Expanding the CRISPR Toolbox for Engineering Lycopene Biosynthesis in Corynebacterium glutamicum.

Lycopene represents one of the central compounds in the carotenoid pathway and it exhibits a potent antioxidant ability with wide potential applications in medicine, food, and cosmetics. The microbial production of lycopene has received increasing concern in recent years. Corynebacterium glutamicum (C. glutamicum) is considered to be a safe and beneficial industrial production platform, naturally endowed with the ability to produce lycopene. However, the scarcity of efficient genetic tools and the challenge of identifying crucial metabolic genes impede further research on C. glutamicum for achieving high-yield lycopene production. To address these challenges, a novel genetic editing toolkit, CRISPR/MAD7 system, was established and developed. By optimizing the promoter, ORI and PAM sequences, the CRISPR/MAD7 system facilitated highly efficient gene deletion and exhibited a broad spectrum of PAM sites. Notably, 25 kb of DNA from the genome was successfully deleted. In addition, the CRISPR/MAD7 system was effectively utilized in the metabolic engineering of C. glutamicum, allowing for the simultaneous knockout of crtEb and crtR genes in one step to enhance the accumulation of lycopene by blocking the branching pathway. Through screening crucial genes such as crtE, crtB, crtI, idsA, idi, and cg0722, an optimal carotenogenic gene combination was obtained. Particularly, cg0722, a membrane protein gene, was found to play a vital role in lycopene production. Therefore, the CBIEbR strain was obtained by overexpressing cg0722, crtB, and crtI while strategically blocking the by-products of the lycopene pathway. As a result, the final engineered strain produced lycopene at 405.02 mg/L (9.52 mg/g dry cell weight, DCW) in fed-batch fermentation, representing the highest reported lycopene yield in C. glutamicum to date. In this study, a powerful and precise genetic tool was used to engineer C. glutamicum for lycopene production. Through the modifications between the host cell and the carotenogenic pathway, the lycopene yield was stepwise improved by 102-fold as compared to the starting strain. This study highlights the usefulness of the CRISPR/MAD7 toolbox, demonstrating its practical applications in the metabolic engineering of industrially robust C. glutamicum.

Circ_0008410 contributes to fibroblast-like synoviocytes dysfunction by regulating miR-149-5p/HIPK2 axis.

Circular RNAs (circRNAs) play functional roles in rheumatoid arthritis (RA) progression. Fibroblast-like synoviocytes (RASFs) are the main effectors in RA development. In this study, we explored the function and mechanism of circ_0008410 in RASFs. qRT-PCR was used to detect the expression of circ_0008410, microRNA-149-5p (miR-149-5p), and homeodomain-interacting protein kinase 2 (HIPK2). Cell counting kit-8, EdU assay, flow cytometry, and transwell assay were performed to evaluate cell proliferation, apoptosis, migration, and invasion. Western blot measured the protein levels of related markers and HIPK2. The levels of IL-1ß, TNF-α, and IL-6 were tested by corresponding ELISA kits and Western blot. The combination between miR-149-5p and circ_0008410 or HIPK2 was detected by dual-luciferase reporter assay or RNA immunoprecipitation (RIP) assay. Our data showed that circ_0008410 and HIPK2 were elevated, while miR-149-5p was downregulated in RA synovial tissues and RASFs. Circ_0008410 promoted RASF proliferation, migration, invasion, and inflammation while inhibiting apoptosis. MiR-149-5p was a target of circ_0008410, and its overexpression could reverse the promoting effects of circ_0008410 on RASF dysfunction. Moreover, miR-149-5p could target HIPK2 to suppress RASF proliferation, migration, invasion, and inflammation. Collectively, circ_0008410 promoted RASF dysfunction via miR-149-5p/HIPK2, which might provide a potential target for RA therapy.

Employing machine learning using ferroptosis-related genes to construct a prognosis model for patients with osteosarcoma.

Identifying effective biomarkers in osteosarcoma (OS) is important for predicting prognosis. We investigated the prognostic value of ferroptosis-related genes (FRGs) in OS. Transcriptome and clinical data were obtained from The Cancer Genome Atlas and Gene Expression Omnibus. FRGs were obtained from the ferroptosis database. Univariate COX regression and LASSO regression screening were performed and an FRG-based prognostic model was constructed, which was validated using the Gene Expression Omnibus cohort. The predictive power of the model was assessed via a subgroup analysis. A nomogram was constructed using clinical markers with independent prognostic significance and risk score results. The CIBERSORT algorithm was used to detect the correlation between prognostic genes and 22 tumor-infiltrating lymphocytes. The expression of prognostic genes in erastin-treated OS cell lines was verified via real-time PCR. Six prognostic FRGs (ACSL5, ATF4, CBS, CDO1, SCD, and SLC3A2) were obtained and used to construct the risk prognosis model. Subjects were divided into high- and low-risk groups. Prognosis was worse in the high-risk group, and the model had satisfactory prediction performance for patients younger than 18 years, males, females, and those with non-metastatic disease. Univariate COX regression analysis showed that metastasis and risk score were independent risk factors for patients with OS. Nomogram was built on independent prognostic factors with superior predictive power and patient benefit. There was a significant correlation between prognostic genes and tumor immunity. Six prognostic genes were differentially expressed in ferroptosis inducer-treated OS cell lines. The identified prognostic genes can regulate tumor growth and progression by affecting the tumor microenvironment.

