Perfecting Targeting in CRISPR.

CRISPR-based genome editing holds promise for genome engineering and other applications in diverse organisms. Defining and improving the genome-wide and transcriptome-wide specificities of these editing tools are essential for realizing their full potential in basic research and biomedical therapeutics. This review provides an overview of CRISPR-based DNA- and RNA-editing technologies, methods to quantify their specificities, and key solutions to reduce off-target effects for research and improve therapeutic applications.

Development of miniature base editors using engineered IscB nickase.

As a miniature RNA-guided endonuclease, IscB is presumed to be the ancestor of Cas9 and to share similar functions. IscB is less than half the size of Cas9 and thus more suitable for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells limits its in vivo applications. Here we describe the engineering of OgeuIscB and its corresponding ωRNA to develop an IscB system that is highly efficient in mammalian systems, named enIscB. By fusing enIscB with T5 exonuclease (T5E), we found enIscB-T5E exhibited comparable targeting efficiency to SpG Cas9 while showing reduced chromosome translocation effects in human cells. Furthermore, by fusing cytosine or adenosine deaminase with enIscB nickase, we generated miniature IscB-derived base editors (miBEs), exhibiting robust editing efficiency (up to 92%) to induce DNA base conversions. Overall, our work establishes enIscB-T5E and miBEs as versatile tools for genome editing.

Interplay between the bacterial protein deacetylase CobB and the second messenger c-di-GMP.

As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.

Antibody Binding Epitope Mapping (AbMap) of Hundred Antibodies in a Single Run.

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.

The structure of occupational diseases in first-third-year piano students.

BACKGROUND: The aim is to analyze the structure of occupational diseases in first-third year piano students studying. METHODS: A total sample of 300 individuals participating in the research process underwent a complete medical examination at the end of each academic year. RESULTS: Among piano students, number of those distributed to special medical groups for health reasons grew from 14 to 38% (p ≤ 0.02). The incidence of diseases of the muscular system increased from 58 to 71% (p ≤ 0.05), with the sharpest rise during the transition from the second to the third year of study. Similar tendencies were obtained for the incidence of hand-related diseases-their overall frequency advanced from 17 to 23% (p ≤ 0.05). The surge of morbidity in the transition from the second to the third year of study can be attributed to the increased educational load experienced by students during this period, as they begin to practice music intensively after theoretical classes. Сorresponding measures directed at preventing occupational diseases, bettering physical education, expanding the rest intervals between piano lessons are to be taken. CONCLUSIONS: The research results indicate that the main occupational diseases of piano students are those related to hands and muscular system. IMPACT: The obtained data suggest that the range of diseases is likely to progress throughout the musical training. The main diseases of piano students are those related to hands and muscular system. The sharpest rise was during the transition from the 2nd to the 3rd year of study.

De novo missense variants in LMBRD2 are associated with developmental and motor delays, brain structure abnormalities and dysmorphic features.

OBJECTIVE: To determine the potential disease association between variants in LMBRD2 and complex multisystem neurological and developmental delay phenotypes. METHODS: Here we describe a series of de novo missense variants in LMBRD2 in 10 unrelated individuals with overlapping features. Exome sequencing or genome sequencing was performed on all individuals, and the cohort was assembled through GeneMatcher. RESULTS: LMBRD2 encodes an evolutionary ancient and widely expressed transmembrane protein with no known disease association, although two paralogues are involved in developmental and metabolic disorders. Exome or genome sequencing revealed rare de novo LMBRD2 missense variants in 10 individuals with developmental delay, intellectual disability, thin corpus callosum, microcephaly and seizures. We identified five unique variants and two recurrent variants, c.1448G>A (p.Arg483His) in three cases and c.367T>C (p.Trp123Arg) in two cases. All variants are absent from population allele frequency databases, and most are predicted to be deleterious by multiple in silico damage-prediction algorithms. CONCLUSION: These findings indicate that rare de novo variants in LMBRD2 can lead to a previously unrecognised early-onset neurodevelopmental disorder. Further investigation of individuals harbouring LMBRD2 variants may lead to a better understanding of the function of this ubiquitously expressed gene.

SARS-CoV-2-specific antibody response characteristics in COVID-19 patients of different ages.

