Biomolecular Experiments Research Articles

#1996 Identification of the biological bases associated with the mTOR-I-related podocytopathy through high-resolution mass spectrometry

Abstract Background and Aims mTOR inhibitors (mTOR-I) often cause podocytopathy with the development of proteinuria (sometimes in the nephrotic range) in kidney transplant patients, but the biological mechanisms underlying this effect have been only partially defined. Method Immortalized human podocytes were treated in vitro with different everolimus (EVE) doses (10, 100, 200, 500 nM) for 24 h. Total cell lysates and supernatants were subjected to high-resolution mass spectrometry (Proteomics analysis). Innovative tests were used for bioinformatics analysis, including the Support Vector machine (SVM) and partial least squares discriminant analysis (PLS-DA). The results were validated in vitro and in human urine samples from 30 patients treated with EVE-based immunosuppressive treatment, using various molecular biology techniques (western blot, ELISA). Results Proteomic evaluation identified more than 7000 differentially expressed proteins. Among these, after a conservative statistical analysis, 78 showed an expression level significantly and directly correlated with EVE dosage. Functional analysis of the selected proteins showed a biological interconnection that involved several pathways, including kinases, cell cycle regulation, epithelial–mesenchymal transition, and protein synthesis. Osteopontin (SPP1) and Polo Like Kinase 2 (PLK2) were two of the most significantly correlated proteins (p < 0.00001, FDR<0.5%). The data were then confirmed through biomolecular experiments in vitro and in vivo in urine samples from kidney transplant recipients who received immunosuppressive treatment with EVE. Conclusion This study, for the first time, analyzed the biological impact of mTOR-I on podocytes, helping to understand not only the molecular basis of its therapeutic effect but also the biological reasons underlying their adverse events (particularly proteinuria). Validation of our data on larger cohorts of patients treated with this category of drugs will help identify new therapeutic targets to minimize adverse effects.

Optimal control pulses for the 1.2-GHz (28.2-T) NMR spectrometers

The ability to measure nuclear magnetic resonance (NMR) spectra with a large sample volume is crucial for concentration-limited biological samples to attain adequate signal-to-noise (S/N) ratio. The possibility to measure with a 5-mm cryoprobe is currently absent at the 1.2-GHz NMR instruments due to the exceedingly high radio frequency power demands, which is four times compared to 600-MHz instruments. Here, we overcome the high-power demands by designing optimal control (OC) pulses with up to 20 times lower power requirements than currently necessary at a 1.2-GHz spectrometer. We show that multidimensional biomolecular NMR experiments constructed using these OC pulses can bestow improvement in the S/N ratio of up to 26%. With the expected power limitations of a 5-mm cryoprobe, we observe an enhancement in the S/N ratio of more than 240% using our OC sequences. This motivates the development of a cryoprobe with a larger volume than the current 3-mm cryoprobes.

