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Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method, we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology is benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We apply this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detect 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of phosphorylation events relevant to mitochondrial and brain biology., (© 2024. The Author(s).)

Glycol sidechains are often used to enhance the performance of organic photoconversion and electrochemical devices. Herein, we study their effects on electronic states and electronic properties. We find that polymer glycolation not only induces more disordered packing, but also results in a higher reorganisation energy due to more localised π-electron density. Transient absorption spectroscopy and femtosecond stimulated Raman spectroscopy are utilised to monitor the structural relaxation dynamics coupled to the excited state formation upon photoexcitation. Singlet excitons are initially formed, followed by polaron pair formation. The associated structural relaxation slows down in glycolated polymers (5 ps vs. 1.25 ps for alkylated), consistent with larger reorganisation energy. This slower vibrational relaxation is found to drive ultrafast formation of the polaron pair state (5 ps vs. 10 ps for alkylated). These results provide key experimental evidence demonstrating the impact of molecular structure on electronic state formation driven by strong vibrational coupling., (© 2024. The Author(s).)

The frequency of unprovoked shark bites is increasing worldwide, leading to a growing pressure for mitigation measures to reduce shark-bite risk while maintaining conservation objectives. Personal shark deterrents are a promising and non-lethal strategy that can protect ocean users, but few have been independently and scientifically tested. In Australia, bull (Carcharhinus leucas), tiger (Galeocerdo cuvier), and white sharks (Carcharodon carcharias) are responsible for the highest number of bites and fatalities. We tested the effects of two electric deterrents (Ocean Guardian's Freedom+ Surf and Freedom7) on the behaviour of these three species. The surf product reduced the probability of bites by 54% across all three species. The diving product had a similar effect on tiger shark bites (69% reduction) but did not reduce the frequency of bites from white sharks (1% increase), likely because the electrodes were placed further away from the bait. Electric deterrents also increased the time for bites to occur, and frequency of reactions and passes for all species tested. Our findings reveal that both Freedom+ Surf and Freedom7 electric deterrents affect shark behaviour and can reduce shark-bite risk for water users, but neither product eliminated the risk of shark bites entirely. The increasing number of studies showing the ability of personal electric deterrents to reduce shark-bite risk highlights personal protection as an effective and important part of the toolbox of shark-bite mitigation measures., (© 2024. The Author(s).)

No-take marine protected areas (MPAs) can mitigate the effects of overfishing, climate change and habitat degradation, which are leading causes of an unprecedented global biodiversity crisis. However, assessing the effectiveness of MPAs, especially in remote oceanic islands, can be logistically challenging and often restricted to relatively shallow and accessible environments. Here, we used a long-term dataset (2010-2019) collected by the DeepSee submersible of the Undersea Hunter Group that operates in Isla del Coco National Park, Costa Rica, to (1) determine the frequency of occurrence of elasmobranch species at two depth intervals (50-100 m; 300-400 m), and (2) investigate temporal trends in the occurrence of common elasmobranch species between 2010 and 2019, as well as potential drivers of the observed changes. Overall, we observed 17 elasmobranch species, 15 of which were recorded on shallow dives (50-100 m) and 11 on deep dives (300-400 m). We found a decreasing trend in the probability of occurrence of Carcharhinus falciformis over time (2010-2019), while other species (e.g. Taeniurops meyeni, Sphyrna lewini, Carcharhinus galapagensis, Triaenodon obesus, and Galeocerdo cuvier) showed an increasing trend. Our study suggests that some species like S. lewini may be shifting their distributions towards deeper waters in response to ocean warming but may also be sensitive to low oxygen levels at greater depths. These findings highlight the need for regional 3D environmental information and long-term deepwater surveys to understand the extent of shark and ray population declines in the ETP and other regions, as most fishery-independent surveys from data-poor countries have been limited to relatively shallow waters., (© 2024. The Author(s).)

