The role of hypertension in the relationship between leisure screen time, physical activity and migraine: a 2-sample Mendelian randomization study.

BACKGROUND: The relationship between lifestyle and migraine is complex, as it remains uncertain which specific lifestyle factors play the most prominent role in the development of migraine, or which modifiable metabolic traits serve as mediators in establishing causality. METHODS: Independent genetic variants strongly associated with 20 lifestyle factors were selected as instrumental variables from corresponding genome-wide association studies (GWASs). Summary-level data for migraine were obtained from the FinnGen consortium (18,477 cases and 287,837 controls) as a discovery set and the GWAS meta-analysis data (26,052 cases and 487,214 controls) as a replication set. Estimates derived from the two datasets were combined using fixed-effects meta-analysis. Two-step univariable MR (UVMR) and multivariable Mendelian randomization (MVMR) analyses were conducted to evaluate 19 potential mediators of association and determine the proportions of these mediators. RESULTS: The combined effect of inverse variance weighted revealed that a one standard deviation (SD) increase in genetically predicted Leisure screen time (LST) was associated with a 27.7% increase (95% CI: 1.14-1.44) in migraine risk, while Moderate or/and vigorous physical activity (MVPA) was associated with a 26.9% decrease (95% CI: 0.61-0.87) in migraine risk. The results of the mediation analysis indicated that out of the 19 modifiable metabolic risk factors examined, hypertension explains 24.81% of the relationship between LST and the risk of experiencing migraine. Furthermore, hypertension and diastolic blood pressure (DBP) partially weaken the association between MVPA and migraines, mediating 4.86% and 4.66% respectively. CONCLUSION: Our research findings indicated that both LST and MVPA in lifestyle have independent causal effects on migraine. Additionally, we have identified that hypertension and DBP play a mediating role in the causal pathway between these two factors and migraine.

Reprogramming the spleen into a functioning 'liver' in vivo.

OBJECTIVE: Liver regeneration remains one of the biggest clinical challenges. Here, we aim to transform the spleen into a liver-like organ via directly reprogramming the splenic fibroblasts into hepatocytes in vivo. DESIGN: In the mouse spleen, the number of fibroblasts was through silica particles (SiO2) stimulation, the expanded fibroblasts were converted to hepatocytes (iHeps) by lentiviral transfection of three key transcriptional factors (Foxa3, Gata4 and Hnf1a), and the iHeps were further expanded with tumour necrosis factor-α (TNF-α) and lentivirus-mediated expression of epidermal growth factor (EGF) and hepatocyte growth factor (HGF). RESULTS: SiO2 stimulation tripled the number of activated fibroblasts. Foxa3, Gata4 and Hnf1a converted SiO2-remodelled spleen fibroblasts into 2×106 functional iHeps in one spleen. TNF-α protein and lentivirus-mediated expression of EGF and HGF further enabled the total hepatocytes to expand to 8×106 per spleen. iHeps possessed hepatic functions-such as glycogen storage, lipid accumulation and drug metabolism-and performed fundamental liver functions to improve the survival rate of mice with 90% hepatectomy. CONCLUSION: Direct conversion of the spleen into a liver-like organ, without cell or tissue transplantation, establishes fundamental hepatic functions in mice, suggesting its potential value for the treatment of end-stage liver diseases.

Colloidal Synthesis of Ternary Copper Halide Nanocrystals for High-Efficiency Deep-Blue Light-Emitting Diodes with a Half-Lifetime above 100 h.

Currently, the blue perovskite light-emitting diodes (PeLEDs) suffer from a compromise in lead toxicity and poor operation stability, and most previous studies have struggled to meet the crucial blue NTSC standard. In this study, electrically driven deep-blue LEDs (∼445 nm) based on zero-dimensional (0D) Cs3Cu2I5 nanocrystals (NCs) were demonstrated with the color coordinates of (0.16, 0.07) and a high external quantum efficiency of ∼1.12%, comparable with the best-performing blue LEDs based on lead-halide perovskites. Encouraged by the remarkable stability of Cs3Cu2I5 NCs against heat and environmental oxygen/moisture, the proposed device was operated in a continuous current mode for 170 h, producing a record half-lifetime of ∼108 h. The device stability was further verified by an aggressive thermal cycling test (300-360-300 K) and a 35-day storage test. Together with the eco-friendly features and facile colloidal synthesis technique, the 0D Cs3Cu2I5 NCs can be therefore regarded as a promising candidate for deep-blue LEDs applications.

