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Wearable, disposable, implantable and lightweight electronic devices all require low power consumption, typically less than 3 V. Polymerized ionic liquids represent an emerging class of dielectric materials which can enable low voltage organic thin film transistor operation (OTFT) while in the solid state, reducing the potential for toxic or flammable electrolyte leaks. Here we show that bottom gate bottom contact OTFTs were successfully fabricated using a library of 12 poly-(styrene)-b-poly(1-(4-vinylbenzyl)-3-butylimidazolium-r-poly(ethylene glycol) methyl ether methacrylate) block copolymers with varying VBBI+ and PEGMA ratios, and can achieve OTFTs that turn on between 0 and 1 V. We demonstrate that the poly(ionic liquid) microstructure dictates the self-assembly which correlates to OTFT performance. Notably, we indicate that changing the surface chemistry of the substrate the poly(ionic liquid) is cast on is less significant in driving block copolymer self-assembly compared to block ratios and the counter ion. We also report that the semiconductor crystalline domains are similar when deposited on the poly(ionic liquids), however the films differ in terms of semiconductor molecular order in the amorphous domains. Overall, we demonstrate that device performance is primarily linked to the poly(ionic liquid) self-assembly, while also highlighting the importance of both the crystalline and amorphous domains in the semiconductor structure of thin films. [ABSTRACT FROM AUTHOR]

Copyright of Journal of the Botanical Research Institute of Texas is the property of Journal of the Botanical Research Institute of Texas and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Multiple peptide resistance factor (MprF) confers resistance to cationic antimicrobial peptides (AMPs) in several pathogens, thereby enabling evasion of the host immune response. The role of MprF in commensals remains, however, uncharacterized. To close this knowledge gap, we used a common gut commensal of animals, Lactiplantibacillus plantarum, and its natural host, the fruit fly Drosophila melanogaster, as an experimental model to investigate the role of MprF in commensal-host interactions. The L. plantarum ΔmprF mutant that we generated exhibited deficiency in the synthesis of lysyl-phosphatidylglycerol (Lys-PG), resulting in increased negative cell surface charge and increased susceptibility to AMPs. Susceptibility to AMPs had no effect on ΔmprF mutant's ability to colonize guts of uninfected flies. However, we observed significantly reduced abundance of the ΔmprF mutant after infection-induced inflammation in the guts of wild-type flies but not flies lacking AMPs. Additionally, we found that the ΔmprF mutant compared to wild-type L. plantarum induces a stronger intestinal immune response in flies due to the increased release of immunostimulatory peptidoglycan fragments, indicating an important role of MprF in promoting host tolerance to commensals. Our further analysis suggests that MprF-mediated lipoteichoic acid modifications are involved in host immunomodulation. Overall, our results demonstrate that MprF, besides its well-characterized role in pathogen immune evasion and virulence, is also an important commensal resilience factor. Author summary: Commensal microbial communities residing in the host intestine often experience the exposure to host immune effectors triggered by the pathogens during infection. Despite these perturbations, intestinal commensals stably colonize the host, thus exhibiting resilience to inflammation. Here, we used the fruit fly Drosophila melanogaster and its gut commensal, Lactiplantibacillus plantarum, as a model to investigate the role of a specific bacterial gene, multiple peptide resistance factor (MprF), in commensal-host interactions during infection. We generated an L. plantarum ΔmprF mutant and demonstrated susceptibility of this mutant to AMPs and deficiency in the colonization of flies after infection. Importantly, ΔmprF mutant efficiently colonized flies that lack AMPs, demonstrating that MprF-mediated resistance to host AMPs is crucial for commensal persistence during infection. Additionally, we discovered that the ΔmprF mutant induced due to an increased release of cell-wall fragments enhanced immune response in flies as compared to wild-type bacteria. Hence, MprF confers two highly-relevant functions for commensal-host association: AMP resistance and immune evasion. Finally, we showed a role of MprF in regulating LTA size, implying a broader role of MprF in the bacterial physiology then previously appreciated. Collectively, our work shows that AMP resistance via MprF-mediated lipid and LTA modifications is essential for commensal resilience during inflammation. [ABSTRACT FROM AUTHOR]

