Correction: Single-cell in vivo imaging of cellular circadian oscillators in zebrafish.

[This corrects the article DOI: 10.1371/journal.pbio.3000435.].

Single-cell in vivo imaging of cellular circadian oscillators in zebrafish.

The circadian clock is a cell-autonomous time-keeping mechanism established gradually during embryonic development. Here, we generated a transgenic zebrafish line carrying a destabilized fluorescent protein driven by the promoter of a core clock gene, nr1d1, to report in vivo circadian rhythm at the single-cell level. By time-lapse imaging of this fish line and 3D reconstruction, we observed the sequential initiation of the reporter expression starting at photoreceptors in the pineal gland, then spreading to the cells in other brain regions at the single-cell level. Even within the pineal gland, we found heterogeneous onset of nr1d1 expression, in which each cell undergoes circadian oscillation superimposed over a cell type-specific developmental trajectory. Furthermore, we found that single-cell expression of nr1d1 showed synchronous circadian oscillation under a light-dark (LD) cycle. Remarkably, single-cell oscillations were dramatically dampened rather than desynchronized in animals raised under constant darkness, while the developmental trend still persists. It suggests that light exposure in early zebrafish embryos has significant effect on cellular circadian oscillations.

Danggui Buxue Tang: A Review of its Major Components.

Context: Danggui Buxue Tang (DBT) is a classical Chinese medicine that practitioners have used for thousands of years. Historically, those practitioners have used 16 prescriptions of DBT but currently are using only three prescriptions. Objective: The review intended to summarize pharmacological profiles of DBT and also clarify the major active chemicals found within it to provide a better understanding of the significance of DBT clinically. Design: The research team performed a narrative review by searching Pubmed databases. The search used the keywords Danggui Buxue Tang, bioactive chemcials, pharmacological functions. Setting: The databases setting were done by Gong Guowei and Zhou Xuan in the Zunyi Medical University, Zhuhai campus. Results: There are multiple results related to the crude fractions isolated from Danggui Buxue Tang, and also included the clinical trails. Conclusions: Thousands of years of clinical experience have ensured the efficacy of TCM treatments, which can determine the direction of basic research. That research can modify formulas at the molecular level to improve targeting and specificity in the treatment of specific diseases. As a result, the discovery and identification of new compounds within the herbal complex can provide useful research ideas and ensure the viability of new drug development.

Topography-Related EEG-fMRI in Surgically Confirmed Epileptic Foci: A Comparison to Spike-Related EEG-fMRI in Clinical Practice.

EEG-fMRI has gained increasing importance in epilepsy pre-surgical diagnosis. However, 40-70% of EEG-fMRI recordings in patients lack interictal epileptiform discharges (IEDs) during the scan, which could be overcome by detecting matching topography maps. We tried to validate this method in clinical settings taking various electroclinical factors into consideration. Eleven patients who had undergone EEG-fMRI during pre-surgical evaluation for drug-resistant epilepsy and who had had clinical long-term video-EEG were studied. Spike-related blood oxygen level-dependent (BOLD) maps were created using IEDs occurring during the EEG-fMRI scan. Separate maps were then generated from IEDs marked on the clinical long-term EEG recordings, which were averaged to produce topographical IED maps and correlated with the EEGs recorded inside the scanner yielding a correlation coefficient time course. Epileptogenic zones were defined by an expert panel during pre-surgical evaluation and validated by an epilepsy surgery resulting in a good outcome. Both techniques' performance was evaluated according to factors including arousal during IED recording, IED topography and lateralization, lesion type, and localization. Topography-related EEG-fMRI yielded more specific results compared to the spike-related method. Superficial lesion location and ipsilateral IED seem to result in a higher concordance of BOLD maps. The polarity of BOLD responses may be lesion-dependent, and both positive and negative BOLD changes may be associated with the irritative zone. Topography-related EEG-fMRI may show improved specificity especially for superficial lesions producing ipsilateral spikes. This method can be used as an alternative either in the absence of spikes during the simultaneous EEG-fMRI acquisition or to sharpen a diffusely activated BOLD-map.

Circadian genes period1b and period2 differentially regulate inflammatory responses in zebrafish.