Age has been found to be one of the main risk factors for the severity and outcome of COVID-19. However, differences in SARS-CoV-2 specific antibody responses among COVID-19 patients of different age groups remain largely unknown. In this study, we analyzed the IgG/IgM responses to 21 SARS-CoV-2 proteins and 197 peptides that fully cover the spike protein against 731 sera collected from 731 COVID-19 patients aged from 1 to We show that there is no overall difference in SARS-CoV-2 antibody responses in COVID-19 patients in the 4 age groups. By antibody response landscape maps, we find that the IgG response profiles of SARS-CoV-2 proteins are positively correlated with age. The S protein linear epitope map shows that the immunogenicity of the S-protein peptides is related to peptide sequence, disease severity and age of the COVID-19 patients. Furthermore, the enrichment analysis indicates that low S1 IgG responses are enriched in patients aged <50 and high S1 IgG responses are enriched in mild COVID-19 patients aged >60. In addition, high responses of non-structural/accessory proteins are enriched in severe COVID-19 patients aged >70. These results suggest the distinct immune response of IgG/IgM to each SARS-CoV-2 protein in patients of different age, which may facilitate a deeper understanding of the immune responses in COVID-19 patients.

New insights into transmission pathways and possible off-target effects of insecticidal dsRNA released by treated plants.

RNAi has shown great potential in controlling pests and pathogens, and dsRNA-based pesticides have been used in different ways. Due to off-target effects, the transmission pathways and possible impacts of dsRNA on non-target organisms after release should be researched. Here, we tested pathways of dsRNA transmission through the rice-hopper-spider food chain and their efficiency for triggering RNAi. The results revealed five new pathways by which plants transfer dsRNA into the environment through the food chain. We found that ingestion of the tissues or guttation droplets of treated plant could trigger both targeted and off-target RNAi both in consumers and predators. Ingestion of consumer hoppers could also result in localized RNAi in the midguts of the predator spiders. Trace amounts of dsRNA were detected in plant root excretions and in hopper honeydew. Cutting the root tips dramatically increased the levels of dsRNA in root excretions. Host shifting experiments proved that hoppers could transfer a trace amount of dsRNA via vomit. With specially designed dsRNAs, we showed that dsRNA sharing matching sequences of 29 bp or 32 bp in length with non-target genes could trigger off-target RNAi, but that dsRNA sharing 13 bp matching sequences could not. We conclude that field-released pesticidal dsRNA could be transmitted via the hydrophilic transport system in plants, and that this may pose a safety risk to non-target animal consumers that are closely related to target pests. Rational use of pesticidal dsRNAs should involve careful consideration of dsRNA design to manage the biosafety risk.

Molecular chaperones and Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive death of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies (LBs). Mutations in PD-related genes lead to neuronal pathogenesis through various mechanisms, with known examples including SNCA/α-synuclein (PAKR1), Parkin (PARK2), PINK1 (PARK6), DJ-1 (PARK7), and LRRK2 (PARK8). Molecular chaperones/co-chaperones are proteins that aid the folding of other proteins into a functionally active conformation. It has been demonstrated that chaperones/co-chaperones interact with PD-related proteins and regulate their function in PD. HSP70, HSP90 and small heat shock proteins can prevent neurodegeneration by regulating α-syn misfolding, oligomerization and aggregation. The function of chaperones is regulated by co-chaperones such as HSP110, HSP40, HOP, CHIP, and BAG family proteins. Parkin, PINK1 and DJ-1 are PD-related proteins which are associated with mitochondrial function. Molecular chaperones regulate mitochondrial function and protein homeostasis by interacting with these PD-related proteins. This review discusses critical molecular chaperones/co-chaperones and PD-related proteins which contribute to the pathogenesis of PD, hoping to provide new molecular targets for therapeutic interventions to thwart the disease progression instead of only bringing symptomatic relief. Moreover, appreciating the critical role of chaperones in PD can also help us screen efficient biomarkers to identify PD at an early stage.

Antibody dynamics to SARS-CoV-2 in asymptomatic COVID-19 infections.

