Single-cell RNA sequencing of murine hearts for studying the development of the cardiac conduction system.

The development of the cardiac conduction system (CCS) is essential for correct heart function. However, critical details on the cell types populating the CCS in the mammalian heart during the development remain to be resolved. Using single-cell RNA sequencing, we generated a large dataset of transcriptomes of ~0.5 million individual cells isolated from murine hearts at six successive developmental corresponding to the early, middle and late stages of heart development. The dataset provides a powerful library for studying the development of the heart's CCS and other cardiac components. Our initial analysis identified distinct cell types between 20 to 26 cell types across different stages, of which ten are involved in forming the CCS. Our dataset allows researchers to reuse the datasets for data mining and a wide range of analyses. Collectively, our data add valuable transcriptomic resources for further study of cardiac development, such as gene expression, transcriptional regulation and functional gene activity in developing hearts, particularly the CCS.

Functional imaging-guided cell selection for evolving genetically encoded fluorescent indicators.

Genetically encoded fluorescent indicators are powerful tools for tracking cellular dynamic processes. Engineering these indicators requires balancing screening dimensions with screening throughput. Herein, we present a functional imaging-guided photoactivatable cell selection platform, Faculae (functional imaging-activated molecular evolution), for linking microscopic phenotype with the underlying genotype in a pooled mutant library. Faculae is capable of assessing tens of thousands of variants in mammalian cells simultaneously while achieving photoactivation with single-cell resolution in seconds. To demonstrate the feasibility of this approach, we applied Faculae to perform multidimensional directed evolution for far-red genetically encoded calcium indicators (FR-GECIs) with improved brightness (Nier1b) and signal-to-baseline ratio (Nier1s). We anticipate that this image-based pooled screening method will facilitate the development of a wide variety of biomolecular tools.

Relationship between acute glucose variability and cognitive decline in type 2 diabetes: A systematic review and meta-analysis.

BACKGROUND: Cognitive decline is one of the most widespread chronic complications of diabetes, which occurs in more than half of the patients with type 2 diabetes (T2DM). Emerging evidences have suggested that glucose variability (GV) is associated with the pathogenesis of diabetic complications. However, the influence of acute GV on cognitive dysfunction in T2DM is still controversial. The aim of the study was to evaluate the association between acute GV and cognitive defect in T2DM, and provide a most recent and comprehensive summary of the evidences in this research field. METHODS: PubMed, Cochrane library, EMBASE, Web of science, Sinomed, China National Knowledge Infrastructure (CNKI), and Wanfang were searched for articles that reported on the association between acute GV and cognitive impairment in T2DM. RESULTS: 9 eligible studies were included, with a total of 1263 patients with T2DM involved. Results showed that summary Fisher's z value was -0.23 [95%CI (-0.39, -0.06)], suggesting statistical significance (P = 0.006). Summary r value was -0.22 [95%CI (-0.37, -0.06)]. A lower cognitive performance was found in the subjects with greater glucose variation, which has statistical significance. Mean amplitude of glycemic excursions (MAGE) was associated with a higher risk of poor functional outcomes. Fisher's z value was -0.35 [95%CI (-0.43, -0.25)], indicating statistical significance (P = 0.011). Sensitivity analyses by omitting individual studies showed stability of the results. CONCLUSIONS: Overall, higher acute GV is associated with an increased risk of cognitive impairment in patients with T2DM. Further studies should be required to determine whether targeted intervention of reducing acute GV could prevent cognitive decline.

A Benchmark Study of Protein-Fragment Complex Structure Calculations with NMR2.

Protein-fragment complex structures are particularly sought after in medicinal chemistry to rationally design lead molecules. These structures are usually derived using X-ray crystallography, but the failure rate is non-neglectable. NMR is a possible alternative for the calculation of weakly interacting complexes. Nevertheless, the time-consuming protein signal assignment step remains a barrier to its routine application. NMR Molecular Replacement (NMR2) is a versatile and rapid method that enables the elucidation of a protein-ligand complex structure. It has been successfully applied to peptides, drug-like molecules, and more recently to fragments. Due to the small size of the fragments, ca < 300 Da, solving the structures of the protein-fragment complexes is particularly challenging. Here, we present the expected performances of NMR2 when applied to protein-fragment complexes. The NMR2 approach has been benchmarked with the SERAPhic fragment library to identify the technical challenges in protein-fragment NMR structure calculation. A straightforward strategy is proposed to increase the method's success rate further. The presented work confirms that NMR2 is an alternative method to X-ray crystallography for solving protein-fragment complex structures.

