Diatoms are single-celled organisms that contribute approximately 20% of the global primary production and play a crucial role in biogeochemical cycles and trophic chains. Despite their ecological importance, our knowledge of microevolution is limited. We developed a model using the SLiM evolutionary framework to address this knowledge gap. As a reference, we used the diatom Pseudo-nitzschia multistriata, which has been extensively studied in the Gulf of Naples. Our model recapitulates what we observe in natural populations, with microevolutionary processes that occur annually during a three-stage bloom phase. Interestingly, we found that non-bloom phases allow the population to maintain sex-generated diversity produced during blooms. This finding suggests that non-bloom phases are critical to counteract bloom-related pressures and mitigate genetic divergence at the species level. Moreover, our model showed that despite the consistent genetic differentiation during bloom phases, the population tends to return to pre-bloom states. While our model is limited to neutral dynamics, our study provides valuable insights into diatoms\' microevolution, paving the way to explore the ecological implications of the life history dynamics of these organisms.

2-Benzylbenzimidazoles, or \"nitazenes\", are a class of novel synthetic opioids (NSOs) that are increasingly being detected alongside fentanyl analogs and other opioids in drug overdose cases. Nitazenes can be 20× more potent than fentanyl but are not routinely tested for during postmortem or clinical toxicology drug screens; thus, their prevalence in drug overdose cases may be under-reported. Traditional analytical workflows utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) often require additional confirmation with authentic reference standards to identify a novel nitazene. However, additional analytical measurements with ion mobility spectrometry (IMS) may provide a path toward reference-free identification, which would greatly accelerate NSO identification rates in toxicology laboratories. Presented here are the first IMS and collision cross section (CCS) measurements on a set of fourteen nitazene analogs using a structures for lossless ion manipulations (SLIM)-orbitrap MS. All nitazenes exhibited two high intensity baseline-separated IMS distributions, which fentanyls and other drug and druglike compounds also exhibit. Incorporating water into the electrospray ionization (ESI) solution caused the intensities of the higher mobility IMS distributions to increase and the intensities of the lower mobility IMS distributions to decrease. Nitazenes lacking a nitro group at the R1 position exhibited the greatest shifts in signal intensities due to water. Furthermore, IMS-MS/MS experiments showed that the higher mobility IMS distributions of all nitazenes possessing a triethylamine group produced fragment ions with m/z 72, 100, and other low intensity fragments while the lower mobility IMS distributions only produced fragment ions with m/z 72 and 100. The IMS, solvent, and fragmentation studies provide experimental evidence that nitazenes potentially exhibit three gas-phase protomers. The cyclic IMS capability of SLIM was also employed to partially resolve four sets of structurally similar nitazene isomers (e.g., protonitazene/isotonitazene, butonitazene/isobutonitazene/secbutonitazene), showcasing the potential of using high-resolution IMS separations in MS-based workflows for reference-free identification of emerging nitazenes and other NSOs.

AbstractLocal adaptation frequently evolves in patches or environments that are connected via migration. In these cases, genomic regions that are linked to a locally adapted locus experience reduced effective migration rates. Via individual-based simulations of a two-patch system, we show that this reduced effective migration results in the accumulation of conditionally deleterious mutations, but not universally deleterious mutations, adjacent to adaptive loci. When there is redundancy in the genetic basis of local adaptation (i.e., genotypic redundancy), turnover of locally adapted polymorphisms allows conditionally deleterious mutation load to be purged. The amount of mutational load that accumulates adjacent to locally adapted loci is dependent on redundancy, recombination rate, migration rate, population size, strength of selection, and the phenotypic effect size of adaptive alleles. Our results highlight the need to be cautious when interpreting patterns of local adaptation at the level of phenotype or fitness, as the genetic basis of local adaptation can be transient, and evolution may confer a degree of maladaptation to nonlocal environments.

During autophagy, potentially toxic cargo is enveloped by a newly formed autophagosome and trafficked to the lysosome for degradation. Ubiquitinated protein aggregates, a key target for autophagy, are identified by multiple autophagy receptors. NBR1 is an archetypal autophagy receptor and an excellent model for deciphering the role of the multivalent, heterotypic interactions made by cargo-bound receptors. Using NBR1 as a model, we find that three critical binding partners - ATG8-family proteins, FIP200, and TAX1BP1 - each bind to a short linear interaction motif (SLiM) within NBR1. Mutational peptide arrays indicate that these binding events are mediated by distinct overlapping determinants, rather than a single, convergent, SLiM. AlphaFold modeling underlines the need for conformational flexibility within the NBR1 SLiM, as distinct conformations mediate each binding event. To test the extent to which overlapping SLiMs exist beyond NBR1, we performed peptide binding arrays on >100 established LC3-interacting regions (LIRs), revealing that FIP200 and/or TAX1BP1 binding to LIRs is a common phenomenon and suggesting LIRs as protein interaction hotspots. Comparative analysis of phosphomimetic peptides highlights that while FIP200 and Atg8-family binding are generally augmented by phosphorylation, TAX1BP1 binding is nonresponsive, suggesting differential regulation of these binding events. In vivo studies confirm that LIR-mediated interactions with TAX1BP1 enhance NBR1 activity, increasing autophagosomal delivery by leveraging an additional LIR from TAX1BP1. In sum, these results reveal a one-to-many binding modality in NBR1, providing key insights into the cooperative mechanisms among autophagy receptors. Furthermore, these findings underscore the pervasive role of multifunctional SLiMs in autophagy, offering substantial avenues for further exploration into their regulatory functions.

