Classic Membrane Research Articles

Ligand-Receptor Interaction-Induced Intracellular Phase Separation: A Global Disruption Strategy for Resistance-Free Lethality of Pathogenic Bacteria.

Addressing the global challenge of bacterial resistance demands innovative approaches, among which multitargeting is a widely used strategy. Current strategies of multitargeting, typically achieved through drug combinations or single agents inherently aiming at multiple targets, face challenges such as stringent pharmacokinetic and pharmacodynamic requirements and cytotoxicity concerns. In this report, we propose a bacterial-specific global disruption approach as a vastly expanded multitargeting strategy that effectively disrupts bacterial subcellular organization. This effect is achieved through a pioneering chemical design of ligand-receptor interaction-induced aggregation of small molecules, i.e., DNA-induced aggregation of a diarginine peptidomimetic within bacterial cells. These intracellular aggregates display affinity toward various proteins and thus substantially interfere with essential bacterial functions and rupture bacterial cell membranes in an "inside-out" manner, leading to robust antibacterial activities and suppression of drug resistance. Additionally, biochemical analysis of macromolecule binding affinity, cytoplasmic localization patterns, and bacterial stress responses suggests that this bacterial-specific intracellular aggregation mechanism is fundamentally different from nonselective classic DNA or membrane binding mechanisms. These mechanistic distinctions, along with the peptidomimetic's selective permeation of bacterial membranes, contribute to its favorable biocompatibility and pharmacokinetic properties, enabling its in vivo antimicrobial efficacy in several animal models, including mice-based superficial wound models, subcutaneous abscess models, and septicemia infection models. These results highlight the great promise of ligand-receptor interaction-induced intracellular aggregation in achieving a globally disruptive multitargeting effect, thereby offering potential applications in the treatment of malignant cells, including pathogens, tumor cells, and infected tissues.

MG53/TRIM72: multi-organ repair protein and beyond.

MG53, a member of the tripartite motif protein family, possesses multiple functionalities due to its classic membrane repair function, anti-inflammatory ability, and E3 ubiquitin ligase properties. Initially recognized for its crucial role in membrane repair, the therapeutic potential of MG53 has been extensively explored in various diseases including muscle injury, myocardial damage, acute lung injury, and acute kidney injury. However, further research has revealed that the E3 ubiquitin ligase characteristics of MG53 also contribute to the pathogenesis of certain conditions such as diabetic cardiomyopathy, insulin resistance, and metabolic syndrome. Moreover, recent studies have highlighted the anti-tumor effects of MG53 in different types of cancer, such as small cell lung cancer, liver cancer, and colorectal cancer; these effects are closely associated with their E3 ubiquitin ligase activities. In summary, MG53 is a multifunctional protein that participates in important physiological and pathological processes of multiple organs and is a promising therapeutic target for various human diseases. MG53 plays a multi-organ protective role due to its membrane repair function and its exertion of anti-tumor effects due to its E3 ubiquitin ligase properties. In addition, the controversial aspect of MG53's E3 ubiquitin ligase properties potentially causing insulin resistance and metabolic syndrome necessitates further cross-validation for clarity.

Steady-state Approximations for Hodgkin-Huxley Cell Models: Towards Multi-scale Models of Uterine Smooth Muscle

The Tong, et al. 2011 publication presents a comprehensive mathematical model of electro- mechanical behaviour in a uterine smooth muscle cell. It incorporates ion channel currents into a classic Hodgkin-Huxley membrane voltage formulation as well as Ca2+ concentration dynamics with pumps and exchangers. Mechanical force generation by way of the Hai-Murphy smooth muscle cell model is computed based on the Ca2+ concentrations. The ‘full’ Tong, 2011 model (FTM) includes nineteen ordinary differential equations for the ion channel activation/inactivation variables out of a total of 105 equations overall. An effort at reducing the FTM into a smaller number of equations for computational efficiency – the ‘reduced’ Tong model (RTM) – aims at reproducing the overall behaviour of the FTM without excessive detail. We present here the CellML implementation of the RTM for simulating the FTM within the context of the reduced model’s primary publication as derived from the Tong, et al. 2011 model.