Isoliensinine suppresses bone loss by targeted inhibition of RANKL-RANK binding

BackgroundOsteoporosis, a systemic metabolic bone disease, is often caused by the disruption of dynamic equilibrium between osteoclasts and osteoblasts. Overactive bone resorption, in which osteoclasts play a major role, is one of the most common and major causes of osteoporosis. Less costly and more effective drug treatments for this disease are needed. Based on the combination of molecular docking techniques and in vitro cell assays, this study aimed to explore the mechanism by which Isoliensinine (ILS) protects the bone loss by inhibiting osteoclast differentiation. MethodsA virtual docking model based on molecular docking technology was used to investigate the interactions between ILS and the Receptor Activator of Nuclear Kappa-B (RANK)/Receptor Activator of Nuclear Kappa-B Ligand (RANKL).In this study, we determined the effective dose of action of ILS to inhibit osteoclast differentiation in vitro and, using bone resorption experiments, RT-CPR and Western Blot investigated the effects of ILS on bone resorption function and normal expression of osteoclast-associated genes and proteins, and validated potential mechanistic pathways. In vivo experiments revealed that ILS could inhibit bone loss through Micro-CT results.Finally, the molecular interaction between ILS and RANK/RANKL was investigated using biomolecular interaction experiments to verify the correctness and accuracy of the computational results. ResultsILS binds to RANK and RANKL proteins, respectively, through virtual molecular docking. The Surface Plasmon Resonance (SPR) experiment results revealed that phosphorylated JNK, ERK, P38, and P65 expression was significantly downregulated when ILS were targeted to inhibit RANKL/RANK binding. At the same time, the expression of IKB-a was significantly increased under the stimulation of ILS, which rescued the degradation of IKB-a. ILS can significantly inhibit the levels of Reactive Oxygen Species (ROS) and Ca2 + concentration in vitro. Finally, the results of Micro-CT showed that ILS can significantly inhibit bone loss in vivo, indicating that ILS has a potential role in the treatment of osteoporosis. ConclusionILS inhibits osteoclast differentiation and bone loss by preventing the normal binding of RANKL/RANK, affecting downstream signaling pathways, including MAPK.NF-KB, ROS, Ca2+, genes, and proteins.

The high-strength and toughness Janus bionic periosteum matching bone development and growth in children

Pediatric periosteum requires suitable strength to stabilize bone injury and match the rapid growth of children's bone, as well as strong osteogenic and angiogenic capacity to promote bone repair. Here, a Janus membrane was constructed based on the natural gradient network of bacterial nano-cellulose (BNC), the molecular chain assembly of fish collagen (Col I) was achieved through the formation of amide bond from the amino group on the collagen and carboxyl group on the BNC surface after selective oxidization by using TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical). In situ synthesis of nano-hydroxyapatite (nHA) was conducted in the looser network of BNC by adjusting pH after addition in sequence of Ca(NO3)2·4H2O and Na3PO4 solution. The Janus membrane possessed Young's modulus and tensile capacity within the ligament range (0.113 GPa and 29.63 MPa, respectively), which allow material-bone fixation and integration. In vitro biomolecular experiments showed that the Janus periosteum promoted the proliferation of bone marrow mesenchymal stem cells and the expression of angiogenic and osteogenic factors. The promotions in angiogenesis and osteogenesis were further verified in vivo. Overall, the Janus biomimetic periosteum met the requirements of growth and development in children, indicating its regenerative potential for the treatment of periosteum-associated diseases in children.

HIV-1 VIF and human APOBEC3G interaction directly observed through molecular specific labeling using a new dual promotor vector

Over the last few decades, protein NMR isotope labeling methods using E. coli based expression have revolutionized the information accessible from biomolecular NMR experiments. Selective labeling of a protein of interest in a multi-protein complex can significantly reduce the number of cross-peaks and allow for study of large protein complexes. However, limitations still remain since some proteins are not stable independently and cannot be separately labeled in either NMR active isotope enriched or unenriched media and reconstituted into a multimeric complex. To overcome this limitation, the LEGO NMR method was previously developed using protein expression plasmids containing T7 or araBAD promoters to separately express proteins in the same E. coli after changing between labeled and unlabeled media.Building on this, we developed a method to label the Human Immunodeficiency Virus type 1 viral infectivity factor (HIV-1 Vif), a monomerically unstable protein, in complex with CBFβ, it’s host binding partner. We designed a dual promoter plasmid containing both T7 and araBAD promoters to independently control the expression of HIV-1 Vif in NMR active isotope enriched media and CBFβ in unenriched media. Using this method, we assigned the backbone resonance and directly observed the binding of HIV-1 Vif with APOBEC3G, a host restriction factor to HIV-1.

Electrospun Fibers Improving Cellular Respiration via Mitochondrial Protection.

