Triboelectric Nanogenerators for the Masses: A Low-Cost Do-It-Yourself Pulsed Ion Source for Sample-Limited Applications.

Triboelectric nanogenerators (TENG) are useful devices for converting mechanical motion into electric current using readily available materials. Though the applications for these devices span across many fields, TENG can be leveraged for mass spectrometry (MS) as inexpensive and effective power supplies for pulsed nanoelectrospray ionization (nESI). The inherently discontinuous spray provided by TENG is particularly useful in scenarios where high sample economy is imperative, as in the case of ultraprecious samples. Previous work has shown the utility of TENG MS as a highly sensitive technique capable of yielding quality spectra from only a few microliters of sample at low micromolar concentrations. As the field of miniaturized, fieldable mass spectrometers grows, it remains critical to develop advanced ion sources with similarly small power requirements and footprints. Here, we present a redesigned TENG ion source with a sub-1000 USD material cost, lower power consumption, reduced footprint, and improved capabilities. We validate the performance of this new device for a diverse set of applications, including lipid double bond localization and native protein analysis.

Triboelectric Nanogenerators: Low-Cost Power Supplies for Improved Electrospray Ionization.

Electrospray ionization (ESI) is one of the most popular methods to generate ions for mass spectrometry (MS). When compared with other ionization techniques, it can generate ions from liquid-phase samples without additives, retaining covalent and non-covalent interactions of the molecules of interest. When hyphenated to liquid chromatography, it greatly expands the versatility of MS analysis of complex mixtures. However, despite the extensive growth in the application of ESI, the technique still suffers from some drawbacks when powered by direct current (DC) power supplies. Triboelectric nanogenerators promise to be a new power source for the generation of ions by ESI, improving on the analytical capabilities of traditional DC ESI. In this review we highlight the fundamentals of ESI driven by DC power supplies, its contrasting qualities to triboelectric nanogenerator power supplies, and its applications to three distinct fields of research: forensics, metabolomics, and protein structure analysis.

Ion Mobility-Mass Spectrometry Reveals the Structures and Stabilities of Biotherapeutic Antibody Aggregates.

Stability is a key critical quality attribute monitored throughout the development of monoclonal antibody (mAb) therapeutics. Minor changes in their higher order structure (HOS) caused by stress or environment may alter mAb aggregation, immunogenicity, and efficacy. In addition, the structures of the resulting mAb aggregates are largely unknown, as are their dependencies on conditions under which they are created. In this report, we investigate the HOS of mAb monomers and dimers under a variety of forced degradation conditions with ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) technologies. We evaluate two model IgG1 antibodies that differ significantly only in their complementarity-determinant regions: IgG1α and IgG1ß. Our data covering both heat- and pH-based forced degradation conditions, aquired on two different IM-MS platforms, show that these mAbs undergo global HOS changes at both monomer and dimer levels upon degradation, but shifts in collision cross section (CCS) differ under pH or heat degradation conditions. In addition, the level of CCS change detected is different between IgG1α and IgG1ß, suggesting that differences in the CDR drive differential responses to degradation that influence the antibody HOS. Dramatically different CIU fingerprints are obtained for IgG1α and IgG1ß monomers and dimers for both degradation conditions. Finally, we constructed a series of computational models of mAb dimers for comparison with experimental CCS values and found evidence for a compact, overlapped dimer structure under native and heat degradation conditions, possibly adopting an inverted or nonoverlapped quaternary structure when produced through pH degredation. We conclude by discussing the potential impact of our findings on ongoing biotherapeutic discovery and development efforts.

Mass Spectrometry Methods for Measuring Protein Stability.

Mass spectrometry is a central technology in the life sciences, providing our most comprehensive account of the molecular inventory of the cell. In parallel with developments in mass spectrometry technologies targeting such assessments of cellular composition, mass spectrometry tools have emerged as versatile probes of biomolecular stability. In this review, we cover recent advancements in this branch of mass spectrometry that target proteins, a centrally important class of macromolecules that accounts for most biochemical functions and drug targets. Our efforts cover tools such as hydrogen-deuterium exchange, chemical cross-linking, ion mobility, collision induced unfolding, and other techniques capable of stability assessments on a proteomic scale. In addition, we focus on a range of application areas where mass spectrometry-driven protein stability measurements have made notable impacts, including studies of membrane proteins, heat shock proteins, amyloidogenic proteins, and biotherapeutics. We conclude by briefly discussing the future of this vibrant and fast-moving area of research.

Collision-Induced Unfolding Reveals Stability Differences in Infliximab Therapeutics under Native and Heat Stress Conditions.

Ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) assays of monoclonal antibody (mAb)-based biotherapeutics have proven sensitive to disulfide bridge structures, glycosylation patterns, and small molecule conjugation levels. Despite promising prior reports detailing the capabilities of IM-MS and CIU to differentiate biosimilars, generic mAb therapeutics, there remain questions surrounding the sensitivity of CIU to mAb structure changes that occur upon stress, the reproducibility of such measurements across IM-MS platforms, and the correlation between CIU and differential scanning calorimetry (DSC) datasets. In this report, we describe a comprehensive IM-MS and CIU dataset acquired for three Infliximabs: Remicade, Inflectra, and Renflexis. We subject each infliximab sample to forced degradation through heat stress and observe broadly similar yet subtly different stability patterns for these three biotherapeutics. We find that CIU is capable of tracking differences in mAb higher-order structure (HOS) imparted during forced heat stress degradation and that DSC is less sensitive to these alterations in comparison. Furthermore, we collected our comprehensive IM-MS and CIU data across two instrument platforms (Waters G2 and Agilent 6560), with both producing similar abilities to differentiate mAbs while also revealing minor differences between the results obtained on the two instruments. Finally, we demonstrate that CIU-based heatmaps and classification allow for rapid assessment of the most differentiating charge states for the analysis of infliximab, and using multiplexed classification, we conservatively estimate a 30-fold improvement in the time required to perform mAb stability and HOS measurements over standard DSC tools.

Metodología de canales endémicos del dengue en Ecuador 2015-2020: Necesidad para planificar y administrar la salud pública / Methodology of endemic channels of dengue, Ecuador 2015-2020: Need to plan and manage public health

El Dengue es la enfermedad de más rápida propagación en el mundo y una permanente amenaza para la salud pública, puesto que la mitad de la población global corre el riesgo de contraer la enfermedad. Ante la gravedad del cuadro de la enfermedad a nivel nacional y mundial, es necesario generar nuevas metodologías de predicción útiles para la adopción de decisiones en salud pública. Por ello, se caracterizó los casos notificados de dengue entre el 2015 y el 2020 en Ecuador. Se hizo un estudio descriptivo retrospectivo, utilizando la base de datos de las gacetas oficiales, caracterizando los casos de dengue. Se elaboraró un canal endémico describiendo la metodología empleada. Se evidenció que la tendencia del dengue en provincias es positiva, lo que indica que en los últimos cinco años se ha incrementado el número de casos, junto a la afectación de un ciclo epidemiológico común en el primer periodo de cada año del último quinquenio. El estudio de los datos actuales de la enfermedad del dengue en Ecuador, su tendencia epidémica y el contexto en que se desarrolla, permiten concluir en la importante necesidad de planificar y administrar la salud pública, aplicando la metodología planteada de elaboración de canales endémicos en la vigilancia de situaciones de interés en salud pública que conlleven a acciones de control de alcance tanto nacional como territorial que se sostengan el tiempo(AU)

Dengue is the fastest spreading disease in the world and a permanent threat to public health, since half of the global population is at risk of contracting the disease. Given the seriousness of the disease at the national and global level, it is necessary to generate new useful prediction methodologies for decision-making in public health. Therefore, the reported cases of dengue between 2015 and 2020 in Ecuador were characterized. A retrospective descriptive study was carried out, using the official gazette database, characterizing dengue cases. An endemic channel was developed describing the methodology used. It was evidenced that the dengue trend in the provinces is positive, which indicates that in the last five years the number of cases has increased, together with the involvement of a common epidemiological cycle in the first period of each year of the last five years. The study of current data on dengue disease in Ecuador, its epidemic trend and the context in which it develops, allow us to conclude on the important need to plan and administer public health, applying the methodology proposed for the elaboration of endemic channels in the surveillance of situations of interest in public health that lead to control actions of both national and territorial scope that are sustained over time(AU)

Enhanced Collision Induced Unfolding and Electron Capture Dissociation of Native-like Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6-1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.43 ± 0.20%. Furthermore, we compare our CCS results to previously reported DTIM values, finding strong agreement across similarly configured instrumentation (average RSD of 0.82 ± 0.73%), and systematic differences for DTIM CCS values commonly used to calibrate traveling-wave IM separators (-3% average RSD). Our CIU experiments reveal that the modified DTIM-MS instrument described here achieves enhanced levels of ion activation when compared with any previously reported IM-MS platforms, allowing for comprehensive unfolding of large multiprotein complex ions as well as interplatform CIU comparisons. Using our modified DTIM instrument, we studied two protein complexes. The enhanced CIU capabilities enable us to study the gas phase stability of the GroEL 7-mer and 14-mer complexes. Finally, we report CIU-ECD experiments for the alcohol dehydrogenase tetramer, demonstrating improved sequence coverage by combining ECD fragmentation integrated over multiple CIU intermediates. Further improvements for such native top-down sequencing experiments were possible by leveraging IM separation, which enabled us to separate and analyze CID and ECD fragmentation simultaneously.