Steady-state Approximations for Hodgkin-Huxley Cell Models: Towards Multi-scale Models of Uterine Smooth Muscle

The Tong, et al. 2011 publication presents a comprehensive mathematical model of electro- mechanical behaviour in a uterine smooth muscle cell. It incorporates ion channel currents into a classic Hodgkin-Huxley membrane voltage formulation as well as Ca2+ concentration dynamics with pumps and exchangers. Mechanical force generation by way of the Hai-Murphy smooth muscle cell model is computed based on the Ca2+ concentrations. The ‘full’ Tong, 2011 model (FTM) includes nineteen ordinary differential equations for the ion channel activation/inactivation variables out of a total of 105 equations overall. An effort at reducing the FTM into a smaller number of equations for computational efficiency – the ‘reduced’ Tong model (RTM) – aims at reproducing the overall behaviour of the FTM without excessive detail. We present here the CellML implementation of the RTM for simulating the FTM within the context of the reduced model’s primary publication as derived from the Tong, et al. 2011 model.

Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

Measuring Expiratory Diaphragm Activity: An Electrifying Tool to Guide Positive End-Expiratory Pressure Strategy in Critically Ill Children?

Measuring Expiratory Diaphragm Activity: An Electrifying Tool to Guide Positive End-Expiratory Pressure Strategy in Critically Ill Children?

Partial osmotic pressures of ions in electrolyte solutions and the Gibbs-Guggenheim uncertainty principle.

The concept of the partial osmotic pressure of ions in an electrolyte solution is critically examined. In principle these can be defined by introducing a solvent-permeable wall and measuring the force per unit area which can certainly be attributed to individual ions. Here I demonstrate that although the total wall force balances the bulk osmotic pressure as required by mechanical equilibrium, the individual partial osmotic pressures are extrathermodynamic quantities dependent on the electrical structure at the wall, and as such they resemble attempts to define individual ion activity coefficients. The limiting case where the wall is a barrier to only one species of ion is also considered, and with ions on both sides the classic Gibbs-Donnan membrane equilibrium is recovered, thus providing a unifying treatment. The analysis can be extended to illustrate how the electrical state of the bulk is affected by the nature of the walls and the container handling history, thus supporting the "Gibbs-Guggenheim uncertainty principle," namely the notion that the electrical state is unmeasurable and usually accidentally determined. Since this uncertainty is conferred also onto individual ion activities, it has implications for the current (2002) IUPAC definition of pH.

Clinical Profile and Risk Factors of Exudative Age-related Macular Degeneration: A Hospital Based Case-control Study

Introduction: Age-Related Macular Degeneration (ARMD) is one of the leading causes of vision loss in the elderly population. ARMD is characterised by progressive degeneration of the retinal pigment epithelial complex and photoreceptors primarily in the macular region of the retina. The prevalence of ARMD in India ranges from 39.5% to 0.3%, as reported in populationbased studies. Numerous risk factors, both modifiable and nonmodifiable, have been identified for this condition. Aim: To determine the clinical profile and risk factors associated with exudative ARMD. Materials and Methods: This is a case-control study conducted in a tertiary eye care hospital in South Kerala from June 2011 to June 2012. The cases were patients attending the Outpatient Department (OPD) and Retina clinic who were diagnosed with wet ARMD. The corresponding control is the next patient seen after the case who is of the same sex and comparable age. Data were analysed using the Statistical Package for Social Sciences (SPSS) version 17.0. Chi-square test was used to elucidate the association between the presence of ARMD and diet, educational status, occupation, smoking, alcoholism, hypertension, diabetes mellitus, cardiovascular disease, obesity, hyperlipidemia, history of cataract surgery, and cataract. Multivariate logistic regression analysis was used to assess these risk factors for cases and controls. Results: A total of 130 subjects consisting of 65 cases and 65 controls participated in the study. The majority of patients were in the age group of 60-69 years. History of hyperlipidemia (Odds Ratio [OR]: 1.649, Confidence Interval [CI]: 0.524-5.191, p=0.042) was significantly associated with the development of exudative ARMD. Hypertension (OR: 1.398, CI: 0.694-2.815, p=0.051), cardiovascular disease (OR: 1.770, CI: 0.274-5.064, p=0.188), Body Mass Index (BMI) >25 (OR: 0.537, CI: 0.254- 1.133, p=0.075), dietary factors (OR: 1.351, CI: 0.461-3.961, p=0.609), smoking (OR: 1.400, CI: 0.484-4.051, p=0.593), alcoholism (OR: 1.400, CI: 0.484-4.051, p=0.593), history of cataract surgery (OR: 1.160, CI: 0.411-3.279, p=0.456), cataract (OR: 1.618, CI: 0.692-3.782, p=0.149) were not found to be significant. The results may vary in the present scenario due to changes that have occurred over the years, especially in the case of smoking and alcoholism, as the prevalence of which has increased. The majority (88.75%) of study subjects have classic Choroidal Neovascular Membrane (CNVM). Conclusion: ARMD was found to have a significant association with hyperlipidemia. Those with hypertension, cardiovascular disease, cataract, smokers, and alcoholics have a higher risk of developing exudative ARMD. By controlling modifiable risk factors like hyperlipidemia and by avoiding smoking and alcoholism, the authors can prevent this potentially blinding condition to some extent.

