Author Correction: CasX enzymes comprise a distinct family of RNA-guided genome editors.

In this Article, owing to issues with the first 30 nucleotides of the sgRNA, which run in the opposite direction, corrections have been made to the Protein Data Bank (PDB) accessions in the 'Data availability' section, and this also affects Figs. 3, 4, Extended Data Fig. 6, Supplementary Table 1 and Supplementary Video 1. The original Article has been corrected online. See the accompanying Amendment for further details.

CasX enzymes comprise a distinct family of RNA-guided genome editors.

The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.

Removing independent noise in systems neuroscience data using DeepInterpolation.

Progress in many scientific disciplines is hindered by the presence of independent noise. Technologies for measuring neural activity (calcium imaging, extracellular electrophysiology and functional magnetic resonance imaging (fMRI)) operate in domains in which independent noise (shot noise and/or thermal noise) can overwhelm physiological signals. Here, we introduce DeepInterpolation, a general-purpose denoising algorithm that trains a spatiotemporal nonlinear interpolation model using only raw noisy samples. Applying DeepInterpolation to two-photon calcium imaging data yielded up to six times more neuronal segments than those computed from raw data with a 15-fold increase in the single-pixel signal-to-noise ratio (SNR), uncovering single-trial network dynamics that were previously obscured by noise. Extracellular electrophysiology recordings processed with DeepInterpolation yielded 25% more high-quality spiking units than those computed from raw data, while DeepInterpolation produced a 1.6-fold increase in the SNR of individual voxels in fMRI datasets. Denoising was attained without sacrificing spatial or temporal resolution and without access to ground truth training data. We anticipate that DeepInterpolation will provide similar benefits in other domains in which independent noise contaminates spatiotemporally structured datasets.

Super-Enhancers and Their Parts: From Prediction Efforts to Pathognomonic Status.

Super-enhancers (SEs) are regions of the genome that play a crucial regulatory role in gene expression by promoting large-scale transcriptional responses in various cell types and tissues. Recent research suggests that alterations in super-enhancer activity can contribute to the development and progression of various disorders. The aim of this research is to explore the multifaceted roles of super-enhancers in gene regulation and their significant implications for understanding and treating complex diseases. Here, we study and summarise the classification of super-enhancer constituents, their possible modes of interaction, and cross-regulation, including super-enhancer RNAs (seRNAs). We try to investigate the opportunity of SE dynamics prediction based on the hierarchy of enhancer single elements (enhancers) and their aggregated action. To further our understanding, we conducted an in silico experiment to compare and differentiate between super-enhancers and locus-control regions (LCRs), shedding light on the enigmatic relationship between LCRs and SEs within the human genome. Particular attention is paid to the classification of specific mechanisms and their diversity, exemplified by various oncological, cardiovascular, and immunological diseases, as well as an overview of several anti-SE therapies. Overall, the work presents a comprehensive analysis of super-enhancers across different diseases, aiming to provide insights into their regulatory roles and may act as a rationale for future clinical interventions targeting these regulatory elements.

VITAMIN D STATUS IN CHILDREN WITH PARALITIC SYNDROMS.

OBJECTIVE: The aim: Determination of serum 25(OH)D in the children with paralytic syndromes and its distribution depending on age, sex, taking anticonvulsant drugs, nutritional status for a period of one year (autumn-spring) of one center. PATIENTS AND METHODS: Materials and methods: There were recruited of 77 children with paralytic syndromes and 73 health children for the same period aged from 1 till 18 years. The study included a scrutiny of medical history and analysis of medical documents, assessment of motor dysfunction by GMFCS, and nutritional status. RESULTS: Results: Among children with paralytic syndromes there were spastic tetraparesis 59.7%, malnutrition 92%, IV-V level of gross motor disfunction 80.5%, antiseizure medications 59.7% and cognitive impairment 77.9%. The variation of serum 25(OH)D is from 6.1 to 76.7 ng/mL with median 18.3 ng/mL in healthy children. The variation of serum 25(OH)D is from 2.2 to 83.0 ng/mL with median 14.8 ng/mL in children with paralytic syndromes (p=0.0103). Vitamin status among them is the following: insufficiency (21-29 ng/mL)-28.7% vs 16.8%; deficiency (<20 ng/mL)-56.1 vs 72.2% (p=0.0300). The 25.9% children with paralytic syndromes and those who have deficiency demonstrate severe deficiency (<10 ng/mL) compare 10.9% in healthy children (p=0.00189). There is a tendency to decrease of serum 25(OH)D in children with paralytic syndrome older 7 years. CONCLUSION: Conclusions: We failed to record a significant difference in the 25(ОН)D between males and females, between different level of GMFCS, and anticonvulsants using. Deficiency of vitamin D in 2.25 times higher in children with paralytic syndromes and severe malnutrition. Additional researches with specific items are need in perspective.

