in complex biological samples. Here, we developed a plug-and-play chemical proteomic approach for high-throughput analyis of PTM-mediated protein complexes. Taking advantage of the glutathione-S-transferase (GST) tag, which is the gold standard for protein purification and has wide access to a variety of proteins of interest (POIs), a glutathione (GSH) group- and photo-cross-linking group-containing trifunctional chemical probe was developed to tag POIs and assembled onto a streptavidin-coated 96-well plate for affinity purification, photo-cross-linking, and proteomics sample preparation in a fully integrated manner. Compared with the previously developed photo-pTyr-scaffold strategy, by assembling the tyrosine phosphorylation (pTyr) binding domain through covalent NHS chemistry, the new plug-and-play strategy using a noncovalent GST-GSH interaction has comparable enrichment efficiency for EGF stimulation-dependent pTyr protein complexes. To further prove its feasibility, we additionally assembled four pTyr-binding domains in the 96-well plate and selectively identified their pTyr-dependent interacting proteins. Importantly, we systematically optimized and applied the plug-and-play approach for exploring protein methylation-mediated protein complexes, which are difficult to be characterized due to their weak binding affinity and the lack of efficient enrichment strategies. We explored a comprehensive protein methylation-mediated interaction network assembled by five protein methylation binding domains including the chromo domain of MPP8, tandem tudor domain of KDM4A, full-length CBX1, PHD domain of RAG2, and tandem tudor domain of TP53BP1 and validated the chromo domain- and tudor domain-mediated interaction with histone H3. Collectively, this plug-and-play approach provides a convenient and generic strategy for exploring PTM-dependent protein complexes for any POIs with the GST tag.

Assessment of Knowledge, Attitude, and Practice Regarding Diabetic Retinopathy in an Urban Population in Northeast China.

Objective: This study aimed to assess the knowledge, attitude, and practice (KAP) of diabetic subjects with diabetic retinopathy (DR) and those without DR (NDR) in an urban community in Northeast China, as well as their risk factors in subjects with DR and NDR. Methods: A community-based survey involving 1,662 subjects was conducted in Fushun, China, between July 2012 and May 2013. The subjects included diabetics with DR (n = 783) and those NDR (n = 879), and questionnaires were completed to collect information about their sociodemographic and healthcare characteristics. A Chi-square test and multiple logistic analyses were performed to analyze the data. Results: Among the DR group, 21.88% had a good knowledge of DR, 94.15% had a positive attitude, and 68.07% followed good practice, whereas 20.98% of the NDR group had a good knowledge of DR, 94.18% had a positive attitude, and 66.92% followed good practice. There was no significant difference in the KAP of the two groups of subjects. In the NDR group, a good level of knowledge was associated with a high-level of education (OR = 0.1, 0.2; p < 0.05), a good attitude was associated with retirement (OR = 0.2; p < 0.05), and good practice was associated with being female, having a high-level of education, and the type of treatment (OR = 0.5, 0.4, 2.3, 3.1; p < 0.05). In the DR group, good practice was associated with older age and retirement (OR = 0.6, 0.4; p < 0.05). Conclusions: There was no significant difference between the DR and NDR subjects in the overall levels of KAP, but both groups showed a poor level of knowledge. Age, gender, education, occupation, and type of treatment were the main factors associated with the KAP scores, more risk factors in the NDR group than in the DR group. There is an urgent need for coordinated educational campaigns with a prioritized focus on the northeast region of China, especially NDR group.

Prevalence of and Risk Factors for Diabetic Retinopathy and Diabetic Macular Edema in Patients with Early- and Late-Onset Diabetes Mellitus.

PURPOSE: The aim of this study was to compare the prevalence of diabetic retinopathy (DR) and diabetic macular edema (DME), as well as their risk factors in patients with early-onset diabetes (EOD, ≤40 years) and late-onset diabetes (LOD, >40 years). METHODS: Patients were recruited from a community-based study, Fushun Diabetic Retinopathy Cohort Study (FS-DIRECT), conducted between July 2012 and May 2013 in China. The presence and severity of DR and DME were determined by a modified Early Treatment Diabetic Retinopathy Study (ETDRS) retinopathy scale of six-field fundus photographs. RESULTS: A total of 1,932 patients (796 male, 41.2%) with gradable fundus photography were included. The prevalence of any DR and DME was 67.0% (95% confidence interval [CI]: 60.3-73.7%) and 39.3% (95% CI: 32.1-46.5%) in the EOD patients, respectively, which were both significantly higher than that in the LOD patients (DR: 41.9%, 39.6-44.2%, p < 0.001; DME: 14.4%, 12.7-16.1%, p < 0.001). Insulin use was associated with both the presence of DR and DME in both EOD and LOD patients. Besides insulin use, a high level of income (odds ratio [OR], 95% CI: 0.05, 0.01-0.51) was negatively associated with DR, and higher high-density lipoprotein (OR, 95% CI: 4.14, 1.44-11.91) was associated with DME among EOD patients. CONCLUSION: In this sample of patients with type 2 diabetes, both prevalence of DR and DME were apparently higher in patients who developed diabetes ≤40 years of age than those who developed it later.

