KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres.

Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer. Here, we describe KaryoCreate (karyotype CRISPR-engineered aneuploidy technology), a system that enables the generation of chromosome-specific aneuploidies by co-expression of an sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to mutant KNL1. We design unique and highly specific sgRNAs for 19 of the 24 chromosomes. Expression of these constructs leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny, with an average efficiency of 8% for gains and 12% for losses (up to 20%) validated across 10 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, frequent in gastrointestinal cancers, promotes resistance to TGF-ß, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe an innovative technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.

Restoring the Sense of Touch Using a Sensorimotor Demultiplexing Neural Interface.

Paralyzed muscles can be reanimated following spinal cord injury (SCI) using a brain-computer interface (BCI) to enhance motor function alone. Importantly, the sense of touch is a key component of motor function. Here, we demonstrate that a human participant with a clinically complete SCI can use a BCI to simultaneously reanimate both motor function and the sense of touch, leveraging residual touch signaling from his own hand. In the primary motor cortex (M1), residual subperceptual hand touch signals are simultaneously demultiplexed from ongoing efferent motor intention, enabling intracortically controlled closed-loop sensory feedback. Using the closed-loop demultiplexing BCI almost fully restored the ability to detect object touch and significantly improved several sensorimotor functions. Afferent grip-intensity levels are also decoded from M1, enabling grip reanimation regulated by touch signaling. These results demonstrate that subperceptual neural signals can be decoded from the cortex and transformed into conscious perception, significantly augmenting function.

Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1.

RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3' untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.

The RNA-binding protein HuR is essential for the B cell antibody response.

Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.

Inflammation-induced formation of fat-associated lymphoid clusters.

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

Harvesting electrons and holes from photodriven symmetry-breaking charge separation within a perylenediimide photosynthetic model dimer.

Understanding how to utilize symmetry-breaking charge separation (SB-CS) offers a path toward increasingly efficient light-harvesting technologies. This process plays a central role in the first step of photosynthesis, in which the dimeric "special pair" of the photosynthetic reaction center enters a coherent SB-CS state after photoexcitation. Previous research on SB-CS in both biological and synthetic chromophore dimers has focused on increasing the efficiency of light-driven processes. In a chromophore dimer undergoing SB-CS, the energy of the radical ion pair product is nearly isoenergetic with that of the lowest excited singlet (S1) state of the dimer. This means that very little energy is lost from the absorbed photon. In principle, the relatively high energy electron and hole generated by SB-CS within the chromophore dimer can each be transferred to adjacent charge acceptors to extend the lifetime of the electron-hole pair, which can increase the efficiency of solar energy conversion. To investigate this possibility, we have designed a bis-perylenediimide cyclophane (mPDI2) covalently linked to a secondary electron donor, peri-xanthenoxanthene (PXX) and a secondary electron acceptor, partially fluorinated naphthalenediimide (FNDI). Upon selective photoexcitation of mPDI2, transient absorption spectroscopy shows that mPDI2 undergoes SB-CS, followed by two secondary charge transfer reactions to generate a PXX•+-mPDI2-FNDI•- radical ion pair having a nearly 3 µs lifetime. This strategy has the potential to increase the efficiency of molecular systems for artificial photosynthesis and photovoltaics.

A multiomic approach to defining the essential genome of the globally important pathogen Corynebacterium diphtheriae.

Diphtheria is a respiratory disease caused by Corynebacterium diphtheriae. While the toxin-based vaccine has helped control outbreaks of the disease since the mid-20th century there has been an increase in cases in recent years, including systemic infections caused by non-toxigenic C. diphtheriae strains. Here we describe the first study of gene essentiality in C. diphtheriae, providing the most-dense Transposon Directed Insertion Sequencing (TraDIS) library in the phylum Actinobacteriota. This high-density library has allowed the identification of conserved genes across the genus and phylum with essential function and enabled the elucidation of essential domains within the resulting proteins including those involved in cell envelope biogenesis. Validation of these data through protein mass spectrometry identified hypothetical and uncharacterized proteins in the proteome which are also represented in the vaccine. These data are an important benchmark and useful resource for the Corynebacterium, Mycobacterium, Nocardia and Rhodococcus research community. It enables the identification of novel antimicrobial and vaccine targets and provides a basis for future studies of Actinobacterial biology.