Laser-induced plasma in water at high pressures up to 40 MPa: A time-resolved study.

The knowledge on the laser-induced plasma emission in water at high pressures is essential for the application of laser-induced breakdown spectroscopy (LIBS) in the deep-sea. In this work, we investigate the spectral features of ionic, atomic and molecular emissions for the plasma in water at different pressures from 1 to 40 MPa. By comparing between the time-resolved spectra and shadowgraph images, we demonstrate that the dynamics of the cavitation bubble at high pressures plays a key role on the characterization of plasma emission. The initial plasma emission depends weakly on the external pressure. As time evolves, the cavitation bubble is more compressed by the higher external pressure, leading to a positive confinement effect to maintain the plasma emission. However, at very high pressures, the bubble collapses extremely fast and even earlier than the cooling of the plasma. The plasma will gain energy from the bubble collapse phase, but quench immediately after the collapse, leading to a sharp reduction in the plasma persistence. These effects caused by bubble dynamics explain well the observed spectral features and are further proved by the temporal evolutions of the plasma temperature and electron density. This work gives not only some insights into the laser-induced plasma and bubble dynamics in high pressure liquids but also better understanding for the application of underwater LIBS in the deep-sea.

CaOH Molecular Emissions in Underwater Laser-Induced Breakdown Spectroscopy: Spatial-Temporal Characteristics and Analytical Performances.

Recently, molecular emissions from the laser-induced plasma in ambient gas have gained increasing interest; however, very little is known about the case in water solutions. In this work, we investigated the spatiotemporal characteristics of molecular emissions, CaOH for instance, in underwater laser-induced breakdown spectroscopy (LIBS) by using time-resolved spectroscopy, spectral-resolved imaging, and shadowgraph techniques. It was shown that clear CaOH molecular bands can be observed in the spectrum at very early times after the laser pulse and presented a much longer lifetime and more homogeneous emission distribution compared with the Ca I and Ca II lines. Such unique characteristics of CaOH molecular emission inspired us to improve the performances of underwater LIBS by using the CaOH molecular bands instead of Ca I and Ca II lines. We demonstrated the excellent quantification results of CaOH with higher stability, less self-absorption, and reduced matrix effect. Meanwhile, the limit of detection (LOD) of Ca with the CaOH molecular band (2.46 ppm) is comparable to that with the atomic line of Ca I (2.07 ppm), and much lower than that with the ionic line of Ca II (13.81 ppm), indicating a good sensitivity of CaOH. This work gives not only some insights into the molecule formation mechanisms in underwater plasmas, but also provides new ideas to improve the analytical performances of underwater LIBS.

A Case Study Comparing Heterogeneous Lysine- and Site-Specific Cysteine-Conjugated Maytansinoid Antibody-Drug Conjugates (ADCs) Illustrates the Benefits of Lysine Conjugation.

Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small molecule effectors. First-generation molecules of this class often employed heterogeneous conjugation chemistry, but many site-specifically conjugated ADCs have been described recently. Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysine-conjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chemistry, we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chemistry, target antigen, and other variables that determine efficacy in a given setting.

Long Noncoding RNA Meg3 Regulates Mafa Expression in Mouse Beta Cells by Inactivating Rad21, Smc3 or Sin3α.

