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The CRISPR-Cas system has enabled the development of sophisticated, multigene metabolic engineering programs through the use of guide RNA-directed activation or repression of target genes. To optimize biosynthetic pathways in microbial systems, we need improved models to inform design and implementation of transcriptional programs. Recent progress has resulted in new modeling approaches for identifying gene targets and predicting the efficacy of guide RNA targeting. Genome-scale and flux balance models have successfully been applied to identify targets for improving biosynthetic production yields using combinatorial CRISPR-interference (CRISPRi) programs. The advent of new approaches for tunable and dynamic CRISPR activation (CRISPRa) promises to further advance these engineering capabilities. Once appropriate targets are identified, guide RNA prediction models can lead to increased efficacy in gene targeting. Developing improved models and incorporating approaches from machine learning may be able to overcome current limitations and greatly expand the capabilities of CRISPR-Cas9 tools for metabolic engineering.

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Multi-modality image registration is an important task in medical imaging because it allows for information from different domains to be correlated. Histopathology plays a crucial role in oncologic surgery as it is the gold standard for investigating tissue composition from surgically excised specimens. Research studies are increasingly focused on registering medical imaging modalities such as white light cameras, magnetic resonance imaging, computed tomography, and ultrasound to pathology images. The main challenge in registration tasks involving pathology images comes from addressing the considerable amount of deformation present. This work provides a framework for deep learning-based multi-modality registration of microscopic pathology images to another imaging modality. The proposed framework is validated on the registration of prostate ex-vivo white light camera snapshot images to pathology hematoxylin-eosin images of the same specimen. A pipeline is presented detailing data acquisition, protocol considerations, image dissimilarity, training experiments, and validation. A comprehensive analysis is done on the impact of pre-processing, data augmentation, loss functions, and regularization. This analysis is supplemented by clinically motivated evaluation metrics to avoid the pitfalls of only using ubiquitous image comparison metrics. Consequently, a robust training configuration capable of performing the desired registration task is found. Utilizing the proposed approach, we achieved a dice similarity coefficient of 0.96, a mutual information score of 0.54, a target registration error of 2.4 mm, and a regional dice similarity coefficient of 0.70.

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In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.

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Robust control over gene translation at arbitrary mRNA targets is an outstanding challenge in microbial synthetic biology. The development of tools that can regulate translation will greatly expand our ability to precisely control genes across the genome. In Escherichia coli, most genes are contained in multi-gene operons, which are subject to polar effects where targeting one gene for repression leads to silencing of other genes in the same operon. These effects pose a challenge for independently regulating individual genes in multi-gene operons. Here, we use CRISPR-dCas13 to address this challenge. We find dCas13-mediated repression exhibits up to 6-fold lower polar effects compared to dCas9. We then show that we can selectively activate single genes in a synthetic multi-gene operon by coupling dCas9 transcriptional activation of an operon with dCas13 translational repression of individual genes within the operon. We also show that dCas13 and dCas9 can be multiplexed for improved biosynthesis of a medically-relevant human milk oligosaccharide. Taken together, our findings suggest that combining transcriptional and translational control can access effects that are difficult to achieve with either mode independently. These combined tools for gene regulation will expand our abilities to precisely engineer bacteria for biotechnology and perform systematic genetic screens.

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Dynamic, multi-input gene regulatory networks (GRNs) are ubiquitous in nature. Multilayer CRISPR-based genetic circuits hold great promise for building GRNs akin to those found in naturally occurring biological systems. We develop an approach for creating high-performing activatable promoters that can be assembled into deep, wide, and multi-input CRISPR-activation and -interference (CRISPRa/i) GRNs. By integrating sequence-based design and in vivo screening, we engineer activatable promoters that achieve up to 1,000-fold dynamic range in an Escherichia coli-based cell-free system. These components enable CRISPRa GRNs that are six layers deep and four branches wide. We show the generalizability of the promoter engineering workflow by improving the dynamic range of the light-dependent EL222 optogenetic system from 6-fold to 34-fold. Additionally, high dynamic range promoters enable CRISPRa systems mediated by small molecules and protein-protein interactions. We apply these tools to build input-responsive CRISPRa/i GRNs, including feedback loops, logic gates, multilayer cascades, and dynamic pulse modulators. Our work provides a generalizable approach for the design of high dynamic range activatable promoters and enables classes of gene regulatory functions in cell-free systems.