A cognitive neurogenetic approach to uncovering the structure of executive functions.

One central mission of cognitive neuroscience is to understand the ontology of complex cognitive functions. We addressed this question with a cognitive neurogenetic approach using a large-scale dataset of executive functions (EFs), whole-brain resting-state functional connectivity, and genetic polymorphisms. We found that the bifactor model with common and shifting-specific components not only was parsimonious but also showed maximal dissociations among the EF components at behavioral, neural, and genetic levels. In particular, the genes with enhanced expression in the middle frontal gyrus (MFG) and the subcallosal cingulate gyrus (SCG) showed enrichment for the common and shifting-specific component, respectively. Finally, High-dimensional mediation models further revealed that the functional connectivity patterns significantly mediated the genetic effect on the common EF component. Our study not only reveals insights into the ontology of EFs and their neurogenetic basis, but also provides useful tools to uncover the structure of complex constructs of human cognition.

Hippocampal Representations of Event Structure and Temporal Context during Episodic Temporal Order Memory.

The hippocampus plays an important role in representing spatial locations and sequences and in transforming representations. How these representational structures and operations support memory for the temporal order of random items is still poorly understood. We addressed this question by leveraging the method of loci, a powerful mnemonic strategy for temporal order memory that particularly recruits hippocampus-dependent computations of spatial locations and associations. Applying representational similarity analysis to functional magnetic resonance imaging activation patterns revealed that hippocampal subfields contained representations of multiple features of sequence structure, including spatial locations, location distance, and sequence boundaries, as well as episodic-like temporal context. Critically, the hippocampal CA1 exhibited spatial transformation of representational patterns, showing lower pattern similarity for items in same locations than closely matched different locations during retrieval, whereas the CA23DG exhibited sequential transformation of representational patterns, showing lower pattern similarity for items in near locations than in far locations during encoding. These transformations enabled the encoding of multiple items in the same location and disambiguation of adjacent items. Our results suggest that the hippocampus can flexibly reconfigure multiplexed event structure representations to support accurate temporal order memory.

Analysis of task-based functional MRI data preprocessed with fMRIPrep.

Functional magnetic resonance imaging (fMRI) is a standard tool to investigate the neural correlates of cognition. fMRI noninvasively measures brain activity, allowing identification of patterns evoked by tasks performed during scanning. Despite the long history of this technique, the idiosyncrasies of each dataset have led to the use of ad-hoc preprocessing protocols customized for nearly every different study. This approach is time consuming, error prone and unsuitable for combining datasets from many sources. Here we showcase fMRIPrep (http://fmriprep.org), a robust tool to prepare human fMRI data for statistical analysis. This software instrument addresses the reproducibility concerns of the established protocols for fMRI preprocessing. By leveraging the Brain Imaging Data Structure to standardize both the input datasets (MRI data as stored by the scanner) and the outputs (data ready for modeling and analysis), fMRIPrep is capable of preprocessing a diversity of datasets without manual intervention. In support of the growing popularity of fMRIPrep, this protocol describes how to integrate the tool in a task-based fMRI investigation workflow.

Retrieval practice facilitates memory updating by enhancing and differentiating medial prefrontal cortex representations.

Updating old memories with new, more current information is critical for human survival, yet the neural mechanisms for memory updating in general and the effect of retrieval practice in particular are poorly understood. Using a three-day A-B/A-C memory updating paradigm, we found that compared to restudy, retrieval practice could strengthen new A-C memories and reduce old A-B memory intrusion, but did not suppress A-B memories. Neural activation pattern analysis revealed that compared to restudy, retrieval practice led to stronger target representation in the medial prefrontal cortex (MPFC) during the final test. Critically, it was only under the retrieval practice condition that the MPFC showed strong and comparable competitor evidence for both correct and incorrect trials during final test, and that the MPFC target representation during updating was predictive of subsequent memory. These results suggest that retrieval practice is able to facilitate memory updating by strongly engaging MPFC mechanisms in memory integration, differentiation and consolidation.