BACKGROUND: The missing asymptomatic COVID-19 infections have been overlooked because of the imperfect sensitivity of the nucleic acid testing (NAT). Globally understanding the humoral immunity in asymptomatic carriers will provide scientific knowledge for developing serological tests, improving early identification, and implementing more rational control strategies against the pandemic. MEASURE: Utilizing both NAT and commercial kits for serum IgM and IgG antibodies, we extensively screened 11 766 epidemiologically suspected individuals on enrollment and 63 asymptomatic individuals were detected and recruited. Sixty-three healthy individuals and 51 mild patients without any preexisting conditions were set as controls. Serum IgM and IgG profiles were further probed using a SARS-CoV-2 proteome microarray, and neutralizing antibody was detected by a pseudotyped virus neutralization assay system. The dynamics of antibodies were analyzed with exposure time or symptoms onset. RESULTS: A combination test of NAT and serological testing for IgM antibody discovered 55.5% of the total of 63 asymptomatic infections, which significantly raises the detection sensitivity when compared with the NAT alone (19%). Serum proteome microarray analysis demonstrated that asymptomatics mainly produced IgM and IgG antibodies against S1 and N proteins out of 20 proteins of SARS-CoV-2. Different from strong and persistent N-specific antibodies, S1-specific IgM responses, which evolved in asymptomatic individuals as early as the seventh day after exposure, peaked on days from 17 days to 25 days, and then disappeared in two months, might be used as an early diagnostic biomarker. 11.8% (6/51) mild patients and 38.1% (24/63) asymptomatic individuals did not produce neutralizing antibody. In particular, neutralizing antibody in asymptomatics gradually vanished in two months. CONCLUSION: Our findings might have important implications for the definition of asymptomatic COVID-19 infections, diagnosis, serological survey, public health, and immunization strategies.

Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA.

RNAi is a potent technique for the knockdown of target genes. However, its potential off-target effects limit the widespread applications in both reverse genetic analysis and genetic manipulation. Previous efforts have uncovered rules underlying specificity of siRNA-based silencing, which has broad applications in humans, but the basis for specificity of dsRNAs, which are better suited for use as insecticides, is poorly understood. Here, we investigated the rules governing dsRNA specificity. Mutational analyses showed that dsRNAs with >80% sequence identity with target genes triggered RNAi efficiently. dsRNAs with ≥16 bp segments of perfectly matched sequence or >26 bp segments of almost perfectly matched sequence with one or two mismatches scarcely distributed (single mismatches inserted between ≥5 bp matching segments or mismatched couplets inserted between ≥8 bp matching segments) also able to trigger RNAi. Using these parameters to predict off-target risk, dsRNAs can be designed to optimize specificity and efficiency, paving the way to the widespread, rational application of RNAi in pest control.

Expansion of GGC repeat in the human-specific NOTCH2NLC gene is associated with essential tremor.

Essential tremor is one of the most common movement disorders. Despite its high prevalence and heritability, the genetic aetiology of essential tremor remains elusive. Up to now, only a few genes/loci have been identified, but these genes have not been replicated in other essential tremor families or cohorts. Here we report a genetic study in a cohort of 197 Chinese pedigrees clinically diagnosed with essential tremor. Using a comprehensive strategy combining linkage analysis, whole-exome sequencing, long-read whole-genome sequencing, repeat-primed polymerase chain reaction and GC-rich polymerase chain reaction, we identified an abnormal GGC repeat expansion in the 5' region of the NOTCH2NLC gene that co-segregated with disease in 11 essential tremor families (5.58%) from our cohort. Clinically, probands that had an abnormal GGC repeat expansion were found to have more severe tremor phenotypes, lower activities of daily living ability. Obvious genetic anticipation was also detected in these 11 essential tremor-positive families. These results indicate that abnormal GGC repeat expansion in the 5' region of NOTCH2NLC gene is associated with essential tremor, and provide strong evidence that essential tremor is a family of diseases with high clinical and genetic heterogeneities.

The role of genetics in Parkinson's disease: a large cohort study in Chinese mainland population.

This study aimed to determine the mutational spectrum of familial Parkinson's disease and sporadic early-onset Parkinson's disease (sEOPD) in a mainland Chinese population and the clinical features of mutation carriers. We performed multiplex ligation-dependent probe amplification assays and whole-exome sequencing for 1676 unrelated patients with Parkinson's disease in a mainland Chinese population, including 192 probands from families with autosomal-recessive Parkinson's disease, 242 probands from families with autosomal-dominant Parkinson's disease, and 1242 sEOPD patients (age at onset ≤ 50). According to standards and guidelines from the American College of Medical Genetics and Genomics, pathogenic/likely pathogenic variants in 23 known Parkinson's disease-associated genes occurred more frequently in the autosomal-recessive Parkinson's disease cohort (65 of 192, 33.85%) than in the autosomal-dominant Parkinson's disease cohort (10 of 242, 4.13%) and the sEOPD cohort (57 of 1242, 4.59%), which leads to an overall molecular diagnostic yield of 7.88% (132 of 1676). We found that PRKN was the most frequently mutated gene (n = 83, 4.95%) and present the first evidence of an SNCA duplication and LRRK2 p.N1437D variant in mainland China. In addition, several novel pathogenic/likely pathogenic variants including LRRK2 (p.V1447M and p.Y1645S), ATP13A2 (p.R735X and p.A819D), FBXO7 (p.G67E), LRP10 (c.322dupC/p.G109Rfs*51) and TMEM230 (c.429delT/p.P144Qfs*2) were identified in our cohort. Furthermore, the age at onset of the 132 probands with genetic diagnoses (median, 31.5 years) was about 14.5 years earlier than that of patients without molecular diagnoses (i.e. non-carriers, median 46.0 years). Specifically, the age at onset of Parkinson's disease patients with pathogenic/likely pathogenic variants in ATP13A2, PLA2G6, PRKN, or PINK1 was significantly lower than that of non-carriers, while the age at onset of carriers with other gene pathogenic/likely pathogenic variants was similar to that of non-carriers. The clinical spectrum of Parkinson's disease-associated gene carriers in this mainland Chinese population was similar to that of other populations. We also detected 61 probands with GBA possibly pathogenic variants (3.64%) and 59 probands with GBA p.L444P (3.52%). These results shed insight into the genetic spectrum and clinical manifestations of Parkinson's disease in mainland China and expand the existing repertoire of pathogenic or likely pathogenic variants involved in known Parkinson's disease-associated genes. Our data highlight the importance of genetic testing in Parkinson's disease patients with age at onset < 40 years, especially in those from families with a recessive inheritance pattern, who may benefit from early diagnosis and treatment.

