Large-scale multiplexed mosaic CRISPR perturbation in the whole organism.

Here, we report inducible mosaic animal for perturbation (iMAP), a transgenic platform enabling in situ CRISPR targeting of at least 100 genes in parallel throughout the mouse body. iMAP combines Cre-loxP and CRISPR-Cas9 technologies and utilizes a germline-transmitted transgene carrying a large array of individually floxed, tandemly linked gRNA-coding units. Cre-mediated recombination triggers expression of all the gRNAs in the array but only one of them per cell, converting the mice to mosaic organisms suitable for phenotypic characterization and also for high-throughput derivation of conventional single-gene perturbation lines via breeding. Using gRNA representation as a readout, we mapped a miniature Perturb-Atlas cataloging the perturbations of 90 genes across 39 tissues, which yields rich insights into context-dependent gene functions and provides a glimpse of the potential of iMAP in genome decoding.

REPAIRx, a specific yet highly efficient programmable A > I RNA base editor.

Programmable A > I RNA editing is a valuable tool for basic research and medicine. A variety of editors have been created, but a genetically encoded editor that is both precise and efficient has not been described to date. The trade-off between precision and efficiency is exemplified in the state of the art editor REPAIR, which comprises the ADAR2 deaminase domain fused to dCas13b. REPAIR is highly efficient, but also causes significant off-target effects. Mutations that weaken the deaminase domain can minimize the undesirable effects, but this comes at the expense of on-target editing efficiency. We have now overcome this dilemma by using a multipronged approach: We have chosen an alternative Cas protein (CasRx), inserted the deaminase domain into the middle of CasRx, and redirected the editor to the nucleus. The new editor created, dubbed REPAIRx, is precise yet highly efficient, outperforming various previous versions on both mRNA and nuclear RNA targets. Thus, REPAIRx markedly expands the RNA editing toolkit and illustrates a novel strategy for base editor optimization.

Programmable C-to-U RNA editing using the human APOBEC3A deaminase.

Programmable RNA cytidine deamination has recently been achieved using a bifunctional editor (RESCUE-S) capable of deaminating both adenine and cysteine. Here, we report the development of "CURE", the first cytidine-specific C-to-U RNA Editor. CURE comprises the cytidine deaminase enzyme APOBEC3A fused to dCas13 and acts in conjunction with unconventional guide RNAs (gRNAs) designed to induce loops at the target sites. Importantly, CURE does not deaminate adenosine, enabling the high-specificity versions of CURE to create fewer missense mutations than RESCUE-S at the off-targets transcriptome-wide. The two editing approaches exhibit overlapping editing motif preferences, with CURE and RESCUE-S being uniquely able to edit UCC and AC motifs, respectively, while they outperform each other at different subsets of the UC targets. Finally, a nuclear-localized version of CURE, but not that of RESCUE-S, can efficiently edit nuclear RNAs. Thus, CURE and RESCUE are distinct in design and complementary in utility.

Modulation of WNT, Activin/Nodal, and MAPK Signaling Pathways Increases Arterial Hemogenic Endothelium and Hematopoietic Stem/Progenitor Cell Formation During Human iPSC Differentiation.

Several differentiation protocols enable the emergence of hematopoietic stem and progenitor cells (HSPCs) from human-induced pluripotent stem cells (iPSCs), yet optimized schemes to promote the development of HSPCs with self-renewal, multilineage differentiation, and engraftment potential are lacking. To improve human iPSC differentiation methods, we modulated WNT, Activin/Nodal, and MAPK signaling pathways by stage-specific addition of small-molecule regulators CHIR99021, SB431542, and LY294002, respectively, and measured the impact on hematoendothelial formation in culture. Manipulation of these pathways provided a synergy sufficient to enhance formation of arterial hemogenic endothelium (HE) relative to control culture conditions. Importantly, this approach significantly increased production of human HSPCs with self-renewal and multilineage differentiation properties, as well as phenotypic and molecular evidence of progressive maturation in culture. Together, these findings provide a stepwise improvement in human iPSC differentiation protocols and offer a framework for manipulating intrinsic cellular cues to enable de novo generation of human HSPCs with functionality in vivo.