Individual-specific and shared representations during episodic memory encoding and retrieval.

Although human memories seem unique to each individual, they are shared to a great extent across individuals. Previous studies have examined, separately, subject-specific and cross-subject shared representations during memory encoding and retrieval, but how shared memories are formed from individually encoded representations is not clearly understood. Using a unique fMRI design involving memory encoding and retrieval, and representational similarity analysis to link representations from different individuals, brain regions, and processing stages, the current study revealed that distributed brain regions showed both subject-specific and shared neural representations during both memory encoding and retrieval. Furthermore, different brain regions showed stage-specific representational strength, with the visual cortex showing greater unique and shared representations during encoding, whereas the left angular gyrus showing greater unique and shared representations during retrieval. The neural representations during encoding were transformed during retrieval, as shown by smaller cross-subject encoding-retrieval similarity (ERS) than cross-subject similarity either during encoding or during retrieval. This cross-subject and cross-stage similarity was found both within and across regions, with strong pattern similarity between the encoded representation in VVC and the retrieved representation in the angular gyrus. Simulation analysis further suggested that these patterns could be achieved by incorporating stage-specific representational strength, and cross-region reinstatement from encoding to retrieval, but not by a common transformation from encoding to retrieval across subjects. Together, our results shed light on how memory representations are encoded and transformed to maintain individual characteristics and at the same time to create shared representations to facilitate interpersonal communication.

Partitioning heritability analyses unveil the genetic architecture of human brain multidimensional functional connectivity patterns.

Resting-state functional connectivity profiles have been increasingly shown to be important endophenotypes that are tightly linked to human cognitive functions and psychiatric diseases, yet the genetic architecture of this multidimensional trait is barely understood. Using a unique sample of 1,704 unrelated, young and healthy Chinese Han individuals, we revealed a significant heritability of functional connectivity patterns in the whole brain and several subnetworks. We further proposed a partitioned heritability analysis for multidimensional functional connectivity patterns, which revealed the common and unique enrichment patterns of the genetic contributions to brain connectivity patterns for several gene sets linked to brain functions, including the genes expressed preferentially in the central nervous system and those associated with intelligence, educational attainment, attention-deficit/hyperactivity disorder, and schizophrenia. These results for the first time reveal the genetic architecture of multidimensional brain connectivity patterns across different networks and advance our understanding of the complex relationship between gene sets, neural networks, and behaviors.

Accurate location and minimally invasive treatment of lumbar lateral recess stenosis with combined SNRB and PTED.

OBJECTIVE: To establish a management strategy for multi-segment lumbar lateral recess stenosis. METHODS: A retrospective study was performed in patients in whom suspected responsible nerve roots underwent sequential selective nerve root block (SNRB). Based on pain remission rate after blocking, the contribution of nerve root compression to symptoms was classified as absolutely (≥70%) or relatively (30-70%) responsible or non-responsible (<30%). Conservative treatment was continued if visual analogue scale (VAS) at 3 days after blocking a single nerve root or VAS at 3 days after blocking multiple nerve roots was ≥50%; otherwise, percutaneous transforaminal endoscopic discectomy (PTED) was performed. Pain and functional scores were evaluated on day 3, 6 months and 1 year after SNRB or PTED. RESULTS: Fifty-seven of 80 patients had a single absolutely responsible root, 20 had 2 responsible roots, and 3 had 3 responsible roots. Among them, 41, 10, and 1 patient underwent PTED, respectively. Both the PTED and conservative groups improved significantly in VAS remission rate and functional scores compared with admission. Moreover, the PTED group had a better VAS remission rate compared with the conservative group. CONCLUSION: A combination of SNRB with PTED was effective for diagnosing and treating multi-segment lumbar lateral recess stenosis.

Spaced Learning Enhances Episodic Memory by Increasing Neural Pattern Similarity Across Repetitions.

