Dual genetic level modification engineering accelerate genome evolution of Corynebacterium glutamicum.

High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the 'dock' of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.

miR-4645-3p attenuates podocyte injury and mitochondrial dysfunction in diabetic kidney disease by targeting Cdk5.

Podocyte injury plays a critical role in the progression of diabetic kidney disease (DKD), but the underlying cellular and molecular mechanisms remain poorly understanding. MicroRNAs (miRNAs) can disrupt gene expression by inducing translation inhibition and mRNA degradation, and recent evidence has shown that miRNAs may play a key role in many kidney diseases. In this study, we identified miR-4645-3p by global transcriptome expression profiling as one of the major downregulated miRNAs in high glucose-cultured podocytes. Moreover, whether DKD patients or STZ-induced diabetic mice, expression of miR-4645-3p was also significantly decreased in kidney. In the podocytes cultured by normal glucose, inhibition of miR-4645-3p expression promoted mitochondrial damage and podocyte apoptosis. In the podocytes cultured by high glucose (30 mM glucose), overexpression of miR-4645-3p significantly attenuated mitochondrial dysfunction and podocyte apoptosis induced by high glucose. Furthermore, we found that miR-4645-3p exerted protective roles by targeting Cdk5 inhibition. In vitro, miR-4645-3p obviously antagonized podocyte injury by inhibiting overexpression of Cdk5. In vivo of diabetic mice, podocyte injury, proteinuria, and impaired renal function were all effectively ameliorated by treatment with exogenous miR-4645-3p. Collectively, these findings demonstrate that miR-4645-3p can attenuate podocyte injury and mitochondrial dysfunction in DKD by targeting Cdk5. Sustaining the expression of miR-4645-3p in podocytes may be a novel strategy to treat DKD.

Low-intensity transcranial ultrasound stimulation improves memory in vascular dementia by enhancing neuronal activity and promoting spine formation.

Memory is closely associated with neuronal activity and dendritic spine formation. Low-intensity transcranial ultrasound stimulation (TUS) improves the memory of individuals with vascular dementia (VD). However, it is unclear whether neuronal activity and dendritic spine formation under ultrasound stimulation are involved in memory improvement in VD. In this study, we found that seven days of TUS improved memory in VD model while simultaneously increasing pyramidal neuron activity, promoting dendritic spine formation, and reducing dendritic spine elimination. These effects lasted for 7 days but disappeared on 14 d after TUS. Neuronal activity and dendritic spine formation strongly corresponded to improvements in memory behavior over time. In addition, we also found that the memory, neuronal activity and dendritic spine of VD mice cannot be restored again by TUS of 7 days after 28 d. Collectively, these findings suggest that TUS increases neuronal activity and promotes dendritic spine formation and is thus important for improving memory in patients with VD.

An Emulated Clinical Trial of Deprescribing Proton Pump Inhibitors in Patients With Cirrhosis.

INTRODUCTION: Proton pump inhibitors (PPI) are overused and carry harms in cirrhosis. Deprescribing is advocated but has not been trialed. METHODS AND FINDINGS: We emulated a clinical trial using Medicare data. All patients were receiving chronic PPI therapy before a compensated cirrhosis diagnosis. We compared the risk death/decompensation over 3 years between continuous users and deprescribers. We find that PPI deprescription is associated with less ascites and that cumulative PPI use is associated with more ascites and encephalopathy. Ultimately, 71% of deprescribers restart PPIs. DISCUSSION: PPI deprescribing has benefits but requires ongoing support and alternative therapies for gastrointestinal symptoms.

Miniature two-photon microscopy for enlarged field-of-view, multi-plane and long-term brain imaging.

We have developed a miniature two-photon microscope equipped with an axial scanning mechanism and a long-working-distance miniature objective to enable multi-plane imaging over a volume of 420 × 420 × 180 µm3 at a lateral resolution of ~1 µm. Together with the detachable design that permits long-term recurring imaging, our miniature two-photon microscope can help decipher neuronal mechanisms in freely behaving animals.

Low-complexity frequency domain frame synchronization for short-reach IM/DD optical fiber transmission systems.