The circadian clock has been shown to regulate various immune processes in different animals. Our previous report demonstrated that the innate immune responses in zebrafish show significant rhythmicity that could be regulated by melatonin. Here, we used diurnal zebrafish to determine the role of circadian genes in the inflammatory responses. Our results indicate that circadian genes exhibit rhythmic oscillations in zebrafish leukocytes, and mutations of the clock genes period1b (per1b) and period2 (per2) considerably affect these oscillations. Using a wounded zebrafish inflammation model, we found that under constant dark conditions (DD), the expression of pro-inflammatory cytokines is significantly downregulated in per1b gene mutant zebrafish and significantly upregulated in the per2 gene mutant zebrafish. Furthermore, using real-time imaging technology, we found that the per1b gene markedly disturbs the rhythmic recruitment of neutrophils toward the injury, whereas the per2 gene does not show a significant effect. Taken together, our results reveal differential functions of the circadian genes per1b and per2 in the inflammatory responses, serving as evidence that circadian rhythms play a vital role in immune processes.

Anti-deadzone adaptive fuzzy dynamic surface control for planar space robot with elastic base and flexible links.

In order to combat the impact of the dead zone and reduce vibration of the space robot's elastic base and flexible links, the trajectory tracking and vibration suppression of a multi-flexible-link free-floating space robot system are addressed. First, the elastic connection between the base and the link is considered as a linear spring. Then the assumed mode approach is used to derive the dynamic model of the flexible system. Secondly, a slow subsystem characterizing the rigid motion and a fast subsystem relating to vibration of the elastic base and multiple flexible links are generated utilizing two-time scale hypotheses of singular perturbation. For the slow subsystem with a dead zone in joint input torque, a dynamic surface control method with adaptive fuzzy approximator is designed. Dynamic surface control scheme is adopted to avoid calculation expansion and to simplify calculation. The fuzzy logic function is applied to approximate uncertain terms of the dynamic equation including the dead zone errors. For the fast subsystem, an optimal linear quadratic regulator controller is used to suppress the vibration of the multiple flexible links and elastic base, ensuring the stability and tracking accuracy of the system. Lastly, the simulation results verify the effectiveness of the proposed control strategy.

The RNA m6A-Binding Protein YTHDC1 Is Downregulated and Associated With M2 Macrophage Infiltration in Muscle-Invasive Bladder Cancer.

Background: Dysregulation of RNA N6-methyladenosine (m6A) modification is indispensable in tumorigenesis. However, in muscle-invasive bladder cancer (MIBC), the key regulators and mechanisms involved in this process remain largely unknown. This study aimed to screen the key m6A regulators and explore its possible role in MIBC. Methods: Aberrantly expressed m6A regulator genes were screened in The Cancer Genome Atlas (TCGA) MIBC cohort (n = 408) and validated using fresh-frozen and formalin-fixed paraffin-embedded (FFPE) specimens collected during this study. Clinicopathological relevance and association with tumor immune infiltration was further assessed. Results: We identified that the expression of YT521-B homology-domain-containing protein 1 (YTHDC1), an m6A RNA-binding protein, was downregulated in tumor tissues compared with adjacent noncancerous tissues in the TCGA MIBC cohort and our clinical samples. Low YTHDC1 expression correlated with short patient survival, advanced pathologic stage, lymph node metastasis, basal-squamous molecular subtype, non-papillary histological type, and certain genetic mutations important to MIBC. Remarkably, YTHDC1 expression exhibited negative association with tumor-infiltrating M2 macrophage abundance in MIBC. Conclusion: Among m6A regulators, we identified that YTHDC1 was downregulated in MIBC and might play an important role in the pathological process in MIBC, especially tumor microenvironment regulation.

Targeting integrin α5 in fibroblasts potentiates colorectal cancer response to PD-L1 blockade by affecting extracellular-matrix deposition.