Performance of heart rate adjusted heart rate variability for risk stratification of sudden cardiac death.

PURPOSE: As a non-invasive tool for the assessment of cardiovascular autonomic function, the predictive value of heart rate variability (HRV) for sudden cardiac death (SCD) risk stratification remains unclear. In this study, we investigated the performance of the individualized heart rate (HR) adjusted HRV (HRVI) for SCD risk stratification in subjects with diverse risks. METHODS: A total of 11 commonly used HRV metrics were analyzed in 192 subjects, including 88 healthy controls (low risk group), 82 hypertrophic cardiomyopathy (HCM) patients (medium risk group), and 22 SCD victims (high risk group). The relationship between HRV metrics and HR was examined with long-term and short-term analysis. The performance HRVI was evaluated by area under the receiver operating characteristic curve (AUC) and covariance of variation (CV). RESULTS: Most of the HRV metrics were exponentially decayed with the increase of HR, while the exponential power coefficients were significantly different among groups. The HRVI metrics discriminated low, medium and high risk subjects with a median AUC of 0.72[0.11], which was considerably higher than that of the traditional long-term (0.63[0.04]) and short-term (0.58[0.05]) HRV without adjustment. The average CV of the HRVI metrics was also significantly lower than traditional short-term HRV metrics (0.09 ± 0.02 vs. 0.24 ± 0.13, p < 0.01). CONCLUSIONS: Subjects with diverse risks of SCD had similar exponential decay relationship between HRV metrics and HR, but with different decaying rates. HRVI provides reliable and robust estimation for risk stratification of SCD.

Characterization and Discrimination of Volatile Compounds in Chilled Tan Mutton Meat during Storage Using HiSorb-TD-GC-MS and E-Nose.

Chilled Tan mutton is currently the mainstream of Tan mutton production and consumption in China, but the reports on chilled meat quality evaluation and shelf-life discrimination by volatiles are limited. This study aimed to investigate the changes of volatile compounds in chilled Tan mutton at four storage stages (1d, 3d, 5d, 7d) in order to differentiate the various storage stages. An analysis protocol was established for the characterization and discrimination of the volatiles in chilled Tan mutton based on high capacity sorptive extraction-thermal desorption-gas coupled with chromatography-mass spectrometry (HiSorb-TD-GC-MS), electronic nose (E-nose), and multivariate statistical analysis. A total of 96 volatile compounds were identified by HiSorb-TD-GC-MS, in which six compounds with relative odor activity value >1 were screened as the key characteristic volatiles in chilled Tan mutton. Four storage stages were discriminated by partial least squares discriminant analysis, and nine differential volatile compounds showed a variable importance for the projection score >1, including octanoic acid, methyl ester, decanoic acid, methyl ester, acetic acid, heptanoic acid, methyl ester, propanoic acid, 2-hydroxy-, methyl ester, (ñ)-, hexanoic acid, propanoic acid, butanoic acid, and nonanoic acid. With the volcano plot analysis, hexadecanoic acid, methyl ester, was the common volatile marker candidate to discriminate chilled stages of Tan mutton. Meanwhile, E-nose could discriminate chilled Tan mutton at different storage stages rapidly and efficiently using linear discriminant analysis. Furthermore, E-nose sensors could obtain comprehensive volatile profile information, especially in esters, acids, and alcohols, which could confirm the potential of E-nose for meat odor recognition. Thus, this analysis protocol could characterize and discriminate the volatiles in chilled Tan mutton during storage.

[Development of Human Vital Signs and Body Posture Monitoring and Positioning Alarm Systems].