BACKGROUND/AIMS: The main pathogenic mechanism of diabetes is a decrease in the number of islet beta cells or a decline in their function. Recent studies have shown that pancreatic long noncoding RNAs (lncRNAs) have a high degree of tissue specificity and may be involved in the maintenance of islet cells function and the development of diabetes. The aim of this study was to investigate the molecular regulatory mechanism of mouse maternal expressed gene 3 (Meg3) in insulin biosynthesis in pancreatic islets. METHODS: Chromatin immunoprecipitation-quantitative polymerase chain reaction (qPCR) and RNA immunoprecipitation-qPCR were used to investigate the molecular mechanism of lncRNA Meg3 in insulin biosynthesis by regulating v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), a mature beta cell marker in the MIN6 beta cell line. Further, the expression levels of Meg3, Ezh2, MafA, Rad21, Smc3, and Sin3α were analyzed in vivo and in vitro by RT-PCR and western blotting. RESULTS: Intranuclear lncRNA Meg3 can bind EZH2, a methyltransferase belonging to the Polycomb repressive complex-2, in pancreatic islet cells. In addition, knockdown of Ezh2 can also inhibit the expression of MafA and Ins2, while expression levels of Rad21, Smc3, and Sin3α are upregulated, by interfering with Ezh2 or Meg3 in pancreatic beta cells. Knockdown of Meg3 resulted in the loss of EZH2 binding and H3K27 trimethylation occupancy of Rad21, Smc3, and Sin3α promoter regions. The inhibition of Rad21, Smc3, or Sin3α, which directly act on the MafA promoter, leads to upregulated expression of MafA in both MIN6 cells and mouse islets. Moreover, the synthesis and secretion of insulin were increased by inhibition of these transcription factors. CONCLUSIONS: Pancreatic lncRNA Meg3 can epigenetically regulate the expression of Rad21, Smc3, and Sin3α via EZH2-driven H3K27 methylation. By inhibiting the expression of Rad21, Smc3, or Sin3α, Meg3 promotes the expression of MafA and affects the production of insulin.

Inhibition of myeloid differentiation factor-2 attenuates obesity-induced cardiomyopathy and fibrosis.

Obesity causes cardiovascular diseases, including cardiac hypertrophy and remodeling, via chronic tissue inflammation. Myeloid differentiation factor-2 (MD2), a binding protein of lipopolysaccharide, is functionally essential for the activation of proinflammatory pathways in endotoxin-induced acute inflammatory diseases. Here we tested the hypothesis that MD2 plays a central role in obesity-induced cardiomyopathy. Wildtype or MD2 knockout mice were fed with a high fat diet (HFD) or normal diet (Control) for total 16weeks, and MD2 inhibitor L6H21 (20mg/kg) or vehicle (1% CMC-Na) were administered from the beginning of the 9th week. HFD induced significant weight gain and cardiac hypertrophy, with increased cardiac fibrosis and inflammation. L6H21 administration or MD2 knockout attenuated HFD-induced obesity, inflammation and cardiac remodeling. In vitro exposure of H9C2 cells to high lipids induced cell hypertrophy with activated JNK/ERK and NF-κB pathways, which was abolished by pretreatment of MD2 inhibitor L6H21. Our results demonstrate that MD2 is essential to obesity-related cardiac hypertrophy through activating JNK/ERK and NF-κB-dependent cardiac inflammatory pathways. Targeting MD2 would be a therapeutic approach to prevent obesity-induced cardiac injury and remodeling.

Blockade of myeloid differentiation protein 2 prevents obesity-induced inflammation and nephropathy.

Obesity is a major and independent risk factor of kidney diseases. The pathogenic mechanisms of obesity-associated renal injury are recognized to at least involve a lipid-rich and pro-inflammatory state of the renal tissues, but specific mechanisms establishing causal relation remain unknown. Saturated fatty acids are elevated in obesity, and known to induce chronic inflammation in kidneys. Myeloid differentiation protein 2 (MD2) is an important protein in lipopolysaccharide-induced innate immunity response and inflammation. We suggested that obesity-associated renal injury is regulated by MD2 thereby driving an inflammatory renal injury. The used three mouse models for in vivo study: MD2 knockout mice (KO) maintained on high fat diet (HFD), wild-type mice on HFD plus L6H21, a specific MD2 inhibitor and KO mice given palmitic acid (PA) by IV injection. The in vitro studies were carried out in cultured renal tubular epithelial cells, mouse mesangial cells and primary macrophages, respectively. The HFD mice presented with increased hyperlipidemia, serum creatinine and proteinuria. Renal tissue from HFD mice had increased fibrosis, inflammatory cytokines, macrophage infiltration, and activation of NF-κB and MAPKs. This HFD-induced renal injury profile was not observed in KO mice or L6H21-treated mice. Mice given PA mimmicked the HFD-induced renal injury profiles, which were prevented by MD2 knockout. The in vitro data further confirmed MD2 mediates PA-induced inflammation. MD2 is causally related with obesity-associated renal inflammatory injury. We believe that MD2 is an attractive target for future therapeutic strategies in obesity-associated kidney diseases.

Downregulation of Long Noncoding RNA Gas5 Affects Cell Cycle and Insulin Secretion in Mouse Pancreatic ß Cells.