The opioid crisis in the United States is being fueled by the rapid emergence of new fentanyl analogs and precursors that can elude traditional library-based screening methods, which require data from known reference compounds. Since reference compounds are unavailable for new fentanyl analogs, we examined if fentanyls (fentanyl + fentanyl analogs) could be identified in a reference-free manner using a combination of electrospray ionization (ESI), high-resolution ion mobility (IM) spectrometry, high-resolution mass spectrometry (MS), and higher-energy collision-induced dissociation (MS/MS). We analyzed a mixture containing nine fentanyls and W-15 (a structurally similar molecule) and found that the protonated forms of all fentanyls exhibited two baseline-separated IM distributions that produced different MS/MS patterns. Upon fragmentation, both IM distributions of all fentanyls produced two high intensity fragments, resulting from amine site cleavages. The higher mobility distributions of all fentanyls also produced several low intensity fragments, but surprisingly, these same fragments exhibited much greater intensities in the lower mobility distributions. This observation demonstrates that many fragments of fentanyls predominantly originate from one of two different gas-phase structures (suggestive of protomers). Furthermore, increasing the water concentration in the ESI solution increased the intensity of the lower mobility distribution relative to the higher mobility distribution, which further supports that fentanyls exist as two gas-phase protomers. Our observations on the IM and MS/MS properties of fentanyls can be exploited to positively differentiate fentanyls from other compounds without requiring reference libraries and will hopefully assist first responders and law enforcement in combating new and emerging fentanyls.

Synthetic cannabinoids, a subclass of new psychoactive substances (NPS), are laboratory-made substances that are chemically similar to those found naturally in the cannabis plant. Many of these substances are illicitly manufactured and have been associated with severe health problems, prompting a need to develop analytical methods capable of characterizing both known and previously undetected compounds. This work focuses on a novel Structures for Lossless Ion Manipulations (SLIM) IM-MS approach to the differentiation and structural characterization of synthetic cannabinoid metabolites, specifically MDA-19/BUTINACA, JWH-018, and JWH-250 isomer groups. These different compound classes are structurally very similar, differing only in the position of one or a few functional groups; this yielded similarity in measured collision cross section (CCS) values. However, the high resolution of SLIM IM provided adequate separation of many of these isomers, such as sodiated JWH-250 metabolites N-4-OH, N-5-OH, and 5-OH, which displayed CCS of 187.5, 182.5, and 202.3 Å2, respectively. In challenging cases where baseline separation was precluded due to nearly identical CCS, such as for JWH-018 isomers, simple derivatization by dansyl chloride selectively reacted with the 6-OH compound to provide differentiation of all isomers using a combination of CCS and m/z. Finally, the opportunity to use this method for structural elucidation of unknowns was demonstrated by using SLIM IM mobility-aligned MS/MS fragmentation. Different MDA-19/BUTINACA isomers were first mobility separated and could then be individually activated, yielding unique fragments for both targeted identification and structural determination. Overall, the described SLIM IM-MS/MS workflow provides significant potential as a rapid screening tool for the characterization of emerging NPS such as synthetic cannabinoids and their metabolites.

Population genetic simulation has emerged as a common tool for investigating increasingly complex evolutionary and demographic models. Software capable of handling high-level model complexity has recently been developed, and the advancement of tree sequence recording now allows simulations to merge the efficiency and genealogical insight of coalescent simulations with the flexibility of forward simulations. However, frameworks utilizing these features have not yet been compared and benchmarked. Here, we evaluate various simulation workflows using the coalescent simulator msprime and the forward simulator SLiM, to assess resource efficiency and determine an optimal simulation framework. Three aspects were evaluated: (1) the burn-in, to establish an equilibrium level of neutral diversity in the population; (2) the forward simulation, in which temporally fluctuating selection is acting; and (3) the final computation of summary statistics. We provide typical memory and computation time requirements for each step. We find that the fastest framework, a combination of coalescent and forward simulation with tree sequence recording, increases simulation speed by over twenty times compared to classical forward simulations without tree sequence recording, although it does require six times more memory. Overall, using efficient simulation workflows can lead to a substantial improvement when modelling complex evolutionary scenarios-although the optimal framework ultimately depends on the available computational resources.