Respiratory symptoms and outcomes among cigar smokers: findings from the Population Assessment of Tobacco and Health (PATH) study waves 2-5 (2014-2019).

BACKGROUND: The mechanisms by which cigarette smoking increases the risk of respiratory disease have been studied. However, less is known about risks of respiratory symptoms and outcomes associated with smoking cigars, and risks by cigar types have not been previously explored. The aim of this study was to examine associations between cigar use, including traditional cigars, cigarillos, filtered cigars, and dual cigar and cigarette use, and functionally important respiratory symptoms (FIRS), lifetime asthma diagnosis, uncontrolled asthma, and new cases of FIRS. METHODS: Data from Waves 2-5 (2014-19) of the Population Assessment of Tobacco and Health (PATH) Study, a nationally representative longitudinal study, were analyzed in two ways. For cross-sectional analysis, the analytic sample included adults 18 and older at each wave, resulting in 44,040 observations. Separately, longitudinal analyses were assessed among adults 18 and older at Wave 2, resulting in 7,930 individuals. Both analyses excluded adults with chronic obstructive pulmonary disease (COPD) or non-asthma respiratory disease. RESULTS: Current established cigarillo smokers had higher odds of having FIRS (Adjusted odds ratio (AOR): 1.72; 95% CI: 1.08, 2.74) compared to never smokers of cigarillos and cigarettes, after adjusting for covariates. Current established filtered cigar smokers had higher odds of asthma diagnosis (AOR: 1.35; 95% CI: 1.10, 1.66) while current established dual smokers of filtered cigars and cigarettes had higher odds of uncontrolled asthma (AOR: 5.13; 95% CI: 1.75, 15.02) compared to never smokers of filtered cigars or cigarettes. Both current established cigar smokers and current established dual smokers of cigarettes and cigars had higher odds of new FIRS compared to never cigar or cigarette smokers (AORs: 1.62; 95% CI: 1.02, 2.60 for exclusive cigars and 2.55; 95% CI 1.57, 4.14 for dual smokers). CONCLUSIONS: This study provides evidence that cigar smokers or dual smokers of cigars and cigarettes have greater odds of FIRS, asthma, and uncontrolled asthma and that new incidence of FIRS is higher among any cigar smokers compared to never cigar or cigarette smokers. Understanding health impacts associated with cigar use provides information for supporting policy development, as well as for designing clinical interventions focused on smoking cessation for cigars.

Patient and caregiver perceptions of the possibility of home blood transfusions.

BACKGROUND: Patients with hematologic malignancies (HM) often develop transfusion dependence. The patient and caregiver burdens associated with the need for frequent transfusions are high. Home blood transfusions has the potential to reduce these burdens, but is not widely practiced in the United States. We designed a qualitative study to evaluate the patient and caregiver perceptions of the potential for a home blood transfusion program. STUDY DESIGN AND METHODS: Eligible patients included Adult (≥18 years) patients who were English speaking and met the definition for transfusion dependence within 3 months of study enrollment. We identified and interviewed eligible participants (patients and caregivers), using a semi-structured interview guide to elicit patient perceptions of the acceptability, barriers, and benefits related to home blood product transfusions. Interviews were audio recorded and transcribed. Results were imported into NVivo 12 (version 12; QSR International, Burlington, VT) for coding and analysis. RESULTS: We recruited participants until we reached thematic saturation, which occurred at 29 participants (20 patients, 9 caregivers). Among the 20 patient participants, nine had MDS (45%) and 11 had acute leukemia (55%). Most of the patients (60%) reported getting one transfusion per week. Four themes emerged when the participants discussed their perception regarding the potential of a home blood transfusion program: (1) current in-person experience, (2) caregiver burden, (3) perceptions of home blood transfusions, and (4) interest in participating in a home blood transfusion program. CONCLUSION: The concept of home blood transfusions was well received and further research to study its implementation is warranted.