ON ISSUE OF HIGH-QUALITY STOMATOLOGICAL SERVICE IN UKRAINE.

OBJECTIVE: The aim: To detect the main problems regarding available high-quality stomatological service in Ukraine and define their main solutions. PATIENTS AND METHODS: Materials and methods: The authors used general scientific methods of synthesis, generalization, scientific data interpreting, systemic approach method, medical statistic method, and analysis of the activity of state and private institutions dealing with stomatological service in Ukraine. The paper is based on the materials of a representative selective study of Ukrainian households, held by the State Committee of Statistics of Ukraine to study people's self-estimation of their health and the availability of certain medical services. RESULTS: Results: Most citizens of Ukraine (60-80%) are treated in the state/public healthcare sector. Though, during the last century, a decrease in dental visits per citizen in the state and public institutions has been noted, as well as a decrease of all medical service types' volume, offered in the mentioned institutions. In Ukraine the observed trends are represented as the decrease in the network institutions number, insufficient budgeting of state/public medical institutions, prevailing commercial characteristics of stomatological service and people's low income, which leads to decreased affordability, and quality of medical service, thus negatively affecting people's health. CONCLUSION: Conclusions: The fundamental studies of the quality assessment show that the medical service requires strong structure, process quality, and result quality. The quality of medical service organization is extremely important and it should be maintained high on all levels of management and treatment processes, regarding the conditions of medical process and resources of medical organizations. Medical service should be patient-centered. To solve the problem, the entire state system of quality management is required in Ukraine.

CHRONIC PAIN AND PHYSICAL THERAPY IN CHILDREN WITH PARALYTIC SYNDROMES: ARE THERE ANY CHANGES DURING LOCKDOWN?

OBJECTIVE: The aim: To evaluate an influence of physical therapy on chronic pain in children with paralytic's syndrome and to maternal emotional status on lockdown time during the COVID-19 pandemic. PATIENTS AND METHODS: Materials and methods: Data from 96 children and their mothers (96 persons) were included in the study. On-site services of physical therapists before the pandemic (2018- 2019) were received by 64 children and by 32 children during quarantine measures due to COVID-19 pandemic (2020). The age of the children ranged from 1 to 6 years, the median age was 3 years and 3 months. RESULTS: Results: We note that there were more boys with paralytic syndromes. Among the leading paralytic syndromes, the most common was spastic tetraparesis. The frequency of children with level III-V motor disorders prevailed. CONCLUSION: Conclusions: The authors consider that physical rehabilitation in children with paralytic syndromes reduces the incidence of moderate chronic pain and improves the emotional state of parents. But, these changes do not occur during the pandemic.

Wide. Fast. Deep: Recent Advances in Multiphoton Microscopy of In Vivo Neuronal Activity.

Multiphoton microscopy (MPM) has emerged as one of the most powerful and widespread technologies to monitor the activity of neuronal networks in awake, behaving animals over long periods of time. MPM development spanned across decades and crucially depended on the concurrent improvement of calcium indicators that report neuronal activity as well as surgical protocols, head fixation approaches, and innovations in optics and microscopy technology. Here we review the last decade of MPM development and highlight how in vivo imaging has matured and diversified, making it now possible to concurrently monitor thousands of neurons across connected brain areas or, alternatively, small local networks with sampling rates in the kilohertz range. This review includes different laser scanning approaches, such as multibeam technologies as well as recent developments to image deeper into neuronal tissues using new, long-wavelength laser sources. As future development will critically depend on our ability to resolve and discriminate individual neuronal spikes, we will also describe a simple framework that allows performing quantitative comparisons between the reviewed MPM instruments. Finally, we provide our own opinion on how the most recent MPM developments can be leveraged at scale to enable the next generation of discoveries in brain function.

On Command Drug Delivery via Cell-Conveyed Phototherapeutics.