In view of the high incidence of malignant diseases such as malignant arrhythmias in the elderly population, accidental injuries such as falls, and the problem of no witnesses when danger occurs, the study developed a human vital signs and body posture monitoring and positioning alarm system. Through the collection and analysis of electrocardiogram (ECG), respiration (RESP) and acceleration (ACC) signals, the system monitors human vital signs and body posture in real time, automatically judges critical states such as malignant arrhythmias and accidental falls on the local device side, and then issues alarm information, opens the positioning function, and uploads physiological information and patient location information through 4G communication. Experiments have shown that the system can accurately determine the occurrence of ventricular fibrillation and falls, and issue position and alarm information.

Genetic characterization of a novel HIV­1 CRF01_AE/ CRF07_BC recombinant form found among men who have sex with men in Baoding City, Hebei Province, China.

Large numbers of unique recombinant forms (URF) of human immunodeficiency virus (HIV-1) have been found among sexual transmission populations in China. Here, we report a novel second-generation URF of HIV-1 named BD201AQ that was isolated from an HIV-1-positive man who was infected through homosexual transmission in Baoding City, Hebei Province, China. Phylogenetic analysis based on the near-full-length genome (NFLG) sequence indicated that BD201AQ formed a monophyletic branch that did not cluster with other HIV-1 subtypes. Recombination analysis showed that the NFLG of BD201AQ had 12 segments, six CRF07_BC and six CRF01_AE segments, with CRF07_BC as the main framework. These findings indicate that the constant emergence of novel recombinant forms should receive more attention and that more measures should be taken to monitor the molecular epidemiological characteristics of HIV-1 and to prevent the spread of HIV-1 infections.

Sulfitobacter maritimus sp. nov., isolated from coastal sediment.

A facultatively anaerobic bacterium, strain S0837T, was isolated from the marine sediment of Jingzi Wharf, Weihai, China. Cells of the novel strain were Gram-stain-negative, non-flagellated, non-gliding, non-pigmented and rod-shaped. Cells were around 0.3-0.5×1.0-1.4 µm in size and often appeared singly. Optimum growth occurred at 33 °C, with 2 % (w/v) NaCl and at pH 7.0-7.5. On the basis of the results of 16S rRNA gene sequences, stain S0837T had the closest relative with Sulfitobacter delicatus KCTC 32183T (98.0 %). Genome sequencing revealed a genome size of 3 785 026 bp, a G+C content of 59.8 mol% and several genes related with sulphur oxidation. The strain shared 98.0 % 16S rRNA sequence similarities with closely related type species and shared ANI value below 95-96 %, dDDH value of showed relatedness of 27.4, 25.2 and 25.2 % respectively with the closely related type species. Strain S0837T had ubiquinone-10 as the sole respiratory quinone, and possessed summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) as the major fatty acid. The major polar lipids were phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. According to the results of the phenotypic, chemotaxonomic characterization, phylogenetic properties and genome analysis, strain S0837T should represent a novel species of the genus Sulfitobacter, for which the name Sulfitobacter maritimus sp. nov. is proposed. The type strain is S0837T (=MCCC 1K04635T=KCTC 72860T).

Interaction between gender and post resuscitation interventions on neurological outcome in an asphyxial rat model of cardiac arrest.