Internal Limiting Membrane Peeling versus Nonpeeling to Prevent Epiretinal Membrane Formation following Vitrectomy for Posterior Segment Open-Globe Injury.

PURPOSE: Approximately 30% of patients with an open-globe injury (OGI) develop a secondary epiretinal membrane (ERM). This study was performed to assess whether internal limiting membrane (ILM) peeling in the treatment of posterior segment OGI prevents ERM formation. METHODS: The medical records of 33 patients who underwent vitrectomy for posterior segment OGI from 2016 to 2019 were retrospectively analyzed. Of these patients, 17 underwent ILM peeling during the vitrectomy and 16 did not. The patients' demographic and surgical data were collected. The associations of ILM peeling with the preoperative findings of posterior segment OGI and development of a postoperative ERM were analyzed. Student's t-test was used to evaluate differences in continuous variables, and the chi-squared test or Fisher's exact test was used for categorical variables. Time-to-event curves were calculated from postestimation Cox proportional hazards models. RESULTS: An ERM developed in three eyes (17.6%) in the ILM peeling group and in eight eyes (50.0%) in the nonpeeling group (p < 0.05). There was no statistically significant difference between the groups in visual acuity at baseline (1.68 vs. 1.58 logMAR, p=0.68) or at final follow-up (0.72 vs. 0.78 logMAR, p=0.66). Median visual acuity significantly improved in both groups (p < 0.001). In the multivariable models, ILM peeling (odds ratio, 0.19; 95% confidence interval, 0.04-0.91; p=0.04) and worse preoperative vision (odds ratio, 0.29; 95% confidence interval, 0.10-0.80; p=0.02) were associated with lower likelihood of ERM formation. CONCLUSION: Preventative treatment with ILM peeling contributed to decreased development of an ERM in patients with OGI involving areas near the fovea.

Stable and EGF-Induced Temporal Interactome Profiling of CBL and CBLB Highlights Their Signaling Complex Diversity.

The epidermal growth factor receptor (EGFR) signal modulates cell proliferation, migration, and survival. Aberrant activation of EGFR constitutes the major cause of various cancers. Receptor ubiquitination and degradation mediated by CBL proteins play negative regulatory roles and control the intensity and duration of the signaling. With the construction of stable cell lines inducibly expressing FLAG-tagged CBL or CBLB, we identified 102 and 82 stable interacting proteins of CBL and CBLB, respectively, through the affinity purification followed by mass spectrometry (AP-MS) approach. Time-resolved profiling at six different time points combined with functional annotations of the temporal interactomes provides insights into the dynamic assembly of signal proteins upon EGFR signaling activation. Comparison between the interactomes of CBL and CBLB indicates their redundant but also complementary functions. Importantly, we validated the stable association of EPS15L1 and ITSN2 and temporal association of TNK2 to both CBL and CBLB through biochemical assays. Collectively, these results offer a useful resource for CBL and CBLB interactomes and highlight their prominent and diverse roles in the EGFR signaling network.

Dataset on capability of suppressing background noise and anti-tilting of divided-aperture differential confocal Raman microscopy system.

The dataset describes the mechanism of suppressing the background noise of the divided-aperture differential confocal Raman microscopy system and the range of tilting angles that the system can handle. On the basis of the confocal microscopy (CM), the divided-aperture confocal microscopy divided the pupil plane of the objective lens into the illumination pupil and collection pupil. Compared with the CM, the divided-aperture confocal microscopy only changes the pupil parameters, according to the partially coherent imaging theory, we simulate and analyze the axial response curves of the divided-aperture confocal system and the traditional confocal system. We also simulated the differential confocal response curve at different tilting angles and get the data for the applicability of the differential confocal response curve to see if there is a single zero-crossing point or a good linearity near the zero-crossing point. The goodness-of-fit (GOF) is used to evaluate the accuracy of linear fitting, and can be used as a simple measure method of linearity. And the closer the GOF value is to 1, the higher fitting accuracy is. Through simulation analysis, we can have a better understanding of the advances of divided-aperture differential confocal Raman microscopy.

Fully Integrated and Multiplexed Sample Preparation Technology for Sensitive Interactome Profiling.