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this Review will serve as a handbook for researchers who are new to the field of bottom-up proteomics., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m / z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS 3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O -fatty acylation in Corynebacterium , termed protein O -mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms' outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O -mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O -mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O -acylated peptides. Using these tools in the model organism Corynebacterium glutamicum , we identified approximately 30 candidate O -mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O -mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O -mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae , Mycobacterium tuberculosis ), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS 1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach., Competing Interests: Conflict of interest The authors declare the following competing financial interest(s): J. J. C. is a consultant for Thermo Fisher Scientific, Seer, and 908 Devices. T. N. A., E. D., A. P., M. Z., D. H., H. S., C. H., A. M., and V. Z. are employees of Thermo Fisher Scientific., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Conventional non-fullerene acceptors (NFAs) typically have planar structures that can enable improved electron mobility and produce more efficient organic photovoltaic devices. A relatively simple A-D-A'-D-A type NFA specifically designed to match with poly(3-hexylthiophene-2,5-diyl) (P3HT) for green-absorbing agrivoltaic applications has been examined using a variety of techniques: microsecond transient absorption spectroscopy, atomic force microscopy, and photoluminescence. Relatively invariant charge carrier decay dynamics are observed in the blend films across a variety of processing solvents. Raman spectroscopy in conjunction with computational studies reveals that this NFA is non-planar and that multiple conformations are present in films, while preserving the crystalline nature of P3HT. The non-planarity of the NFA therefore creates a dispersive acceptor environment, irrespective of processing solvent, and this leads to the observed relative invariance in charge carrier decay dynamics and high tolerance to morphological variation. The findings presented in this work highlight the potential of non-planar materials as acceptors in organic photovoltaic devices., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA., Competing Interests: Conflict of interest J. J. C. is a consultant for Thermo Fisher Scientific, 908 Devices, and Seer. All other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bismuth-based coordination complexes are advantageous over other metal complexes, as bismuth is the heaviest nontoxic element with high spin-orbit coupling and potential optoelectronics applications. Herein, four bismuth halide-based coordination complexes [Bi 2 Cl 6 (phen-thio) 2 ] ( 1 ), [Bi 2 Br 6 (phen-thio) 2 ] ( 2 ), [Bi 2 I 6 (phen-thio) 2 ] ( 3 ), and [Bi 2 I 6 (phen-Me) 2 ] ( 4 ) were synthesized, characterized, and subjected to detailed photophysical studies. The complexes were characterized by single-crystal X-ray diffraction, powder X-ray diffraction, and NMR studies. Spectroscopic analyses of 1 - 4 in solutions of different polarities were performed to understand the role of the organic and inorganic components in determining the ground- and excited-state properties of the complexes. The photophysical properties of the complexes were characterized by ground-state absorption, steady-state photoluminescence, microsecond time-resolved photoluminescence, and absorption spectroscopy. Periodic density functional theory (DFT) calculations were performed on the solid-state structures to understand the role of the organic and inorganic parts of the complexes. The studies showed that changing the ancillary ligand from chlorine (Cl) and bromine (Br) to iodine (I) bathochromically shifts the absorption band along with enhancing the absorption coefficient. Also, changing the halides (Cl, Br to I) affects the photoluminescent quantum yields of the ligand-centered (LC) emissive state without markedly affecting the lifetimes. The combined results confirmed that ground-state properties are strongly influenced by the inorganic part, and the lower-energy excited state is LC. This study paves the way to design novel bismuth coordination complexes for optoelectronic applications by rigorously choosing the ligands and bismuth salt.

A new family of molecules obtained by coupling Tröger's base unit with dicyanovinylene-terminated oligothiophenes of different lengths has been synthesized and characterized by steady-state stationary and transient time-resolved spectroscopies. Quantum chemical calculations allow us to interpret and recognize the properties of the stationary excited states as well as the time-dependent mechanisms of singlet-to-triplet coupling. The presence of the diazocine unit in Tröger's base derivatives is key to efficiently producing singlet-to-triplet intersystem crossing mediated by the role of the nitrogen atoms and of the almost orthogonal disposition of the two thiophene arms. Spin-orbit coupling-mediated interstate intersystem crossing (ISC) is activated by a symmetry-breaking process in the first singlet excited state with partial charge transfer character. This mechanism is a characteristic of these molecular triads since the independent dicyanovinylene-oligothiophene branches do not display appreciable ISC. These results show how Tröger's base coupling of organic chromophores can be used to improve the ISC efficiency and tune their photophysics.

Aldehydes are important synthons for DNA-encoded library (DEL) construction, but the development of a DNA-compatible method for the oxidation of alcohols to aldehydes remains a significant challenge in the field of DEL chemistry. We report that a copper/TEMPO catalyst system enables the solution-phase DNA-compatible oxidation of DNA-linked primary activated alcohols to aldehydes. The semiaqueous, room-temperature reaction conditions afford oxidation of benzylic, heterobenzylic, and allylic alcohols in high yield, with DNA compatibility verified by mass spectrometry, qPCR, Sanger sequencing, and ligation assays. Subsequent transformations of the resulting aldehydes demonstrate the potential of this method for robust library diversification.

Mass spectrometry (MS)-based proteomics is a powerful technology to globally profile protein abundances, activities, interactions, and modifications. The extreme complexity of proteomics samples, which often contain hundreds of thousands of analytes, necessitates continuous development of MS techniques and instrumentation to improve speed, sensitivity, precision, and accuracy, among other analytical characteristics. Here, we systematically evaluated the Orbitrap Ascend Tribrid mass spectrometer in the context of shotgun proteomics, and we compared its performance to that of the previous generation of Tribrid instruments─the Orbitrap Eclipse. The updated architecture of the Orbitrap Ascend includes a second ion-routing multipole (IRM) in front of the redesigned C-trap/Orbitrap and a new ion funnel that allows gentler ion introduction, among other changes. These modifications in Ascend hardware configuration enabled an increase in parallelizable ion injection time during higher-energy collisional dissociation (HCD) Orbitrap tandem MS (FTMS 2 ) analysis of ∼5 ms. This enhancement was particularly valuable in the analyses of limited sample amounts, where improvements in sensitivity resulted in up to 140% increase in the number of identified tryptic peptides. Further, analysis of phosphorylated peptides enriched from the K562 human cell line yielded up to ∼50% increase in the number of unique phosphopeptides and localized phosphosites. Strikingly, we also observed a ∼2-fold boost in the number of detected N-glycopeptides, likely owing to the improvements in ion transmission and sensitivity. In addition, we performed the multiplexed quantitative proteomics analyses of TMT11-plex labeled HEK293T tryptic peptides and observed 9-14% increase in the number of quantified peptides. In conclusion, the Orbitrap Ascend consistently outperformed its predecessor the Orbitrap Eclipse in various bottom-up proteomic analyses, and we anticipate that it will generate reproducible and in-depth datasets for numerous proteomic applications.