We propose a low-complexity frequency domain frame synchronization method for short-reach intensity modulation and direct detection (IM/DD) systems. A four-level pulse amplitude modulation-training sequence (PAM4-TS) is specially designed for the proposed method, which has an obvious peak in the amplitude spectrum that is higher than the normal signal. The proposed method comprises a coarse synchronization stage and a fine synchronization stage. Firstly, the coarse synchronization stage takes advantage of the feature of PAM4-TS to obtain the approximate position of the frame head by identifying the peak value in amplitude spectrum of the segmented received signal. Then, the fine synchronization stage calculates the correlation between the coarse synchronization result and PAM4-TS by multiplying the two in the frequency domain. Compared with the traditional sliding window correlation method realized in the time domain, both simulation and experimental results of C-band 50 Gbit/s PAM4 transmission demonstrate that the proposed method reduces the multiplication complexity by up to about 96.01% without any additional performance penalty.

Is Phenotypic Evolution Affected by Spiders' Construction Behaviors?

What happens when organisms actively modify their environment? Clarifying the role of construction behavior on a macroevolutionary scale is crucial to interpreting phenotypic evolution. Spiders, an extremely successful group of animals exhibiting a wide variety of functional, morphological, and behavioral diversity, are ideal candidates to test whether animal behaviors are related to their phenotypic evolution. Herein, we reconstructed the phylogenomic relationships of 303 spiders covering 105 families with 99 newly developed molecular markers that universally apply across Araneae, and explicitly tested the potential link between construction behavior and somatic evolution based on extensive morphological data from 3,342 extant species and 682 fossil species. Our dated molecular phylogeny provides the backbone for analyses, revealing the behavioral and ecological processes behind these spiders' morphological adaptations. Evolutionary model analyses showed the artifacts constructed by spiders, especially the suspending webs, expand the spider's ability to inhabit different habitats. These artifacts have more substantial impacts on their somatic traits than habitats and promote different trajectories of morphological evolution. Specifically, for spiders, silk-lined burrowing produced larger bodies, relatively shorter legs, and longer patellae, while web-building produced smaller bodies, relatively longer legs, and shorter patellae, and hunting promoted an intermediate morphological size. Molecular convergence analysis showed that genes related to morphogenesis or response to stimulus and stress are enriched in spiders with construction behavior. Our study demonstrated that the construction behavior of an animal plays a crucial role in determining the direction and intensity of the selection pressure imposed on it and provides strong evidence that construction behaviors are associated with phenotypic evolution on macroevolutionary timescales. [Araneae; body size; habitat change; molecular marker; leg length; phylogenomics.].

Swertiamarin relieves radiation-induced intestinal injury by limiting DNA damage.

Radiotherapy is the conventional treatment for pelvic abdominal tumors. However, it can cause some damage to the small intestine and colorectal, which are very sensitive to radiation. Radiation-induced intestinal injury (RIII) affects the prognosis of radiotherapy, causing sequelae of loss of function and long-term damage to patients' quality of life. Swertiamarin is a glycoside that has been reported to prevent a variety of diseases including but not limited to diabetes, hypertension, atherosclerosis, arthritis, malaria, and abdominal ulcers. However, its therapeutic effect and mechanism of action on RIII have not been established. We investigated whether swertiamarin has a protective effect against RIII. In this article, we use irradiator to create cellular and mouse models of radiation damage. Preventive administration of swertiamarin could reduce ROS and superoxide anion levels to mitigate the cellular damage caused by radiation. Swertiamarin also attenuated RIII in mice, as evidenced by longer survival, less weight loss and more complete intestinal barrier. We also found an increase in the relative abundance of primary bile acids in irradiated mice, which was reduced by both FXR agonists and swertiamarin, and a reduction in downstream interferon and inflammatory factors via the cGAS-STING pathway to reduce radiation-induced damage.

Identification and characterization of an L-type lectin from obscure puffer Takifugu obscurus in response to bacterial infection.

L-type lectins (LTLs) contain a carbohydrate recognition domain homologous to leguminous lectins, and have functions in selective protein trafficking, sorting and targeting in the secretory pathway of animals. In this study, a novel LTL, designated as ToERGIC-53, was cloned and identified from obscure puffer Takifugu obscurus. The open reading frame of ToERGIC-53 contained 1554 nucleotides encoding 517 amino acid residues. The deduced ToERGIC-53 protein consisted of a signal peptide, a leguminous lectin domain (LTLD), a coiled-coil region, and a transmembrane region. Quantitative real-time PCR showed that ToERGIC-53 was expressed in all examined tissues, with the highest expression level in the liver. The expression of ToERGIC-53 was significantly upregulated after infection with Vibrio harveyi and Staphylococcus aureus. Recombinant ToERGIC-53-LTLD (rToERGIC-53-LTLD) protein could not only agglutinate and bind to one Gram-positive bacterium (S. aureus) and three Gram-negative bacteria (V. harveyi, V. parahaemolyticus and Aeromonas hydrophila), but also bind to glycoconjugates on the surface of bacteria such as lipopolysaccharide, peptidoglycan, mannose and galactose. In addition, rToERGIC-53-LTLD inhibited the growth of bacteria in vitro. All these results suggested that ToERGIC-53 might be a pattern recognition receptor involved in antibacterial immune response of T. obscurus.