BACKGROUND: Evidence shows that long non-coding RNAs (lncRNAs) are involved in individual development, cell differentiation, cell cycle processes and other important life processes and are closely related to major human diseases, including diabetes. Recent studies have reported that lncRNAs are involved in ß cell functions and that lncRNA Gas5 levels decreased in T2DM patients' serum. The purpose of this study was to clarify the role of lncRNA Gas5 in mouse ß cell functions in vitro and in vivo. METHODS: lncRNA Gas5 expression in T2DM and normal mouse tissues was analyzed using qRT-PCR. RNAi, qRT-PCR, Annexin V-FITC assays, western blot, GSIS and RIA were performed to detect the effects of lncRNA Gas5 on insulin synthesis and secretion in vitro and in vivo. RESULTS: The lncRNA Gas5 level was significantly decreased in db/db mice. However, lncRNA Gas5 expression was relatively high in the pancreas of normal mice. Knockdown of lncRNA Gas5 expression led to cell cycle G1 arrest and impaired insulin synthesis and secretion in Min6 cells. Further, knockdown of lncRNA Gas5 expression in primary isolated islets resulted in decreased expression of insulin gene and transcription factors, Pdx1 and MafA. These results indicate that lncRNA Gas5 might perform as a new regulator, maintaining ß cell identity and function by affecting insulin synthesis and secretion.

Effects of Drug-Antibody Ratio on Pharmacokinetics, Biodistribution, Efficacy, and Tolerability of Antibody-Maytansinoid Conjugates.

Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of ∼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but for those with an average DAR of ∼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.

Downregulation of Long Noncoding RNA Meg3 Affects Insulin Synthesis and Secretion in Mouse Pancreatic Beta Cells.

Increasing evidence indicates that long noncoding RNAs (lncRNAs) are involved in diverse biological process. Mouse maternal expressed gene 3 (Meg3) is an imprinted gene and essential for development. Here, we explored the relationship between Meg3 and the function of mouse beta cells in vitro and in vivo. Real-time PCR analyses revealed that Meg3 was more abundantly expressed in Balb/c mouse islets than exocrine glands. Moreover, the expression of Meg3 in islets was decreased in T1DM (NOD female mice) and T2DM (db/db mice) models. Meg3 expression was modulated dynamically by glucose in Min6 cells and isolated mouse islets. The function role of Meg3 was investigated in Min6 cells and normal mouse by knockdown of Meg3 using small interfering RNA. After suppression of Meg3 expression in vitro, insulin synthesis and secretion were impaired and the rate of beta cells apoptosis was increased. Moreover, knockdown of Meg3 in vivo led to the impaired glucose tolerance and decreased insulin secretion, consisted with the reduction of insulin positive cells areas by immunochemistry assays. Notably, islets from Meg3 interference groups showed significant decrease of Pdx-1 and MafA expression in mRNA and protein levels. These results indicate that Meg3 may function as a new regulator of maintaining beta cells identity via affecting insulin production and cell apoptosis. J. Cell. Physiol. 231: 852-862, 2016. © 2015 Wiley Periodicals, Inc.

Understanding How the Stability of the Thiol-Maleimide Linkage Impacts the Pharmacokinetics of Lysine-Linked Antibody-Maytansinoid Conjugates.

Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.

Downregulation of lncRNA TUG1 affects apoptosis and insulin secretion in mouse pancreatic ß cells.

BACKGROUND: Increasing evidence indicates that long noncoding RNAs (IncRNAs) perform specific biological functions in diverse processes. Recent studies have reported that IncRNAs may be involved in ß cell function. The aim of this study was to characterize the role of IncRNA TUG1 in mouse pancreatic ß cell functioning both in vitro and in vivo. METHODS: qRT-PCR analyses were performed to detect the expression of lncRNA TUG1 in different tissues. RNAi, MTT, TUNEL and Annexin V-FITC assays and western blot, GSIS, ELISA and immunochemistry analyses were performed to detect the effect of lncRNA TUG1 on cell apoptosis and insulin secretion in vitro and in vivo. RESULTS: lncRNA TUG1 was highly expressed in pancreatic tissue compared with other organ tissues, and expression was dynamically regulated by glucose in Nit-1 cells. Knockdown of lncRNA TUG1 expression resulted in an increased apoptosis ratio and decreased insulin secretion in ß cells both in vitro and in vivo . Immunochemistry analyses suggested decreased relative islet area after treatment with lncRNA TUG1 siRNA. CONCLUSION: Downregulation of lncRNA TUG1 expression affected apoptosis and insulin secretion in pancreatic ß cells in vitro and in vivo. lncRNA TUG1 may represent a factor that regulates the function of pancreatic ß cells.