At mammalian neuronal synapses, synaptic vesicle (SV) glycoproteins are essential for robust neurotransmission. Asparagine (N)-linked glycosylation is required for delivery of the major SV glycoproteins synaptophysin and SV2A to SVs. Despite this key role for N-glycosylation, the molecular compositions of SV N-glycans are largely unknown. In this study, we combined organelle isolation techniques and high-resolution mass spectrometry to characterize N-glycosylation at synapses and SVs from mouse brain. Detecting over 2,500 unique glycopeptides, we found that SVs harbor a distinct population of oligomannose and highly fucosylated N-glycans. Using complementary fluorescence methods, we identify at least one highly fucosylated N-glycan enriched in SVs compared with synaptosomes. High fucosylation was characteristic of SV proteins, plasma membrane proteins, and cell adhesion molecules with key roles in synaptic function and development. Our results define the N-glycoproteome of a specialized neuronal organelle and inform timely questions in the glycobiology of synaptic pruning and neuroinflammation., Competing Interests: Declaration of interests J.J.C. is a consultant for Thermo Fisher Scientific, Seer, and 908 Devices., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Organic photovoltaics are remarkably close to reaching a landmark power conversion efficiency of 20%. Given the current urgent concerns regarding climate change, research into renewable energy solutions is crucially important. In this perspective article, we highlight several key aspects of organic photovoltaics, ranging from fundamental understanding to implementation, that need to be addressed to ensure the success of this promising technology. We cover the intriguing ability of some acceptors to undergo efficient charge photogeneration in the absence of an energetic driving force and the effects of the resulting state hybridization. We explore one of the primary loss mechanisms of organic photovoltaics-non-radiative voltage losses-and the influence of the energy gap law. Triplet states are becoming increasingly relevant owing to their presence in even the most efficient non-fullerene blends, and we assess their role as both a loss mechanism and a potential strategy to enhance efficiency. Finally, two ways in which the implementation of organic photovoltaics can be simplified are addressed. The standard bulk heterojunction architecture could be superseded by either single material photovoltaics or sequentially deposited heterojunctions, and the attributes of both are considered. While several important challenges still lie ahead for organic photovoltaics, their future is, indeed, bright.

Narrow bandgap conjugated polymers are a heavily studied class of organic semiconductors, but their excited states usually have a very short lifetime, limiting their scope for applications. One approach to overcome the short lifetime is to populate long-lived triplet states for which relaxation to the ground state is forbidden. However, the triplet lifetime of narrow bandgap polymer films is typically limited to a few microseconds. Here, we investigated the effect of film morphology on triplet dynamics in red-emitting conjugated polymers based on the classic benzodithiophene monomer unit with the solubilizing alkyl side chains C 16 and C 2 C 6 and then used Pd porphyrin sensitization as a further strategy to change the triplet dynamics. Using transient absorption spectroscopy, we demonstrated a 0.45 ms triplet lifetime for the more crystalline nonsensitized polymer C 2 C 6 , 2-3 orders of magnitude longer than typically reported, while the amorphous C 16 had only a 5 μs lifetime. The increase is partly due to delaying bimolecular electron-hole recombination in the more crystalline C 2 C 6, where a higher energy barrier for charge recombination is expected. A triplet lifetime of 0.4 ms was also achieved by covalently incorporating 5% of Pd porphyrin into the C 16 polymer, which introduced extra energy transfer steps between the polymer and porphyrin that delayed triplet dynamics and increased the polymer triplet yield by 7.9 times. This work demonstrates two synthetic approaches to generate the longest-lived triplet excited states in narrow bandgap conjugated polymers, which is of necessity in a wide range of fields that range from organic electronics to sensors and bioapplications.

One of the main assumptions in the design of new conjugated polymer materials for their use in organic electronics is that higher coplanarity leads to greater conjugation along the polymer backbone. Conventionally, a more planar monomer structure induces a larger backbone coplanarity, thus leading to a greater overlap of the carbon π-orbitals and therefore a higher degree of π-electron delocalisation. However, here we present a case that counters the validity of this assumption. Different diselenophene-based polymers were studied where one polymer possesses two selenophene rings fused together to create a more rigid, planar structure. The effects of this greater polymer coplanarity were examined using Raman spectroscopy and theoretical calculations. Raman spectra showed a large difference between the vibrational modes of the fused and unfused polymers, indicating very different electronic structures. Resonance Raman spectroscopy confirmed the rigidity of the fused selenophene polymer and also revealed, by studying the excitation profiles of the different bands, the presence of two shorter, uncoupled conjugation pathways. Supported by Density Functional Theory (DFT) calculations, we have demonstrated that the reason for this lack of conjugation is a distortion of the selenophene rings due to the induced planarity, forming a new truncated conjugation pathway through the selenophene β-position and bypassing the beneficial α-position. This effect was studied using DFT in an ample range of derivatives, where substitution of the selenium atom with other heteroatoms still maintained the same unconventional conjugation-planarity relationship, confirming the generality of this phenomenon. This work establishes an important structure-property relationship for conjugated polymers that will help rational design of more efficient organic electronics materials., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Isobaric tagging facilitates multiplexed experiments that can determine sequences and relative amounts of peptides in biological samples using tandem mass spectrometry (MS n ). Limited reporter ion generation limits quantitative accuracy and precision. As reporter ions are susceptible to unintended fragmentation and scattering by high-energy collisions, we activated peptides with IR photons and prevented successive dissociation of generated reporter ions with ion parking, which altogether boosted reporter ion yield by up to 55%. Even so, unintended co-isolation of contaminating peaks in MS 2 experiments distorts reporter ion intensities and can distort quantitative information. MS 3 experiments address contamination by generating reporter ions via collisional activation (HCD) of one or more peptide product ions rather than the isolated peptide precursor ion. Because HCD performance is related to m / z , activation of multiple synchronously isolated product ions generates less than optimal reporter ion intensities. In this work, we show that using infrared multiphoton dissociation, which is not dependent on m / z , to generate reporter ions from 10 synchronously isolated peptide product ions results in a 2.4-fold increase in reporter ion intensities, significantly enhancing the sensitivity and dynamic range of quantitation via isobaric tagging.

