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The WRAMP structure is a protein network associated with tail-end actomyosin contractility, membrane retraction, and directional persistence during cell migration. A marker of WRAMP structures is melanoma cell adhesion molecule (MCAM) which dynamically polarizes to the cell rear. However, factors that mediate MCAM polarization are still unknown. In this study, BioID using MCAM as bait identifies the ERM family proteins, moesin, ezrin, and radixin, as WRAMP structure components. We also present a novel image analysis pipeline, Protein Polarity by Percentile ("3P"), which classifies protein polarization using machine learning and facilitates quantitative analysis. Using 3P, we find that depletion of moesin, and to a lesser extent ezrin, decreases the proportion of cells with polarized MCAM. Furthermore, although copolarized MCAM and ERM proteins show high spatial overlap, 3P identifies subpopulations with ERM proteins closer to the cell periphery. Live-cell imaging confirms that MCAM and ERM protein polarization is tightly coordinated, but ERM proteins enrich at the cell edge first. Finally, deletion of a juxtamembrane segment in MCAM previously shown to promote ERM protein interactions impedes MCAM polarization. Our findings highlight the requirement for ERM proteins in recruitment of MCAM to WRAMP structures and an advanced computational tool to characterize protein polarization.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron dysfunction and loss. A portion of ALS cases are caused by mutation of the proteasome shuttle factor Ubiquilin 2 ( UBQLN2 ), but the molecular pathway leading from UBQLN2 dysfunction to disease remains unclear. Here, we demonstrate that UBQLN2 regulates the domesticated gag-pol retrotransposon 'paternally expressed gene 10 (PEG10)' in human cells and tissues. In cells, the PEG10 gag-pol protein cleaves itself in a mechanism reminiscent of retrotransposon self-processing to generate a liberated 'nucleocapsid' fragment, which uniquely localizes to the nucleus and changes the expression of genes involved in axon remodeling. In spinal cord tissue from ALS patients, PEG10 gag-pol is elevated compared to healthy controls. These findings implicate the retrotransposon-like activity of PEG10 as a contributing mechanism in ALS through the regulation of gene expression, and restraint of PEG10 as a primary function of UBQLN2., Competing Interests: HB, JH, JR, SL, WC, CE, GH, JT, AM, EU, CL No competing interests declared, AW The University of Colorado, Boulder, has a patent pending for the use of PEG10 inhibitors on which the author is an inventor, (© 2023, Black, Hanson et al.)

Myosin heavy chain 7b (MYH7b) is an evolutionarily ancient member of the sarcomeric myosin family, which typically supports striated muscle function. However, in mammals, alternative splicing prevents MYH7b protein production in cardiac and most skeletal muscles and limits expression to a subset of specialized muscles and certain nonmuscle environments. In contrast, MYH7b protein is abundant in python cardiac and skeletal muscles. Although the MYH7b expression pattern diverges in mammals versus reptiles, MYH7b shares high sequence identity across species. So, it remains unclear how mammalian MYH7b function may differ from that of other sarcomeric myosins and whether human and python MYH7b motor functions diverge as their expression patterns suggest. Thus, we generated recombinant human and python MYH7b protein and measured their motor properties to investigate any species-specific differences in activity. Our results reveal that despite having similar working strokes, the MYH7b isoforms have slower actin-activated ATPase cycles and actin sliding velocities than human cardiac β-MyHC. Furthermore, python MYH7b is tuned to have slower motor activity than human MYH7b because of slower kinetics of the chemomechanical cycle. We found that the MYH7b isoforms adopt a higher proportion of myosin heads in the ultraslow, super-relaxed state compared with human cardiac β-MyHC. These findings are supported by molecular dynamics simulations that predict MYH7b preferentially occupies myosin active site conformations similar to those observed in the structurally inactive state. Together, these results suggest that MYH7b is specialized for slow and energy-conserving motor activity and that differential tuning of MYH7b orthologs contributes to species-specific biological roles., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Global commerce and transportation facilitate the spread of invasive species. The African big-headed ant, Pheidole megacephala (Fabricius), has achieved worldwide distribution through globalization. Since the late 19th century, Taiwan has served as a major seaport because of its strategic location. The population genetic structure of P. megacephala in Taiwan is likely to be shaped by international trade and migration between neighboring islands. In this study, we investigated the population genetics of P. megacephala colonies sampled from four geographical regions in Taiwan and elucidated the population genetic structures of P. megacephala sampled from Taiwan, Okinawa, and Hawaii. We observed a low genetic diversity of P. megacephala across regions in Taiwan. Moreover, we noted low regional genetic differentiation and did not observe isolation by distance, implying that long-distance jump dispersal might have played a crucial role in the spread of P. megacephala . We sequenced the partial cytochrome oxidase I gene and observed three mitochondrial haplotypes (TW1-TW3). TW1 and TW3 most likely originated from populations within the species' known invasive range, suggesting that secondary introduction is the predominant mode of introduction for this invasive ant. TW2 represents a novel haplotype that was previously unreported in other regions. P. megacephala populations from Taiwan, Okinawa, and Hawaii exhibited remarkable genetic similarity, which may reflect their relative geographic proximity and the historical connectedness of the Asia-Pacific region., Competing Interests: The authors declare no competing financial interests., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 h quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p -coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J.C.C. is a co-founder and equity holder in Prometheus Materials Inc. All other authors declare no competing interests., (© 2022 The Authors. Published by Elsevier B.V. on behalf of International Metabolic Engineering Society.)