Inhibition of mitogen-activated protein kinases/nuclear factor κB-dependent inflammation by a novel chalcone protects the kidney from high fat diet-induced injuries in mice.

The prevalence of obesity has increased dramatically worldwide leading to increases in obesity-related complications, such as obesity-related glomerulopathy (ORG). Obesity is a state of chronic, low-grade inflammation, and increased inflammation in the adipose and kidney tissues has been shown to promote the progression of renal damage in obesity. Current therapeutic options for ORG are fairly limited and, as a result, we are seeing increased rates of progression to end-stage renal disease. Chalcones are a class of naturally occurring compounds with various pharmacological properties. 1-(3,4-Dihydroxyphenyl)-3-(2-methoxyphenyl)prop-2-en-1-one (L2H17) is a chalcone that we have previously synthesized and found capable of inhibiting the lipopolysaccharide-induced inflammatory response in macrophages. In this study, we investigated L2H17's effect on obesity-induced renal injury using palmitic acid-induced mouse peritoneal macrophages and high fat diet-fed mice. Our results indicate that L2H17 protects against renal injury through the inhibition of the mitogen-activated protein kinase/nuclear factor κB pathways significantly by decreasing the expression of proinflammatory cytokines and cell adhesion molecules and improving kidney histology and pathology. These findings lead us to believe that L2H17, as an anti-inflammatory agent, can be a potential therapeutic option in treating ORG.

Attenuation of inflammatory response by a novel chalcone protects kidney and heart from hyperglycemia-induced injuries in type 1 diabetic mice.

High glucose-induced inflammatory response in diabetic complications plays an important role in disease occurrence and development. With inflammatory cytokines and signaling pathways as important mediators, targeting inflammation may be a new avenue for treating diabetic complications. Chalcones are a class of natural products with various pharmacological activities. Previously, we identified L2H17 as a chalcone with good anti-inflammatory activity, inhibiting LPS-induced inflammatory response in macrophages. In this study, we examined L2H17's effect on hyperglycemia-induced inflammation both in mouse peritoneal macrophages and a streptozotocin-induced T1D mouse model. Our results indicate that L2H17 exhibits a strong inhibitory effect on the expression of pro-inflammatory cytokines, cell adhesion molecules, chemokines and macrophage adhesion via modulation of the MAPK/NF-κB pathway. Furthermore, in vivo oral administration of L2H17 resulted in a significant decrease in the expression of pro-inflammatory cytokines and cell adhesion molecules, contributing to a reduction of key markers for renal and cardiac dysfunction and improvements in fibrosis and pathological changes in both renal and cardiac tissues of diabetic mice. These findings provide the evidence supporting targeting MAPK/NF-κB pathway may be effective therapeutic strategy for diabetic complications, and suggest that L2H17 may be a promising anti-inflammatory agent with potential as a therapeutic agent in the treatment of renal and cardiac diabetic complications.

Statistics of the distribution of the abundance of molecules with various drug loads in maytansinoid antibody-drug conjugates.

The maytansinoid antibody-drug conjugates (ADCs) in clinical development for cancer therapy each contain a derivative of the microtubule-targeting agent, maytansine, covalently attached to the antibody via an engineered linker. A sample of any of these conjugates contains molecules with different numbers of maytansinoid molecules, or "drug" loads, the relative abundance of which can be determined by mass spectrometry. We examined the accuracy of the Poisson distribution and the binomial distribution in predicting the relative abundance of species with different drug loads for three antibody-maytansinoid conjugates with different antibodies and linker-maytansinoid pairings. We used variance, calculated from the experimental mass distribution data, as the parameter to determine the optimal value n of the binomial distribution number of trials. The accuracy of the Poisson distribution in predicting distribution of the species abundance in these conjugates varied among the conjugates. In contrast, the accuracy of the binomial distribution was similar for all three conjugates and comparable to the best accuracy of the Poisson distribution, as supported by a paired t-test.