Herein, the use of red blood cells (RBCs) as carriers of cytoplasmically interned phototherapeutic agents is described. Photolysis promotes drug release from the RBC carrier thereby providing the means to target specific diseased sites. This strategy is realized with a vitamin B12-taxane conjugate (B12-TAX), in which the drug is linked to the vitamin via a photolabile CoC bond. The conjugate is introduced into mouse RBCs (mRBCs) via a pore-forming/pore-resealing procedure and is cytoplasmically retained due to the membrane impermeability of B12. Photolysis separates the taxane from the B12 cytoplasmic anchor, enabling the drug to exit the RBC carrier. A covalently appended Cy5 antenna sensitizes the conjugate (Cy5-B12-TAX) to far red light, thereby circumventing the intense light absorbing properties of hemoglobin (350-600 nm). Microscopy and imaging flow cytometry reveal that Cy5-B12-TAX-loaded mRBCs act as drug carriers. Furthermore, intravital imaging of mice furnish a real time assessment of circulating phototherapeutic-loaded mRBCs as well as evidence of the targeted photorelease of the taxane upon photolysis. Histopathology confirms that drug release occurs in a well resolved spatiotemporal fashion. Finally, acoustic angiography is employed to assess the consequences of taxane release at the tumor site in Nu/Nu-tumor-bearing mice.

Two-photon frequency division multiplexing for functional in vivo imaging: a feasibility study.

Recently, we presented a new approach to create high-speed amplitude modulation of femtosecond laser pulses and tag multiple excitation beams with specific modulation frequencies. In this work, we discuss the utility of this method to record calcium signals in brain tissue with two-photon frequency-division multiplexing (2P-FDM) microscopy. While frequency-multiplexed imaging appears slightly inferior in terms of image quality as compared to conventional two-photon laser scanning microscopy due to shot noise-induced cross-talk between frequency channels, applying this technique to record average signals from regions of interest (ROI) such as neuronal cell bodies was found to be promising. We use phase information associated with each pixel or waveform within a selected ROI to phase-align and recombine the signals into one extended amplitude-modulated waveform. This procedure narrows the frequency detection window, effectively decreasing noise contributions from other frequency channels. Using theoretical analysis, numerical simulations, and in vitro imaging, we demonstrate a reduction of cross-talk by more than an order of magnitude and predict the usefulness of 2P-FDM for functional studies of brain activity.

The replication initiator of the cholera pathogen's second chromosome shows structural similarity to plasmid initiators.

The conserved DnaA-oriC system is used to initiate replication of primary chromosomes throughout the bacterial kingdom; however, bacteria with multipartite genomes evolved distinct systems to initiate replication of secondary chromosomes. In the cholera pathogen, Vibrio cholerae, and in related species, secondary chromosome replication requires the RctB initiator protein. Here, we show that RctB consists of four domains. The structure of its central two domains resembles that of several plasmid replication initiators. RctB contains at least three DNA binding winged-helix-turn-helix motifs, and mutations within any of these severely compromise biological activity. In the structure, RctB adopts a head-to-head dimeric configuration that likely reflects the arrangement in solution. Therefore, major structural reorganization likely accompanies complex formation on the head-to-tail array of binding sites in oriCII. Our findings support the hypothesis that the second Vibrionaceae chromosome arose from an ancestral plasmid, and that RctB may have evolved additional regulatory features.

Amplitude modulation of femtosecond laser pulses in the megahertz range for frequency-multiplexed two-photon imaging.

We present a frequency-multiplexed multi-site two-photon imaging method utilizing amplitude modulation of femtosecond laser pulses in the MHz range to tag each excitation beam and the corresponding fluorescence signals with specific frequencies. The frequency tags are generated with an interferometric scheme employing acousto-optic deflectors (AODs) to achieve precise spatial overlap of femtosecond laser pulses with periodically varying phase shift. Creating matching excitation beam patterns in each interferometer arm using multiple AOD driving frequencies, and subsequently overlapping these matching patterns, results in multiple encoded excitation beams with unique beat frequencies available for scanning. As a proof-of-concept, we demonstrate multiplexed two-photon image acquisition using test samples, and compare the performance of this approach to conventional two-photon laser scanning microscopy.

Standardised Measurements for Monitoring and Comparing Multiphoton Microscope Systems.

The goal of this protocol is to enable better characterisation of multiphoton microscopy hardware across a large user base. The scope of this protocol is purposefully limited to focus on hardware, touching on software and data analysis routines only where relevant. The intended audiences are scientists using and building multiphoton microscopes in their laboratories. The goal is that any scientist, not only those with optical expertise, can test whether their multiphoton microscope is performing well and producing consistent data over the lifetime of their system.

Drawing Parallels between SARS, MERS, and COVID-19: A Comparative Overview of Epidemiology, Pathogenesis, and Pathological Features.