Cellular respiration is the prerequisite for cell survival and functions, and mitochondrial function and microcirculation oxygen supply are essential for cellular respiration. However, in diabetic fracture, cellular respiration of bone marrow stem cells (BMSCs) is disrupted because of the dysfunction of mitochondria and microcirculation disorders. Here, the electrospun fibers of GelMA loaded with Hif-1 pathway activator (DFO) are constructed to improve the cellular respiration of BMSCs via protecting mitochondrial function and reconstructing microcirculation. The sequential process of electrospinning and UV crosslinking endowed the electrospun fibers with breathability and the biomechanical properties like the periosteum. In vitro biomolecular experiments showed that by crosslinking grafted polyethylene glycol acrylate liposomes loaded with DFO, the functional electrospun fibers can release DFO locally to activate Hif-1 in BMSCs, which can regulate the balance of Bcl-2/Bax to protect mitochondria and upregulate the expression of VEGF to reconstruct microcirculation. Animal experiments confirmed that the functional electrospun fibers can promote the recovery of diabetic fracture in vivo. These suggested that the functional electrospun fibers can improve cellular respiration for cell survival and functions of BMSCs. This study provides a new treatment strategy for diabetic fracture and other tissue regeneration on basis of cellular respiration improvement.

PROXIMA-1 beamline for macromolecular crystallography measurements at Synchrotron SOLEIL.

The undulator beamline PROXIMA-1 at Synchrotron SOLEIL scheduled itsfirst users in March 2008. The endstation is dedicated to biomolecular crystallography experiments, with a layout designed to favour anomalous data recording and studies of crystals with large cell dimensions. In 12 years, the beamline has accommodated 4267 shifts of 8 h and more than 6300 visitors. By the end of 2020, it saw 1039 identified published scientific papers referring to1415 coordinates deposited in the Protein Data Bank. The current paper describes the PROXIMA-1 beamline, including the recent specific implementations developed for the sample environment. The setup installed in the experimental station contains numerous beam-shaping equipment, a chi-geometry three-axis goniometer, a single-photon-counting pixel-array X-ray detector, combined with a medium-throughput sample exchange robot. As part of a standard experimental scheme, PROXIMA-1 can also be accessed via `mail-in' services or remotely.

Labeling Preferences of Diazirines with Protein Biomolecules.

Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.

BioAFMviewer: An interactive interface for simulated AFM scanning of biomolecular structures and dynamics.

We provide a stand-alone software, the BioAFMviewer, which transforms biomolecular structures into the graphical representation corresponding to the outcome of atomic force microscopy (AFM) experiments. The AFM graphics is obtained by performing simulated scanning over the molecular structure encoded in the corresponding PDB file. A versatile molecular viewer integrates the visualization of PDB structures and control over their orientation, while synchronized simulated scanning with variable spatial resolution and tip-shape geometry produces the corresponding AFM graphics. We demonstrate the applicability of the BioAFMviewer by comparing simulated AFM graphics to high-speed AFM observations of proteins. The software can furthermore process molecular movies of conformational motions, e.g. those obtained from servers which model functional transitions within a protein, and produce the corresponding simulated AFM movie. The BioAFMviewer software provides the platform to employ the plethora of structural and dynamical data of proteins in order to help in the interpretation of biomolecular AFM experiments.

Cloning short DNA into plasmids by one-step PCR.

BackgroundPlasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one‐step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10–15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo.MethodsThe pEGFP‐N1‐HA plasmid was constructed by one‐step PCR and transformation. Cells were transfected with pEGFP‐N1‐HA and pEGFP‐N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA‐GFP fusion protein was detected by confocal microscopy.ResultsThe pEGFP‐N1‐HA plasmid was successfully constructed and HA expression in cells.ConclusionsFree from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction.Key pointsSignificant findings of the studyA method to clone short DNA into plasmids was found.What this study addsOur study provides a flexible and economical option to clone short DNA into plasmids.

Geometric Characterization of Data Sets with Unique Reduced Gröbner Bases.