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix–turn–helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR–Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2–5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2–RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR–Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.The helix–turn–helix domain of an anti-CRISPR-associated (Aca) protein represses transcription of anti-CRISPR (Acr)-encoding genes and inhibits their translation to protein via distinct binding modes to DNA and RNA, respectively. [ABSTRACT FROM AUTHOR]

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage—all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection. Cas12 nucleases offer powerful tools for RNA-guided DNA detection. Here, Jiao et al. show that these same nucleases can be harnessed for sensitive and specific RNA detection by reprogramming their associated RNA processing factors called tracRNAs. [ABSTRACT FROM AUTHOR]

Much is unknown about the impact of the COVID-19 pandemic on the carceral experience. Firsthand accounts, however, can illuminate how the virus traveled through correctional institutions and how operational changes intended to mitigate virus transmission altered daily life for incarcerated people. Analyzing semi-structured interviews with 53 individuals released from state prisons, county jails, and halfway houses in a northeastern U.S. state between October 2020 and June 2021, this study explores the shifting conditions of confinement in the early months of the pandemic, as well as how incarcerated individuals experienced the pandemic in terms of their mental and physical health, safety, and trust in correctional institutions. Interviewees described the spread of COVID-19 in correctional environments and how it led to several pandemic-related changes, such as stoppages or adjustments to programs, visits, recreation time, and movement around the facility. The data ultimately suggest that pandemic lockdown measures, instituted to prevent virus transmission, led to environments akin to solitary confinement and compromised the ability of incarcerated individuals to connect with loved ones, exacerbated their fears about contracting the virus, and further eroded their trust in correctional institutions. [ABSTRACT FROM AUTHOR]

Understanding how tropical forests respond to abiotic environmental changes is critical for preserving biodiversity, mitigating climate change, and maintaining ecosystem services in the coming century. To evaluate the relative roles of the abiotic environment and human disturbance on Central African tree community composition, we employ tree inventory data, remotely sensed climatic data, and soil nutrient data collected from 30 1‐ha plots distributed across a large‐scale observational experiment in forests that had been differently impacted by logging and hunting in northern Republic of Congo. We show that the composition of Afrotropical plant communities at this scale responds to human disturbance more than to climate, with particular sensitivities to hunting and distance to the nearest village (a proxy for other human activities, including tree‐cutting and gathering). These findings contrast neotropical predictions, highlighting the unique ecological, evolutionary, and anthropogenic history of Afrotropical forests. [ABSTRACT FROM AUTHOR]

This document provides an analysis of pharmacy-based buprenorphine dispensing in the United States from 2019 to 2021. Buprenorphine is an FDA-approved medication for opioid use disorder (MOUD) treatment. The study found that the number of buprenorphine prescriptions dispensed increased by 3.66% during this period, while the number of individuals receiving prescriptions increased by 11.07%. However, there were still significant variations between states in terms of buprenorphine dispensing, indicating potential unmet needs. The study suggests that efforts should be made to increase the number of clinicians prescribing buprenorphine and promote linkage to MOUD in healthcare and community settings. [Extracted from the article]

Alkaline polymer membrane electrochemical energy conversion devices offer the prospect of using non-platinum group catalysts. However, their cationic functionalities are currently not sufficiently stable for vapor-phase applications, such as fuel cells. Herein, we report 1,6-diazabicyclo[4.4.4]tetradecan-1,6-ium (in-DBD), a cationic proton cage, that is orders of magnitude more resistant to hydroxide-promoted degradation than state-of-the-art organic cations under ultra-dry conditions and elevated temperature, and the first organic cation-hydroxide to persist at critically low hydration levels (< 10% RH at 80 °C). This high stability against hydroxide-promoted degradation is due to the unique combination of endohedral protection and intra-bridgehead hydrogen bonding that prevents the removal of the inter-cavity proton and lowers the susceptibility to Hofmann elimination. We anticipate this discovery will facilitate a step-change in the advancement of materials and electrochemical devices utilizing anion-exchange membranes based on in-DBD that will enable stable operation under extreme alkaline conditions. Organic cations are typically unstable under low-humidity, alkaline conditions. Here, the authors report an organic cation featuring a proton protected within a cage that can withstand vapor-phase alkaline conditions. [ABSTRACT FROM AUTHOR]