Differential molecular responses of hemolymph and hepatopancreas of swimming crab, Portunus trituberculatus, infected with Ameson portunus (Microsporidia).

Ameson portunus (Microsporidia) has caused serious economic losses to the aquaculture industry of swimming crab, Portunus trituberculatus. The hemolymph and hepatopancreas are the main immune organs of P. trituberculatus, and the main sites of A. portunus infection. Elucidating the response characteristics of hemolymph and hepatopancreas to microsporidian infection facilitates the development of microsporidiosis prevention and control strategy. This study performed comparative transcriptomic analysis of hemolymph (PTX/PTXA) and hepatopancreas (PTG/PTGA) of P. trituberculatus uninfected and infected with A. portunus. The results showed that there were 223 and 1309 differentially expressed genes (DEGs) in PTX/PTXA and PTG/PTGA, respectively. The lysosome pathway was significantly enriched after the invasion of the hemolymph by A. portunus. Also, immune-related genes were all significantly up-regulated in the hemolymph and hepatopancreas, suggesting that the invasion by A. portunus may activate host immune responses. Unlike hemolymph, antioxidant and detoxification-related genes were also significantly up-regulated in the hepatopancreas. Moreover, metabolism-related genes were significantly down-regulated in the hepatopancreas, suggesting that energy synthesis, resistance to pathogens, and regulation of oxidative stress were suppressed in the hepatopancreas. Hemolymph and hepatopancreas have similarity and tissue specificity to microsporidian infection. The differential genes and pathways identified in this study can provide references for the prevention and control of microsporidiosis.

Characterization of the Ictalurid herpesvirus 1 immediate-early gene ORF24 and its potential role in transcriptional regulation in yeast.

Herpesviruses adhere to a precise temporal expression model in which immediate-early (IE) genes play a crucial role in regulating the viral life cycle. However, there is a lack of functional research on the IE genes in Ictalurid herpesvirus 1 (IcHV-1). In this study, we identified the IcHV-1 ORF24 as an IE gene via a metabolic inhibition assay, and subcellular analysis indicated its predominant localisation in the nucleus. To investigate its function, we performed yeast reporter assays using an ORF24 fusion protein containing the Gal4-BD domain and found that BD-ORF24 was able to activate HIS3/lacZ reporter genes without the Gal4-AD domain. Our findings provide concrete evidence that ORF24 is indeed an IE gene that likely functions as a transcriptional regulator during IcHV-1 infection. This work contributes to our understanding of the molecular mechanisms underlying fish herpesvirus IE gene expression.

Advantages of transformer and its application for medical image segmentation: a survey.

PURPOSE: Convolution operator-based neural networks have shown great success in medical image segmentation over the past decade. The U-shaped network with a codec structure is one of the most widely used models. Transformer, a technology used in natural language processing, can capture long-distance dependencies and has been applied in Vision Transformer to achieve state-of-the-art performance on image classification tasks. Recently, researchers have extended transformer to medical image segmentation tasks, resulting in good models. METHODS: This review comprises publications selected through a Web of Science search. We focused on papers published since 2018 that applied the transformer architecture to medical image segmentation. We conducted a systematic analysis of these studies and summarized the results. RESULTS: To better comprehend the benefits of convolutional neural networks and transformers, the construction of the codec and transformer modules is first explained. Second, the medical image segmentation model based on transformer is summarized. The typically used assessment markers for medical image segmentation tasks are then listed. Finally, a large number of medical segmentation datasets are described. CONCLUSION: Even if there is a pure transformer model without any convolution operator, the sample size of medical picture segmentation still restricts the growth of the transformer, even though it can be relieved by a pretraining model. More often than not, researchers are still designing models using transformer and convolution operators.

Effect of lanthanum nitrate on adipogenesis in mice.