Cost-effectiveness of installing modules for remote reading of natural gas consumption based on a pilot project

The gas sector in the EU is very developed and advanced, but there are still some steps and measures to improve this sector. One of the measures for the improvement, but also for the collection of data on energy consumption for an easy transition to a low carbon economy, are the projects of installing smart meters. This paper analyzes a pilot project of installing modules for remote reading of natural gas consumption on existing membrane gas meters. In the pilot project observed, modules for remote reading were installed on more than 10,000 existing meters, and their use, activation, and as well as the benefits that are achieved by using them are described in detail. These modules use the increasingly popular Sigfox network, which has proven to be an excellent tool for data transmission with low energy consumption, which can greatly simplify the digitization of many energy sectors. The installation of modules for remote reading on existing meters represents a novelty improvement of classic membrane meters towards modern and increasingly sought-after smart meters, which has proven to be time effective because, unlike some smart meter installation projects, this approach requires a very short installation time.The results of the financial and sensitivity analysis have shown that with the currently valid legal regulation the financial savings achieved by energy supplier due to exemption from paying penalties are more than sufficient to cover the investment and operating costs of the entire investment. In case the legislation changes or is different, this paper proposes the concept of transferring partial financial value of energy savings from end consumers to energy supplier in order to cover the investment and operating costs of implementation.

Radio-Histological Correlation of Lung Features in Severe COVID-19 Through CT-Scan and Lung Ultrasound Evaluation.

BackgroundPatients with coronavirus disease 2019 (COVID-19) can develop severe bilateral pneumonia leading to respiratory failure. Lung histological samples were scarce due to the high risk of contamination during autopsies. We aimed to correlate histological COVID-19 features with radiological findings through lung ultrasound (LU)-guided postmortem core needle biopsies (CNBs) and computerized tomography (CT) scans.MethodologyWe performed an observational prospective study, including 30 consecutive patients with severe COVID-19. The thorax was divided into 12 explorations regions to correlate LU and CT-scan features. Histological findings were also related to radiological features through CNBs.ResultsMean age was 62.56 ± 13.27 years old, with 96.7% male patients. Postmortem LU-guided CNBs were performed in 13 patients. Thirty patients were evaluated with both thoracic LU and chest CT scan, representing a total of 279 thoracic regions explored. The most frequent LU finding was B2-lines (49.1%). The most CT-scan finding was ground-glass opacity (GGO, 29%). Pathological CT-scan findings were commonly observed when B2-lines or C-lines were identified through LU (positive predictive value, PPV, 87.1%). Twenty-five postmortem echo-guided histological samples were obtained from 12 patients. Histological samples showed diffuse alveolar damage (DAD) (75%) and chronic interstitial inflammation (25%). The observed DAD was heterogeneous, showing multiple evolving patterns of damage, including exudative (33.3%), fibrotic (33.3%), and organizing (8.3%) phases. In those patients with acute or exudative pattern, two lesions were distinguished: classic hyaline membrane; fibrin “plug” in alveolar space (acute fibrinous organizing pneumonia, AFOP). C-profile was described in 33.3% and presented histological signs of DAD and lung fibrosis. The predominant findings were collagen deposition (50%) and AFOP (50%). B2-lines were identified in 66.7%; the presence of hyaline membrane was the predominant finding (37.5%), then organizing pneumonia (12.5%) and fibrosis (37.5%). No A-lines or B1-lines were observed in these patients.ConclusionLU B2-lines and C-profile are predominantly identified in patients with severe COVID-19 with respiratory worsening, which correspond to different CT patterns and histological findings of DAD and lung fibrosis.