Model selection based on experimental data is an important challenge in biological data science. Particularly when collecting data is expensive or time-consuming, as it is often the case with clinical trial and biomolecular experiments, the problem of selecting information-rich data becomes crucial for creating relevant models. We identify geometric properties of input data that result in an unique algebraic model, and we show that if the data form a staircase, or a so-called linear shift of a staircase, the ideal of the points has a unique reduced Gröbner basis and thus corresponds to a unique model. We use linear shifts to partition data into equivalence classes with the same basis. We demonstrate the utility of the results by applying them to a Boolean model of the well-studied lac operon in E. coli.

Loss of function in SAGA deubiquitinating module caused by Sgf73 H93A mutation: A molecular dynamics study

The Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating module (DUBm), comprising Ubp8, Sgf11, Sus1, and Sgf73, functions as a deubiquitinase. Data from recent biomolecular experiments have indicated that the H93A mutation in Sgf73 abrogates the enzyme function without interfering with module formation. Interestingly Sgf73 H93 residue is neither involved in the active site nor near the ubiquitin substrate binding site but is capable of influencing the active site through an allosteric mechanism. In this study, molecular dynamics simulations were used to analyze the structural discrepancy between wild-type and H93A mutant of the SAGA DUBm to reveal the interprotein communication pathway linking the mutation site to the catalytic site. We concluded that H93A mutation directly impairs Sgf73's zinc finger motif and further induces a downward movement of Sgf11's N-terminal long α-helix. The structural influence gradually propagates toward Sgf11's C-terminal end and results in the rearrangement of the catalytic triad of Ubp8 protein. The repositioned catalytic Cys-His-Asn triad could no longer execute the deubiquitinating function.

Reliable Biomolecular Structural Modelling with Small-Angle Scattering

The small-angle scattering (SAS) of X-rays or neutrons from biomolecules in solution can provide (1) unique structural insights, (2) information on the range of potential conformations present, (3) and powerful restraints for integrative/hybrid structural modelling (IHM). The rotational and ensemble averaging of the signal presents multiple challenges to the reliable utilisation of SAS data: (1) demonstrating SAS profile represents the species of interest, (2) reliably evaluating model fits, and (3) over-interpretation and over-fitting of data. A community driven project has been underway for more than a decade now to define the information that should be made available regarding sample quality, data acquisition and reduction, presentation of scattering data and validation, and modelling for biomolecular SAS experiments. These guidelines (Trewhella et al. (2017) Acta D73, 710) provide a reporting framework so that “readers can independently assess the quality of the data and the basis for any interpretations presented” concerning the results of bimolecular SAS experiments, including the generation or testing of 3D models. The validation task forces for SAS (Trewhella et al. (2013) Structure 21, 875) and for IHM (Sali et al. (2015) Structure 23, 1156) of the world-wide Protein Data Bank are additionally addressing the complex issues concerning the archiving and validation of IHM models that depend upon computational methods and disparate data from multiple techniques (Burley et al. (2017) Structure 25, 1317). Finally, the question of how many parameters are justified by the model evidence inherent in the SAS profile is an important question to answer when modelling conformational ensembles (Potrzebowski, Trewhella and Andr⌈ (2018) in review PLOS Computational Biology). This presentation will address the recommended reporting framework and its rationale, the reliable incorporation of SAS data in IHM, and the strengths and limits of SAS in ensemble modelling.

An expression signature model to predict lung adenocarcinoma-specific survival.

BackgroundThe current TNM staging system plays a central role in lung adenocarcinoma (LUAD) prognosis. However, it may not adequately stratify the risk of tumor recurrence. With the aid of gene expression profiling, we identified 31 lncRNAs whose expressions in tumor tissues could be used as a risk indicator for the guidance of lung cancer therapy. This exploratory analysis may shed new light on identification of potential prognostic factors.Materials and methodsA survival prediction scoring model was developed from the data that are publicly available in The Cancer Genome Atlas (TCGA) LUAD RNA Sequencing dataset. Multivariate Cox regression analysis and Kaplan–Meier analysis were performed on a cohort of 254 stage I lung carcinoma patients with survival records.ResultsOur model indicates that the panels comprising 31 lncRNAs are highly associated with overall survival (OS): 18.9% (95% CI: 10.4%–34.5%) and 89.5% (95% CI: 80.7%–99.2%) for the high- and low-risk group, respectively. The specificity and sensitivity of the model are verified, which show that the area under receiver operating characteristic curve yields 0.881, meaning our model has good accuracy and it is feasible for further applications.ConclusionThe 31-lncRNA model might be able to predict OS in patients with LUAD with high accuracy. Its further applications in biomolecular experiments using clinical samples with independent cohorts of patients are needed to verify the results.