The bimolecular recombination characteristics of conjugated polymer poly[(4,4'-bis(2-ethylhexyl)dithieno[3,2- b :2',3'- d ]silole)-2,6-diyl-alt-(2,5-bis 3-tetradecylthiophen-2-yl thiazolo 5,4- d thiazole)-2,5diyl] (PDTSiTTz) blended with the fullerene series PC60BM, ICMA, ICBA, and ICTA have been investigated using microsecond and femtosecond transient absorption spectroscopy, in conjunction with electroluminescence measurements and ambient photoemission spectroscopy. The non-Langevin polymer PDTSiTTz allows an inspection of intrinsic bimolecular recombination rates uninhibited by diffusion, while the low oscillator strengths of fullerenes allow polymer features to dominate, and we compare our results to those of the well-known polymer Si-PCPDTBT. Using μs-TAS, we have shown that the trap-limited decay dynamics of the PDTSiTTz polaron becomes progressively slower across the fullerene series, while those of Si-PCPDTBT are invariant. Electroluminescence measurements showed an unusual double peak in pristine PDTSiTTz, attributed to a low energy intragap charge transfer state, likely interchain in nature. Furthermore, while the pristine PDTSiTTz showed a broad, low-intensity density of states, the ICBA and ICTA blends presented a virtually identical DOS to Si-PCPDTBT and its blends. This has been attributed to a shift from a delocalized, interchain highest occupied molecular orbital (HOMO) in the pristine material to a dithienosilole-centered HOMO in the blends, likely a result of the bulky fullerenes increasing interchain separation. This HOMO localization had a side effect of progressively shifting the polymer HOMO to shallower energies, which was correlated with the observed decrease in bimolecular recombination rate and increased "trap" depth. However, since the density of tail states remained the same, this suggests that the traditional viewpoint of "trapping" being dominated by tail states may not encompass the full picture and that the breadth of the DOS may also have a strong influence on bimolecular recombination., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Ion-ion reactions are valuable tools in mass-spectrometry-based peptide and protein sequencing. To boost the generation of sequence-informative fragment ions from low charge-density precursors, supplemental activation methods, via vibrational and photoactivation, have become widely adopted. However, long-lived radical peptide cations undergo intramolecular hydrogen atom transfer from c-type ions to z • -type ions. Here we investigate the degree of hydrogen transfer for thousands of unique peptide cations where electron transfer dissociation (ETD) was performed and was followed by beam-type collisional activation (EThcD), resonant collisional activation (ETcaD), or concurrent infrared photoirradiation (AI-ETD). We report on the precursor charge density and the local amino acid environment surrounding bond cleavage to illustrate the effects of intramolecular hydrogen atom transfer for various precursor ions. Over 30% of fragments from EThcD spectra comprise distorted isotopic distributions, whereas over 20% of fragments from ETcaD have distorted distributions and less than 15% of fragments derived from ETD and AI-ETD reveal distorted isotopic distributions. Both ETcaD and EThcD give a relatively high degree of hydrogen migration, especially when D, G, N, S, and T residues were directly C-terminal to the cleavage site. Whereas all postactivation methods boost the number of c- and z • -type fragment ions detected, the collision-based approaches produce higher rates of hydrogen migration, yielding fewer spectral identifications when only c- and z • -type ions are considered. Understanding hydrogen rearrangement between c- and z • -type ions will facilitate better spectral interpretation.

We report DNA-scaffolded synergistic catalysis, a concept that combines the diverse reaction scope of synergistic catalysis with the ability of DNA to precisely preorganize abiotic groups and undergo stimuli-triggered conformational changes. As an initial demonstration of this concept, we focus on Cu-TEMPO-catalyzed aerobic alcohol oxidation, using DNA as a scaffold to hold a copper cocatalyst and an organic radical cocatalyst (TEMPO) in proximity. The DNA-scaffolded catalyst maintained a high turnover number upon dilution and exhibited 190-fold improvement in catalyst turnover number relative to the unscaffolded cocatalysts. By incorporating the cocatalysts into a DNA hairpin-containing scaffold, we demonstrate that the rate of the synergistic catalytic reaction can be controlled through a reversible DNA conformational change that alters the distance between the cocatalysts. This work demonstrates the compatibility of synergistic catalytic reactions with DNA scaffolding, opening future avenues in reaction discovery, sensing, responsive materials, and chemical biology.