Rational design of mixed nanomicelle eye drops with structural integrity investigation

Chitosan oligosaccharide-stearic acid-Valyl-Valyl-Valine/1-2-Dioleoyl-sn-glycero-3-phosphoethanolamine (CSO-SA-VVV5:2/DOPE) nanomicelles were rationally designed and developed for topical drug delivery to the posterior segment of the eye. The new ligand of VVV selected by computer-aided design exhibited better peptide transporter 1 active targeting in human conjunctival epithelial cells (HConEpiC) than other ligands mentioned in this project. The classic membrane fusion lipid of DOPE was indicated to facilitate the stability and lysosomal escape of the mixed nanomicelles. Förster Resonance Energy Transfer was used to investigate the integrity of mixed nanomicelles in HConEpiC after passing through cell monolayer as well as in ocular tissues after topical administration. The results indicated that mixed nanomicelles could keep more intact micellar structure than CSO-SA nanomicelles in transit. These findings suggested that CSO-SA-VVV5:2/DOPE nanomicelles could overcome multiple ocular barriers and offer an efficient strategy for drug delivery from ocular surface to the posterior segment of the eye. Statement of significanceOcular drug delivery systems face multiple physiological barriers in delivering drugs to the posterior segment of the eye by topical administration. In this study, new ligand of Valyl-Valyl-Valine was selected with computer-aided design for active targeting to peptide transporter 1 and anchored onto nanomicelles. Chitosan oligosaccharide-stearic acid- Valyl-Valyl-Valine/1-2-Dioleoyl-sn-glycero-3-phosphoethanolamine nanomicelles were rational designed. The mixed nanomicelles exhibited better active targeting ability and lysosomal escape. Nanomicellar integrity analysis with fluorescence resonance energy transfer technique demonstrated that mixed nanomicelles significantly enhanced cell permeability and exhibited more intact micellar structure in transit. These results suggested that the mixed nanomicelle eye drops have the potential to deliver drugs from ocular surface to the posterior segment of the eye.

Abstract PO-101: CmP signaling network unveils novel biomarkers for triple negative breast cancer in African American women

Abstract Breast cancer is the most commonly diagnosed cancer worldwide and remains the second leading cause of cancer death. While breast cancer mortality has steadily declined over the past decades through medical advances, an alarming disparity in breast cancer mortality has emerged between African American women (AAW) and Caucasian American women (CAW); and new evidence suggests more aggressive behavior of triple-negative breast cancer (TNBC) in AAW may contribute to racial differences in tumor biology and mortality. Progesterone (PRG) is capable of exerting its cellular effects through either its classic, non-classic or combined responses through binding to either classic nuclear PRG receptors (nPRs) or non-classic membrane PRG receptors (mPRs), warranting both pathways an equally important status in PRG-mediated signaling. In our previous report, we demonstrated that the CCM signaling complex (CSC) consisting of CCM1, CCM2, and CCM3 proteins can couple both nPRs and mPRs signaling cascades to form a CSC-mPRs-PRG-nPRs (CmPn) signaling network in nPR positive(+) breast cancer cells. In this report, we furthered our research by establishing the CSC-mPRs-PRG (CmP) signaling network in nPR(-) breast cancer cells, demonstrating that a common core mechanism exists, regardless of nPR(+/-) cell type. This is the first report stating that inducible expression patterns exist between CCMs and major mPRs in TNBC cells. Furthermore, we firstly show mPRs in TNBC cells are localized in the nucleus and participate in nucleocytoplasmic shuttling in a coordinately synchronized fashion with CCM proteins under steroid actions, following the same cellular distribution as other well-defined steroid hormone receptors. Finally, for the first time, we deconvoluted the CmP signalosome by using multi-omics approaches, which helped us understand key factors within the CmP network, and identify 21 specific biomarkers with potential clinical applications associated with AAW-TNBC tumorigenesis. These novel biomarkers could have immediate clinical implications to dramatically improve health disparities among AAW-TNBCs. Citation Format: Johnathan Abou-Fadel, Jun Zhang. CmP signaling network unveils novel biomarkers for triple negative breast cancer in African American women [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-101.

