ABSTRACT
The epigenome is a complex framework through which gene expression is precisely and flexibly modulated to incorporate heritable memory and responses to environmental stimuli. It governs diverse cellular processes, including cell fate, disease, and aging. The need to understand this system and precisely control gene expression outputs for therapeutic purposes has precipitated the development of a diverse set of epigenetic editing tools. Here, we review the existing toolbox for targeted epigenetic editing, technical considerations of the current technologies, and opportunities for future development. We describe applications of therapeutic epigenetic editing and their potential for treating disease, with a discussion of ongoing delivery challenges that impede certain clinical interventions, particularly in the brain. With simultaneous advancements in available engineering tools and appropriate delivery technologies, we predict that epigenetic editing will increasingly cement itself as a powerful approach for safely treating a wide range of disorders in all tissues of the body.
ABSTRACT
Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit's utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Synthetic Biology , Gene Editing/methods , Synthetic Biology/methods , Humans , Students , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , SchoolsABSTRACT
Significance: Photoacoustic computed tomography (PACT), a hybrid imaging modality combining optical excitation with acoustic detection, has rapidly emerged as a prominent biomedical imaging technique. Aim: We review the challenges and advances of PACT, including (1) limited view, (2) anisotropy resolution, (3) spatial aliasing, (4) acoustic heterogeneity (speed of sound mismatch), and (5) fluence correction of spectral unmixing. Approach: We performed a comprehensive literature review to summarize the key challenges in PACT toward practical applications and discuss various solutions. Results: There is a wide range of contributions from both industry and academic spaces. Various approaches, including emerging deep learning methods, are proposed to improve the performance of PACT further. Conclusions: We outline contemporary technologies aimed at tackling the challenges in PACT applications.
Subject(s)
Photoacoustic Techniques , Tomography, X-Ray Computed , Photoacoustic Techniques/methods , Humans , Tomography, X-Ray Computed/methods , Image Processing, Computer-Assisted/methods , Anisotropy , Deep LearningABSTRACT
The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.
Subject(s)
Endoplasmic Reticulum , Organelles , RNA, Viral , SARS-CoV-2 , Virus Replication , SARS-CoV-2/physiology , SARS-CoV-2/ultrastructure , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , RNA, Viral/metabolism , RNA, Viral/genetics , Virus Replication/physiology , Humans , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , Endoplasmic Reticulum/ultrastructure , Organelles/virology , Organelles/metabolism , Organelles/ultrastructure , Chlorocebus aethiops , Vero Cells , Animals , COVID-19/virology , COVID-19/metabolism , Viral Proteins/metabolism , Viral Proteins/genetics , Microscopy, Fluorescence , Viral Replication Compartments/metabolism , RNA, Double-Stranded/metabolismABSTRACT
Embryonic stem cells (ESCs) can self-organize in vitro into developmental patterns with spatial organization and molecular similarity to that of early embryonic stages. This self-organization of ESCs requires transmission of signaling cues, via addition of small molecule chemicals or recombinant proteins, to induce distinct embryonic cellular fates and subsequent assembly into structures that can mimic aspects of early embryonic development. During natural embryonic development, different embryonic cell types co-develop together, where each cell type expresses specific fate-inducing transcription factors through activation of non-coding regulatory elements and interactions with neighboring cells. However, previous studies have not fully explored the possibility of engineering endogenous regulatory elements to shape self-organization of ESCs into spatially-ordered embryo models. Here, we hypothesized that cell-intrinsic activation of a minimum number of such endogenous regulatory elements is sufficient to self-organize ESCs into early embryonic models. Our results show that CRISPR-based activation (CRISPRa) of only two endogenous regulatory elements in the genome of pluripotent stem cells is sufficient to generate embryonic patterns that show spatial and molecular resemblance to that of pre-gastrulation mouse embryonic development. Quantitative single-cell live fluorescent imaging showed that the emergence of spatially-ordered embryonic patterns happens through the intrinsic induction of cell fate that leads to an orchestrated collective cellular motion. Based on these results, we propose a straightforward approach to efficiently form 3D embryo models through intrinsic CRISPRa-based epigenome editing and independent of external signaling cues. CRISPRa-Programmed Embryo Models (CPEMs) show highly consistent composition of major embryonic cell types that are spatially-organized, with nearly 80% of the structures forming an embryonic cavity. Single cell transcriptomics confirmed the presence of main embryonic cell types in CPEMs with transcriptional similarity to pre-gastrulation mouse embryos and revealed novel signaling communication links between different embryonic cell types. Our findings offer a programmable embryo model and demonstrate that minimum intrinsic epigenome editing is sufficient to self-organize ESCs into highly consistent pre-gastrulation embryo models.