Intensity-detection-based guided-mode-resonance optofluidic biosensing system for rapid, low-cost, label-free detection

We present an intensity-detection-based optofluidic biosensing system with a disposable guided-mode-resonance (GMR) biosensor chip for low-cost, rapid, label-free biosensing. The developed sensing system consists of a low-cost spectrally limited light-emitting diode (LED) as the light source and a photodetector as the optical receiver. This detection instrument can transform the GMR wavelength shift induced by the change in the local refractive index (RI) near the chip’s grating surface into change in the light intensity, thus enabling label-free, real-time detection. RI measurement experiments in both the transmission and reflection modes are performed to study the corresponding sensing mechanisms and compare detection performance. The results indicate that this sensing system operating in the reflection mode can achieve a superior RI resolution of 4.10×10-5 RIU over a wide linear detection range of 0.04 RIU. Label-free biomolecular interaction experiments show that a low limit of detection (LOD) of 7.5×10−8g/ml for dinitro-phenyl (DNP)/anti-DNP interactions can be achieved, thus confirming the our system’s sensitivity. The present investigation demonstrates a low-cost, compact, high-performance optofluidic biosensing system for rapid and sensitive biochemical detection that can be utilized for a wide variety of applications.

Lanthanide MOFs detect solvents

With the help of a little light, a metal-organic framework (MOF) containing photoluminescent lanthanides can uniquely identify and measure the concentrations of multiple solvents, a research team reports (Chem 2017, DOI: j.chempr.2017.02.010). Applied to a dipstick, the material could enable rapid identification of environmental contaminants on-site rather than require responders to send samples to a lab. The approach could also solve the tricky analytical problem of determining how much H2O contaminates D2O, an issue for isotopic labeling in biomolecular experiments and calibration for spectroscopic methods such as nuclear magnetic resonance. Led by University of Texas, Austin, chemistry professor Simon M. Humphrey, the team investigated a MOF composed of tris(p-carboxylato)triphenylphosphine and europium(III), gadolinium(III), and terbium(III). Each lanthanide has a unique emission spectrum: EuIII emits red, GdIII emits ultraviolet, and TbIII emits green light. But when solvent molecules bind within the...

Compressed sensing: Reconstruction of non‐uniformly sampled multidimensional NMR data

AbstractNuclear magnetic resonance (NMR) spectroscopy is widely used across the physical, chemical, and biological sciences. A core component of NMR studies is multidimensional experiments, which enable correlation of properties from one or more NMR‐active nuclei. In high‐resolution biomolecular NMR, common nuclei are 1H, 15N, and 13C, and triple resonance experiments using these three nuclei form the backbone of NMR structural studies. In other fields, a range of other nuclei may be used. Multidimensional NMR experiments provide unparalleled information content, but this comes at the price of long experiment times required to achieve the necessary resolution and sensitivity. Non‐uniform sampling (NUS) techniques to reduce the required data sampling have existed for many decades. Recently, such techniques have received heightened interest due to the development of compressed sensing (CS) methods for reconstructing spectra from such NUS datasets. When applied jointly, these methods provide a powerful approach to dramatically improve the resolution of spectra per time unit and under suitable conditions can also lead to signal‐to‐noise ratio improvements. In this review, we explore the basis of NUS approaches, the fundamental features of NUS reconstruction using CS and applications based on CS approaches including the benefits of expanding the repertoire of biomolecular NMR experiments into higher dimensions. We discuss some of the recent algorithms and software packages and provide practical tips for recording and processing NUS data by CS.