Modelling orthographic similarity effects in recognition memory reveals support for open bigram representations of letter coding.

A variety of letter string representations has been proposed in the reading literature to account for empirically established orthographic similarity effects from masked priming studies. However, these similarity effects have not been explored in episodic memory paradigms and very few memory models have employed orthographic representation of words. In the current work, through two recognition memory experiments employing word and pseudoword stimuli respectively, we empirically established a set of key orthographic similarity effects for the first time in recognition memory - namely the substitution effect, transposition effect and reverse effect in recognition memory of words and pseudowords, and a start-letter importance in recognition memory of words. Subsequently, we compared orthographic representations from the reading literature including slot coding, closed-bigram, open-bigram and the overlap model. Each of these representations was situated in a global matching model and fitted to recognition performance via Luce's choice rule in a hierarchical Bayesian framework. Model selection results showed support for the open-bigram representation in both experiments.

The British Journal of Clinical Pharmacology: The first 50 years.

The British Journal of Clinical Pharmacology celebrates its 50th anniversary of publication in 2023. Here four previous Editors-in-Chief and the current Editor reflect on the Journal's history and the changes that have occurred during that time.

Transient intermediate in the formation of an amorphous metal-organic framework.

Amorphous metal-organic frameworks are rarely formed via direct synthesis. Our limited understanding of their atomic assembly in solution prevents full exploitation of their unique structural complexity. Here, we use in situ synchrotron X-ray absorption spectroscopy with sub-second time resolution to probe the formation of the amorphous Fe-BTC framework. Using a combination of spectral fingerprinting, linear combination analysis, and principal component analysis coupled with kinetic analyses, we reveal a multi-stage formation mechanism that, crucially, proceeds via the generation of a transient intermediate species.

A scoping review on bovine tuberculosis highlights the need for novel data streams and analytical approaches to curb zoonotic diseases.

Zoonotic diseases represent a significant societal challenge in terms of their health and economic impacts. One Health approaches to managing zoonotic diseases are becoming more prevalent, but require novel thinking, tools and cross-disciplinary collaboration. Bovine tuberculosis (bTB) is one example of a costly One Health challenge with a complex epidemiology involving humans, domestic animals, wildlife and environmental factors, which require sophisticated collaborative approaches. We undertook a scoping review of multi-host bTB epidemiology to identify trends in species publication focus, methodologies, and One Health approaches. We aimed to identify knowledge gaps where novel research could provide insights to inform control policy, for bTB and other zoonoses. The review included 532 articles. We found different levels of research attention across episystems, with a significant proportion of the literature focusing on the badger-cattle-TB episystem, with far less attention given to tropical multi-host episystems. We found a limited number of studies focusing on management solutions and their efficacy, with very few studies looking at modelling exit strategies. Only a small number of studies looked at the effect of human disturbances on the spread of bTB involving wildlife hosts. Most of the studies we reviewed focused on the effect of badger vaccination and culling on bTB dynamics with few looking at how roads, human perturbations and habitat change may affect wildlife movement and disease spread. Finally, we observed a lack of studies considering the effect of weather variables on bTB spread, which is particularly relevant when studying zoonoses under climate change scenarios. Significant technological and methodological advances have been applied to bTB episystems, providing explicit insights into its spread and maintenance across populations. We identified a prominent bias towards certain species and locations. Generating more high-quality empirical data on wildlife host distribution and abundance, high-resolution individual behaviours and greater use of mathematical models and simulations are key areas for future research. Integrating data sources across disciplines, and a "virtuous cycle" of well-designed empirical data collection linked with mathematical and simulation modelling could provide additional gains for policy-makers and managers, enabling optimised bTB management with broader insights for other zoonoses.

Divergence in Cigarette Discontinuation Rates by Use of Electronic Nicotine Delivery Systems (ENDS): Longitudinal Findings From the United States PATH Study Waves 1-6.