FLASH Genome Editing Pipeline: An Efficient and High-Throughput Method to Construct Arrayed CRISPR Library for Plant Functional Genomics.

CRISPR/Cas9 genome editing is a revolutionary technology for plant functional genomics and crop breeding. In this system, the Cas9 nuclease is directed by a guide RNA (gRNA) to cut the DNA target and introduce mutation through error-prone DNA break repair. Owing to its simplicity, CRISPR/Cas9-mediated targeted gene knockout is widely used for high-throughput genetic screening in animal cell cultures and bacteria. However, high-throughput genetic screening using CRISPR/Cas9 is still challenging in plants. We recently established a new approach, named the FLASH genome editing pipeline, to construct an arrayed CRISPR library in plants. In this pipeline, a set of 12 PCR fragments with different lengths (referred to as FLASH tags) are used to index the Cas9/gRNA vectors. Subsequently, a mixture of 12 Agrobacterium strains, in which each strain contained a FLASH-tag indexed vector, was transformed into rice plants. As a result, a unique link between the target gene/gRNA and FLASH tag is generated, which allows reading gRNA information in bacterial strains and gene-edited plants using regular PCR and gel electrophoresis. This protocol includes step-by-step instructions for gRNA design, high throughput assembly of FLASH-tag indexed Cas9/gRNA plasmids, Agrobacterium-mediated transformation of 12 indexed plasmids, and fast assignment of target gene information in primary transformants. The arrayed CRISPR library described here is suitable for small- to large-scale genetic screening and allows fast and comprehensive gene function discovery in plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assembly of FLASH-tag-indexed Cas9/gRNA plasmids Basic Protocol 2: Preparation of the Cas9/gRNA plasmid library Basic Protocol 3: Library preparation of Agrobacterium strains and mixing FLASH-tag indexed strains Basic Protocol 4: Grouped transformation and assignments of gRNA information of gene-edited plants.

Neuroprognostication value of serum neurofilament light chain for out-of-hospital cardiac arrest: A systematic review and meta-analysis.

BACKGROUND: Neurofilament light chain (NfL) is a novel biomarker for the assessment of neurological function after cardiac arrest (CA). Although meta-analysis has confirmed its predictive value, it has not conducted a more detailed analysis of its research. We conducted a meta-analysis to evaluate the relationship between serum NfL level and neurological prognosis in patients with spontaneous circulation recovery after CA, and subgroup analysis was conducted according to sample collection time, time to assess neurological function, study design, whether TTM was received, the method of specimen determination, and the presence of neurological disease in patients. To analyze the influence of these factors on the predictive value of serum NfL. METHODS: Published Cochrane reviews and an updated, extended search of MEDLINE, Cochrane Library, Embase, Scopus, ClinicalKey, CINAHL, and Web of Science for relevant studies until March 2022 were assessed through inclusion and exclusion criteria. The standard mean difference and 95% confidence interval were calculated using the random-effects model or fixed-effects model to assess the association between one variable factor NfL level and the outcome of CA patients. Subgroup analysis according to sample collection time was performed. The prognosis analysis and publication bias were also assessed using Egger's and Begg's tests. RESULTS: Among 1209 related articles for screening, 6 studies (1360 patients) met the inclusion criteria and were selected for meta-analysis. The level of serum NfL in the good prognosis group (CPC1-2, CPC: cerebral performance category score) was significantly lower than that in the poor prognosis group (CPC3-5)SMD(standardized mean difference) = 0.553, 95%CI(confidence interval) = 0.418-0.687, I2 = 65.5% P<0.05). And this relationship also exists at each sampling time point (NfL specimens were collected on admission: SMD:0.48,95%CI:0.24-0.73; Samples were collected 24 hours after CA: SMD:0.60,95%CI:0.32-0.88;Specimens were obtained 48 hours after CA: SMD:0.51, 95%CI:0.18-0.85;Specimens were obtained 72 hours after CA: SMD:0.59, 95%CI:0.38-0.81). CONCLUSION: NfL may play a potential neuroprognostication role in postcardiac arrest patients with spontaneous circulation, regardless of when the sample was collected after CA.