BACKGROUND: One reason patients with cancer cannot benefit from immunotherapy is the lack of immune cell infiltration in tumor tissues. Cancer-associated fibroblasts (CAFs) are emerging as central players in immune regulation that shapes tumor microenvironment (TME). Earlier we reported that integrin α5 was enriched in CAFs in colorectal cancer (CRC), however, its role in TME and cancer immunotherapy remains unclear. Here, we aimed to investigate the role for integrin α5 in fibroblasts in modulating antitumor immunity and therapeutic efficacy combined with checkpoint blockade in CRC. METHODS: We analyzed the CRC single-cell RNA sequencing (scRNA-seq) database to define the expression of ITGA5 in CRC tumor stroma. Experimentally, we carried out in vivo mouse tumor xenograft models to confirm the targeting efficacy of combined α5ß1 inhibition and anti-Programmed death ligand 1 (PD-L1) blockade and in vitro cell-co-culture assay to investigate the role of α5 in fibroblasts in affecting T-cell activity. Clinically, we analyzed the association between α5 expression and infiltrating T cells and evaluated their correlation with patient survival and immunotherapy prognosis in CRC. RESULTS: We revealed that ITGA5 was enriched in FAP-CAFs. Both ITGA5 knockout fibroblasts and therapeutic targeting of α5 improved response to anti-PD-L1 treatment in mouse subcutaneous tumor models. Mechanistically, these treatments led to increased tumor-infiltrating CD8+ T cells. Furthermore, we found that α5 in fibroblasts correlated with extracellular matrix (ECM)-related genes and affected ECM deposition in CRC tumor stroma. Both in vivo analysis and in vitro culture and cell killing experiment showed that ECM proteins and α5 expression in fibroblasts influence T-cell infiltration and activity. Clinically, we confirmed that high α5 expression was associated with fewer CD3+ T and CD8+ T cells, and tissues with low α5 and high CD3+ T levels correlated with better patient survival and immunotherapy response in a CRC cohort with 29 patients. CONCLUSIONS: Our study identified a role for integrin α5 in fibroblasts in modulating antitumor immunity by affecting ECM deposition and showed therapeutic efficacy for combined α5ß1 inhibition and PD-L1 blockade in CRC.

Fecal microbiota transplantation improves VPA-induced ASD mice by modulating the serotonergic and glutamatergic synapse signaling pathways.

Autism spectrum disorder (ASD) is a complex behavioral disorder diagnosed by social interaction difficulties, restricted verbal communication, and repetitive behaviors. Fecal microbiota transplantation (FMT) is a safe and efficient strategy to adjust gut microbiota dysbiosis and improve ASD-related behavioral symptoms, but its regulatory mechanism is unknown. The impact of the microbiota and its functions on ASD development is urgently being investigated to develop new therapeutic strategies for ASD. We reconstituted the gut microbiota of a valproic acid (VPA)-induced autism mouse model through FMT and found that ASD is in part driven by specific gut dysbiosis and metabolite changes that are involved in the signaling of serotonergic synapse and glutamatergic synapse pathways, which might be associated with behavioral changes. Further analysis of the microbiota showed a profound decrease in the genera Bacteroides and Odoribacter, both of which likely contributed to the regulation of serotonergic and glutamatergic synapse metabolism in mice. The engraftment of Turicibacter and Alistipes was also positively correlated with the improvement in behavior after FMT. Our results suggested that successful transfer of the gut microbiota from healthy donors to ASD mice was sufficient to improve ASD-related behaviors. Modulation of gut dysbiosis by FMT could be an effective approach to improve ASD-related behaviors in patients.

Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis.

Two alternative cell-surface display systems were developed in Pichia pastoris using the alpha-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins alpha-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with alpha-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with alpha-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using alpha-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with alpha-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.

Strong static magnetic field delayed the early development of zebrafish.

One of the major topics in magnetobiology is the biological effects of strong static magnetic field (SMF) on living organisms. However, there has been a paucity of the comprehensive study of the long-term effects of strong SMF on an animal's development. Here, we explored this question with zebrafish, an excellent model organism for developmental study. In our research, zebrafish eggs, just after fertilization, were exposed to a 9.0 T SMF for 24 h, the critical period of post-fertilization development from cleavage to segmentation. The effects of strong SMF exposure on the following developmental progress of zebrafish were studied until 6 days post-fertilization (dpf). Results showed that 9.0 T SMF exposure did not influence the survival or the general developmental scenario of zebrafish embryos. However, it slowed down the developmental pace of the whole animal, and the late developers would catch up with their control peers after the SMF was removed. We proposed a mechanical model and deduced that the development delaying effect was caused by the interference of SMF in microtubule and spindle positioning during mitosis, especially in early cleavages. Our research data provide insights into how strong SMF influences the developing organisms through basic physical interactions with intracellular macromolecules.

Comparison of Resting-State Brain Activation Detected by BOLD, Blood Volume and Blood Flow.