INTRODUCTION: We compare real-world trends in population-level cigarette discontinuation rates among adults (ages ≥21) who smoked cigarettes, by electronic nicotine delivery systems (ENDS) use. AIMS AND METHODS: U.S nationally representative data from adults in the Population Assessment of Tobacco and Health (PATH) Study (2013/14-2021, Waves 1-6) who smoked cigarettes in the past 30 days (P30D) were analyzed (n = 13 640). The exposure was P30D ENDS use. The outcome was P30D cigarette discontinuation at biennial follow-up. Weighted trend analyses were conducted to test for differences in cigarette discontinuation trends by ENDS use. RESULTS: Between 2013/14 and 2015/16, cigarette discontinuation rates were both 16% for those who used ENDS and for those who did not; between 2018/19 and 2021, rates were ~30% for those who used ENDS and ~20% for those who did not; the time by ENDS use interaction was significant. CONCLUSIONS: The relationship between adults' ENDS use and cigarette discontinuation in the context of an expanded ENDS marketplace, new tobacco regulatory actions, and COVID-19 differs from the relationship in earlier years. IMPLICATIONS: It is important for public health decisions to be informed by research based on the contemporary ENDS marketplace and circumstances.

Fire frequency drives decadal changes in soil carbon and nitrogen and ecosystem productivity.

Fire frequency is changing globally and is projected to affect the global carbon cycle and climate. However, uncertainty about how ecosystems respond to decadal changes in fire frequency makes it difficult to predict the effects of altered fire regimes on the carbon cycle; for instance, we do not fully understand the long-term effects of fire on soil carbon and nutrient storage, or whether fire-driven nutrient losses limit plant productivity. Here we analyse data from 48 sites in savanna grasslands, broadleaf forests and needleleaf forests spanning up to 65 years, during which time the frequency of fires was altered at each site. We find that frequently burned plots experienced a decline in surface soil carbon and nitrogen that was non-saturating through time, having 36 per cent (±13 per cent) less carbon and 38 per cent (±16 per cent) less nitrogen after 64 years than plots that were protected from fire. Fire-driven carbon and nitrogen losses were substantial in savanna grasslands and broadleaf forests, but not in temperate and boreal needleleaf forests. We also observe comparable soil carbon and nitrogen losses in an independent field dataset and in dynamic model simulations of global vegetation. The model study predicts that the long-term losses of soil nitrogen that result from more frequent burning may in turn decrease the carbon that is sequestered by net primary productivity by about 20 per cent of the total carbon that is emitted from burning biomass over the same period. Furthermore, we estimate that the effects of changes in fire frequency on ecosystem carbon storage may be 30 per cent too low if they do not include multidecadal changes in soil carbon, especially in drier savanna grasslands. Future changes in fire frequency may shift ecosystem carbon storage by changing soil carbon pools and nitrogen limitations on plant growth, altering the carbon sink capacity of frequently burning savanna grasslands and broadleaf forests.

Greater target or lure variability? An exploration on the effects of stimulus types and memory paradigms.

In recognition memory, the variance of the target distribution is almost universally found to be greater than that of the lure distribution. However, these estimates commonly come from long-term memory paradigms where words are used as stimuli. Two exceptions to this rule have found evidence for greater lure variability: a short-term memory task (Yotsumoto et al., Memory & Cognition, 36, 282-294 2008) and in an eyewitness memory paradigm (Wixted et al., Cognitive Psychology, 105, 81-114 2018). In the present work, we conducted a series of recognition memory experiments using different stimulus (faces vs. words) along with different paradigms (long-term vs. short-term paradigms) to evaluate whether either of these conditions would result in greater variability in lure items. Greater target variability was observed across stimulus types and memory paradigms. This suggests that factors other than stimuli and retention interval might be responsible for cases where variability is less for targets than lures.

Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

Developing a high fidelity, low cost simulation model for retrosigmoid craniotomy and microvascular decompression of the trigeminal nerve.