Tracing the identity of Parmigiano Reggiano "Prodotto di Montagna - Progetto Territorio" cheese using NMR spectroscopy and multivariate data analysis.

BACKGROUND: Nuclear magnetic resonance (NMR) spectroscopy is one of the well-established tools for food metabolomic analysis, as it proved to be very effective in authenticity and quality control of dairy products, as well as to follow product evolution during processing and storage. The analytical assessment of the EU mountain denomination label, specifically for Parmigiano Reggiano "Prodotto di Montagna - Progetto Territorio" (Mountain-CQ) cheese, has received limited attention. Although it was established in 2012 the EU mountain denomination label has not been much studied from an analytical point of view. Nonetheless, tracing a specific profile for the mountain products is essential to support the value chain of this specialty. RESULTS: The aim of the study was to produce an identity profile for Parmigiano Reggiano "Prodotto di Montagna - Progetto Territorio" (Mountain-CQ) cheese, and to differentiate it from Parmigiano Reggiano PDO samples (conventional-PDO) using 1H NMR spectroscopy coupled with multivariate data analysis. Three different approaches were applied and compared. First, the spectra-as-such were analysed after proper preprocessing. For the other two approaches, Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) was used for signals resolution and features extraction, either individually on manually-defined spectral intervals or by reapplying MCR-ALS on the whole spectra with selectivity constraints using the reconstructed "pure profiles" as initial estimates and targets. All approaches provided comparable information regarding the samples' distribution, as in all three cases the separation between the two product categories conventional-PDO and Mountain-CQ could be highlighted. Moreover, a novel MATLAB toolbox for features extraction via MCR-ALS was developed and used in synergy with the Chenomx library, allowing for a putative identification of the selected features. SIGNIFICANCE: A first identity profile for Parmigiano Reggiano "Prodotto di Montagna - Progetto Territorio" obtained by interpreting the metabolites signals in NMR spectroscopy was obtained. Our workflow and toolbox for generating the features dataset allows a more straightforward interpretation of the results, to overcome the limitations due to dimensionality and to peaks overlapping, but also to include the signals assignment and matching since the early stages of the data processing and analysis.

Scellpam: an R package/C++ library to perform parallel partitioning around medoids on scRNAseq data sets.

BACKGROUND: Partitioning around medoids (PAM) is one of the most widely used and successful clustering method in many fields. One of its key advantages is that it only requires a distance or a dissimilarity between the individuals, and the fact that cluster centers are actual points in the data set means they can be taken as reliable representatives of their classes. However, its wider application is hampered by the large amount of memory needed to store the distance matrix (quadratic on the number of individuals) and also by the high computational cost of computing such distance matrix and, less importantly, by the cost of the clustering algorithm itself. RESULTS: Therefore, new software has been provided that addresses these issues. This software, provided under GPL license and usable as either an R package or a C++ library, calculates in parallel the distance matrix for different distances/dissimilarities ([Formula: see text], [Formula: see text], Pearson, cosine and weighted Euclidean) and also implements a parallel fast version of PAM (FASTPAM1) using any data type to reduce memory usage. Moreover, the parallel implementation uses all the cores available in modern computers which greatly reduces the execution time. Besides its general application, the software is especially useful for processing data of single cell experiments. It has been tested in problems including clustering of single cell experiments with up to 289,000 cells with the expression of about 29,000 genes per cell. CONCLUSIONS: Comparisons with other current packages in terms of execution time have been made. The method greatly outperforms the available R packages for distance matrix calculation and also improves the packages that implement the PAM itself. The software is available as an R package at https://CRAN.R-project.org/package=scellpam and as C++ libraries at https://github.com/JdMDE/jmatlib and https://github.com/JdMDE/ppamlib The package is useful for single cell RNA-seq studies but it is also applicable in other contexts where clustering of large data sets is required.

CRISPR-Cas9 Library Screening Identifies Novel Molecular Vulnerabilities in KMT2A-Rearranged Acute Lymphoblastic Leukemia.