Characterization and proteomics of chicken seminal plasma extracellular vesicles.

In mammals, seminal plasma extracellular vesicles (SPEVs) can regulate sperm motility and capacitation. The characteristics and functions of SPEVs in avians have been rarely reported. In this study, chicken SPEVs were isolated and characterized by transmission and scanning electron microscopy (TEM/SEM) and nanoparticle tracking analysis (NTA); furthermore, seven extracellular vesicle (EVs) marker proteins were detected by Western blot (WB). TEM revealed that chicken SPEVs had a classic bilayer membrane structure. NTA confirmed that the size of SPEVs was 30-250nm, and concentration ranged from 8.0 E+11-8.5 E+11 particles/ml. There were 3073 SPEVs proteins identified by deep sequencing, including 2794 intracellular proteins and 279 extracellular proteins. The overlap rate of proteomes between chicken SPEVs and vesicles reported in the Vesiclepedia database reached 86%, and 360 new proteins that had not been reported by the ExoCarta and Vesiclepedia databases were identified in chicken SPEV proteomes. Gene Ontology (GO) analysis revealed that chicken SPEV proteins were mainly enriched in supplying energy and transporting protein. There were 4 IFT family proteins speculated to play an important role in sperm composition and function. Our data were compared with two previously published studies on the proteomics of chicken seminal plasma (SP) and hen uterine fluid, and some overlapping proteins described in chicken SPEVs had been identified in hen uterine fluid (545) and chicken SP (284). In conclusion, these findings will increase our understanding of the content and composition of proteome in SPEVs and provide new insights into the important role of the SPEV regulation in sperm functions.

Bringing rafts to life: Lessons learned from lipid organization across diverse biological membranes

The ability of lipids to drive lateral organization is a remarkable feature of membranes and has been hypothesized to underlie the architecture of cells. Models for lipid rafts and related domains were originally based on the mammalian plasma membrane, but the nature of heterogeneity in this system is still not fully resolved. However, the concept of lipid-driven organization has been highly influential across biology, and has led to discoveries in organisms that feature a diversity of lipid chemistries and physiological needs. Here we review several emerging and instructive cases of membrane organization in non-mammalian systems. In bacteria, several types of membrane domains that act in metabolism and signaling have been elucidated. These widen our view of what constitutes a raft, but also introduce new questions about the relationship between organization and function. In yeast, observable membrane organization is found in both the plasma membrane and the vacuole. The latter serves as the best example of classic membrane phase partitioning in a living system to date, suggesting that internal organelles are important membranes to investigate across eukaryotes. Finally, we highlight plants as powerful model systems for complex membrane interactions in multicellular organisms. Plant membranes are organized by unique glycosphingolipids, supporting the importance of carbohydrate interactions in organizing lateral domains. These examples demonstrate that membrane organization is a potentially universal phenonenon in biology and argue for the continued broadening of lipid physical chemistry research into a wide range of systems.

Anti-glomerular Basement Membrane Disease: What Have We Learned?