Mammalian cells respond to dsRNA in multiple manners. One key response to dsRNA is the activation of PKR, an eIF2α kinase, which triggers translational arrest and the formation of stress granules. However, the process of PKR activation in cells is not fully understood. In response to increased endogenous or exogenous dsRNA, we observed that PKR forms novel cytosolic condensates, referred to as dsRNA-induced foci (dRIFs). dRIFs contain dsRNA, form in proportion to dsRNA, and are enhanced by longer dsRNAs. dRIFs enrich several other dsRNA-binding proteins, including ADAR1, Stau1, NLRP1, and PACT. Strikingly, dRIFs correlate with and form before translation repression by PKR and localize to regions of cells where PKR activation is initiated. We hypothesize that dRIF formation is a mechanism that cells use to enhance the sensitivity of PKR activation in response to low levels of dsRNA or to overcome viral inhibitors of PKR activation.

Frankliniella occidentalis is among the most economically significant pests of greenhouse crops, whose resistance to conventional insecticides has created demand for biopesticides such as essential oils. We assessed the fumigant toxicity of linalool against F. occidentalis , F. insularis , and Solanum lycopersicum . Thrips were fumigated with polyacrylamide hydrogels containing either ( R )-linalool, ( S )-linalool, racemic linalool, or a binary mixture of ( R )-linalool with one of twelve adjuvants (i.e., peppermint, cedarwood, neem, clove, coconut, jojoba, soybean, olive, α-terpineol, 1,8-cineole, trans-anethole, or ( R )-pulegone). Solanum lycopersicum seedlings were exposed to ( R )-linalool or a mixture of ( R )-linalool and peppermint oil via conditioned hydrogels or foliar spray. For F. insularis , ( R )-linalool was more toxic than ( S )-linalool, with LC 50 values of 11.7 mg/L air and 16.7 mg/L air, respectively. Similarly for F. occidentalis , ( R )-linalool was more toxic than ( S )-linalool, with LC 50 values of 29.0 mg/L air and 34.9 mg/L air, respectively. Peppermint oil and α-terpineol were the only synergists, while the other adjuvants exhibited varying degrees of antagonism. All seedling treatments demonstrated phytotoxicity, but symptoms were most severe for foliar sprays and mixtures containing peppermint oil. While hydrogels conditioned in linalool may be a favorable substitute to conventional insecticides, the cross-resistance demonstrated herein indicates that expectations should be metered.

Tools for refined cell-specific targeting have significantly contributed to understanding the characteristics and dynamics of distinct cellular populations by brain region. While advanced cell-labeling methods have accelerated the field of neuroscience, specifically in brain mapping, there remains a need to quantify and analyze the data. Here, by modifying a toolkit that localizes electrodes to brain regions (SHARP-Track; Slice Histology Alignment, Registration, and Probe-Track analysis), we introduce a post-imaging analysis tool to map histological images to established mouse brain atlases called SHARCQ (Slice Histology Alignment, Registration, and Cell Quantification). The program requires MATLAB, histological images, and either a manual or automatic cell count of the unprocessed images. SHARCQ simplifies the post-imaging analysis pipeline with a step-by-step GUI. We demonstrate that SHARCQ can be applied for a variety of mouse brain images, regardless of histology technique. In addition, SHARCQ rectifies discrepancies in mouse brain region borders between atlases by allowing the user to select between the Allen Brain Atlas or the digitized and modified Franklin-Paxinos Atlas for quantifying cell counts by region. SHARCQ produces quantitative and qualitative data, including counts of brain-wide region populations and a 3D model of registered cells within the atlas space. In summary, SHARCQ was designed as a neuroscience post-imaging analysis tool for cell-to-brain registration and quantification with a simple, accessible interface. All code is open-source and available for download (https://github.com/wildrootlab/SHARCQ)., (Copyright © 2022 Lauridsen et al.)