PURPOSE: Previous clinical studies have suggested an effect of gender on outcome after out-of-hospital cardiac arrest, but the results are conflicting and there is no uniform agreement regarding gender differences in survival and prognosis. The present study was aimed to investigate the interaction between gender and post resuscitation interventions on neurological outcome in an asphyxial rat model of cardiac arrest. METHODS: Asphyxia was induced by blocking the endotracheal tube in 120 adult Sprague-Dawley rats (60 males and 60 females) at the same age. Cardiopulmonary resuscitation (CPR) was started after 5 min of untreated cardiac arrest. Animals were randomized into one of the three post resuscitation care intervention groups (n = 40, 20 males) immediately after resuscitation: (1) normothermic control (NC): ventilated with 2% N2/98% O2 for 1 h under normothermia; (2) targeted temperature management (TTM): ventilated with 2% N2/98% O2 for 1 h under hypothermia; (3) hydrogen inhalation (HI): ventilated with 2% H2/98% O2 for 1 h under normothermia. Physiological variables were recorded during the 5 h post resuscitation monitoring period. Neurological deficit score (NDS) and accumulative survival were used to assess 96 h outcomes. Mutual independence analysis and Mantel-Haenszel stratified analysis were used to explore the associations among gender, intervention and survival. RESULTS: The body weights of female rats were significantly lighter than males, but CPR characteristics did not differ between genders. Compared with male rats, females had significantly lower mean arterial pressure, longer onset time of the electroencephalogram (EEG) burst and time to normal EEG trace (TTNT) in the NC group; relatively longer TTNT in the TTM group; and substantially longer TTNT, lower NDSs, and higher survival in the HI group. Mutual independence analysis revealed that both gender and intervention were associated with neurological outcome. Mantel-Haenszel stratified analysis demonstrated that female rats had significantly higher survival rate than males when adjusted for the confounder intervention. CONCLUSION: In this rat model cardiac arrest and CPR, gender did not affect resuscitation but associated with neurological outcome. The superiority of female rats in neurological recovery was affected by post resuscitation interventions and female rats were more likely to benefit from hydrogen therapy.

The effect and mechanism of 19S proteasome PSMD11/Rpn6 subunit in D-Galactose induced mimetic aging models.

Regulating proteasome activity is a potent therapeutic aspect of age-related hearing loss, which has been proven to protect neurons from age-related damaging. PSMD11, subunit of the 19S proteasome regulatory particle, is known to mainly up-regulate proteasome activity and prolong aging. However, the mechanism of PSMD11 in age-related hearing loss has not been deeply explored. In the present study, we explore the function and mechanism of PSMD11 protecting neurons in d-Galactose (D-Gal) mimetic aging models. Age-related pathologies were detected by Taq-PCR, ABR, Transmission electron microscopy, toluidine blue and ß-galactosidase staining. The relative expressions of the proteins were explored by Western blotting, oxyblot, immunoprecipitation and immunofluorescence. Flow cytometry was used to manifest the oxidative state. We discovered that proteasome activity was impaired with aging, and that ROS and toxic protein accumulated in D-Gal induced aging models. PSMD11 changed with aging, and was associated with the metabolism of proteasome activity in the D-Gal treated models. Moreover, the knockdown or overexpression of PSMD11 was sufficient to change the oxidative state caused by D-Gal. Our results also demonstrated that PSMD11 could bond to AMPKα1/2 in the auditory cortex and PC12 cells, and AMPKα2 but not AMPKα1 was efficient to regulate the function of PSMD11. Deeper insights into the mechanisms of regulating PSMD11 for the anti-aging process are needed, and may offer novel therapeutic methods for central presbycusis.

De novo transcriptome sequencing and analysis of the cuttlefish (Sepiella japonica) with different embryonic developmental stages.

In this study, embryos of Sepiella japonica from eye primordium formation to the larval growing stage were collected and used for RNA-Seq analysis. A total of 183,542,186 clean reads were assembled de novo into 58,054 unigenes consisting of 54,118,228 bp, with the average length at 932 bp and the N50 at 1667 bp. 21,469 (36.98%) unigenes were annotated at least in one of four databases including non-redundant protein (NR), Swiss-Prot, clusters of orthologous groups of proteins (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 4460 (7.68%) unigenes were annotated in all databases. Analysis of differentially expressed genes (DEGs) was carried out on embryos at Eye primordium formation stage (SJ1), organ differentiation stage (SJ2), and hatching stage (SJ3). Overall, the current study provided the de novo assembly of S. japonica transcriptome and identified the DEGs and pathways during embryonic development, which will provide a fundamental genetic resource for further functional research.

[A heart rate detection method for wearable electrocardiogram with the presence of motion interference].