Since the first clinicopathologic description by Ernest Goodpasture of a patient whom he considered to have died of influenza in 1919, substantial progress has been made in our knowledge of anti-glomerular basement membrane disease. This has led to a significant decrease in the morbidity and mortality associated with this disease. In this paper, we aim to review the literature that has enhanced our understanding of classic anti-glomerular basement membrane disease and its clinic-pathologic variants in the key areas of immunopathogenesis and histopathology. We also summarize varied clinical presentations and therapeutic strategies.

A Closed-Form Solution of Prestressed Annular Membrane Internally-Connected with Rigid Circular Plate and Transversely-Loaded by Central Shaft

In this paper, we analytically dealt with the usually so-called prestressed annular membrane problem, that is, the problem of axisymmetric deformation of the annular membrane with an initial in-plane tensile stress, in which the prestressed annular membrane is peripherally fixed, internally connected with a rigid circular plate, and loaded by a shaft at the center of this rigid circular plate. The prestress effect, that is, the influence of the initial stress in the undeformed membrane on the axisymmetric deformation of the membrane, was taken into account in this study by establishing the boundary condition with initial stress, while in the existing work by establishing the physical equation with initial stress. By creating an integral expression of elementary function, the governing equation of a second-order differential equation was reduced to a first-order differential equation with an undetermined integral constant. According to the three preconditions that the undetermined integral constant is less than, equal to, or greater than zero, the resulting first-order differential equation was further divided into three cases to solve, such that each case can be solved by creating a new integral expression of elementary function. Finally, a characteristic equation for determining the three preconditions was deduced in order to make the three preconditions correspond to the situation in practice. The solution presented here could be called the extended annular membrane solution since it can be regressed into the classic annular membrane solution when the initial stress is equal to zero.

PCI Membranes buys Pentair product line

PCI Membranes has completed the strategic acquisition of Pentair's X-Flow Classic Tubular Membrane, including assets and customers.

Probing Acetonitrile Solvation in Perfluorsulfonate Ion Exchange Membrane By in-Situ FTIR

In-situ FTIR is a successful tool to study water absorption and interactions within the Perfluorsulfonate Ion Exchange Membrane (PFSA)(1–4). The versatility of the technique can reveal in-depth information related to proton transport in PFSA, including proton dissociation and solvation. Another desirable feature is that experiments under controlled water content(λ) is also possible. Studies of PFSA membranes equilibrated with different λ was fundamental to understand membrane behavior(5). The volatile nature of acetonitrile and many organic solvents adds to the inaccuracy of many classic membrane characterization methods. To overcome this, techniques tailored for membrane incorporated with organic solvents are necessary. In this study, acetonitrile and lithiated PFSA were chosen to revisit the in-situ IR technique. The goal of this work is to examine acetonitrile’s role in membrane swelling and to create a ‘slow-motion picture’ of how acetonitrile molecules behave upon being imbibed into the membrane, especially at low λ. The vibrational signal of cyanide group (~2250 cm-1)does not overlap with other signals from the membrane and is known to respond to dipole interactions by shifting to higher wavenumber(6). This feature makes an acetonitrile-swollen system an even more favorable candidate for in-situ IR study compared to its aqueous counterpart. For studying proton transport in membrane, initial water buildup in membrane yields a weak and broad signal, making the already obscure -OH stretching region even more complicated to analyze. Without interference from hydrogen bonding, lithium ion solvation with cyanide can be clearly observed. Coupled with monitoring -SO3- area, it is totally feasible to simultaneously see both cation dissociation from membrane (Fig.1A) and cation solvation in solvent (Fig.1B) in a real-time manner to compare with fully swollen state. Acknowledgement We gratefully acknowledge the support of this work by the U.S. Department of Energy, Office of Electricity Delivery and Energy Reliability (Dr. Imre Gyuk). We also thank 3M for providing membranes. References Elabd YA, Baschetti MG, Barbari TA. Time-resolved Fourier transform infrared/attenuated total reflection spectroscopy for the measurement of molecular diffusion in polymers. J Polym Sci Part B Polym Phys. 2003;41(22):2794–807.Hallinan DT, Elabd YA. Diffusion of Water in Nafion Using Time-Resolved Fourier Transform Infrared−Attenuated Total Reflectance Spectroscopy. J Phys Chem B. 2009 ;113(13):4257–66.Hallinan DT, Elabd YA. Diffusion and Sorption of Methanol and Water in Nafion Using Time-Resolved Fourier Transform Infrared−Attenuated Total Reflectance Spectroscopy. J Phys Chem B. 2007 ;111(46):13221–30.Kunimatsu K, Bae B, Miyatake K, Uchida H, Watanabe M. ATR-FTIR Study of Water in Nafion Membrane Combined with Proton Conductivity Measurements during Hydration/Dehydration Cycle. J Phys Chem B. 2011 21;115(15):4315–21.Zawodzinski TA, Derouin C, Radzinski S, Sherman RJ, Smith VT, Springer TE, et al. Water Uptake by and Transport Through Nafion® 117 Membranes. J Electrochem Soc. 1993 ;140(4):1041–7.Barthel J, Buchner R, Wismeth E. FTIR Spectroscopy of Ion Solvation of LiClO4 and LiSCN in Acetonitrile, Benzonitrile, and Propylene Carbonate. J Solut Chem. 2000 ;29(10):937–54. Figure.1A: SO stretching shift due to solvation; Figure.1B: Normalized CN stretching area upon acetonitrile buildup in the membrane. Blue dashline indicates corresponding fully swollen state. Figure 1