Affinity purification coupled to mass spectrometry (AP-MS) is a popular approach for deciphering the architecture of protein interaction networks. Protein lysates (100 µg) are typically required for multistep sample processing in large volumes, which often causes sample loss and reduces the MS analysis sensitivity. Herein, we reported a fully integrated spintip-based AP-MS technology, termed FISAP, for multiplexed and sensitive interactome profiling. The FISAP device can be easily employed for routine use by introducing AP beads into a C18 StageTip. Taking advantage of the switchable functionalization of the C18 matrix by sodium dodecyl sulfate, all the sample preparation steps encompassing peptide or antibody-based AP, reduction, alkylation, tryptic digestion, tandem mass tag (TMT) labeling, and desalting can be performed in a single tip with a benchtop centrifuge in 4 h. Using a biotinylated tyrosine phosphorylated (pTyr) peptide as an affinity ligand, we mapped the pTyr-dependent interactome of the pY191 motif on the immune receptor CD28 cytoplasmic domain. When processing 50 µg of protein lysates, FISAP showed a comparable interactome identification performance but better quantification performance and lower background interference compared to the traditional tube-based method. Furthermore, a cost-effective on-column TMT labeling protocol was established and integrated into the FISAP pipeline with increased sensitivity. Compared to the tube-based method, the usage of a synthetic peptide probe and a TMT reagent was both reduced by 20 times. As low as 1 µg of protein lysates could be applied for interactome profiling. Finally, we expanded the applicability of the FISAP technology to epitope tag-based AP-MS for profiling the ILK/PINCH/Parvin complex using 100 times less protein lysate than a previous report. Collectively, FISAP is an easy-to-use and sensitive technology for quantitatively profiling protein complexes when the starting material and affinity reagent are the limitation, especially for applications in biomedical research and chemical biology.

Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics.

Signaling complexes are often organized in a spatiotemporal manner and on a minute timescale. Proximity labeling based on engineered ascorbate peroxidase APEX2 pioneered in situ capture of spatiotemporal membrane protein complexes in living cells, but its application to cytosolic proteins remains limited due to the high labeling background. Here, we develop proximity labeling probes with increased labeling selectivity. These probes, in combination with label-free quantitative proteomics, allow exploring cytosolic protein assemblies such as phosphotyrosine-mediated protein complexes formed in response to minute-scale EGF stimulation. As proof-of-concept, we systematically profile the spatiotemporal interactome of the EGFR signaling component STS1. For STS1 core complexes, our proximity proteomics approach shows comparable performance to affinity purification-mass spectrometry-based temporal interactome profiling, while also capturing additional-especially endosomally-located-protein complexes. In summary, we provide a generic approach for exploring the interactome of mobile cytosolic proteins in living cells at a temporal resolution of minutes.

Interocular Asymmetry of Visual Field Loss, Intraocular Pressure, and Corneal Parameters in Primary Open-Angle Glaucoma.

OBJECTIVES: This study aimed to assess the association between the corneal biomechanical parameters and visual field (VF) loss in patients with asymmetric primary open-angle glaucoma (POAG). METHODS: A total of 89 POAG patients (50 males, 56.2%) with asymmetric VF loss, aged 65.2 ± 13.3 years, were enrolled in this study. Asymmetric VF loss was defined as an interocular difference of the global index mean deviation (MD) >2 dB. Intraocular pressure (IOP), central corneal thickness (CCT), and corneal biomechanical parameters such as maximum amplitude at the apex of highest concavity (def ampl HC) were measured. The worse eye was defined as the eye with a smaller MD. RESULTS: The worse eyes had lower MD (-11.9 ± 6.7 dB vs. -5.3 ± 5.0 dB; p < 0.001) and higher IOP (14.6 ± 3.3 vs.13.9 ± 2.6 mm Hg, p = 0.04) than the better eyes. There was no significant difference between the 2 groups for CCT. The interocular difference of MD (IDMD) was negatively correlated with the interocular difference of IOP (r = -0.22, p = 0.04), while positively correlated with the interocular difference of def ampl HC (r = 0.27, p = 0.01). In patients with moderate asymmetric VF loss (IDMD ≥6 dB), def ampl HC of the worse eyes group (1.07 ± 0.12 mm) was significantly lower than the better eyes group (1.10 ± 0.11 mm, p = 0.02). CONCLUSION: Asymmetric POAG was associated with asymmetry in IOP and corneal biomechanical parameters but not in CCT. Lower deflection amplitude and higher IOP were found in eyes with more severe VF damage in POAG patients.

Association between Mean Ocular Perfusion Pressure and Diabetic Retinopathy in a Northeastern Chinese Population.