The global lockdown to mitigate COVID-19 pandemic health risks has altered human interactions with nature. Here, we report immediate impacts of changes in human activities on wildlife and environmental threats during the early lockdown months of 2020, based on 877 qualitative reports and 332 quantitative assessments from 89 different studies. Hundreds of reports of unusual species observations from around the world suggest that animals quickly responded to the reductions in human presence. However, negative effects of lockdown on conservation also emerged, as confinement resulted in some park officials being unable to perform conservation, restoration and enforcement tasks, resulting in local increases in illegal activities such as hunting. Overall, there is a complex mixture of positive and negative effects of the pandemic lockdown on nature, all of which have the potential to lead to cascading responses which in turn impact wildlife and nature conservation. While the net effect of the lockdown will need to be assessed over years as data becomes available and persistent effects emerge, immediate responses were detected across the world. Thus, initial qualitative and quantitative data arising from this serendipitous global quasi-experimental perturbation highlights the dual role that humans play in threatening and protecting species and ecosystems. Pathways to favorably tilt this delicate balance include reducing impacts and increasing conservation effectiveness., Competing Interests: Authors declare no competing interests., (© 2021 Published by Elsevier Ltd.)

Understanding interfacial charge transfer processes such as trap-mediated recombination and injection into charge transport layers (CTLs) is crucial for the improvement of perovskite solar cells. Herein, we reveal that the chemical binding of charge transport layers to CH 3 NH 3 PbI 3 defect sites is an integral part of the interfacial charge injection mechanism in both n-i-p and p-i-n architectures. Specifically, we use a mixture of optical and X-ray photoelectron spectroscopy to show that binding interactions occur via Lewis base interactions between electron-donating moieties on hole transport layers and the CH 3 NH 3 PbI 3 surface. We then correlate the extent of binding with an improvement in the yield and longer lifetime of injected holes with transient absorption spectroscopy. Our results show that passivation-mediated charge transfer has been occurring undetected in some of the most common perovskite configurations and elucidate a key design rule for the chemical structure of next-generation CTLs.

Background: Tri-axial accelerometers have been used to remotely describe and identify in situ behaviours of a range of animals without requiring direct observations. Datasets collected from these accelerometers (i.e. acceleration, body position) are often large, requiring development of semi-automated analyses to classify behaviours. Marine fishes exhibit many "burst" behaviours with high amplitude accelerations that are difficult to interpret and differentiate. This has constrained the development of accurate automated techniques to identify different "burst" behaviours occurring naturally, where direct observations are not possible., Methods: We trained a random forest machine learning algorithm based on 624 h of accelerometer data from six captive yellowtail kingfish during spawning periods. We identified five distinct behaviours (swim, feed, chafe, escape, and courtship), which were used to train the model based on 58 predictive variables., Results: Overall accuracy of the model was 94%. Classification of each behavioural class was variable; F 1 scores ranged from 0.48 (chafe) - 0.99 (swim). The model was subsequently applied to accelerometer data from eight free-ranging kingfish, and all behaviour classes described from captive fish were predicted by the model to occur, including 19 events of courtship behaviours ranging from 3 s to 108 min in duration., Conclusion: Our findings provide a novel approach of applying a supervised machine learning model on free-ranging animals, which has previously been predominantly constrained to direct observations of behaviours and not predicted from an unseen dataset. Additionally, our findings identify typically ambiguous spawning and courtship behaviours of a large pelagic fish as they naturally occur.

Efficient charge photogeneration in conjugated polymers typically requires the presence of a second component to act as electron acceptor. Here, we report a novel low band-gap conjugated polymer with a donor/orthogonal acceptor motif: poly-2,6-(4,4-dihexadecyl-4 H -cyclopenta [2,1- b :3,4- b ']dithiophene)- alt -2,6-spiro [cyclopenta[2,1- b :3,4- b ']dithiophene-4,9'-fluorene]-2',7'-dicarbonitrile, referred to as PCPDT-sFCN . The role of the orthogonal acceptor is to spatially isolate the LUMO from the HOMO, allowing for negligible exchange energy between electrons in these orbitals and minimising the energy gap between singlet and triplet charge transfer states. We employ ultrafast and microsecond transient absorption spectroscopy to demonstrate that, even in the absence of a separate electron acceptor, PCPDT-sFCN shows efficient charge photogeneration in both pristine solution and film. This efficient charge generation is a result of an isoenergetic singlet/triplet charge transfer state equilibrium acting as a reservoir for charge carrier formation. Furthermore, clear evidence of enhanced triplet populations, which form in less than 1 ps, is observed. Using group theory, we show that this ultrafast triplet formation is due to highly efficient, quantum mechanically allowed intersystem crossing between the bright, initially photoexcited local singlet state and the triplet charge transfer state. Remarkably, the free charges that form via the charge transfer state are extraordinarily long-lived with millisecond lifetimes, possibly due to the stabilisation imparted by the spatial separation of PCPDT-sFCN 's donor and orthogonal acceptor motifs. The efficient generation of long-lived charge carriers in a pristine polymer paves the way for single-material applications such as organic photovoltaics and photodetectors., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Targeted functionalization of 3D perovskite with a 2D passivation layer via R-NH 3 I treatment has emerged as an effective strategy for enhancing both the efficiency and chemical stability of ABX 3 perovskite solar cells, but the underlying mechanisms behind these improvements remain unclear. Here, we assign a passivation mechanism where R-NH 3 I reacts with excess PbI 2 in the MAPbI 3 film and unsaturated PbI 6 octahedra to form (R-NH 3 ) 2 (MA) n -1 Pb n I 3 n +1 . Crucially, we show that precise control of the 2D (R-NH 3 ) 2 (MA) n -1 Pb n I 3 n +1 layer underpins performance improvements: n = 1 yields over a 2-fold improvement in hole injection to the HTL; n > 1 deteriorates hole injection. Ultrafast transient absorption spectroscopy suggests this n -dependence is rooted in the fact that fast (<6 ns) hole injection does not occur between the 3D and 2D layers. These results help explain contemporary empirical findings in the field and set out an important design rule for the further optimization of multidimensional perovskite optoelectronics.