Assessment of biosimilarity under native and heat-stressed conditions: rituximab, bevacizumab, and trastuzumab originators and biosimilars.

Biosimilars are highly similar to, but not identical with, their originator products. As a result, structural differences between originators and biosimilars can be difficult to detect and characterize without the appropriate analytical tools. Therefore, we first focus on identifying initial structural differences between rituximab, bevacizumab, and trastuzumab originator and biosimilar pairs and later address how these differences change after applying thermal stress at 40 °C with orbital shaking for 4 weeks. Prior to incubation, we detected comparable secondary and tertiary structures for each pair and identified different levels of soluble aggregates, charge variants, and molecular weight variants due to differences in glycoforms and the number of C-terminal lysine groups. Over the course of incubation, we compared differences in charge variants and unfolding patterns. Taken together, our study provides a comparability exercise, providing information on the minor differences present between originator and biosimilar products and how those differences are impacted by stress.

Multifaceted assessment of rituximab biosimilarity: The impact of glycan microheterogeneity on Fc function.

Biosimilars are poised to reduce prices and increase patient access to expensive, but highly effective biologic products. However, questions still remain about the degree of similarity and scarcity of information on biosimilar products from outside of the US/EU in the public domain. Thus, as an independent entity, we performed a comparative analysis between the innovator, Rituxan® (manufactured by Genentech/Roche), and a Russian rituximab biosimilar, Acellbia® (manufactured by Biocad). We evaluated biosimilarity of these two products by a variety of state-of-the-art analytical mass spectrometry techniques, including tandem MS mapping, HX-MS, IM-MS, and intact MS. Both were found to be generally similar regarding primary and higher order structure, though differences were identified in terms of glycoform distribution levels of C-terminal Lys, N-terminal pyroGlu, charge variants and soluble aggregates. Notably, we confirmed that the biosimilar had a higher level of afucosylated glycans, resulting in a stronger FcγIIIa binding affinity and increased ADCC activity. Taken together, our work provides a comprehensive comparison of Rituxan® and Acellbia®.

An Algorithm for Building Multi-State Classifiers Based on Collision-Induced Unfolding Data.

Collision-induced unfolding (CIU) has emerged as a valuable method for distinguishing iso-cross-sectional protein ions through their distinct gas-phase unfolding trajectories. CIU shows promise as a high-throughput, structure-sensitive screening technique with potential applications in drug discovery and biotherapeutic characterization. We recently developed a CIU classification workflow to support screening applications that utilized CIU data acquired from a single protein charge state to distinguish immunoglobulin (IgG) subtypes and membrane protein lipid binding. However, distinguishing highly similar protein structures, such as those associated with biotherapeutics, can be challenging. Here, we present an expansion of this classification method that includes CIU data from multiple charge states, or indeed any perturbation to protein structure that differentially affects CIU, into a combined classifier. Using this improved method, we are able to improve the accuracy of existing, single-state classifiers for IgG subtypes and develop an activation-state-sensitive classifier for selected Src kinase inhibitors when data from a single charge state was insufficient to do so. Finally, we employ the combination of multiple charge states and stress conditions to distinguish a highly similar innovator/biosimilar biotherapeutic pair, demonstrating the potential of CIU as a rapid screening tool for drug discovery and biotherapeutic analysis.

A Modified Drift Tube Ion Mobility-Mass Spectrometer for Charge-Multiplexed Collision-Induced Unfolding.

Collision-induced unfolding (CIU) of protein ions and their noncovalent complexes offers relatively rapid access to a rich portfolio of biophysical information, without the need to tag or purify proteins prior to analysis. Such assays have been characterized extensively for a range of therapeutic proteins, proving exquisitely sensitive to alterations in protein sequence, structure, and post-translational modification state. Despite advantages over traditional probes of protein stability, improving the throughput and information content of gas-phase protein unfolding assays remains a challenge for current instrument platforms. In this report, we describe modifications to an Agilent 6560 drift tube ion mobility-mass spectrometer in order to perform robust, simultaneous CIU across all precursor ions detected. This approach dramatically increases the speed associated with typical CIU assays, which typically involve mass selection of narrow m/ z regions prior to collisional activation, and thus their development requires a comprehensive assessment of charge-stripping reactions that can unintentionally pollute CIU data with chemical noise when more than one precursor ion is allowed to undergo simultaneous activation. By studying the unfolding and dissociation of intact antibody ions, a key analyte class associated with biotherapeutics, we reveal a predictive relationship between the precursor charge state, the amount of buffer components bound to the ions of interest, and the amount of charge stripping detected. We then utilize our knowledge of antibody charge stripping to rapidly capture CIU data for a range of antibody subclasses and subtypes across all charge states simultaneously, demonstrating a strong charge state dependence on the information content of CIU. Finally, we demonstrate that CIU data collection times can be further reduced by scanning fewer voltage steps, enabling us to optimize the throughput of our improved CIU methods and confidently differentiate antibody variant ions using ∼20% of the data typically collected during CIU. Taken together, our results characterize a new instrument platform for biotherapeutic stability measurements with dramatically improved throughput and information content.