With heat treatments to control drywood termites (Blattodea: Kalotermitidae), the presence of heat sinks causes heat to be distributed unevenly throughout the treatment areas. Drywood termites may move to galleries in heat sink areas to avoid exposure to lethal temperatures. Our studies were conducted in Crytotermes brevis -infested condominiums in Honolulu, Hawaii to reflect real-world condominium scenarios; either a standard heat treatment performed by a heat remediation company, or an improved heat treatment was used. For improved treatments, heated air was directed into the toe-kick voids of C. brevis infested cabinets to reduce heat sink effects and increase heat penetration into these difficult-to-heat areas. Eight thermistor sensors placed inside the toe-kick voids, treatment zone, embedded inside cabinets' sidewalls, and in a wooden cube recorded target temperatures of above 46 °C or 50 °C for 120 min. Pre-treatment and follow-up inspections were performed at 6 months posttreatment to monitor termite inactivity using visual observations and by recording the numbers of spiked peaks on a microwave technology termite detection device (Termatrac). In improved treatment condominiums, significantly higher numbers of spiked peaks were recorded at pre-treatment as compared to 6 months posttreatment. Efficacious heat treatment protocols using the improved methods are proposed.

Carboxysomes, prototypical bacterial microcompartments (BMCs) found in cyanobacteria, are large (~1 GDa) and essential protein complexes that enhance CO 2 fixation. While carboxysome biogenesis has been elucidated, the activity dynamics, lifetime, and degradation of these structures have not been investigated, owing to the inability of tracking individual BMCs over time in vivo. We have developed a fluorescence-imaging platform to simultaneously measure carboxysome number, position, and activity over time in a growing cyanobacterial population, allowing individual carboxysomes to be clustered on the basis of activity and spatial dynamics. We have demonstrated both BMC degradation, characterized by abrupt activity loss followed by polar recruitment of the deactivated complex, and a subclass of ultraproductive carboxysomes. Together, our results reveal the BMC life cycle after biogenesis and describe the first method for measuring activity of single BMCs in vivo., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful and widely used tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing the recruitment process quantitatively. Here, we introduce a diffusion model that explicitly accounts for the unique sizes and shapes of individual nuclei and uses two variables: D eff , the effective coefficient of diffusion, and F, the fraction of mobile protein that accumulates at sites of DNA damage. Our model quantitatively describes the accumulation of three test proteins, poly-ADP-ribose polymerases 1 and 2 (PARP1/2) and histone PARylation factor 1. D eff for PARP1, as derived by our approach, is 6× greater than for PARP2 and in agreement with previous literature reports using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Our data indicate that histone PARylation factor 1 arrives at sites of DNA damage independently of either PARP. Importantly, our model, which can be applied to existing data, allows for the direct comparison of the coefficient of diffusion for any DNA repair protein between different cell types, obtained in different laboratories and by different methods, and also allows for the interrogation of cell-to-cell variability., (Copyright © 2019. Published by Elsevier Inc.)

Background: Accumulating evidence indicate that circulating microRNAs (miRNAs) are useful independent non-invasive biomarkers, with unique miRNA signatures defined for various pathophysiological conditions. However, there are no established universal housekeeping miRNAs for the normalisation of miRNAs in body fluids. We have previously identified an oestrogen-responsive miRNA, miR-494, in regulating the anticoagulant, Protein S, in HuH-7 liver cells. Moreover, increased thrombotic risk associated with elevated circulating oestrogen levels is frequently observed in pregnant women and oral contraceptive users. In order to identify other oestrogen-responsive miRNAs, including miR-494, that may be indicative of increased thrombotic risk in plasma, we used nanoString analysis to identify robust and stable endogenous reference miRNAs for the study of oestrogen-responsive miRNAs in plasma., Results: We compared the plasma miRNA expression profile of individuals with: (1) Low circulating oestrogens (healthy men and non-pregnant women not taking oral contraceptives), (2) High circulating synthetic oestrogens, (women taking oral contraceptives) and (3) High circulating natural oestrogens (pregnant females >14 weeks gestation). From the nanoString analyses, 11 candidate reference miRNAs which exhibited high counts and not significantly differentially expressed between groups were selected for validation using realtime quantitative polymerase chain reaction (RT-qPCR) and digital droplet PCR (DDPCR) in pooled plasma samples, and the stability of their expression evaluated using NormFinder and BestKeeper algorithms. Four miRNAs (miR-25-5p, miR-188-5p, miR-222-3p and miR-520f) demonstrated detectable stable expression between groups and were further analysed by RT-qPCR in individual plasma samples, where miR-188-5p and miR-222-3p expression were identified as a stable pair of reference genes. The miRNA reference panel consisting of synthetic spike-ins cel-miR-39 and ath-miR159a, and reference miRNAs, miR-188-5p and miR-222-3p was useful in evaluating fold-change of the pregnancy-associated miRNA, miR-141-3p, between groups., Conclusion: The miRNA reference panel will be useful for normalising qPCR data comparing miRNA expression between men and women, non-pregnant and pregnant females, and the potential effects of endogenous and synthetic oestrogens on plasma miRNA expression.