The dynamic electrocardiogram (ECG) collected by wearable devices is often corrupted by motion interference due to human activities. The frequency of the interference and the frequency of the ECG signal overlap with each other, which distorts and deforms the ECG signal, and then affects the accuracy of heart rate detection. In this paper, a heart rate detection method that using coarse graining technique was proposed. First, the ECG signal was preprocessed to remove the baseline drift and the high-frequency interference. Second, the motion-related high amplitude interference exceeding the preset threshold was suppressed by signal compression method. Third, the signal was coarse-grained by adaptive peak dilation and waveform reconstruction. Heart rate was calculated based on the frequency spectrum obtained from fast Fourier transformation. The performance of the method was compared with a wavelet transform based QRS feature extraction algorithm using ECG collected from 30 volunteers at rest and in different motion states. The results showed that the correlation coefficient between the calculated heart rate and the standard heart rate was 0.999, which was higher than the result of the wavelet transform method ( r = 0.971). The accuracy of the proposed method was significantly higher than the wavelet transform method in all states, including resting (99.95% vs. 99.14%, P < 0.01), walking (100% vs. 97.26%, P < 0.01) and running (100% vs. 90.89%, P < 0.01). The absolute error [0 (0, 1) vs. 1 (0, 1), P < 0.05] and relative error [0 (0, 0.59) vs. 0.52 (0, 0.72), P < 0.05] of the proposed method were significantly lower than the wavelet transform method during running state. The method presented in this paper shows high accuracy and strong anti-interference ability, and is potentially used in wearable devices to realize real-time continuous heart rate monitoring in daily activities and exercise conditions.

Association between heart rate variability indices and features of spontaneous ventricular tachyarrhythmias in mice.

Direct evidence is limited for the association between heart rate variability (HRV) indices and ventricular tachyarrhythmias (VTAs). While galectin-3 (Gal-3) is regarded as a causal factor for cardiac remodelling and a biomarker for arrhythmias, its regulation on VTAs and HVR is unknown. Using aged transgenic (TG) mice with cardiac overexpression of ß2 -adrenoceptors and spontaneous VTAs, we studied whether changes in HRV indices correlated with the severity of VTAs, and whether Gal-3 gene knockout (KO) in TG mice might limit VTA. Body-surface ECG was recorded (10-minute period) in 9- to 10-month-old mice of non-transgenic (nTG), TG and TG × Gal-3 knockout (TG/KO). Time-domain, frequency-domain and nonlinear-domain HRV indices were calculated using the R-R intervals extracted from ECG signals and compared with frequency of VTAs. TG and TG/KO mice developed frequent VTAs and showed significant changes in certain time-domain and nonlinear-domain HRV indices relative to nTG mice. The severity of VTAs in TG and TG/KO mice in combination, estimated by VTA counts and arrhythmia score, was significantly correlated with certain time-domain and nonlinear-domain HRV indices. In conclusion, significant changes in HRV indices were evident and correlated with the severity of spontaneous VTAs in TG mice. The frequency of VTA and HRV indices were largely comparable between TG and TG/KO mice. Deletion of Gal-3 in TG mice altered certain HRV indices implying influence by neuronally localized Gal-3 on autonomic nervous activity.

[Recent advances in external cardiac defibrillation techniques].

As an important medical electronic equipment for the cardioversion of malignant arrhythmia such as ventricular fibrillation and ventricular tachycardia, cardiac external defibrillators have been widely used in the clinics. However, the resuscitation success rate for these patients is still unsatisfied. In this paper, the recent advances of cardiac external defibrillation technologies is reviewed. The potential mechanism of defibrillation, the development of novel defibrillation waveform, the factors that may affect defibrillation outcome, the interaction between defibrillation waveform and ventricular fibrillation waveform, and the individualized patient-specific external defibrillation protocol are analyzed and summarized. We hope that this review can provide helpful reference for the optimization of external defibrillator design and the individualization of clinical application.

Correction to: Exercise-induced circulating extracellular vesicles protect against cardiac ischemia-reperfusion injury.