Resting-state brain activity has been widely investigated using blood oxygenation level dependent (BOLD) contrast techniques. However, BOLD signal changes reflect a combination of the effects of cerebral blood flow (CBF), cerebral blood volume (CBV), as well as the cerebral metabolic rate of oxygen (CMRO2). In this study, resting-state brain activation was detected and compared using the following techniques: (a) BOLD, using a gradient-echo echo planar imaging (GE-EPI) sequence; (b) CBV-weighted signal, acquired using gradient and spin echo (GRASE) based vascular space occupancy (VASO); and (c) CBF, using pseudo-continuous arterial spin labeling (pCASL). Reliable brain networks were detected using VASO and ASL, including sensorimotor, auditory, primary visual, higher visual, default mode, salience and left/right executive control networks. Differences between the resting-state activation detected with ASL, VASO and BOLD could potentially be due to the different temporal signal-to-noise ratio (tSNR) and the short post-labeling delay (PLD) in ASL, along with differences in the spin-echo readout of VASO. It is also possible that the dynamics of spontaneous fluctuations in BOLD, CBV and CBF could differ due to biological reasons, according to their location within the brain.

Effects of circadian clock protein Per1b on zebrafish visual functions.

The circadian clock is an endogenous and entrainable time-keeping mechanism with a period of approximately 24 h, operated by transcription/translation feedback loops composed of circadian clock genes and their proteins. The visual system displays robust circadian changes. Relatively little, however, is known about the mechanisms underlying visual circadian rhythmicity. Zebrafish period1b (per1b), as a canonical circadian clock gene, is involved in circadian regulation. Here, we observed that zebrafish per1b mutants exhibit visual defects including reduced behavioral contrast sensitivity and significant retinal dopaminergic deficiency. Further, partially damaged dopaminergic interplexiform cells in wild-type larvae also led to reduced behavioral contrast sensitivity, while exogenous dopamine administration effectively restored the contrast sensitivity of per1b mutants. Taken together, these results suggest that retinal dopaminergic deficiency derived from loss of per1b results in visual defects in zebrafish. ABBREVIATIONS: per1b, period1b; per, period; per1, period1; per2, period2; per3, period3; ERG, electroretinogram; DA-IPCs, dopaminergic interplexiform cells; IRBP, interphotoreceptor retinoid binding protein; MS-222, methane-sulfonate; USTC, University of Science and Technology of China; OKR, optokinetic response; dpf, day postfertilization; 6-OHDA, 6-hydroxydopamine; TH, tyrosine hydroxylase; DA, dopaminergic; INL, inner nuclear; IPL, innerplexiform layers; hpf, hours postfertilization; cpd, cycle per degree; ADHD, attention deficit and hyperactivity disorder.

Zebrafish Lacking Circadian Gene per2 Exhibit Visual Function Deficiency.

The retina has an intrinsic circadian clock, but the importance of this clock for vision is unknown. Zebrafish offer many advantages for studying vertebrate vision and circadian rhythm. Here, we explored the role of zebrafish per2, a light-regulated gene, in visual behavior and the underlying mechanisms. We observed that per2 mutant zebrafish larvae showed decreased contrast sensitivity and visual acuity using optokinetic response (OKR) assays. Using a visual motor response (VMR) assay, we observed normal OFF responses but abnormal ON responses in mutant zebrafish larvae. Immunofluorescence showed that mutants had a normal morphology of cone photoreceptor cells and retinal organization. However, electron microscopy showed that per2 mutants displayed abnormal and decreased photoreceptor ribbon synapses with arciform density, which resulted in retinal ON pathway defect. We also examined the expression of three cone opsins by quantitative real-time PCR (qRT-PCR), and the expression of long-wave-sensitive opsin (opn1lw) and short-wave-sensitive opsin (opn1sw) was reduced in mutant zebrafish larvae. qRT-PCR analyses also showed a down-regulation of the clock genes cry1ba and bmal1b in the adult eye of per2 mutant zebrafish. This study identified a mechanism by which a clock gene affects visual function and defined important roles of per2 in retinal information processing.

Combining Partial Directed Coherence and Graph Theory to Analyse Effective Brain Networks of Different Mental Tasks.

PURPOSE: The aim of this study is to qualify the network properties of the brain networks between two different mental tasks (play task or rest task) in a healthy population. METHODS AND MATERIALS: EEG signals were recorded from 19 healthy subjects when performing different mental tasks. Partial directed coherence (PDC) analysis, based on Granger causality (GC), was used to assess the effective brain networks during the different mental tasks. Moreover, the network measures, including degree, degree distribution, local and global efficiency in delta, theta, alpha, and beta rhythms were calculated and analyzed. RESULTS: The local efficiency is higher in the beta frequency and lower in the theta frequency during play task whereas the global efficiency is higher in the theta frequency and lower in the beta frequency in the rest task. SIGNIFICANCE: This study reveals the network measures during different mental states and efficiency measures may be used as characteristic quantities for improvement in attentional performance.