Nsp2 has the potential to be a drug target revealed by global identification of SARS-CoV-2 Nsp2-interacting proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health threat since December 2019, and there is still no highly effective drug to control the pandemic. To facilitate drug target identification for drug development, studies on molecular mechanisms, such as SARS-CoV-2 protein interactions, are urgently needed. In this study, we focused on Nsp2, a non-structural protein with largely unknown function and mechanism. The interactome of Nsp2 was revealed through the combination of affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), and 84 proteins of high-confidence were identified. Gene ontology analysis demonstrated that Nsp2-interacting proteins are involved in several biological processes such as endosome transport and translation. Network analysis generated two clusters, including ribosome assembly and vesicular transport. Bio-layer interferometry (BLI) assay confirmed the bindings between Nsp2- and 4-interacting proteins, i.e. STAU2 (Staufen2), HNRNPLL, ATP6V1B2, and RAP1GDS1 (SmgGDS), which were randomly selected from the list of 84 proteins. Our findings provide insights into the Nsp2-host interplay and indicate that Nsp2 may play important roles in SARS-CoV-2 infection and serve as a potential drug target for anti-SARS-CoV-2 drug development.

The binding epitope of sintilimab on PD-1 revealed by AbMap.

PD-1 plays an important role as an immune checkpoint. Sintilimab is a newly approved PD-1 antibody for cancer immunotherapy with an unknown binding epitope on PD-1. In this study, to elucidate the molecular mechanism by which sintilimab blocks PD-1 activation, we applied Antibody binding epitope Mapping (AbMap) to identify the binding epitope of sintilimab. An epitope was successfully identified, i.e. SLAPKA, aa 127-132. By constructing a series of point mutations, the dominant residues S127, L128, A129, P130, and A132 of PD-1 were further validated by western blot analysis, biolayer interferometry, and flow cytometry. Structural analysis showed that the epitope is partially within the binding interface of PD-1 and PD-L1, and this epitope also partially overlaps with that of nivolumab and pembrolizumab. These results demonstrate that sintilimab can attenuate PD-1 activation by directly competing with the interaction between PD-1 and PD-L1 through binding with the key residues of the FG loop on PD-1. This study also demonstrates the high efficiency and accuracy of AbMap for determining the binding epitope of therapeutic antibodies.

Diagnostic and Therapeutic Potential of Exosomal MicroRNAs for Neurodegenerative Diseases.

Neurodegenerative disorders (NDs) are characterized by a gradual loss of neurons and functions that eventually leads to progressive neurological impairment. In view of the heavy burden on the healthcare system, efficient and reliable biomarkers for early diagnosis and therapeutic treatments to reverse the progression of NDs are in urgent need. There has been an increasing interest in using exosomal miRNAs as biomarkers or targeted therapies for neurological diseases recently. In this review, we overviewed the updated studies on exosomal miRNAs as biomarkers and potential therapeutic approaches in NDs, as well as their association with the pathophysiology of this group of disorders, especially Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). The exosomal miRNAs that are commonly dysregulated across different NDs or are commonly used as therapeutic candidates were also identified and summarized. In summary, the feasibility of exosomal miRNAs as biomarkers and potential targeted therapy for NDs has been verified. However, due to the limitations of existing studies and the discrepancies across different studies, high quality laboratory and clinical investigations are still required.