PITX2 Enhances the Regenerative Potential of Dystrophic Skeletal Muscle Stem Cells.

Duchenne muscular dystrophy (DMD), one of the most lethal genetic disorders, involves progressive muscle degeneration resulting from the absence of DYSTROPHIN. Lack of DYSTROPHIN expression in DMD has critical consequences in muscle satellite stem cells including a reduced capacity to generate myogenic precursors. Here, we demonstrate that the c-isoform of PITX2 transcription factor modifies the myogenic potential of dystrophic-deficient satellite cells. We further show that PITX2c enhances the regenerative capability of mouse DYSTROPHIN-deficient satellite cells by increasing cell proliferation and the number of myogenic committed cells, but importantly also increasing dystrophin-positive (revertant) myofibers by regulating miR-31. These PITX2-mediated effects finally lead to improved muscle function in dystrophic (DMD/mdx) mice. Our studies reveal a critical role for PITX2 in skeletal muscle repair and may help to develop therapeutic strategies for muscular disorders.

Copper-Based Metal-Organic Porous Materials for CO2 Electrocatalytic Reduction to Alcohols.

The electrocatalytic reduction of CO2 has been investigated using four Cu-based metal-organic porous materials supported on gas diffusion electrodes, namely, (1) HKUST-1 metal-organic framework (MOF), [Cu3 (µ6 -C9 H3 O6 )2 ]n ; (2) CuAdeAce MOF, [Cu3 (µ3 -C5 H4 N5 )2 ]n ; (3) CuDTA mesoporous metal-organic aerogel (MOA), [Cu(µ-C2 H2 N2 S2 )]n ; and (4) CuZnDTA MOA, [Cu0.6 Zn0.4 (µ-C2 H2 N2 S2 )]n . The electrodes show relatively high surface areas, accessibilities, and exposure of the Cu catalytic centers as well as favorable electrocatalytic CO2 reduction performance, that is, they have a high efficiency for the production of methanol and ethanol in the liquid phase. The maximum cumulative Faradaic efficiencies for CO2 conversion at HKUST-1-, CuAdeAce-, CuDTA-, and CuZnDTA-based electrodes are 15.9, 1.2, 6, and 9.9 %, respectively, at a current density of 10 mA cm-2 , an electrolyte-flow/area ratio of 3 mL min cm-2 , and a gas-flow/area ratio of 20 mL min cm-2 . We can correlate these observations with the structural features of the electrodes. Furthermore, HKUST-1- and CuZnDTA-based electrodes show stable electrocatalytic performance for 17 and 12 h, respectively.

A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate.

The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the transcription factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5(+) satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine.

Bis(1,10-phenanthroline-κN,N')bis-(thio-cyanato-κN)cadmium.

The title compound, [Cd(NCS)(2)(C(12)H(8)N(2))(2)], has been obtained from the decomposition reaction of dithio-oxamide in a dimethyl-formamide solution containing 1,10-phenanthroline (phen) and Cd(NO(3))(2)·4H(2)O. Its crystal structure is formed by monuclear Cd(II) entities in which the metal atom is sited on a twofold rotation axis. The Cd(II) atom is six-coordinated in the form of a distorted octa-hedron by two chelating phenanthroline mol-ecules and two thio-cyanate anions coordinated through their N atoms. In the crystal, C-H⋯N hydrogen bonds are established between the phenanthroline and thio-cyanate ligands of neighbouring complexes.

An exploratory study of the facets of Type A personality and scores on the Machiavellian Behavior (MACH-B) Scale.

The relationship of two facets of Type A personality, Achievement Striving and Impatience-Irritability, with Mach-B scores was investigated. Mach-B scores were not correlated with Achievement Striving and were positively correlated (r = .37; p < .01) with Impatience-Irritability in a sample of 51 hospital employees including physicians (n = 12), nurses (n = 12), administrative staff (n = 13), and custodial services employees (n = 14).