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Bioelectrical slow waves are fundamental to maintaining the normal motility of the gastrointestinal tract. Slow wave abnormalities are associated with several major digestive disorders. High-resolution electrical mapping arrays have been used to investigate pathological slow wave abnormalities. However, conventional electrode substrate materials are opaque with high mechanical modulus, which leads to non-compliance and sub-par contact with the organ, without additional manipulations. Here we developed highly conformal and transparent conducting polymer electrode arrays using the extrusion wet-printing technique. The performance of electrodes for the electrophysiological recording of the gastric slow wave was validated using in a pig model, against a previously validated reference array over 100 s recording window. The conducting polymer electrodes registered comparable frequency to the reference array ( 3.31±0.20 cpm vs. 3.27±0.07 cpm, p = 0.067), with lower amplitude ( 372±237 vs. ), and signal to noise ratio ( 10.92±7.83 vs. [Formula: see text]). Further adjustments to the deposition parameters and contact material will improve the performance of the conducting polymer array for future experimental applications. Clinical Relevance- These conducting polymer electrodes provide better compliance and minimized mechanical mismatch to the gut tissue thus allowing long-term monitoring and stimulation of the gut. This could be potentially extended to other organs as well.

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In response to viral infection, neutrophils release inflammatory mediators as part of the innate immune response, contributing to pathogen clearance through virus internalization and killing. Pre-existing co- morbidities correlating to incidence of severe COVID-19 are associated with chronic airway neutrophilia. Furthermore, examination of COVID-19 explanted lung tissue revealed a series of epithelial pathologies associated with the infiltration and activation of neutrophils, indicating neutrophil activity in response to SARS- CoV-2 infection. To determine the impact of neutrophil-epithelial interactions on the infectivity and inflammatory responses to SARS-CoV-2 infection, we developed a co-culture model of airway neutrophilia. SARS-CoV-2 infection of the airway epithelium alone does not result in a notable pro-inflammatory response from the epithelium. The addition of neutrophils induces the release of proinflammatory cytokines and stimulates a significantly augmented pro-inflammatory response subsequent SARS-CoV-2 infection. The resulting inflammatory response is polarized with differential release from the apical and basolateral side of the epithelium. Additionally, the integrity of the epithelial barrier is impaired with notable epithelial damage and infection of basal stem cells. This study reveals a key role for neutrophil-epithelial interactions in determining inflammation and infectivity in response to SARS-CoV-2 infection.

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BACKGROUND: Disruption of alveolar epithelial cell (AEC) differentiation is implicated in distal lung diseases such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma that impact morbidity and mortality worldwide. Elucidating underlying disease pathogenesis requires a mechanistic molecular understanding of AEC differentiation. Previous studies have focused on changes of individual transcription factors, and to date no study has comprehensively characterized the dynamic, global epigenomic alterations that facilitate this critical differentiation process in humans. RESULTS: We comprehensively profiled the epigenomic states of human AECs during type 2 to type 1-like cell differentiation, including the methylome and chromatin functional domains, and integrated this with transcriptome-wide RNA expression data. Enhancer regions were drastically altered during AEC differentiation. Transcription factor binding analysis within enhancer regions revealed diverse interactive networks with enrichment for many transcription factors, including NKX2-1 and FOXA family members, as well as transcription factors with less well characterized roles in AEC differentiation, such as members of the MEF2, TEAD, and AP1 families. Additionally, associations among transcription factors changed during differentiation, implicating a complex network of heterotrimeric complex switching in driving differentiation. Integration of AEC enhancer states with the catalog of enhancer elements in the Roadmap Epigenomics Mapping Consortium and Encyclopedia of DNA Elements (ENCODE) revealed that AECs have similar epigenomic structures to other profiled epithelial cell types, including human mammary epithelial cells (HMECs), with NKX2-1 serving as a distinguishing feature of distal lung differentiation. CONCLUSIONS: Enhancer regions are hotspots of epigenomic alteration that regulate AEC differentiation. Furthermore, the differentiation process is regulated by dynamic networks of transcription factors acting in concert, rather than individually. These findings provide a roadmap for understanding the relationship between disruption of the epigenetic state during AEC differentiation and development of lung diseases that may be therapeutically amenable.