OBJECTIVE: To evaluate the association between diabetic retinopathy (DR) and mean ocular perfusion pressure (MOPP) in patients with type 2 diabetes mellitus (T2DM). METHODS: Patients from the Fushun Diabetic Retinopathy Cohort Study (FS-DIRECT), a community-based prospective cohort study conducted in northeast China, were included in this study. The presence and severity of DR were determined by grading fundus photographs according to the Early Treatment Diabetic Retinopathy Study (ETDRS) retinopathy scale. Systolic and diastolic blood pressure (SBP and DBP) were recorded using an electronic sphygmomanometer. Intraocular pressure (IOP) was measured using an iCare rebound tonometer. MOPP was calculated using the formula MOPP = 2/3 [DBP + 1/3 (SBP - DBP)] - IOP. RESULTS: In total, 1,857 patients who had gradable fundus photography and MOPP data were enrolled in this study. Male patients had a higher MOPP than female patients (52.25 ± 8.75 vs. 50.96 ± 8.74 mmHg, P = 0.002). Overall, both male and female patients with any type of DR, non-proliferative DR (NPDR), or non-sight-threatening DR (non-STDR) had significantly higher MOPP relative to patients without DR. Increased MOPP (per 1 mmHg) was in turn associated with the presence of any type of DR [odds ratio ( OR) = 1.03, 95% confidence interval ( CI) : 1.02-1.04], NPDR ( OR= 1.03 95% CI: 1.02-1.04), and non-STDR ( OR= 1.03, 95% CI: 1.01-1.04) after adjusting for confounders. Increased MOPP (per 1 mmHg) was also associated with an increased likelihood of macular edema ( OR= 1.02 , 95% CI: 1.01-1.04). CONCLUSIONS: The results suggest that increased MOPP was associated with DR and macular edema in northeastern Chinese patients with T2DM.

AutoProteome Chip System for Fully Automated and Integrated Proteomics Sample Preparation and Peptide Fractionation.

With recent advances in LC-MS systems, current MS-based proteomics has an increasing need for automated, high-throughput sample preparation with neglectable sample loss. In this study, we developed a microfluidic system for fully automated proteomics sample preparation. All of the required proteomics sample preparation steps for both protein digestion and peptide fractionation are fully integrated into a disposable plastic chip device (named AutoProteome Chip). The AutoProteome Chip packed with mixed-mode ion exchange beads and C18 membrane in tandem could be fabricated with very low cost and high stability in organic reagents. Benefiting from its low backpressure, the AutoProteome Chip could be precisely driven by gas pressure, which could be easily multiplexed. As low as 2 ng of standard protein BSA could be trapped into the AutoProteome chip and processed within 2 h. Fully automated processing of 10 µg of protein extracts of HEK 293T cells achieved more than 97% of digestion efficiency with missed cleavage less than 2 and comparable performance with conventional approaches. More than 4700 proteins could be readily identified within 80 min of LC-MS analysis with good label-free quantification performance (Pearson correlation coefficient >0.99). Furthermore, deep proteome profiling by integrated high-pH RP fractionation in the same AutoProteome Chip resulted in more than 7500 proteins being identified from only 20 µg of protein extracts of HEK 293T cells and comparable reprodicibility as single-shot analysis. The AutoProteome Chip system provided a valuable prototype for developing a fully automated proteome analysis workflow and for proteomic applications with high demand for processing throughput, reproducibility, and sensitivity.

High-Throughput and Integrated Chemical Proteomic Approach for Profiling Phosphotyrosine Signaling Complexes.

Phosphotyrosine (pTyr) signaling complexes are important resources of biomarkers and drug targets which often need to be profiled with enough throughput. Current profiling approaches are not feasible to meet this need due to either biased profiling by antibody-based detection or low throughput by traditional affinity purification-mass spectrometry approach (AP-MS), as exemplified by our previously developed photo-pTyr-scaffold approach. To address these limitations, we developed a 96-well microplate-based sample preparation and fast data independent proteomic analysis workflow. By assembling the photo-pTyr-scaffold probe into a 96-well microplate, we achieved steric hindrance-free photoaffinity capture of pTyr signaling complexes, selective enrichment under denaturing conditions, and efficient in-well digestion in a fully integrated manner. EGFR signaling complex proteins could be efficiently captured and identified by using 300 times less cell lysate and 100 times less photo-pTyr-scaffold probe as compared with our previous approach operated in an Eppendorf tube. Furthermore, the lifetime of the photo-pTyr-scaffold probe in a 96-well microplate was significantly extended from 1 week up to 1 month. More importantly, by combining with high-flow nano LC separation and data independent acquisition on the Q Exactive HF-X mass spectrometer, LC-MS time could be significantly reduced to only 35 min per sample without increasing sample loading amount and compromising identification and quantification performance. This new high-throughput proteomic approach allowed us to rapidly and reproducibly profile dynamic pTyr signaling complexes with EGF stimulation at five time points and EGFR inhibitor treatment at five different concentrations. We are therefore optimized for its generic application in biomarkers discovery and drug screening in a high-throughput fashion.