Join us on a journey through the complex and ever-expanding universe of CRISPR approaches for genome editing in bacteria. Discover what is available, current technical challenges, successful implementation of these tools and the regulatory framework around their use. [ABSTRACT FROM AUTHOR]

Several interventional therapies are in development to treat functional tricuspid regurgitation. Most have failed to achieve adequate efficacy, as animal models of this lesion are lacking. We developed a new image-guided technique in swine, by tethering the tricuspid valve chordae using echo-guided chordal encircling snares. Five swine underwent baseline echocardiographic assessment of tricuspid valve function, followed by echo-guided placement of snares that encircle the chordae inserting into the anterior and posterior tricuspid valve leaflets. Tethering these snares and stabilizing them on the right ventricle caused the regurgitant fraction to increase from 8.48±5.38% to 48.76±12.5%, and the valve tenting area to increase from 60.26±52.19 to 160.9±86.92 mm2. Image-guided chordal encircling snares could reproducibly induce clinically significant levels of functional tricuspid regurgitation and create a valve geometry like that seen in patients, providing a new animal model for use to study novel interventional devices. [ABSTRACT FROM AUTHOR]

Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs. [ABSTRACT FROM AUTHOR]

Background: Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of death of premature neonates. NEC is associated with prematurity, a hyperinflammatory response, and dysregulation of intestinal barrier function. We hypothesize that patients with NEC will have, and continue to have after recovery, an increased hyperinflammatory intestinal response compared to those patients without NEC.Methods: Neonates with NEC, those that have recovered from NEC, and those without NEC undergoing intestinal resections had specimens collected and snap frozen or generated into enteroids. The enteroids were treated with 100ug/mL lipopolysaccharide (LPS) and subjected to 24 hr of hypoxia together, then compared with untreated controls. Expression of Tumor Necrosis Factor (TNF-α) and interleukin 8 (IL-8) were evaluated via RT-qPCR and ELISA to measure inflammatory response. ANOVA determined statistical significance (p< 0.05).Results: There was no difference in inflammatory markers in recovered NEC tissue compared to non-NEC tissue on RTqPCR (p=0.701 TNF-α and 0.861 IL-8). However, recovered NEC enteroids demonstrate elevated levels of inflammatory markers after treatment compared to non-NEC enteroids after treatment on RTqPCR (p=0.0485 TNF-α, p=0.0057 IL-8) and ELISA (p=0.0354 TNF-α, p=0.0011 IL-8). Recovered NEC enteroids that underwent treatment demonstrated increased inflammatory markers compared to recovered NEC enteroids without treatment on RTqPCR (p=0.0045 TNF-α, p=0.0002 IL-8) and ELISA (p=0.034 TNF-α, p=0.0002 IL-8) suggesting a heightened inflammatory response to a second hit.Conclusion: Intestinal tissue resected from neonates with NEC has an elevated hyperinflammatory response compared to neonates recovered from NEC and neonates without NEC. Enteroids generated from patients that have recovered from NEC have a heightened inflammatory response in response to NEC inducing stimuli compared to controls. This tendency towards an increased hyperinflammatory state may be correlated with an infant's proclivity to develop NEC and demonstrates the significance of a second hit on this tissue creating a heightened inflammatory response. This could be correlated with the impact and trajectory of an illness post recovery from NEC. [ABSTRACT FROM AUTHOR]