In acute lymphoblastic leukemia (ALL), chromosomal translocations involving the KMT2A gene represent highly unfavorable prognostic factors and most commonly occur in patients less than 1 year of age. Rearrangements of the KMT2A gene drive epigenetic changes that lead to aberrant gene expression profiles that strongly favor leukemia development. Apart from this genetic lesion, the mutational landscape of KMT2A-rearranged ALL is remarkably silent, providing limited insights for the development of targeted therapy. Consequently, identifying potential therapeutic targets often relies on differential gene expression, yet the inhibition of these genes has rarely translated into successful therapeutic strategies. Therefore, we performed CRISPR-Cas9 knock-out screens to search for genetic dependencies in KMT2A-rearranged ALL. We utilized small-guide RNA libraries directed against the entire human epigenome and kinome in various KMT2A-rearranged ALL, as well as wild-type KMT2A ALL cell line models. This screening approach led to the discovery of the epigenetic regulators ARID4B and MBD3, as well as the receptor kinase BMPR2 as novel molecular vulnerabilities and attractive therapeutic targets in KMT2A-rearranged ALL.

Alteration of Mitochondrial Transcript Expression in Arabidopsis thaliana Using a Custom-Made Library of Pentatricopeptide Repeat Proteins.

Pentatricopeptide repeat (PPR) proteins are considered a potential tool for manipulating organelle gene expression in plants because they can recognise a wide range of different RNA sequences, and the molecular basis for this sequence recognition is partially known and understood. A library of redesigned PPR proteins related to restorer-of-fertility proteins was created and transformed into plants in order to target mitochondrial transcripts. Ninety different variants tested in vivo showed a wide range of phenotypes. One of these lines, which displayed slow growth and downward curled leaves, showed a clear reduction in complex V. The phenotype was due to a specific cleavage of atp1 transcripts induced by a modified PPR protein from the library, validating the use of this library as a source of mitochondrial 'mutants'. This study is a step towards developing specific RNA targeting tools using PPR proteins that can be aimed at desired targets.

Inhibitor Library Screening of SH2 Domains Through Denaturation-Based Assays.

Screening of inhibitor libraries for candidate ligands is an important step in the drug discovery process. Thermal denaturation-based screening strategies are built on the premise that a protein-ligand complex has an altered stability profile compared to the protein alone. As such, these assays provide an accessible and rapid methodology for stratifying ligands that directly engage with the protein target of interest. Here, we describe three denaturation-based strategies for examining protein-inhibitor binding, in the context of SH2 domains. This includes conventional dye-based Thermal Shift Assays (TSA), nonconventional labeled ligand-based TSA, and Cellular Thermal Shift Assays (CETSA). Conventional dye-based TSA reports on the fluorescence of an external hydrophobic dye as it interacts with heat-exposed nonpolar protein surfaces as the temperature is incrementally increased. By contrast, nonconventional-labeled ligand TSA involves a fluorescence-tagged probe (phosphopeptide for SH2 domains) that is quenched as it dissociates from the protein during the denaturation process. CETSA involves monitoring the presence of the protein via Western blotting as the temperature is increased. In all three approaches, performing the assay in the presence of a candidate ligand can alter the melting profile of the protein. These assays offer primary screening tools to examine SH2 domain inhibitors libraries with varying chemical motifs, and a subset of the advantages and limitations of each approach is also discussed.

Construction of Naïve and Immune Human Fab Phage Display Library.

Phage display has been applied successfully for the rapid isolation of monoclonal antibodies against various targets including infectious diseases, autoantigens, cancer markers, and even small molecules. The main component in any phage display experiment is the availability of an antibody library to carry out the selection process of target-specific antibodies through an iterative process termed as biopanning. To generate human antibody libraries, the antibody repertoire can be obtained from human peripheral blood mononuclear cell (PBMC) or directly from cell-sorted B-cell populations. The choice of antibody isotype is dictated by the nature of the library. Naïve libraries would utilize IgM repertoires, whereas the IgG repertoire is commonly used for immune libraries. Antibody genes are amplified through polymerase chain reaction (PCR) and paired in a combinatorial fashion to expand the diversity of the cloned library repertoire. The protocol here describes the use of a two-step cloning method that can be applied for the construction of either a naïve or immune human antibody library in Fab format followed by the subsequent panning.

Construction of Human Immune and Naive scFv Phage Display Libraries.