Background: Since November 2019, when the novel coronavirus arose in Wuhan City, over 188 million people worldwide have been infected with COVID-19. It is the third coronavirus outbreak in the twenty-first century. Until now, practically all coronavirus epidemics have occurred due to zoonotic spread from an animal or transitional host or through the consumption of their products. Coronaviruses can infect humans and cause severe illness and even death. Material and Methods: This review was designed to help us recognize and harmonize the similarities and differences between these three coronaviridae family members. Result: Measures aimed at containing the epidemic should be emphasized in this circumstance. Prioritizing and planning these activities require an understanding of the particulars of these three viruses. Given the pandemic's enormous death toll and rapid spread, we should be cognizant of the parallels and differences between these three viruses. Additionally, this pandemic warns us to be cautious against the possibility of a future pandemic. Conclusion: We highlight the fundamental characteristics of coronaviruses that are critical for recognizing coronavirus epidemiology, pathogenesis, and pathological features that reveal numerous significant pathological attributes and evolutionary patterns in the viral genome that aid in better understanding and anticipating future epidemics.

Experimental Validation and Prediction of Super-Enhancers: Advances and Challenges.

Super-enhancers (SEs) are cis-regulatory elements of the human genome that have been widely discussed since the discovery and origin of the term. Super-enhancers have been shown to be strongly associated with the expression of genes crucial for cell differentiation, cell stability maintenance, and tumorigenesis. Our goal was to systematize research studies dedicated to the investigation of structure and functions of super-enhancers as well as to define further perspectives of the field in various applications, such as drug development and clinical use. We overviewed the fundamental studies which provided experimental data on various pathologies and their associations with particular super-enhancers. The analysis of mainstream approaches for SE search and prediction allowed us to accumulate existing data and propose directions for further algorithmic improvements of SEs' reliability levels and efficiency. Thus, here we provide the description of the most robust algorithms such as ROSE, imPROSE, and DEEPSEN and suggest their further use for various research and development tasks. The most promising research direction, which is based on topic and number of published studies, are cancer-associated super-enhancers and prospective SE-targeted therapy strategies, most of which are discussed in this review.

Efficacy and safety of the combined metabolic medication, containing inosine, nicotinamide, riboflavin and succinic acid, for the treatment of diabetic neuropathy: a multicenter randomized, double-blind, placebo-controlled parallel group clinical trial (CYLINDER).

INTRODUCTION: Antioxidants may have positive impact on diabetic polyneuropathy (DPN), presumably due to alleviation of oxidative stress. We aimed to evaluate the efficacy and safety of combination of antioxidants: succinic acid, inosine, nicotinamide, and riboflavin (SINR) in the treatment of DPN. RESEARCH DESIGN AND METHODS: In a double-blind, placebo-controlled clinical trial, men and women aged 45-74 years with type 2 diabetes and symptomatic DPN, with initial Total Symptom Score (TSS) ˃5, were randomized into experimental (n=109) or placebo (n=107) group. Patients received study medication/placebo intravenously for 10 days, followed by oral administration for 75 days. Statistical significance was defined as a two-tailed p<0.05. RESULTS: In SINR group, mean TSS change after 12 weeks was -2.65 (±1.46) vs -1.73 (±1.51) in the placebo group (p<0.0001; t-test). Reduction of symptoms in the SINR group was achieved regardless of hemoglobin A1c levels, but better results were observed in patients with initial TSS <7.5. The analysis of TSS subscores revealed statistically significant between-group differences by dynamics of the intensity of paresthesia and of numbness starting from day 11 (p=0.035 and p=0.001, respectively; mixed model); by day 57, statistically significant between-group differences were detected also by dynamics of burning intensity (p=0.005; mixed model). Study limitations are small effect size, moderate proportion of patients with severe DPN symptoms, subjective assessment of outcomes, exclusion of participants who received injectable glucose-lowering medications other than insulins, and patients with uncontrolled and type 1 diabetes. CONCLUSIONS: The combination of SINR effectively alleviates DPN symptoms in patients with type 2 diabetes. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT04649203; Unique Protocol ID: CTF-III-DM-2019).

Light-Triggered Drug Release from Red Blood Cells Suppresses Arthritic Inflammation.

Arthritis is a leading cause of disability in adults, which can be intensely incapacitating. The location and intensity of the pain is both subjective and challenging to manage. Consequently, patient-directed delivery of anti-inflammatories is an essential component of future therapeutic strategies for the management of this disorder. We describe the design and application of a light responsive red blood cell (RBC) conveyed dexamethasone (Dex) construct that enables targeted drug delivery upon illumination of the inflamed site. The red wavelength (650 nm) responsive nature of the phototherapeutic was validated using tissue phantoms mimicking the light absorbing properties of various skin types. Furthermore, photoreleased Dex has the same impact on cellular responses as conventional Dex. Murine RBCs containing the photoactivatable therapeutic display comparable circulation properties as fluorescently labelled RBCs. In addition, a single dose of light-targeted Dex delivery is 5-fold more effective in suppressing inflammation than the parent drug, delivered serially over multiple days. These results are consistent with the notion that the circulatory system be used as an on-command drug depot, providing the means to therapeutically target diseased sites both efficiently and effectively.