In contrast to events at the cell leading edge, rear-polarized mechanisms that control directional cell migration are poorly defined. Previous work described a new intracellular complex, the Wnt5a-receptor-actomyosin polarity (WRAMP) structure, which coordinates the polarized localization of MCAM, actin, and myosin IIB in a Wnt5a-induced manner. However, the polarity and function for the WRAMP structure during cell movement were not determined. Here we characterize WRAMP structures during extended cell migration using live-cell imaging. The results demonstrate that cells undergoing prolonged migration show WRAMP structures stably polarized at the rear, where they are strongly associated with enhanced speed and persistence of directional movement. Strikingly, WRAMP structures form transiently, with cells displaying directional persistence during periods when they are present and cells changing directions randomly when they are absent. Cells appear to pause locomotion when WRAMP structures disassemble and then migrate in new directions after reassembly at a different location, which forms the new rear. We conclude that WRAMP structures represent a rear-directed cellular mechanism to control directional migration and that their ability to form dynamically within cells may control changes in direction during extended migration., (© 2017 Connacher et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Background: Reproductive division of labor is one of the key features of social insects. Queens are adapted for reproduction while workers are adapted for foraging and colony maintenance. In many species, however, workers retain functional ovaries and can lay unfertilized male eggs or trophic eggs. Here we report for the first time on the occurrence of physogastric workers and apparent worker reproduction in the invasive yellow crazy ant Anoplolepis gracilipes (Fr. Smith). We further examined the reproductive potential and nutritional role of physogastric workers through multidisciplinary approaches including morphological characterization, laboratory manipulation, genetic analysis and behavioral observation., Results: Egg production with two types of eggs, namely reproductive and trophic eggs, by physogastric workers was found. The reproductive egg was confirmed to be haploid and male-destined, suggesting that the workers produced males via arrhenotokous parthenogenesis as no spermatheca was discovered. Detailed observations suggested that larvae were mainly fed with trophic eggs. Along with consumption of trophic eggs by queens and other castes as part of their diet, the vital role of physogastric workers as "trophic specialist" is confirmed., Conclusion: We propose that adaptive advantages derived from worker reproduction for A. gracilipes may include 1) trophic eggs provisioned by physogastric workers likely assist colonies of A. gracilipes in overcoming unfavorable conditions such as paucity of food during critical founding stage; 2) worker-produced males are fertile and thus might offer an inclusive fitness advantage for the doomed orphaned colony.

One of the prime limiting factors of optical imaging in biological applications is the diffusion of light by tissue, which prevents focusing at depths greater than the optical diffusion limit (typically ?1??mm). To overcome this challenge, wavefront shaping techniques that use a spatial light modulator (SLM) to correct the phase of the incident wavefront have recently been developed. These techniques are able to focus light through scattering media beyond the optical diffusion limit. However, the low speeds of typically used liquid crystal SLMs limit the focusing speed. Here, we present a method using a digital micromirror device (DMD) and an electro-optic modulator (EOM) to measure the scattering-induced aberrations, and using a liquid crystal SLM to apply the correction to the illuminating wavefront. By combining phase modulation from an EOM with the DMD’s ability to provide selective illumination, we exploit the DMD’s higher refresh rate for phase measurement. We achieved focusing through scattering media in less than 8 ms, which is sufficiently short for certain in vivo applications, as it is comparable to the speckle correlation time of living tissue.