Shotgun proteomics techniques infer the presence and quantity of proteins using peptide proxies produced by cleavage of the proteome with a protease. Most protein quantitation strategies assume that multiple peptides derived from a protein will behave quantitatively similar across treatment groups, but this assumption may be false due to (1) heterogeneous proteoforms and (2) technical artifacts. Here we describe a strategy called peptide correlation analysis (PeCorA) that detects quantitative disagreements between peptides mapped to the same protein. PeCorA fits linear models to assess whether a peptide's change across treatment groups differs from all other peptides assigned to the same protein. PeCorA revealed that ∼15% of proteins in a mouse microglia stress data set contain at least one discordant peptide. Inspection of the discordant peptides shows the utility of PeCorA for the direct and indirect detection of regulated post-translational modifications (PTMs) and also for the discovery of poorly quantified peptides. The exclusion of poorly quantified peptides before protein quantity summarization decreased false-positives in a benchmark data set. Finally, PeCorA suggests that the inactive isoform of prothrombin, a coagulation cascade protease, is more abundant in plasma from COVID-19 patients relative to non-COVID-19 controls. PeCorA is freely available as an R package that works with arbitrary tables of quantified peptides.

COVID-19 restrictions have led to an unprecedented global hiatus in anthropogenic activities, providing a unique opportunity to assess human impact on biological systems. Here, we describe how a national network of acoustic tracking receivers can be leveraged to assess the effects of human activity on animal movement and space use during such global disruptions. We outline variation in restrictions on human activity across Australian states and describe four mechanisms affecting human interactions with the marine environment: 1) reduction in economy and trade changing shipping traffic; 2) changes in export markets affecting commercial fisheries; 3) alterations in recreational activities; and 4) decline in tourism. We develop a roadmap for the analysis of acoustic tracking data across various scales using Australia's national Integrated Marine Observing System (IMOS) Animal Tracking Facility as a case study. We illustrate the benefit of sustained observing systems and monitoring programs by assessing how a 51-day break in white shark ( Carcharodon carcharias) cage-diving tourism due to COVID-19 restrictions affected the behaviour and space use of two resident species. This cessation of tourism activities represents the longest break since cage-diving vessels started day trips in this area in 2007. Long-term monitoring of the local environment reveals that the activity space of yellowtail kingfish ( Seriola lalandi ) was reduced when cage-diving boats were absent compared to periods following standard tourism operations. However, white shark residency and movements were not affected. Our roadmap is globally applicable and will assist researchers in designing studies to assess how anthropogenic activities can impact animal movement and distributions during regional, short-term through to major, unexpected disruptions like the COVID-19 pandemic., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Elsevier Ltd. All rights reserved.)

We performed RNA-seq and high-resolution mass spectrometry on 128 blood samples from COVID-19-positive and COVID-19-negative patients with diverse disease severities and outcomes. Quantified transcripts, proteins, metabolites, and lipids were associated with clinical outcomes in a curated relational database, uniquely enabling systems analysis and cross-ome correlations to molecules and patient prognoses. We mapped 219 molecular features with high significance to COVID-19 status and severity, many of which were involved in complement activation, dysregulated lipid transport, and neutrophil activation. We identified sets of covarying molecules, e.g., protein gelsolin and metabolite citrate or plasmalogens and apolipoproteins, offering pathophysiological insights and therapeutic suggestions. The observed dysregulation of platelet function, blood coagulation, acute phase response, and endotheliopathy further illuminated the unique COVID-19 phenotype. We present a web-based tool (covid-omics.app) enabling interactive exploration of our compendium and illustrate its utility through a machine learning approach for prediction of COVID-19 severity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Photoactivation and photodissociation have long proven to be useful tools in tandem mass spectrometry, but implementation often involves cumbersome and potentially dangerous configurations. Here, we redress this problem by using a fiber-optic cable to couple an infrared (IR) laser to a mass spectrometer for robust, efficient, and safe photoactivation experiments. Transmitting 10.6 μm IR photons through a hollow-core fiber, we show that such fiber-assisted activated ion-electron transfer dissociation (AI-ETD) and IR multiphoton dissociation (IRMPD) experiments can be carried out as effectively as traditional mirror-based implementations. We report on the transmission efficiency of the hollow-core fiber for conducting photoactivation experiments and perform various intact protein and peptide analyses to illustrate the benefits of fiber-assisted AI-ETD, namely, a simplified system for irradiating the two-dimensional linear ion trap volume concurrent with ETD reactions to limit uninformative nondissociative events and thereby amplify sequence coverage. We also describe a calibration scheme for the routine analysis of IR laser alignment and power through the fiber and into the dual cell quadrupolar linear ion trap. In all, these advances allow for a more robust, straightforward, and safe instrumentation platform, permitting implementation of AI-ETD and IRMPD on commercial mass spectrometers and broadening the accessibility of these techniques.