Cell-free expression, purification and characterization of Drosophila melanogaster odorant receptor OR42a and its co-receptor

Olfactory receptors (OR), a group of classic membrane proteins, plays a vital role in insect reproduction and acclimatization. Deciphering the molecular mechanism of insect olfaction could enhance pest control and environmental protection. Studies on ORs have faced a major bottleneck due to the notoriously strong hydrophobicity of ORs, which results in difficult expression in heterologous cell systems. Here, we demonstrated that insect ORs could be functionally produced using the E. coli cell-free protein synthesis system (CFPS), in which the highest yield of total ORs is 350 μg per 1 ml reaction. We tested the effects of detergent types and concentrations on soluble expression of ORs. The ORs showed a classic α-helical infrared spectrum. Quartz crystal microbalance (QCM) was used to demonstrate that ORs fold correctly and respond to their ligands. This is the first report that insect OR42a could be functionally produced in vitro. This approach may facilitate the development of biomimetic olfactory biosensors and may also be utilized for drug positioning and development, environmental protection and agriculture.

CCN3 and DLL1 co-regulate osteogenic differentiation of mouse embryonic fibroblasts in a Hey1-dependent manner

Notch signaling pathway is one of the most important pathways to regulate intercellular signal transduction and is crucial in the regulation of bone regeneration. Nephroblastoma overexpressed (NOV or CCN3) serves as a non-canonical secreted ligand of Notch signaling pathway and its role in the process of osteogenic differentiation of mesenchymal stem cells (MSCs) was undefined. Here we conducted a comprehensive study on this issue. In vivo and in vitro studies have shown that CCN3 significantly inhibited the early and late osteogenic differentiation of mouse embryonic fibroblasts (MEFs), the expression of osteogenesis-related factors, and the subcutaneous ectopic osteogenesis of MEFs in nude mice. In mechanism studies, we found that CCN3 significantly inhibited the expression of BMP9 and the activation of BMP/Smad and BMP/MAPK signaling pathways. There was also a mutual inhibition between CCN3 and DLL1, one of the classic membrane protein ligands of Notch signaling pathway. Additionally, we further found that Hey1, the target gene shared by BMP and Notch signaling pathways, partially reversed the inhibitory effect of CCN3 on osteoblastic differentiation of MEFs. In summary, our findings suggested that CCN3 significantly inhibited the osteogenic differentiation of MEFs. The inhibitory effect of CCN3 was mainly through the inhibition of BMP signaling and the mutual inhibition with DLL1, so as to inhibit the expression of Hey1, the target gene shared by BMP and Notch signaling pathways.