Although religion and crime occupies a relatively small space in contemporary criminological research, religious beliefs, and practices are central to classical social theory. This study uses fixed effects models to analyze three waves of panel data from the National Longitudinal Study of Adolescent to Adult Health to examine the relationship between a multidimensional measure of religiosity and several forms of delinquency. The results indicate that the relationship between religiosity and delinquency is offense-specific. While religiosity is inversely related to substance use in the presence of social bonds and predictors of delinquency, its relationship with non-violent delinquency is mixed. Finally, religiosity is not associated with violent offending. The results suggest that renewed interest in religion among criminologists is warranted. [ABSTRACT FROM AUTHOR]

Capturing an individual cell's transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected cellular transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed to base pair with sensed transcripts, converting them into guide RNAs. The guide RNAs then direct a Cas9 base editor to target an introduced DNA target. The extent of base editing can then be read in the future by sequencing. We use this approach, called TIGER (transcribed RNAs inferred by genetically encoded records), to record heterologous and endogenous transcripts in individual bacterial cells. TIGER can quantify relative expression, distinguish single-nucleotide differences, record multiple transcripts simultaneously and read out single-cell phenomena. We further apply TIGER to record metabolic bet hedging and antibiotic resistance mobilization in Escherichia coli as well as host cell invasion by Salmonella. Through RNA recording, TIGER connects current cellular states with past transcriptional states to decipher complex cellular responses in single cells. RNA transcripts in bacteria are recorded as DNA modifications using a base editor. [ABSTRACT FROM AUTHOR]

CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing. [ABSTRACT FROM AUTHOR]

Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection1. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of ‘aromatic clamp’ residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.Structural analysis of Cas12a2, a CRISPR-associated nuclease that nonspecifically cleaves ssRNA, ssDNA and dsDNA, reveals a complete activation pathway involved in the abortive infection system protecting cells against invasion. [ABSTRACT FROM AUTHOR]

Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1,2. Several RNA-targeting CRISPR–Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases3–5. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR–Cas systems and create additional opportunities for CRISPR technologies.RNA targeting by the Sulfuricurvum type V single-effector nuclease SuCas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA—after recognition of an RNA target through an activating protospacer-flanking sequence, SuCas12a2 efficiently degrades ssRNA, ssDNA and dsDNA. [ABSTRACT FROM AUTHOR]

CRISPR technologies comprising a Cas nuclease and a guide RNA (gRNA) can utilize multiple gRNAs to enact multi-site editing or regulation in the same cell. Nature devised a highly compact means of encoding gRNAs in the form of CRISPR arrays composed of conserved repeats separated by targeting spacers. However, the capacity to acquire new spacers keeps the arrays longer than necessary for CRISPR technologies. Here, we show that CRISPR arrays utilized by the Cas9 nuclease can be shortened without compromising and sometimes even enhancing targeting activity. Using multiplexed gene repression in E. coli, we found that each region could be systematically shortened to varying degrees before severely compromising targeting activity. Surprisingly, shortening some spacers yielded enhanced targeting activity, which was linked to folding of the transcribed array prior to processing. Overall, shortened CRISPR-Cas9 arrays can facilitate multiplexed editing and gene regulation from a smaller DNA footprint across many bacterial applications of CRISPR technologies. [ABSTRACT FROM AUTHOR]

Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms, also known as DRESS, is a rare, delayed and potentially life-threatening reaction following exposure to certain pharmaceutical agents. Aromatic anticonvulsants and allopurinol are the most frequent causative agents. However, various reports of antibiotic-induced DRESS are available, such as antituberculosis drugs, vancomycin, and sulfonamides. Patient presentation is typically broad, with symptomology ranging from cutaneous to visceral involvement. DRESS has also been implicated in the reactivation of latent viral infection of human herpes virus-6 (HHV-6), Epstein-Barr virus (EBV) and cytomegalovirus (CMV). DRESS is a poorly understood phenomenon with devastating effects if left untreated, which potentiates the need for further investigation and understanding. In this report, we present a patient with DRESS displaying both cutaneous and visceral pathologic processes, as well as discuss the need for a better understanding of this rare but serious adverse drug reaction to ensure appropriate and effective treatment in a timely manner. [ABSTRACT FROM AUTHOR]