Spaced learning has been shown consistently to benefit memory compared with massed learning, yet the neural representations and processes underlying the spacing effect are still poorly understood. In particular, two influential models (i.e., the encoding variability hypothesis and the study-phase retrieval hypothesis) could both model behavioral performance very well, but they make opposite hypotheses regarding the spacing effect's neural mechanisms. The present study attempted to provide empirical neural evidence to adjudicate these competing hypotheses. Using spatiotemporal pattern similarity (STPS) analysis of EEG data, this study investigated whether and how repetition lags (massed/short-spaced/long-spaced) modulated the STPS's contribution to episodic memory encoding in male and female human participants. The results revealed that greater item-specific STPS in the right frontal electrodes at 543-727 ms after stimulus onset was associated with better memory performance. More importantly, this STPS was larger under the spaced-learning condition than the massed-learning condition and partially mediated the spacing effect on memory performance. In addition, we found that massed learning was associated with stronger repetition suppression in the N400 component that reflected momentary retrieval strength, but reduced activity in the late positive component that was associated with memory retrieval. These results suggest that spaced learning improves long-term memory by increasing retrieval effort and enhancing the pattern reinstatement of prior neural representations, which may be achieved by reducing the momentary retrieval strength as the extended repetition lags might help to eliminate the residual representation in working memory.SIGNIFICANCE STATEMENT As one of the most ubiquitous and fundamental phenomena in the history of memory research, the spacing effect provides an important window into understanding how enduring memory is formed in the brain and how different practice strategies could modulate these mechanisms to affect memory performance. By leveraging the neural representational analysis on scalp EEG data, the current study provides the first empirical data to show that spaced learning enhances memory by improving the spatiotemporal similarity that occurs at a late time window. Our results support the study-phase retrieval hypothesis but not the encoding variability hypothesis and emphasize the role of neural pattern reinstatement in strengthening memory via repeated study.

Multiple interactive memory representations underlie the induction of false memory.

Theoretical and computational models such as transfer-appropriate processing (TAP) and global matching models have emphasized the encoding-retrieval interaction of memory representations in generating false memories, but relevant neural mechanisms are still poorly understood. By manipulating the sensory modalities (visual and auditory) at different processing stages (learning and test) in the Deese-Roediger-McDermott task, we found that the auditory-learning visual-test (AV) group produced more false memories (59%) than the other three groups (42∼44%) [i.e., visual learning visual test (VV), auditory learning auditory test (AA), and visual learning auditory test (VA)]. Functional imaging results showed that the AV group's proneness to false memories was associated with (i) reduced representational match between the tested item and all studied items in the visual cortex, (ii) weakened prefrontal monitoring process due to the reliance on frontal memory signal for both targets and lures, and (iii) enhanced neural similarity for semantically related words in the temporal pole as a result of auditory learning. These results are consistent with the predictions based on the TAP and global matching models and highlight the complex interactions of representations during encoding and retrieval in distributed brain regions that contribute to false memories.

Reduced Fidelity of Neural Representation Underlies Episodic Memory Decline in Normal Aging.

Emerging studies have emphasized the importance of the fidelity of cortical representation in forming enduring episodic memory. No study, however, has examined whether there are age-related reductions in representation fidelity that can explain memory declines in normal aging. Using functional MRI and multivariate pattern analysis, we found that older adults showed reduced representation fidelity in the visual cortex, which accounted for their decreased memory performance even after controlling for the contribution of reduced activation level. This reduced fidelity was specifically due to older adults' poorer item-specific representation, not due to their lower activation level and variance, greater variability in neuro-vascular coupling, or decreased selectivity of categorical representation (i.e., dedifferentiation). Older adults also showed an enhanced subsequent memory effect in the prefrontal cortex based on activation level, and their prefrontal activation was associated with greater fidelity of representation in the visual cortex and better memory performance. The fidelity of cortical representation thus may serve as a promising neural index for better mechanistic understanding of the memory declines and its compensation in normal aging.

Neural Global Pattern Similarity Underlies True and False Memories.

UNLABELLED: The neural processes giving rise to human memory strength signals remain poorly understood. Inspired by formal computational models that posit a central role of global matching in memory strength, we tested a novel hypothesis that the strengths of both true and false memories arise from the global similarity of an item's neural activation pattern during retrieval to that of all the studied items during encoding (i.e., the encoding-retrieval neural global pattern similarity [ER-nGPS]). We revealed multiple ER-nGPS signals that carried distinct information and contributed differentially to true and false memories: Whereas the ER-nGPS in the parietal regions reflected semantic similarity and was scaled with the recognition strengths of both true and false memories, ER-nGPS in the visual cortex contributed solely to true memory. Moreover, ER-nGPS differences between the parietal and visual cortices were correlated with frontal monitoring processes. By combining computational and neuroimaging approaches, our results advance a mechanistic understanding of memory strength in recognition. SIGNIFICANCE STATEMENT: What neural processes give rise to memory strength signals, and lead to our conscious feelings of familiarity? Using fMRI, we found that the memory strength of a given item depends not only on how it was encoded during learning, but also on the similarity of its neural representation with other studied items. The global neural matching signal, mainly in the parietal lobule, could account for the memory strengths of both studied and unstudied items. Interestingly, a different global matching signal, originated from the visual cortex, could distinguish true from false memories. The findings reveal multiple neural mechanisms underlying the memory strengths of events registered in the brain.