Evaluation of Targets for Maytansinoid ADC Therapy Using a Novel Radiochemical Assay.

PURPOSE: Many antibody-drug conjugates (ADCs) become active only after antigen-mediated internalization and release of the cytotoxic agent via antibody degradation. Quantifying these processes can provide critical information on the suitability of a particular receptor target or antibody for ADC therapy by providing insight into the amount of cytotoxic agent released. We describe a simple and inexpensive radiolabel assay to monitor this process in cultured cancer cells. METHODS: Monoclonal antibodies were trace-labeled at their lysine residues by treatment with the N-hydroxysuccinimide ester of [(3)H]propionic acid. Human cancer cell cultures were treated with the labeled antibody at concentrations sufficient to saturate the targeted antigen. After washing to remove unbound antibody, cells were incubated and analyzed for antigen expression, conjugate degradation and catabolite formation. Results were compared with data obtained from similar assays run with radiolabeled antibody-[(3)H]maytansinoid conjugates ([(3)H]AMCs). To exemplify the method, studies were conducted with a panel of [(3)H]propionamide-antibodies to evaluate processing efficiency in EGFR-expressing SCCHN cell lines, and in NHL cell lines expressing the B-cell targets CD19, CD20, CD22 and CD37. RESULTS: Use of the [(3)H]propionamide-antibody assay yielded cell-mediated processing results similar to those obtained with corresponding maytansinoid ADCs. Further exploration allowed comparison of expression levels, antigen-dependent degradation, and catabolite formation across a panel of EGFR-expressing SCCHN cell lines, and for multiple targets in various B-cell cancer indications. CONCLUSIONS: The [(3)H]propionamide-antibody assay described here is a sensitive, facile method which enables rapid and robust assessment of relative antibody processing amounts for target, antibody, and cell line evaluation.

Modeling and Analysis of Bio-Inspired, Reconfigurable, Piezo-Driven Vibration Isolator for Spacecraft.

The positioning accuracy of spacecraft in orbit is easily affected by low-frequency micro-vibrations of the environment and internal disturbances caused by the payload. Inspired by the neck structure of birds, this study devised a piezo-driven active vibration isolation unit with high stiffness. First, a dynamic model and two-sensor feedback control method for the isolation unit were developed, and the isolation mechanism and anti-disturbance characteristics were analyzed. Further, the stability of the closed-loop was verified. Simulation models of serial and parallel systems based on the proposed vibration isolation unit were implemented to demonstrate its feasibility. The results indicate that the proposed isolation units can provide excellent low-frequency vibration isolation performance and inertial stability and that they can effectively resist the internal disturbance of the payload. Moreover, its performance can be further improved via serial or parallel reconfiguration that facilitates its adaptation to the varied isolation requirements of spacecraft.

Unmasking Chemokine-Inducing Specificity in Oligosaccharide Biomaterial to Promote Hair Growth.

Hair loss affects over 50 million people worldwide with limited therapeutic options. Despite evidence highlighting the vital role of local immune cells in regulating the life cycle of hair follicles (HFs), accurate regulation of immunocytes to directly promote hair growth remains unachieved. Here, inspired by the physiological feedback in the skin immunity to suppress microbe-triggered inflammation, an oligosaccharide biomaterial with "unmasked" specific activity is developed to recruit regulatory T (Treg ) cells around HFs, leading to accelerated hair growth in mice. By processing the glucomannan polysaccharide via controllable enzymatic cleavage, a series of oligosaccharide fractions with more specific chemokine-inducing functions is obtained. Notably, a hexasaccharide-based fraction (OG6) stimulates macrophages to selectively express Treg -chemoattractant C-C Motif Chemokine Ligand 5 (CCL5) through a mannose receptor-mediated endocytosis and NOD1/2-dependent signaling, as evidenced by molecular docking, inhibition assays, and a Foxp3-reporter mouse model. Intradermal delivery of OG6 to the depilated mouse skin promotes Treg mobilization around HFs and stimulates de novo regeneration of robust hairs. This study demonstrates that unmasking precise immunomodulatory functions in oligosaccharides from their parental polysaccharide can potentially solve the long-lasting dilemma with polysaccharide biomaterials that are widely renowned for versatile activities yet high heterogeneity, opening new avenues to designing glycan-based therapeutic tools with improved specificity and safety.