To evaluate the effects of the juvenile hormone analogue pyriproxyfen on colonies of the Pharaoh ant Monomorium pharaonis (L.), peanut oil containing different concentrations (0.3, 0.6, or 0.9%) of pyriproxyfen was fed to monogynous (1 queen, 500 workers, and 0.1 g of brood) and polygynous (8 queens, 50 workers, and 0.1 g of brood) laboratory colonies of M. pharaonis. Due to its delayed activity, pyriproxyfen at all concentrations resulted in colony elimination. Significant reductions in brood volume were recorded at weeks 3 - 6, and complete brood mortality was observed at week 8 in all treated colonies. Brood mortality was attributed to the disruption of brood development and cessation of egg production by queens. All polygynous colonies exhibited significant reduction in the number of queens present at week 10 compared to week 1. Number of workers was significantly lower in all treated colonies compared to control colonies at week 8 due to old-age attrition of the workers without replacement. At least 98.67 ± 1.33% of workers were dead at week 10 in all treated colonies. Thus, treatment with slow acting pyriproxyfen at concentrations of 0.3 - 0.9% is an effective strategy for eliminating Pharaoh ant colonies.

Phase distortions due to scattering in random media restrict optical focusing beyond one transport mean free path. However, scattering can be compensated for by applying a correction to the illumination wavefront using spatial light modulators. One method of obtaining the wavefront correction is by iterative determination using an optimization algorithm. In the past, obtaining a feedback signal required either direct optical access to the target region, or invasive embedding of molecular probes within the random media. Here, we propose using ultrasonically encoded light as feedback to guide the optimization dynamically and non-invasively. In our proof-of-principle demonstration, diffuse light was refocused to the ultrasound focal zone, with a focus-to-background ratio of more than one order of magnitude after 600 iterations. With further improvements, especially in optimization speed, the proposed method should find broad applications in deep tissue optical imaging and therapy.

Background: Acquired protein S (PS) deficiency is highly associated with elevated circulating estrogen levels resulting from pregnancy, oral contraceptives, and estrogen replacement therapy; however, the mechanism of estrogen-mediated acquired PS deficiency remains poorly understood. Increasing evidence indicates that estrogen receptor signaling can indirectly modulate the expression of target genes at the post-transcriptional level by modulating the expression of microRNAs (miRNAs), and miRNAs have also been demonstrated to be involved in the regulation of hemostasis., Objectives: To investigate the mechanism of estrogen-mediated downregulation of PROS1 expression by the microRNA miR-494., Methods: Computational analyses of the PROS1 3'-untranslated region (UTR) were performed to identify putative miRNA-binding sites, and direct targeting of the PROS1 3'-UTR by miR-494 was determined with dual luciferase reporter assays in HuH-7 cells. Reporter vectors containing the PROS1 3'-UTR sequence with deleted miR-494-binding sites were also analyzed with luciferase reporter assays. The effects of estrogen on miR-494 and PROS1 mRNA levels in HuH-7 cells were determined by quantitative real-time PCR, and estrogen-mediated changes to secreted PS levels in culture supernatant of HuH-7 cells were measured with an ELISA., Results: The PROS1 3'-UTR sequence contains three putative miR-494-binding sites. miR-494 directly targets PROS1, and miR-494 levels are upregulated following estrogen treatment in HuH-7 liver cells in association with downregulated PROS1 mRNA and PS levels., Conclusions: The results from this study provide the first evidence for miRNA downregulation of PROS1 by miR-494, and suggest that miR-494 is involved in the mechanism of estrogen-mediated downregulation of PS expression., (© 2013 International Society on Thrombosis and Haemostasis.)

We describe theoretical and experimental demonstration for optical detection of ultrasound using a spectral hole engraved in cryogenically cooled rare-earth ion-doped solids. Our method utilizes the dispersion effects due to the spectral hole to perform phase-to-amplitude modulation conversion. Like previous approaches using spectral holes, it has the advantage of detection with large étendue. The method also has the benefit that high sensitivity can be obtained with moderate absorption contrast for the spectral holes.

A method for characterizing the phase response of spatial light modulators (SLMs) by using a Sagnac interferometer is proposed and demonstrated. The method represents an improvement over conventional diffraction-based or interferometric techniques by providing a simple and accurate phase measurement while taking advantage of the inherent phase stability of a Sagnac interferometer. As a demonstration, the phase response of a commercial liquid crystal on a silicon SLM is characterized and then linearized by using a programmable lookup table. The transverse phase profile over the SLM surface is also measured.