Transcript level is a key factor affecting RNAi efficiency.

Efficiency is the basis for the application of RNA interference (RNAi) technology. Actually, RNAi efficiency varies greatly among insect species, tissues and genes. Previous efforts have revealed the mechanisms for variation among insect species and tissues. Here, we investigated the reason for variable efficiency among the target genes in the same insect. First, we tested the genes sampled randomly from Tribolium castaneum, Locusta migratoria and Drosophila S2 cells for both their expression levels and sensitivity to RNAi. The results indicated that the genes with higher expression levels were more sensitive to RNAi. Statistical analysis showed that the correlation coefficients between transcript levels and knockdown efficiencies were 0.8036 (n = 90), 0.7255 (n = 18) and 0.9505 (n = 13), respectively in T. castaneum, L. migratoria and Drosophila S2 cells. Subsequently, ten genes with varied expression level in different tissues (midgut and carcass without midgut) of T. castaneum were tested. The results indicated that the higher knockdown efficiency was always obtained in the tissue where the target gene expressed higher. In addition, three genes were tested in different developmental stages, larvae and pupae of T. castaneum. The results found that when the expression level increased after insect pupation, these genes became more sensitive to RNAi. Thus, all the proofs support unanimously that transcript level is a key factor affecting RNAi sensitivity. This finding allows for a better understanding of the RNAi efficiency variation and lead to effective or efficient use of RNAi technology.

Research progress in rapid eye movement sleep behavior disorder. / å¿«é€Ÿçœ¼åŠ¨ç¡çœ è¡Œä¸ºéšœç¢çš„ç ”ç©¶è¿›å±•.

Rapid eye movement (REM) sleep behavior disorder (RBD) is characterized by abnormal dream acting behavior such as vocalization and twitching related to dream content during REM sleep. The diagnosis requires polysomnography demonstrating a loss of normal skeletal muscle atonia during REM sleep. Both idiopathic RBD and secondary RBD are highly related to synucleinopathy including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Almost all idiopathic RBD patients will develop synucleinopathy after a few years. Therefore, RBD may be an early marker in the progression of synucleinopathy.

CRISPR/Cas9-mediated whole genomic wide knockout screening identifies mitochondrial ribosomal proteins involving in oxygen-glucose deprivation/reperfusion resistance.

Recanalization therapy by intravenous thrombolysis or endovascular therapy is critical for the treatment of cerebral infarction. However, the recanalization treatment will also exacerbate acute brain injury and even severely threatens human life due to the reperfusion injury. So far, the underlying mechanisms for cerebral ischaemia-reperfusion injury are poorly understood and effective therapeutic interventions are yet to be discovered. Therefore, in the research, we subjected SK-N-BE(2) cells to oxygen-glucose deprivation/reperfusion (OGDR) insult and performed a pooled genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) knockout screen to discover new potential therapeutic targets for cerebral ischaemia-reperfusion injury. We used Metascape to identify candidate genes which might involve in OGDR resistance. We found that the genes contributed to OGDR resistance were primarily involved in neutrophil degranulation, mitochondrial translation, and regulation of cysteine-type endopeptidase activity involved in apoptotic process and response to oxidative stress. We then knocked down some of the identified candidate genes individually. We demonstrated that MRPL19, MRPL32, MRPL52 and MRPL51 inhibition increased cell viability and attenuated OGDR-induced apoptosis. We also demonstrated that OGDR down-regulated the expression of MRPL19 and MRPL51 protein. Taken together, our data suggest that genome-scale screening with Cas9 is a reliable tool to analyse the cellular systems that respond to OGDR injury. MRPL19 and MRPL51 contribute to OGDR resistance and are supposed to be promising targets for the treatment of cerebral ischaemia-reperfusion damage.

Rapid response flexible humidity sensor for respiration monitoring using nano-confined strategy.

Development of wearable devices for continuous respiration monitoring is of great importance for evaluating human health. Here, we propose a new strategy to achieve rapid respiration response by confining conductive polymers into 1D nanowires which facilitates the water molecules absorption/desorption and maximizes the sensor response to moisture. The nanowires arrays were fabricated through a low-cost nanoscale printing approach on flexible substrate. The nanoscale humidity sensor shows a high sensitivity (5.46%) and ultrafast response (0.63 s) when changing humidity between 0% and 13% and can tolerate 1000 repetitions of bending to a curvature radius of 10 mm without influencing its performance. Benefited by its fast response and low power assumption, the humidity sensor was demonstrated to monitor human respiration in real time. Different respiration patterns including normal, fast and deep respiration can be distinguished accurately.