ABSTRACT
CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.
Subject(s)
Metabolic Engineering , T-Lymphocytes , Humans , Gene Expression Profiling , Metabolic Engineering/methods , RNA , TranscriptomeABSTRACT
[This corrects the article DOI: 10.1117/1.JBO.29.S1.S11507.].
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Objective: To evaluated the safety and feasibility of dissection of lymph nodes posterior to right recurrent laryngeal nerve (â ¥B compartment) in endoscopic thyroidectomy through gasless axillary posterior approach. Methods: A total of 350 cases with right lobe papillary thyroid carcinoma (PTC) who underwent endoscopic lobectomy, isthmusectomy and central compartment neck dissection via gasless axillary posterior approach based at the Department of General Surgery, Nanfang Hospital, Southern Medical University from June 2020 to December 2022 were retrospectively analyzed. Summarize the clinical, pathological characteristics, and postoperative complications of the patients. SPSS 25.0 was used for statistical analysis of the data. Results: All 350 patients underwent endoscopic surgery successfully, with no conversion to open surgery. There were 303 females and 47 males, with an average age of (36.3±9.2) years. Of those, 287 patients were in pT1a stage, 62 in pT1b stage, and one patient in pT2 stage. There was no T3 or T4 stage patient. The mean numbers of yielded lymph nodes in right central compartment and â ¥B compartment were 8.11±4.65 (range, 1-31) and 2.62±1.86 (range, 1-12), respectively. â ¥B compartment metastasis was detected in 52 (14.86%) of 350 patients. The incidence of transient recurrent laryngeal nerve injury was 0.86%(3/350). Postoperative hematoma occurred in three patients (0.86%). Conclusion: The dissection of â ¥B compartment in endoscopic thyroidectomy through gasless axillary posterior approach is safe and feasible in selected PTC patients.
Subject(s)
Recurrent Laryngeal Nerve , Thyroid Neoplasms , Female , Male , Humans , Adult , Middle Aged , Retrospective Studies , Thyroidectomy , Lymph Nodes , Thyroid Cancer, Papillary/surgery , Thyroid Neoplasms/surgeryABSTRACT
The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.
ABSTRACT
Significance: Wavefront shaping (WFS) can compensate for distortions by optimizing the wavefront of the input light or reversing the transmission matrix of the media. It is a promising field of research. A thorough understanding of principles and developments of WFS is important for optical research. Aim: To provide insight into WFS for researchers who deal with scattering in biomedicine, imaging, and optical communication, our study summarizes the basic principles and methods of WFS and reviews recent progress. Approach: The basic principles, methods of WFS, and the latest applications of WFS in focusing, imaging, and multimode fiber (MMF) endoscopy are described. The practical challenges and prospects of future development are also discussed. Results: Data-driven learning-based methods are opening up new possibilities for WFS. High-resolution imaging through MMFs can support small-diameter endoscopy in the future. Conclusion: The rapid development of WFS over the past decade has shown that the best solution is not to avoid scattering but to find ways to correct it or even use it. WFS with faster speed, more optical modes, and more modulation degrees of freedom will continue to drive exciting developments in various fields.