Methods of forward feature selection based on the aggregation of classifiers generated by single attribute.

Compared to backward feature selection (BFS) method in gene expression data analysis, forward feature selection (FFS) method can obtain an expected feature subset with less iteration. However, the number of FFS method is considerably less than that of BFS method. More efficient FFS methods need to be developed. In this paper, two FFS methods based on the pruning of the classifier ensembles generated by single attribute are proposed for gene selection. The main contributions are as follows: (1) a new loss function, p-insensitive loss function, is proposed to overcome the disadvantage of the margin Euclidean distance loss function in the pruning of classifier ensembles; (2) two FFS methods based on the margin Euclidean distance loss function and the p-insensitive loss function, named as FFS-ACSA1 and FFS-ACSA2 respectively, are proposed; (3) the comparison experiments on four gene expression datasets show that FFS-ACSA2 obtains the best results among three FFS methods (i.e. signal-to-noise ratio (SNR), FFS-ACSA1 and FFS-ACSA2), and is competitive to the famous support vector machine-based recursive feature elimination (SVM-RFE), while FFS-ACSA1 is unstable.

Improving the computational efficiency of recursive cluster elimination for gene selection.

The gene expression data are usually provided with a large number of genes and a relatively small number of samples, which brings a lot of new challenges. Selecting those informative genes becomes the main issue in microarray data analysis. Recursive cluster elimination based on support vector machine (SVM-RCE) has shown the better classification accuracy on some microarray data sets than recursive feature elimination based on support vector machine (SVM-RFE). However, SVM-RCE is extremely time-consuming. In this paper, we propose an improved method of SVM-RCE called ISVM-RCE. ISVM-RCE first trains a SVM model with all clusters, then applies the infinite norm of weight coefficient vector in each cluster to score the cluster, finally eliminates the gene clusters with the lowest score. In addition, ISVM-RCE eliminates genes within the clusters instead of removing a cluster of genes when the number of clusters is small. We have tested ISVM-RCE on six gene expression data sets and compared their performances with SVM-RCE and linear-discriminant-analysis-based RFE (LDA-RFE). The experiment results on these data sets show that ISVM-RCE greatly reduces the time cost of SVM-RCE, meanwhile obtains comparable classification performance as SVM-RCE, while LDA-RFE is not stable.

[Yeast cell surface display and its application of enzymatic synthesis in non-aqueous phase].

Yeast surface display involves that the exogenous protein, which was fused with the yeast outer shell cell wall protein, was genetically anchored on the yeast cell surface. It has been widely used in expression and screening of functional protein. Here, we focused on the construction of lipase-displaying systems and its application in enzymatic biosynthesis, such as fatty acid methyl esters, short-chain flavour esters and sugar esters applications, and so on.

[Synthesis of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in non-aqueous phase].

An enzyme-displaying yeast as a whole-cell biocatalyst seemed an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, we tried to use a recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) to catalyze the synthesis of short chain flavor esters in n-heptane. We studied some major influential factors of esterification reactions, such as carbon chain length of the substrates, alcohol structure, enzyme concentration, substrates concentration, molar ratio of the substrates. The acid conversions were determined by titration and gas chromatography analysis. About ten kinds of esters were synthesized successfully, and the acid conversions of eight esters reached as high as 90% after reaction for 6 h. The result also indicated that ethanol and hexanoic acid were the most suitable substrates for this whole-cell catalyst. Under the optimal reaction conditions (the amount of lipase 20 g/L (306.0 U/g-dry cell), hexanoic acid concentration 0.8 mol/L, the molar ratio of hexanoic acid to ethanol 1:1.1), hexanoic acid conversion reached 97.3% after reaction for 1.5 h. To our knowledge, the CALB-displaying P. pastoris whole-cell biocatalyst showed good tolerance for high substrates concentration and exhibited high reaction rate on esterification of short chain flavor esters among the present enzyme/cell reported. Thus, CALB-displaying P pastoris whole-cell biocatalyst was promising in commercial application for flavor esters synthesis in non-aqueous phase.