Antibody phage display is a widely used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. In order to successfully generate antibodies using phage display, the basis is the construction of high-quality antibody gene libraries. Here, we describe detailed methods for the construction of such high-quality immune and naive scFv gene libraries of human origin. These protocols were used to develop human naive (e.g., HAL9/10) and immune libraries, which resulted in thousands of specific antibodies for all kinds of applications.

Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification.

An important contributor to the successful generation of recombinant affinity reagents via phage display is a large and diverse library. We describe, herein, the application of Kunkel mutagenesis and rolling circle amplification (RCA) to the construction of a 1.1 × 1011 member library, with only 26 electroporations, and isolation of low- to sub-nanomolar monobodies to a number of protein targets, including human COP9 signalosome subunit 5 (COPS5), HIV-1 Rev. binding protein-like protein (HRBL), X-ray repair cross-complementing 5/6 (Ku70/80) heterodimer, the receptor-binding domain (RBD) of SARS-CoV-2, and transforming growth factor beta 1 (TGF-ß1).

Selection of Affibody Affinity Proteins from Phagemid Libraries.

Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.

Estimating DNA-Binding Specificities of Transcription Factors Using SELEX-Seq.

Here we provide an updated protocol for the Systematic Evolution of Ligands followed by massively parallel sequencing (SELEX-seq) method to study protein-DNA interaction specificities. This in vitro method is used to characterize DNA-binding specificities of transcription factors (TFs). The procedure is based on cycles of immunoprecipitation of protein-DNA complexes, starting with a randomized DNA library of defined fragment length, followed by massively parallel sequencing. The updated protocol includes aspects of experimental design and procedure as well as basic instructions on data analysis.

Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Synthesis of (-)-Cotylenol, a 14-3-3 Molecular Glue Component.

Small molecules that modulate the 14-3-3 protein-protein interaction (PPI) network represent valuable therapeutics and tool compounds. However, access has been lost to 14-3-3 PPI molecular glues of the cotylenin class, leading to investigations into the practical chemical syntheses of congeners and analogues. Here we report a concise synthesis of (-)-cotylenol via a 10-step asymmetric entry into a diversifiable 5-8-5 core. This route features a mild Liebeskind-Srogl fragment coupling that tolerates unprecedented steric hindrance to produce a highly congested ketone, and a tandem Claisen-ene cascade that establishes the 8-membered ring. Late-stage control of stereochemistry and functionality leads to (-)-cotylenol and sets the stage for focused library synthesis.

Comparative efficacy of intraoperative radiotherapy and external boost irradiation in early-stage breast cancer: a systematic review and meta-analysis.

External boost radiotherapy (EBRT) and intraoperative radiotherapy (IORT) are shown to be effective in patients with early-stage breast cancer. However, the difference between IORT and EBRT for patients' prognosis remains to be elucidated. The purpose of this meta-analysis is to investigate differences in local recurrence (LR), distant metastases, disease free survival (DFS), and overall survival (OS) between these two therapies. We searched the Cochrane Library, PubMed, Web of Science and Embase, from inception to Jan 10th, 2022. We used The Cochrane risk-of-bias assessment tool to assess the risk of bias of the included studies, and the STATA15.0 tool was used for the meta-analyses. Eight studies were ultimately included. Meta-analysis demonstrated that there was an inconsistent finding in the long-term risk of LR between the two radiotherapies, and there was no significant difference in short-term risk of LR, the metastasis rate, DFS, and OS IORT would be more convenient, less time-consuming, less costly, and more effective at reducing side effects and toxicity. However, these benefits must be balanced against the potential for increased risk of LR in the long term.

DNA-Compatible Cyanomethylation of (Hetero)aryl Halides or Triflates under a Tandem Reaction for DNA-Encoded Library Synthesis.

A DNA-compatible reaction has been developed for the cyanomethylation of (hetero)aryl halides or triflates via a tandem process involving palladium-mediated Suzuki-Miyaura coupling and base-promoted isoxazole fragmentation. This one-pot protocol employs easily accessible starting materials, exhibits a wide substrate scope, and results in no significant DNA damage. Additionally, the resulting (hetero)arylacetonitriles can be converted into the corresponding carboxylic acids, which may be utilized for the synthesis of DNA-encoded chemical libraries.