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OBJECTIVE: To identify the risk factors associated with antimicrobial use on the initial acquisition of carbapenem-resistant Klebsiella pneumoniae (CRKP) in elderly intensive care unit (ICU) patients.METHODS: Respiratory secretion, blood, urine, anal swab and peritoneal drainage samples from all elderly patients with non-colonised CRKP who had been hospitalised from January 2021 to December 2022 were collected, and screened for CRKP colonisation using surveillance culture at the time of the first ICU admission and weekly thereafter in Zhejiang Provincial Hospital of Chinese Medicine, Zhejiang, China. Cumulative antibiotic variables included duration of antibiotic use, total amount of antimicrobials received in grams, total antibiotic consumption (defined daily dose) and the types of antimicrobial exposure. A time-dependent model based on Cox regression analysis was used to investigate the effect of each variable on the initial acquisition of CRKP infection or colonisation.RESULTS: Of 214 patients, 44 were infected or had CRKP colonies and death rate was 34.1%. males were the risk factor for acquiring CRKP in culture (HR 2.12, 95% CI 1.06-4.21; P = 0.033). It is notable that the hazard of acquiring CRKP increased by 9% with every single-point increase in the APACHE II score (HR 1.09, 95% CI 1.01-1.18; P = 0.025). The hazard of acquiring CRKP doubled when carbapenems were administered (HR 1.81, 95% CI 1.42-2.30; P < 0.001), In contrast, exposure to quinolone antimicrobials had a smaller effect on acquiring CRKP (HR 1.07; 95% CI 1.01-1.14; P = 0.024).CONCLUSION: This study found that male sex, APACHE II score and exposure to quinolones and carbapenems were independent risk factors for acquiring CRKP.
Subject(s)
Klebsiella Infections , Klebsiella pneumoniae , Humans , Male , Aged , Klebsiella Infections/drug therapy , Klebsiella Infections/epidemiology , Drug Resistance, Bacterial , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Carbapenems/pharmacology , Carbapenems/therapeutic use , Risk Factors , Intensive Care UnitsABSTRACT
BACKGROUND: To study the efficacy and nephrotoxicity of polymyxin B in the treatment of elderly patients with carbapenem-resistant organism (CRO) infection. METHODS: The clinical and microbiological data of patients with CRO-infected sepsis treated with polymyxin B were retrospectively analyzed. The effective rate, bacterial clearance, incidence and recovery rate of acute renal injury (AKI) and prognosis-related indicators in AKI at different stages were compared. RESULTS: The effective rate of 215 elderly patients with CRO infection treated with polymyxin was 50.7%. The total bacterial clearance rate was 44.2%, the total incidence of AKI was 37.2%, the recovery rate of AKI was 35%, and the incidence range of polymyxin B-related AKI was 10.2-37.2%. Logistic multivariate regression analysis showed that the predictors of AKI in elderly patients were high APACHE II score, long duration of polymyxin, chronic renal insufficiency and ineffective outcome; the ROC curve showed that the cutoff value for predicting AKI was a serum creatinine concentration of 73 mmol/L before polymyxin B use, and the AUC was 0.931. CONCLUSIONS: Rational use of polymyxin B is safe and effective in elderly patients with CRO infection, and its effective outcome can improve the recovery rate of AKI.
Subject(s)
Acute Kidney Injury , Bacterial Infections , Humans , Aged , Polymyxin B/adverse effects , Anti-Bacterial Agents/adverse effects , Retrospective Studies , Bacterial Infections/drug therapy , Acute Kidney Injury/chemically induced , Carbapenems/adverse effectsABSTRACT
Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.
Subject(s)
Gene Editing , Genome , Animals , Mice , Genome/genetics , Genetic Therapy , Epigenome , Genes, Reporter , CRISPR-Cas Systems/geneticsABSTRACT
A three-dimensional (3D) holographic display (3DHD) can preserve all the volumetric information about an object. However, the poor fidelity of 3DHD constrains its applications. Here, we present an ultrahigh-fidelity 3D holographic display that uses scattering for homogenization of angular spectrum. A scattering medium randomizes the incident photons and homogenizes the angular spectrum distribution. The redistributed field is recorded by a photopolymer film with numerous modulation modes and a half-wavelength scale pixel size. We have experimentally improved the contrast of a focal spot to 6 × 106 and tightened its spatial resolution to 0.5 micrometers, respectively ~300 and 4.4 times better than digital approaches. By exploiting the spatial multiplexing ability of the photopolymer and the transmission channel selection capability of the scattering medium, we have realized a dynamic holographic display of 3D spirals consisting of 20 foci across 1 millimeter × 1 millimeter × 26 millimeters with uniform intensity.