The liver in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

BACKGROUND: The ongoing outbreak of COVID-19 is associated with higher levels of morbidity and mortality among patients with comorbidities, including the metabolic syndrome. Liver impairment has been reported in up to 54% of hospitalized patients with COVID-19. The impact of COVID-19 on a preexisting chronic liver disease is an actively studied area of research. The contribution of our study is towards determining the predictors of severity and the outcome of liver injury among hospitalized patients with COVID-19 infection, including patients with a preexisting liver disease and COVID-19. METHODS: This single center retrospective cohort study included all patients ≥18 years, admitted in Sheba Medical Center with confirmed COVID-19 infection. Demographic, clinical and laboratory data were obtained using the MDClone platform and rechecked after data decryption using electronic health records. RESULTS: Of 382 patients with COVID-19, 66.4% had increased liver biochemistry. Mild increase was observed in 76.7%. The higher level of fibrosis-4 (FIB-4) at admission was independently associated with higher mortality rate. Preexisting liver disease was detected in 15.4% patients. Most common etiology was nonalcoholic fatty liver disease (78.7%). The mortality of hospitalized patients with preexisting liver disease was 16.7% compared to 6.8% in patients without preexisting liver disease (RR = 2.792, P = 0.01). In multivariate analysis, liver disease adjusted to age and BMI was associated with mortality with high statistical significance. CONCLUSIONS: Patients with preexisting chronic liver disease were at a higher risk of mortality. The FIB-4 level at admission was associated with worse prognosis. These findings should be reevaluated in a larger cohort of patients.

High-performance method for identification of super enhancers from ChIP-Seq data with configurable cloud virtual machines.

A universal method for rapid identifying super-enhancers which are large domains of multiple closely-spaced enhancers is proposed. The method applies configurable cloud virtual machines (cVMs) and the rank-ordering of super-enhancers (ROSE) algorithm. To identify super-enhancers a сVM-based analysis of the ChIP-seq binding patterns of the active enhancer-associated mark is employed. The use of the proposed method is described step-by-step: configuration of cVM; ChIP-seq data alignment; peak calling; ROSE algorithm; interpretation of the results on a client machine. The method was validated for the search of super-enhancers using the H3K27ac mark in the sample datasets of a cell line (human MCF-7), mouse tissue (heart), and human tissue (adrenal gland). The total analysis cycle time of raw ChIP-seq data ranges from 15 to 48 min, depending on the number of initial short reads. Depending on the data processing step and availability of multi-threading, a cVM can be scaled up to a multi-CPU configuration with large amount of RAM. An important feature of the method is that it can run on a client machine that has low-performance with virtually any OS. The proposed method allows for simultaneous and independent processing of different sample datasets on multiple clones of a single cVM.•Cloud VMs were used for rapid processing of ChIP-seq data to identify super-enhancers.•The method can use a low-performance computer with virtually any OS on it.•It can be scaled up for parallel processing of individual sample datasets on their own VMs for rapid high-throughput processing.

Synergistic suppression of t(8;21)-positive leukemia cell growth by combining oridonin and MAPK1/ERK2 inhibitors.

One of the most common chromosomal translocations in acute myeloid leukemia is t(8;21)(q22;q22), which results in the appearance of abnormal transcripts encoding for the fusion protein RUNX1-ETO. Therefore, this oncoprotein is considered to be a pertinent and promising target for treating t(8;21) leukemia. Previously, we have shown that downregulation of RUNX1-ETO leads to activation of intracellular signaling pathways enhancing cell survival and determined that the protein ERK2 can mediate activation of most of these pathways. Here we used a combination of oridonin (natural tetracycline diterpenoid), which has been shown to exhibit anti-RUNX1-ETO activity, and ERK2 kinase inhibitors. We found that treatment of leukemic t(8;21)-positive Kasumi-1 cells with oridonin cause decrease of phosphorylated ERK1/2. Treatment of these cells with ERK2 inhibitors makes them more sensitive to RUNX1-ETO inhibition with oridonin. Therefore we postulate that simultaneous inhibition of RUNX1-ETO and ERK2 cause synergistic effect on survival of leukemic cells.