In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light‐dependent reaction and CO2 fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer‐DNA (T‐DNA) insertion mutant line SALK_008491, previously known as nhd1‐1. Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non‐photochemical quenching (NPQ). Interestingly, an independent nhd1 null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F2 pool, we identified an ~14‐kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of NHD1. Besides NHD1, which encodes for a putative plastid Na+/H+ antiporter, the stromal NAD‐dependent D‐3‐phosphoglycerate dehydrogenase 3 (PGDH3) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow‐up studies employing respective single mutants and complementation with overlapping transformation‐competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion‐carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T‐DNA lines. Although second‐site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T‐DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress. [ABSTRACT FROM AUTHOR]

A personal narrative is presented which explores the author's experience of grieving the loss of their father and explores the common stages and challenges of grief and loss.

Copyright of Canadian Journal of Chemistry is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Accurate, rapid, and multiplexed nucleic acid detection is of great value for applications in biomedicine and agriculture. Here, we demonstrated a one‐step Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic method for the digital and multiplexed detection of both single‐stranded (ss) and double‐stranded (ds) DNA on a handheld smartphone device. ssDNA targets exhibited faster reaction kinetics of this single‐step CRISPR–Cas12a assay than dsDNA counterparts. Under optimized conditions, picomolar levels of ssDNA targets can be detected in 96‐well plates by using a benchtop plate reader. The detection sensitivity can be further improved to 5 fM when running the reaction in a microwell‐based digital assay chip. Multiplexed detection of hepatitis B virus and human papillomavirus DNA markers was demonstrated on the smartphone‐based platform. Finally, the one‐step CRISPR–Cas12a assay performed robustly in human serum samples with a recovery rate of ssDNA detection between 96% and 105.6%, suggesting a high potential for clinical diagnostic applications in point‐of‐care settings. [ABSTRACT FROM AUTHOR]

CRISPR-Cas adaptive immune systems in bacteria and archaea utilize short CRISPR RNAs (crRNAs) to guide sequence-specific recognition and clearance of foreign genetic material. Multiple crRNAs are stored together in a compact format called a CRISPR array that is transcribed and processed into the individual crRNAs. While the exact processing mechanisms vary widely, some CRISPR-Cas systems, including those encoding the Cas9 nuclease, rely on a trans-activating crRNA (tracrRNA). The tracrRNA was discovered in 2011 and was quickly co-opted to create single-guide RNAs as core components of CRISPR-Cas9 technologies. Since then, further studies have uncovered processes extending beyond the traditional role of tracrRNA in crRNA biogenesis, revealed Cas nucleases besides Cas9 that are dependent on tracrRNAs, and established new applications based on tracrRNA engineering. In this review, we describe the biology of the tracrRNA and how its ongoing characterization has garnered new insights into prokaryotic immune defense and enabled key technological advances. [ABSTRACT FROM AUTHOR]

As we move toward roll‐to‐roll processing of organic photovoltaics (OPVs), it is important to validate the process and the ink formulations. A poly[[9‐(1‐octylnonyl)‐9Hcarbazole‐2,7‐diyl]‐2,5‐thiophenediyl‐2,1,3‐benzothiadiazole‐4,7 diyl2,5thiophenediyl]:[6,6]‐phenyl C71 butyric acid methyl ester (PCDTBT:PC71BM)‐based OPV with an increase in power conversion efficiency (PCE) from 4.6% up to 5.4% by incorporating bis(tri‐hexylsiloxy) silicon phthalocyanine ((3HS)2‐SiPc) NIR absorbing ternary additives on indium tin oxide (ITO)/polyethyleneterapthalate (PET) flexible substrate with active area of 1 cm2 under simulated AM 1.5G one sun irradiation. Maximum PCE of the ternary OPVs was obtained with 5 wt% of (3HS)2‐SiPc added when processing the active layer by spin coating, whereas the addition of 10 wt% of (3HS)2‐SiPc is required for the maximization of PCE when processing the films by blade coating. The importance of processing conditions when optimizing the concentration of ternary additives in bulk heterojunction (BHJ) OPVs is demonstrated. [ABSTRACT FROM AUTHOR]

CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes. Technologies that can halt the spread of gene drives would be highly useful in controlling or reverting their effect. Here the authors use the anti-CRISPR protein AcrIIA4 to inactivate drives in A. gambiae. [ABSTRACT FROM AUTHOR]

Since the reemergence of St. Louis Encephalitis (SLE) Virus (SLEV) in the Southwest United States, identified during the 2015 outbreak in Arizona, SLEV has been seasonally detected within Culex spp. populations throughout the Southwest United States. Previous work revealed the 2015 outbreak was caused by an importation of SLEV genotype III, which had only been detected previously in Argentina. However, little is known about when the importation occurred or the transmission and genetic dynamics since its arrival into the Southwest. In this study, we sought to determine whether the annual detection of SLEV in the Southwest is due to enzootic cycling or new importations. To address this question, we analyzed 174 SLEV genomes (142 sequenced as part of this study) using Bayesian phylogenetic analyses to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Maricopa and Riverside counties form two distinct populations with little evidence of inter-county transmission since the onset of the outbreak. Alternatively, it appears that in 2019, Yuma and Clark counties experienced annual importations of SLEV that originated in Riverside and Maricopa counties. Finally, the earliest representatives of SLEV genotype III in the Southwest form a polytomy that includes both California and Arizona samples. We propose that the initial outbreak most likely resulted from the importation of a population of SLEV genotype III variants, perhaps in multiple birds, possibly multiple species, migrating north in 2013, rather than a single variant introduced by one bird. [ABSTRACT FROM AUTHOR]

An 11-year-old girl presented to a dermatology clinic with multiple pink-white linear patches on her thighs. The rash was asymptomatic but would occasionally flare up and become more red and prominent. A punch biopsy was performed, which revealed an increase in elastic fibers and interstitial mast cells in the mid-upper reticular dermis. The diagnosis was linear focal elastosis (LFE), a rare dermal elastic condition. LFE can be clinically mistaken for other conditions such as striae distensae or pseudoxanthoma elasticum (PXE), but histopathological examination can help differentiate between them. It is important to distinguish LFE from PXE due to potential systemic complications associated with PXE. [Extracted from the article]

Seed gut passage times, the time from ingestion to defecation, and frugivore movement patterns determine patterns of seed deposition across the landscape and are thus crucial parameters to quantify in wild populations. Recent advancements in satellite and telemetry technologies mean that animal movement patterns are readily quantifiable in increasingly high resolution. However, data on wild frugivore gut passage times are scarce to non-existent due to the difficulty of monitoring seed ingestion and defecation in natural habitats; therefore, GPT estimates are often extrapolated from captive species whose diets and activity patterns may have limited transferability to free-ranging populations. Here we develop, trial and deploy a suite of model seeds to address this shortfall in wild African forest elephants Loxodonta cyclotis – one of the most effective seed dispersers in the tropics. We use a combination of 'active' seed mimics, which indirectly measure gut passage through recording temperature fluctuations, and 'passive' seed mimics, which serve to mark the point of defecation to allow gut passage estimation from time-stamped GPS collar data. In doing so, we present the first ever GPT estimates from wild forest elephants: mean = 39.8 h (min = 16.6 h; max = 113.7 h). The estimates were derived exclusively from passive seed mimics (plastic beads and modified native seeds) as all active seed mimics were rejected by the focal elephant. The methods described are translatable to other free-ranging, GPS-collared, species and if widely adopted, will begin to address the current gap in our understanding of seed dispersal by wild frugivores. [ABSTRACT FROM AUTHOR]