BACKGROUND: Trigeminal neuralgia is a very painful condition that may require a surgical approach as treatment, which is typically retrosigmoid craniotomy followed by microvascular decompression. Due to the limited margin for error when operating in the small triangular window of the cerebellopontine angle and the infrequency of this condition, the operating room can present a difficult learning environment for surgical trainees. Our aim is to create a synthetic, low-cost, high-fidelity, and largely reusable simulation model that will enable neurosurgical trainees to practice these procedural steps in a safe learning environment. MATERIALS AND METHODS: Design-based research was employed to develop the model through iterative micro-cycles, with expert evaluation from an educational and clinical team. The model was made from easy to source materials without advanced technology where sustainability, reproduction at scale and cost where significant considerations. RESULTS: Our model effectively simulates a retrosigmoid craniotomy and microvascular decompression of the trigeminal nerve. The model consists of two distinct parts that are made of synthetic materials. Part A is a single-use, moulded portion of the skull, while part B depicts the cerebellopontine angle and some of its internal anatomical and pathological structures crucial to carrying out all the steps to this procedure. Part A sits ergonomically flush on top of Part B, with both parts subsequently clamped to the table. CONCLUSIONS: As a proof of concept, we report the development and utilisation of a novel, low-cost, replicable retrosigmoid craniotomy and microvascular decompression of the trigeminal nerve simulation model.

Development of a Tag/Catcher-mediated capsid virus-like particle vaccine presenting the conserved Neisseria gonorrhoeae SliC antigen that blocks human lysozyme.

Virus-like particles (VLPs) are promising nanotools for the development of subunit vaccines due to high immunogenicity and safety. Herein, we explored the versatile and effective Tag/Catcher-AP205 capsid VLP (cVLP) vaccine platform to address the urgent need for the development of an effective and safe vaccine against gonorrhea. The benefits of this clinically validated cVLP platform include its ability to facilitate unidirectional, high-density display of complex/full-length antigens through an effective split-protein Tag/Catcher conjugation system. To assess this modular approach for making cVLP vaccines, we used a conserved surface lipoprotein, SliC, that contributes to the Neisseria gonorrhoeae defense against human lysozyme, as a model antigen. This protein was genetically fused at the N- or C-terminus to the small peptide Tag enabling their conjugation to AP205 cVLP, displaying the complementary Catcher. We determined that SliC with the N-terminal SpyTag, N-SliC, retained lysozyme-blocking activity and could be displayed at high density on cVLPs without causing aggregation. In mice, the N-SliC-VLP vaccines, adjuvanted with AddaVax or CpG, induced significantly higher antibody titers compared to controls. In contrast, similar vaccine formulations containing monomeric SliC were non-immunogenic. Accordingly, sera from N-SliC-VLP-immunized mice also had significantly higher human complement-dependent serum bactericidal activity. Furthermore, the N-SliC-VLP vaccines administered subcutaneously with an intranasal boost elicited systemic and vaginal IgG and IgA, whereas subcutaneous delivery alone failed to induce vaginal IgA. The N-SliC-VLP with CpG (10 µg/dose) induced the most significant increase in total serum IgG and IgG3 titers, vaginal IgG and IgA, and bactericidal antibodies.

Consistent physiological, ecological and evolutionary effects of fire regime on conservative leaf economics strategies in plant communities.

The functional response of plant communities to disturbance is hypothesised to be controlled by changes in environmental conditions and evolutionary history of species within the community. However, separating these influences using direct manipulations of repeated disturbances within ecosystems is rare. We evaluated how 41 years of manipulated fire affected plant leaf economics by sampling 89 plant species across a savanna-forest ecotone. Greater fire frequencies created a high-light and low-nitrogen environment, with more diverse communities that contained denser leaves and lower foliar nitrogen content. Strong trait-fire coupling resulted from the combination of significant intraspecific trait-fire correlations being in the same direction as interspecific trait differences arising through the turnover in functional composition along the fire-frequency gradient. Turnover among specific clades helped explain trait-fire trends, but traits were relatively labile. Overall, repeated burning led to reinforcing selective pressures that produced diverse plant communities dominated by conservative resource-use strategies and slow soil nitrogen cycling.