Acquiring and processing ultrafast biomolecular 2D NMR experiments using a referenced-based correction.

Thanks to their special spatiotemporal encoding/decoding scheme, ultrafast (UF) NMR sequences can deliver arbitrary 2D spectra following a single excitation. Regardless of their nature, these sequences have in common their tracing of a path in the [Formula: see text]-[Formula: see text] plane, that will deliver the spectrum being sought after a 1D Fourier transformation versus [Formula: see text]. This need to simultaneously digitize two domains, tends to impose bandwidth limitations along all spectral axes. Along the [Formula: see text]/[Formula: see text] dimension this problem is exacerbated by the fact that odd and even time points are not equispaced, and by additional artifacts such as time shifts between time points sampled while under the action of positive and negative decoding gradients. As a result, odd and even [Formula: see text] points are typically Fourier transformed separately, halving the potential spectral width along this dimension. While this halving of the [Formula: see text] span can be overcome by an interlaced Fourier transform, this post-processing is seldom used because of its sensitivity to hardware inaccuracies requiring even finer corrections of the even/odd [Formula: see text] data points. These corrections have so far been done manually, but are challenging to implement when dealing with low signal-to-noise ratio signals like those associated with biomolecular NMR experiments. This study introduces an algorithm for an automatic correction of all even/odd ultrafast NMR inconsistencies, based on the acquisition of a reference scan on the solvent. This algorithm was verified experimentally using an [Formula: see text]-[Formula: see text] UF-HSQC variant on ubiquitin at 600MHz. Features of this method as well as of the interlaced Fourier transformation in general, are discussed.

An Atomic Force Microscope with Dual Actuation Capability for Biomolecular Experiments.

We report a modular atomic force microscope (AFM) design for biomolecular experiments. The AFM head uses readily available components and incorporates deflection-based optics and a piezotube-based cantilever actuator. Jetted-polymers have been used in the mechanical assembly, which allows rapid manufacturing. In addition, a FeCo-tipped electromagnet provides high-force cantilever actuation with vertical magnetic fields up to 0.55 T. Magnetic field calibration has been performed with a micro-hall sensor, which corresponds well with results from finite element magnetostatics simulations. An integrated force resolution of 1.82 and 2.98 pN, in air and in DI water, respectively was achieved in 1 kHz bandwidth with commercially available cantilevers made of Silicon Nitride. The controller and user interface are implemented on modular hardware to ensure scalability. The AFM can be operated in different modes, such as molecular pulling or force-clamp, by actuating the cantilever with the available actuators. The electromagnetic and piezoelectric actuation capabilities have been demonstrated in unbinding experiments of the biotin-streptavidin complex.

Effects of radiation damping for biomolecular NMR experiments in solution: a hemisphere concept for water suppression.

Abundant solvent nuclear spins, such as water protons in aqueous solution, cause radiation damping in NMR experiments. It is important to know how the effect of radiation damping appears in high-resolution protein NMR because macromolecular studies always require very high magnetic field strengths with a highly sensitive NMR probe that can easily cause radiation damping. Here, we show the behavior of water magnetization after a pulsed-field gradient (PFG) using nutation experiments at 900 MHz with a cryogenic probe: when water magnetization is located in the upper hemisphere (having +Z component, parallel to the external magnetic field), dephasing of the magnetization by a PFG effectively suppresses residual water magnetization in the transverse plane. In contrast, when magnetization is located in the lower hemisphere (having -Z component), the small residual transverse component remaining after a PFG is still sufficient to induce radiation damping. Based on this observation, we designed 1H-15N HSQC experiments in which water magnetization is maintained in the upper hemisphere, but not necessarily along Z, and compared them with the conventional experiments, in which water magnetization is inverted during the t1 period. The result demonstrates moderate gain of signal-to-noise ratio, 0-28%. Designing the experiments such that water magnetization is maintained in the upper hemisphere allows shorter pulses to be used compared to the complete water flip-back and, thereby, is useful as a building block of protein NMR pulse programs in solution.