The baculovirus IE1 gene encodes a multifunctional protein that is essential for both DNA replication and RNA transcription of the virus. Prior to viral DNA replication, IE1 promotes early gene transcription when localized in hr-dependent foci. During viral DNA replication, the IE1 foci expand and fuse to generate the virogenic stroma (VS) with IE1 found in the VS reticulum. To explore the IE1 structural features essential for this coordinated localization, we constructed various IE1 mutants based on three putative domains (N, I, and C). We determined that a BDI motif located in the intrinsic disorder region (IDR) between the N and I domains acts as a nuclear localization signal, whereas BDII and HLH in the C domain are required for VS localization in infected cells or for chromosomal association in uninfected mitotic cells. Deletion of the SLiM (short linear motif) located in the I domain restrains both nuclear- and VS localization. Intra-molecular fluorescence resonance energy transfer (FRET) probes of IE1 mutants revealed a conformational change of the I-C two-domain fragment during infection, which was inhibited by aphidicolin, suggesting that IE1 undergoes a stage-dependent conformational change. Further, homo-dimerization of the I domain and stage-dependent conformational changes require an intact SLiM. Mutational analysis of SLiM revealed that VS localization and chromosomal association were retained following S291A and S291E substitutions, but hr-dependent focus formation differed between the two mutations. These results suggest that coordinated IE1 localization is controlled by SLiM-dependent conformational changes that are potentially switched by the phosphorylation state of the SLiM.

The amenability of traveling wave ion mobility spectrometry (TWIMS) to extended separation pathlengths has prompted a recent surge of interest concerning the technique. While promising, the optimization of ion transmission, particularly when analyzing increasingly disparate species, remains an obstacle in TWIMS. To address this issue, we evaluated a suite of dynamic TW profiles using an original TW structures for lossless ion manipulations (TW-SLIM) platform developed at Washington State University. Inspired by the range of gradient elution profiles used in traditional chromatography, three distinct square TW profiles were evaluated: a static approach which represents a traditional waveform, a dual approach which consists of two distinct TW profiles within a given separation event; and a ramp approach which varies TW speed and amplitude at a fixed rate during separation. The three waveform profiles were evaluated in terms of their impact on separation (quantified as resolution) and sensitivity (quantified using signal-to-noise ratio (SNR), and ion abundance). Concerning separation, the highest resolution (R) was observed when operating with the static waveform (R = 7.92); however, the ramp waveform performed comparably (R = 7.70) under similar conditions. Regarding SNR, optimum waveform profiles were species dependent. Bradykinin2+ displayed the largest gains in SNR (36.6% increase) when ramping TW speed, while the gains were greatest (33.5% increase) for tetraoctylammonium when modulating TW amplitude with the static waveform. Lastly, significant (>10%) increases in the abundance of tetraoctylammonium ions were observed exclusively when utilizing a ramped waveform. The present set of experiments outline the results and challenges related to optimizing separations using alternative TW profiles and provides insight concerning TW-SLIM method development which may be tailored to enhance select analytical metrics.

Traveling wave ion mobility experiments using planar electrode structures (e.g., structures for lossless ion manipulation, TW-SLIM) leverage the mature manufacturing capabilities of printed circuit boards (PCBs). With routine levels of mechanical precision below 150 μm, the conceptual flexibility afforded by PCBs for use as planar ion guides is expansive. To date, the design and construction of TW-SLIM platforms require considerable legacy expertise, especially with respect to simulation and circuit layout strategies. To lower the barrier of TW-SLIM implementation, we introduce Python-based interactive tools that assist in graphical layout of the core electrode footprints for planar ion guides with minimal user inputs. These scripts also export the exact component locations and assignments for direct integration into KiCad and SIMION for PCB finalization and ion flight simulations. The design concepts embodied in the set of scripts comprising SLIM Pickins (PCB CAD generation) and pigsim (SIMION workspace generation) build upon the lessons learned in the independent development of the research-grade TW-SLIM platforms in operation at WSU. Due to the inherent flexibility of the PCB manufacturing process and the time devoted to board layouts prior to manufacturing, both scripts serve to enable rapid, iterative design considerations. Because only a few predefined parameters are necessary (i.e., the TW-SLIM monomer width, x position following a TW Turn, and y position following a TW Turn) it is possible to design the exact component layouts and accompanying simulation space in a manner of minutes. There is no known limitation to the board layout capacities of the scripts, and the size of a designed layout is ultimately constrained by the abilities of the final PCB design and simulation tools, KiCad and SIMION, to accommodate the thousands of electrodes comprising the final design (i.e., RAM and software overhead). Toward removing the barriers to exploring new SLIM tracks and the likelihood of layout errors that require considerable revision and engineering time, the SLIM Pickins and pigsim tools (included as Supporting Information) allow the user to quickly design a length of planar ion guide, simulate its abilities to confine and transmit ions, compare hypothetical board outlines to given vacuum chamber dimensions, and generate a near-production ready PCB CAD file. In addition to these tools, this report outlines a series of cost-saving strategies with respect to vacuum feedthroughs and vacuum chamber design for TW ion mobility experiments using planar ion guides.