Modified oligonucleotides represent a promising avenue for drug development, with small interfering RNAs (siRNA) and microRNAs gaining traction in the therapeutic market. Mass spectrometry (MS)-based analysis offers many benefits for characterizing modified nucleic acids. Negative electron transfer dissociation (NETD) has proven valuable in sequencing oligonucleotide anions, particularly because it can retain modifications while generating sequence-informative fragments. We show that NETD can be successfully implemented on a widely available quadrupole-Orbitrap-linear ion trap mass spectrometer that uses a front-end glow discharge source to generate radical fluoranthene reagent cations. We characterize both unmodified and modified ribonucleic acids and present the first application of activated-ion negative electron transfer dissociation (AI-NETD) to nucleic acids. AI-NETD achieved 100% sequence coverage for both a 6-mer (5'-rGmUrArCmUrG-3') with 2'- O -methyl modifications and a 21-mer (5'-rCrArUrCrCrUrCrUrArGrArGrGrArUrArGrArArUrG-3'), the luciferase antisense siRNA. Both NETD and AI-NETD afforded complete sequence coverage of these molecules while maintaining a relatively low degree of undesired base-loss products and internal products relative to collision-based methods.

Designing chromophores for biological applications requires a fundamental understanding of how the chemical structure of a chromophore influences its photophysical properties. We here describe the synthesis of a library of BODIPY dyes, exploring diversity at various positions around the BODIPY core. The results show that the nature and position of substituents have a dramatic effect on the spectroscopic properties. Substituting in a heavy atom or adjusting the size and orientation of a conjugated system provides a means of altering the spectroscopic profiles with high precision. The insight from the structure-activity relationship was applied to devise a new BODIPY dye with rationally designed photochemical properties including absorption towards the near-infrared region. The dye also exhibited switch-on fluorescence to enable visualisation of cells with high signal-to-noise ratio without washing-out of unbound dye. The BODIPY-based probe is non-cytotoxic and compatible with staining procedures including cell fixation and immunofluorescence microscopy., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The active layer of organic solar cells typically possesses a complex morphology, with amorphous donor/acceptor mixed domains present in addition to purer, more crystalline domains. These crystalline domains may represent an energy sink for free charges that aids charge separation and suppresses bimolecular recombination. The first step in exploiting this behavior is the identification and characterization of charges located in these different domains. Herein, the generation and recombination of both bulk and interfacial polarons are demonstrated in the dual electron donor/acceptor polymer XIND using transient absorption spectroscopy. The absorption spectra of XIND bulk polarons, present in pristine polymer domains, are clearly distinguishable from those of polarons present at the donor/acceptor interface. Furthermore, it is shown that photogenerated polarons are transferred from the interface to the bulk. These findings support the energy sink hypothesis and offer a way to maximize morphology relationships to enhance charge generation and suppress recombination.

Controlling the activity of biomolecules with light-triggered photocages is an important research tool in the life sciences. We describe here a coumarin photocage that unusually combines the biocompatible optical properties of strong absorption at a long wavelength close to 500 nm and high photolysis quantum yields. The favourable properties are achieved by synthetically installing on the photocage scaffold a diethyl amino styryl moiety and a thionoester group rather than the lactone typical for coumarins. The photocage's photophysics are analysed with microsecond transient absorption spectroscopy to reveal the nature of the excited state in the photolysis pathway. The excited state is found to be strongly dependent on solvent polarity with a triplet state formed in DMSO and a charge-separated state in water that is likely due to aggregation. A long triplet lifetime is also correlated with a high photolysis quantum yield. Our study on the biocompatible photocage reveals fundamental insight for designing advanced photocages such as longer wavelengths in different solvent conditions tailored for applications in basic and applied research.

Interfacial layers are frequently used in organic solar cells performing various functions, including blocking surface recombination, improving selectivity of charge carrier extraction, modification of the work function of the contact materials, and enhancing light absorption within the photoactive layer through an optical cavity effect. The aim of this work is to investigate the origin of performance enhancement of bulk heterojunction solar cells using various electron and hole interfacial layers, with a particular focus on separating the contributions of work function modification and reduced recombination to the improvement of the open circuit voltage ( V oc ). Solar cells using poly[ N-9'-hepta-decanyl-2,7-carbazole- alt-5,5-(4',7'-di-2-thienyl-2',1',3'-benzothiadiazole)]:[6,6]-phenyl C 70 -butyric acid methyl ester (1:4) active layers were prepared with a combination of polymeric, metal oxide, and polyelectrolyte electron and/or hole interfacial layers. Four device structures with (i) no interfacial layers (reference); (ii) only hole; (iii) only electron; (iv) both electron and hole interfacial layers were fabricated and compared using current-voltage, transient photovoltage, and charge extraction measurements. The voltage gains (Δ V oc ) at matched charge density attributed to work function modification (Δ V oc h or Δ V oc e ) are distinguished from the increase in V oc arising from increased charge carrier density. At the hole contact, Δ V oc h was 0.21 V by using a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) hole interfacial layer, whereas Δ V oc e was 0.29 V on the electron contact using a polyethoxylate imine-TiO x interfacial layer compared to reference devices. The electron lifetime also improved by orders of magnitude with the use of either electron or hole contact layers, contributing to a further 0.35-0.38 V increase in the open circuit voltage (Δ V oc rec ) because of increased charge density. The increased charge carrier lifetime is proposed to originate from the larger spatial separation of the electrons and holes in the device because of the increased internal field. Using both an electron and a hole interfacial layer did not significantly increase the charge carrier lifetime compared to single interfacial layer devices; therefore, the V oc did not increase significantly. The findings presented clarify the role of interfacial layers in organic solar cells and provide new insights into using time-resolved charge extraction techniques to understand the influence of interfacial layers on the open circuit voltage.