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Objective: To analyze the epidemiological characteristics of reinfection of 2019-nCoV and influencing factors, and provide evidence for effective prevention and control of COVID-19 epidemic. Methods: The incidence data of COVID-19 in Ningbo from January 1, 2020 to November 30, 2022 were collected from the infectious disease surveillance system of Chinese information system for disease control and prevention. The incidence of reinfection of 2019-nCoV was investigated by using questionnaire. logistic regression analysis was used to analyze the influences of gender, age, time interval from the first infection, history of underlying disease, 2019-nCoV vaccination dose and disease severity on the reinfection. Results: A total of 897 previous 2019-nCoV infection cases were investigated, of which 115 experienced the reinfection of 2019-nCoV, the reinfection rate was 12.82%. The interval between the two infections M(Q1, Q3) was 1 052 (504, 1 056) days. Univariate analysis showed that age, 2019-nCoV vaccination dose, history of underlying disease, type of 2019-nCoV variant causing the first infection, time interval from the first infection and severity of the first infection were associated with the reinfection rate (all P<0.05). Multivariate logistic regression analysis showed that the risk for reinfection in age group 30- years was higher than that in age group ≥60 years (OR=2.10, 95%CI: 1.11-3.97). No reinfection occurred in those with time interval from the first infection of <6 months, and the risk for reinfection was higher in those with the time interval of ≥12 months than in those with the time interval of 6- months (OR=6.68, 95%CI: 3.46-12.90). The risk for reinfection was higher in the common or mild cases than in the asymptomatic cases (OR=2.64, 95%CI: 1.18-5.88; OR=2.79, 95%CI: 1.27-6.11). Conclusion: The time interval from the first infection was an important influencing factor for the reinfection of 2019-nCoV, and the probability of the reinfection within 6 months was low.
Subject(s)
COVID-19 , Epidemics , Reinfection , Adult , Humans , Middle Aged , Asian People/statistics & numerical data , COVID-19/epidemiology , COVID-19/prevention & control , Reinfection/epidemiology , Reinfection/etiology , Reinfection/prevention & control , SARS-CoV-2 , China/epidemiology , Risk Factors , Time FactorsABSTRACT
The pleiotropic benefits of statins in cardiovascular diseases that are independent of their lipid-lowering effects have been well documented, but the underlying mechanisms remain elusive. Here we show that simvastatin significantly improves human induced pluripotent stem cell-derived endothelial cell functions in both baseline and diabetic conditions by reducing chromatin accessibility at transcriptional enhanced associate domain elements and ultimately at endothelial-to-mesenchymal transition (EndMT)-regulating genes in a yes-associated protein (YAP)-dependent manner. Inhibition of geranylgeranyltransferase (GGTase) I, a mevalonate pathway intermediate, repressed YAP nuclear translocation and YAP activity via RhoA signaling antagonism. We further identified a previously undescribed SOX9 enhancer downstream of statin-YAP signaling that promotes the EndMT process. Thus, inhibition of any component of the GGTase-RhoA-YAP-SRY box transcription factor 9 (SOX9) signaling axis was shown to rescue EndMT-associated endothelial dysfunction both in vitro and in vivo, especially under diabetic conditions. Overall, our study reveals an epigenetic modulatory role for simvastatin in repressing EndMT to confer protection against endothelial dysfunction.
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Successful implementation of adoptive cell therapy (ACT) of cancer requires comprehensively addressing biological and practical challenges. This approach has been largely overlooked, resulting in a gap between the potential of ACT and its actual effectiveness. We summarize the most promising technical strategies in creating an "ideal" ACT product, focusing on chimeric antigen receptor (CAR)-engineered cells. Since many requirements for effective ACT are common to most cancers, what we outline here might have a broader impact.