Aim: Large trees [≥ 70 cm diameter at breast height (DBH)] contribute disproportionately to aboveground carbon stock (AGC) across the tropics but may be vulnerable to changing climate and human activities. Here we determine the distribution, drivers and threats to large trees and high carbon forest. Location: Central Africa. Time period: Current. Major taxa studied: Trees. Methods: Using Gabon's new National Resource Inventory of 104 field sites, AGC was calculated from 67,466 trees from 578 species and 97 genera. Power and Michaelis–Menten models assessed the contribution of large trees to AGC. Environmental and anthropogenic drivers of AGC, large trees, and stand variables were modelled using Akaike's information criterion (AIC) weights to calculate average regression coefficients for all p ossible models. Results: Mean AGC for trees ≥ 10 cm DBH in Gabonese forestlands was 141.7 Mg C/ha, with averages of 166.6, 171.3 and 96.6 Mg C/ha in old growth, concession and secondary forest. High carbon forests occurred where large trees are most abundant: 31% of AGC was stored in large trees (2.3% of all stems). Human activities largely drove variation in AGC and large trees, but climate and edaphic conditions also determined stand variables (basal area, tree height, wood density, stem density). AGC and large trees increased with distance from human settlements; AGC was 40% lower in secondary than primary and concession forests and 33% higher in protected than non‐managed areas. Main conclusions: AGC and large trees were negatively associated with human activities, highlighting the importance of forest management. Redefining large trees as ≥ 50 cm DBH (4.3% more stems) would account for 20% more AGC. This study demonstrates that protecting relatively undisturbed forests can be disproportionately effective in conserving carbon and suggests that including sustainable forestry in programs like reduced emissions for deforestation and forest degradation could maintain carbon dense forests in logging concessions that are a large proportion of remaining Central African forests. [ABSTRACT FROM AUTHOR]

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5′ vs. 3′ untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems. [ABSTRACT FROM AUTHOR]

This study examines race socialization, defined as the process whereby individuals learn about the meaning and significance of race and racism. With data from the 2001–2004 National Survey of American Life-Adolescent Supplement (NSAL-A), we analyze responses to the Comprehensive Race Socialization Inventory (CRSI) among 1,170 African American and Caribbean Black adolescents between the ages of 13 and 17 years. The CRSI captures sources, frequency, content of messages, and prevalence of the most useful message, among other components (e.g., onset and recency, anticipatory socialization, and socializing behaviors). We find 90% of respondents report someone talked with them about what being Black means. In addition, most respondents report receiving messages from all four sources specified in the CRSI (i.e., parents, relatives, friends, and other adults). We find little evidence to suggest sources differ by sex, age, ethnicity, or U.S. region. The question assessing the most useful message is open-ended. Over 60% of respondents recall content supporting "Race equality," maintaining a "Positive self-attitude," or emphasizing "Black pride" as the most useful message. We conclude race socialization is commonplace among Black adolescents because racism necessitates it. [ABSTRACT FROM AUTHOR]

CRISPR-Cas immune systems in bacteria and archaea record prior infections as spacers within each system's CRISPR arrays. Spacers are normally derived from invasive genetic material and direct the immune system to complementary targets as part of future infections. However, not all spacers appear to be derived from foreign genetic material and instead can originate from the host genome. Their presence poses a paradox, as self-targeting spacers would be expected to induce an autoimmune response and cell death. In this review, we discuss the known frequency of self-targeting spacers in natural CRISPR-Cas systems, how these spacers can be incorporated into CRISPR arrays, and how the host can evade lethal attack. We also discuss how self-targeting spacers can become the basis for alternative functions performed by CRISPR-Cas systems that extend beyond adaptive immunity. Overall, the acquisition of genome-targeting spacers poses a substantial risk but can aid in the host's evolution and potentially lead to or support new functionalities. [ABSTRACT FROM AUTHOR]