We have previously identified extensive glycation, bound fatty acids and increased quantities of protein aggregates in commercially available recombinant HSA (rHSA) expressed in Oryza sativa (Asian rice) (OsrHSA) when compared to rHSA from other expression systems. We propose these differences may alter some attributes of nanoparticles fabricated with OsrHSA, as studies have associated greater quantities of aggregates with increased nanoparticle diameters. To determine if this is the case, nanoparticles were fabricated with OsrHSA from various suppliers using ethanol desolvation and subsequent glutaraldehyde cross-linking. All nanoparticles fabricated with OsrHSA showed larger diameters of approximately 20 to 90nm than particles fabricated with either defatted bovine serum albumin (DF-BSA) (100.9 ± 2.8nm) or human plasma albumin (pHSA) (112.0 ± 4.0nm). It was hypothesized that the larger nanoparticle diameters were due to the presence of bound fatty acids and this was confirmed through defatting OsrHSA prior to particle fabrication which yielded particles with diameters similar to those fabricated with pHSA. For additional conformation, DF-BSA was incubated with dodecanoic acid prior to desolvation yielding particles with significantly larger diameters. Further studies showed the increased nanoparticle diameters were due to the bound fatty acids modulating electrostatic interactions between albumin nanoparticles during the desolvation and not changes in protein structure, stability or generation of additional albumin oligomers. Finally the presence of dodecanoic acid was shown to improve doxorubicin loading efficiency onto preformed albumin nanoparticles.

The presence of energetically low-lying triplet states is a hallmark of organic semiconductors. Even though they present a wealth of interesting photophysical properties, these optically dark states significantly limit optoelectronic device performance. Recent advances in emissive charge-transfer molecules have pioneered routes to reduce the energy gap between triplets and "bright" singlets, allowing thermal population exchange between them and eliminating a significant loss channel in devices. In conjugated polymers, this gap has proved resistant to modification. Here, we introduce a general approach to reduce the singlet-triplet energy gap in fully conjugated polymers, using a donor-orthogonal acceptor motif to spatially separate electron and hole wave functions. This new generation of conjugated polymers allows for a greatly reduced exchange energy, enhancing triplet formation and enabling thermally activated delayed fluorescence. We find that the mechanisms of both processes are driven by excited-state mixing between π-π*and charge-transfer states, affording new insight into reverse intersystem crossing.

Reduction kinetics of oxidized dyes absorbed on semiconductor surfaces and immersed in redox active electrolytes has been mainly modeled based on the free energy difference between the oxidation potential of the dye and the redox potential of the electrolyte. Only a few mechanisms have been demonstrated to enhance the kinetics by other means. In this work, the rate constant of the reduction of oxidized porphyrin dye is enhanced by attaching non-conjugated carbazole triphenylamine moiety using iodine/triiodide and tris(2,2'-bispyridinium)cobalt II/III electrolytes. These results are obtained using transient absorption spectroscopy by selectively probing the regeneration kinetics at the porphyrin radical cation and the carbazole triphenylamine radical cation absorption wavelengths. The enhancement in the reduction kinetics is not attributed to changes in the driving force, but to the more exposed dye cation radical orbitals of the dichromophoric dye. The results are important for the development of high efficiency photo-electrochemical devices with minimalized energy loss at electron transfer interfaces.

Microflow digital imaging (MDI) has become a widely accepted method for assessing sub-visible particles in pharmaceutical formulations however, to date; no data have been presented on the utility of this methodology when formulations include opaque vaccine adjuvants. This study evaluates the ability of MDI to assess sub-visible particles under these conditions. A Fluid Imaging Technologies Inc. FlowCAM® instrument was used to assess a number of sub-visible particle types in solution with increasing concentrations of AddaVax™, a nanoscale squalene-based adjuvant. With the objective (10X) used and the limitations of the sensor resolution, the instrument was incapable of distinguishing between sub-visible particles and AddaVax™ droplets at particle sizes less than 5 μm. The instrument was capable of imaging all particle types assessed (polystyrene beads, borosilicate glass, cellulose, polyethylene protein aggregate mimics, and lysozyme protein aggregates) at sizes greater than 5 μm in concentrations of AddaVax™ up to 50% (vol:vol). Reduced edge gradients and a decrease in measured particle sizes were noted as adjuvant concentrations increased. No significant changes in particle counts were observed for polystyrene particle standards and lysozyme protein aggregates, however significant reductions in particle counts were observed for borosilicate (80% of original) and cellulose (92% of original) particles. This reduction in particle counts may be due to the opaque adjuvant masking translucent particles present in borosilicate and cellulose samples. Although the results suggest that the utility of MDI for assessing sub-visible particles in high concentrations of adjuvant may be highly dependent on particle morphology, we believe that further investigation of this methodology to assess sub-visible particles in challenging formulations is warranted.

This study reports evidence of dispersive transport in planar PbS colloidal quantum dot heterojunction-based devices as well as the effect of incorporating a MoO3 hole selective layer on the charge extraction behavior. Steady state and transient characterization techniques are employed to determine the complex recombination processes involved in such devices. The addition of a selective contact drastically improves the device efficiency up to 3.15% (especially due to increased photocurrent and decreased series resistance) and extends the overall charge lifetime by suppressing the main first-order recombination pathway observed in device without MoO3. The lifetime and mobility calculated for our sulfur-rich PbS-based devices are similar to previously reported values in lead-rich quantum dots-based solar cells. Nevertheless, strong Shockley-Read-Hall mechanisms appear to keep restricting charge transport, as the equilibrium voltage takes more than 1 ms to be established.