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A common outcome of antibiotic exposure in patients and in vitro is the evolution of a hypermutator phenotype that enables rapid adaptation by pathogens. While hypermutation is a robust mechanism for rapid adaptation, it requires trade-offs between the adaptive mutations and the more common "hitchhiker" mutations that accumulate from the increased mutation rate. Using quantitative experimental evolution, we examined the role of hypermutation in driving the adaptation of Pseudomonas aeruginosa to colistin. Metagenomic deep sequencing revealed 2,657 mutations at ≥5% frequency in 1,197 genes and 761 mutations in 29 endpoint isolates. By combining genomic information, phylogenetic analyses, and statistical tests, we showed that evolutionary trajectories leading to resistance could be reliably discerned. In addition to known alleles such as pmrB, hypermutation allowed identification of additional adaptive alleles with epistatic relationships. Although hypermutation provided a short-term fitness benefit, it was detrimental to overall fitness. Alarmingly, a small fraction of the colistin-adapted population remained colistin susceptible and escaped hypermutation. In a clinical population, such cells could play a role in reestablishing infection upon withdrawal of colistin. We present here a framework for evaluating the complex evolutionary trajectories of hypermutators that applies to both current and emerging pathogen populations.

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BACKGROUND: Facebook has a comprehensive set of policies intended to inhibit promotion and sales of tobacco products. Their effectiveness has yet to be studied. METHODS: Leading tobacco brands (388) were identified via Nielsen and Ranker databases and 108 were found to maintain brand-sponsored Facebook pages. Key indicators of alignment with Facebook policy were evaluated. RESULTS: Purchase links (eg, 'shop now' button) on brand-sponsored pages were found for hookah tobaccos (41%), e-cigarettes (74%), smokeless (50%) and cigars (31%). Sales promotions (eg, discount coupons) were present in hookah tobacco (48%), e-cigarette (76%) and cigar (69%) brand-sponsored pages. While conventional cigarettes did not maintain brand-sponsored pages, they were featured in 80% of online tobacco vendors' Facebook pages. The requirement for age gating, to exclude those <18 from viewing tobacco promotion, was absent in hookah tobacco (78%), e-cigarette (62%) and cigar (21%) brand-sponsored pages and for 90% of online tobacco stores which promote leading cigarette brands (eg, Marlboro, Camel). Many of the brand-sponsored tobacco product pages had thousands of 'likes'. CONCLUSIONS: It is laudable that Facebook has policies intended to interdict tobacco promotion throughout its platform. Nevertheless, widespread tobacco promotion and sales were found at variance with the company's policies governing advertising, commerce, page content and under age access. Vetting could be improved by automated screening in partnership with human reviewers.

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In this study we have examined the biochemical attributes of the redox systems that regulate human sperm function using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium, monosodium salt (WST-1), lucigenin and luminol-peroxidase as probes. WST-1 was readily reduced by human spermatozoa in the presence of an intermediate electron acceptor (IEA) or NAD(P)H. The IEA-mediated activity resembled a previously described trans-membrane NADH oxidase in being inhibited by capsaicin, superoxide dismutase (SOD) and N-ethyl maleimide, but differed in its sensitivity to p-chloromercuriphenylsulphonic acid (pCMBS). The NAD(P)H-induced WST-1 reduction resembled the superficial oxidase described previously, in its sensitivity to pCMBS, but differed in its suppression by capsaicin. Lucigenin was reduced by human spermatozoa in a manner that could be inhibited by SOD and stimulated by NAD(P)H or 12-myristate, 13-acetate phorbol ester. A23187 also stimulated human spermatozoa via a diphenylene iodonium-sensitive pathway detectable with luminol-peroxidase but not lucigenin. Defective sperm populations recovered from the low-density region of Percoll gradients were characterized by high levels of redox activity that was only discernable with lucigenin. We conclude that human spermatozoa possess multiple plasma membrane redox systems that are involved to varying extents in the physiological control and pathological disruption of sperm function. Their distinct pharmacological profiles should significantly assist attempts to resolve and characterize these systems.

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