The original version of this article unfortunately contained a mistake.

SGK1 inhibits PM2.5-induced apoptosis and oxidative stress in human lung alveolar epithelial A549 cells.

Emerging evidence demonstrated that particulate matter 2.5 (PM2.5) is an important environmental risk factor for lung diseases. Serum- and glucocorticoid-inducible kinase 1(SGK1) was reported to be a crucial factor for cell survival. However, the role of SGK1 in PM2.5-induced cell injury is still unclear. In this work, we firstly found that the expression of SGK1 was decreased in PM2.5-treated human lung alveolar epithelial (A549) cells by western blot. In addition, overexpression of SGK1 significantly attenuated A549 cell apoptosis and reduced the reactive oxygen species (ROS) generation induced by PM2.5. Moreover, we found that PM2.5 exposure significantly promoted the ERK1/2 activation and inhibited the AKT activation, whereas overexpression of SGK1 could reverse that. Finally, the results of the rescue experiment showed that MK2206 (AKT inhibitor) could rescue the impact of SGK1 on A549 cell apoptosis, while PD98059 (ERK1/2 inhibitor) could not further aggravate the impact. Taken together, our results suggest that SGK1 inhibits PM2.5-induced cell apoptosis and ROS generation via ERK1/2 and AKT signaling pathway in human lung alveolar epithelial A549 cells.

Combining early post-resuscitation EEG and HRV features improves the prognostic performance in cardiac arrest model of rats.

OBJECTIVE: Early and reliable prediction of neurological outcome remains a challenge for comatose survivors of cardiac arrest (CA). The purpose of this study was to evaluate the predictive ability of EEG, heart rate variability (HRV) features and the combination of them for outcome prognostication in CA model of rats. METHODS: Forty-eight male Sprague-Dawley rats were randomized into 6 groups (n=8 each) with different cause and duration of untreated arrest. Cardiopulmonary resuscitation was initiated after 5, 6 and 7min of ventricular fibrillation or 4, 6 and 8min of asphyxia. EEG and ECG were continuously recorded for 4h under normothermia after resuscitation. The relationships between features of early post-resuscitation EEG, HRV and 96-hour outcome were investigated. Prognostic performances were evaluated using the area under receiver operating characteristic curve (AUC). RESULTS: All of the animals were successfully resuscitated and 27 of them survived to 96h. Weighted-permutation entropy (WPE) and normalized high frequency (nHF) outperformed other EEG and HRV features for the prediction of survival. The AUC of WPE was markedly higher than that of nHF (0.892 vs. 0.759, p<0.001). The AUC was 0.954 when WPE and nHF were combined using a logistic regression model, which was significantly higher than the individual EEG (p=0.018) and HRV (p<0.001) features. CONCLUSIONS: Earlier post-resuscitation HRV provided prognostic information complementary to quantitative EEG in the CA model of rats. The combination of EEG and HRV features leads to improving performance of outcome prognostication compared to either EEG or HRV based features alone.

Noncoding RNAs in Muscle Atrophy.

Denervation, disuse, fasting, and various diseases could induce skeletal muscle atrophy, which results in the decline of life quality and increase of the mortality risk for patients. Noncoding RNAs (ncRNAs) are implicated important in regulating gene expression. Thus, ncRNAs, especially microRNAs and long noncoding RNAs (lncRNAs), have gained widespread attention as crucial players in numerous physiological and pathological processes, including skeletal muscle atrophy. In this review, we comprehensively described the potential of circulating microRNAs as biomarkers, summarized the profiling of microRNAs and lncRNAs in atrophying muscles, as well as discussed the effects and underlying mechanisms of microRNA machinery proteins, microRNAs, and lncRNAs in skeletal muscle atrophy. Considering the large quantity and variety of ncRNAs, the understanding of ncRNAs in muscle atrophy is still very limited. Future studies are needed to elucidate the possibility of ncRNAs as diagnosis biomarkers and therapeutic targets in muscle atrophy.