Class 2 CRISPR–Cas systems, such as Cas9 and Cas12, have been widely used to target DNA sequences in eukaryotic genomes. However, class 1 CRISPR–Cas systems, which represent about 90% of all CRISPR systems in nature, remain largely unexplored for genome engineering applications. Here, we show that class 1 CRISPR–Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control. We repurpose type I variants of class 1 CRISPR–Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells. This study demonstrates the use of Cascade as a CRISPR-based technology for targeted eukaryotic gene regulation, highlighting class 1 CRISPR–Cas systems for further exploration. Type I CRISPR–Cas systems, the largest group of CRISPR systems in nature, can be repurposed for DNA targeting and gene regulation in human cells [ABSTRACT FROM AUTHOR]

Genome editing is essential for probing genotype–phenotype relationships and for enhancing chemical production and phenotypic robustness in industrial bacteria. Currently, the most popular tools for genome editing couple recombineering with DNA cleavage by the CRISPR nuclease Cas9 from Streptococcus pyogenes. Although successful in some model strains, CRISPR-based genome editing has been slow to extend to the multitude of industrially relevant bacteria. In this review, we analyze existing barriers to implementing CRISPR-based editing across diverse bacterial species. We first compare the efficacy of current CRISPR-based editing strategies. Next, we discuss alternatives when the S. pyogenes Cas9 does not yield colonies. Finally, we describe different ways bacteria can evade editing and how elucidating these failure modes can improve CRISPR-based genome editing across strains. Together, this review highlights existing obstacles to CRISPR-based editing in bacteria and offers guidelines to help achieve and enhance editing in a wider range of bacterial species, including non-model strains. [ABSTRACT FROM AUTHOR]

Rationale: Cell‐free transcription‐translation (TXTL) is becoming a popular technology to prototype and engineer biological systems outside living organisms. TXTL relies commonly on a cytoplasmic extract that provides the molecular components necessary to recapitulate gene expression in vitro, where most of the available systems are derived from E. coli. The proteinic and enzymatic composition of lysates, however, is typically unknown. In this work, we analyzed by mass spectrometry the molecular constituents of the all‐E. coli TXTL platform myTXTL prepared from the E. coli strain BL21 Rosetta2. Methods: Standard TXTL reactions were assembled and executed for 10–12 hours at 29°C. In addition to a no‐DNA control, four DNA programs were executed in separate reactions to synthesize the reporter protein deGFP as well as the phages MS2, phix174 and T7. The reactions were treated according to standard procedures (trypsin treatment, cleaning) before performing liquid chromatography/mass spectrometry (LC/MS). Data analysis was performed using Sequest and protein identification using Scaffold. Results: A total of 500–800 proteins were identified by LC/MS in the blank reactions. We organized the most abundant protein sets into several categories pertaining, in particular, to transcription, translation and ATP regeneration. The synthesis of deGFP was easily measured. The major structural proteins that compose the three phages MS2, phix174 and T7 were also identified. Conclusions: Mass spectrometry is a practical tool to characterize biochemical solutions as complex as a cell‐free TXTL reaction and to determine the presence of synthesized proteins. The data presented demonstrate that the composition of TXTL based on lysates can be used to validate some underlying molecular mechanisms implicated in cell‐free protein synthesis. The composition of the lysate shows significant differences with respect to similar studies on other E. coli strains. [ABSTRACT FROM AUTHOR]

To build complex genetic networks with predictable behaviors, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher‐order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely used to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA‐based transcriptional regulation, using small transcription activating RNAs (STARs) and clustered regularly interspaced short palindromic repeats interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell‐free transcription‐translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ordinary differential equation‐based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity. Modular genetic regulators are vital for the construction of predictable synthetic networks. In this study, two RNA‐based genetic regulators were individually characterized and parameterized revealing a timescale mismatch. Simulations and experiments demonstrated these regulators could modularly combine to create a pulse generator network in a cell‐free transcription‐translation system. [ABSTRACT FROM AUTHOR]