Lanthanum (La) is widely used in modern industry and agriculture because of its unique physicochemical properties and is broadly exposed in the population. Some studies have shown that La may have some effects on adipogenesis, but there is a lack of related in vivo evidence. In this study, the effects of La(NO3 )3 on adipogenesis and its associated mechanism were studied using C57BL/6J mouse model. The results showed that La(NO3 )3 exposure caused a decrease in body weight and the percentage of fat content in mice. In addition, the adipose marker molecules and specific adipogenic transcription factors decreased in both white adipose tissue (WAT) and brown adipose tissue (BAT). Detection of signaling pathway-related molecules revealed that canonical wnt/ß-catenin pathway-related molecules were upregulated in both adipose tissues. In summary, in vivo exposure to La(NO3 )3 might inhibited adipogenesis in mice, possibly through upregulation of the canonical Wnt/ß-catenin signaling pathway.

Insights into the differential molecular response of non-germinated and germinated spores of Ameson portunus in vitro by comparative transcriptome analysis.

Ameson portunus, the recently discovered causative agent of "toothpaste disease" of pond-cultured swimming crabs in China has caused enormous economic losses in aquaculture. Understanding the process of spore germination is helpful to elucidate the molecular mechanism of its invasion of host cells. Here, we obtained mature and germinating spores by isolation and purification and in vitro stimulation, respectively. Then, non-germinated and germinated spores were subjected to the comparative transcriptomic analysis to disclose differential molecular responses of these two stages. The highest germination rate, i.e., 71.45 %, was achieved in 0.01 mol/L KOH germination solution. There were 9,609 significantly differentially expressed genes (DEGs), with 685 up-regulated and 8,924 down-regulated DEGs. The up-regulated genes were significantly enriched in ribosome pathway, and the down-regulated genes were significantly enriched in various metabolic pathways, including carbohydrate metabolism, amino acid metabolism and other metabolism. The results suggested that spores require various carbohydrates and amino acids as energy to support their life activities during germination and synthesize large amounts of ribosomal proteins to provide sites for DNA replication, transcription, translation and protein synthesis of the spores of A. portunus within the host cells. Functional genes related to spore germination, such as protein phosphatase CheZ and aquaporin, were also analyzed. The analysis of transcriptome data and identification of functional genes will help to understand the process of spore germination and invasion.

Role of TRAF6 from obscure puffer (Takifugu obscurus) in immune response against Edwardsiella tarda infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a vital molecule of inflammatory signaling pathways in innate immune response against pathogens. To elucidate its role in defense against Edwardsiella tarda infection in teleost fish, TRAF6 homologue was identified from obscure puffer (Takifugu obscurus) and functionally analyzed in this study. The obscure puffer TRAF6 (ToTRAF6) is a protein of 565 amino acids containing conserved RING domain, zinc finger-TRAF and MATH_TRAF6 domain. ToTRAF6 mRNA distributed in various healthy tissues of obscure puffer and was upregulated in the immune related tissues after E. tarda infection. ToTRAF6 protein was localized in the cytoplasm and aggregate as dots around the nuclei in FHM cells. The overexpression of ToTRAF6 in FHM cells decreased the quantity of E. tarda and induced the significant upregulation of downstream MAPK signaling pathway genes. These data suggest that ToTRAF6 is a key molecule of MAPK signaling pathway in defense against E. tarda infection.

A portable intelligent hydrogel platform for multicolor visual detection of HAase.

Hyaluronidase (HAase) is an important endoglycosidase involved in numerous physiological and pathological processes, such as apoptosis, senescence, and cancer progression. Simple, convenient, and sensitive detection of HAase is important for clinical diagnosis. Herein, an easy-to-operate multicolor visual sensing strategy was developed for HAase determination. The proposed sensor was composed of an enzyme-responsive hydrogel and a nanochromogenic system (gold nanobipyramids (AuNBPs)). The enzyme-responsive hydrogel, formed by polyethyleneimine-hyaluronic acid (PEI-HA), was specifically hydrolyzed with HAase, leading to the release of platinum nanoparticles (PtNPs). Subsequently, PtNPs catalyzed the mixed system of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 to produce TMB2+ under acidic conditions. Then, TMB2+ effectively etched the AuNBPs and resulted in morphological changes in the AuNBPs, accompanied by a blueshift in the localized surface plasmon resonance peak and vibrant colors. Therefore, HAase can be semiquantitatively determined by directly observing the color change of AuNBPs with the naked eye. On the basis of this, the method has a linear detection range of HAase concentrations between 0.6 and 40 U/mL, with a detection limit of 0.3 U/mL. In addition, our designed multicolor biosensor successfully detected the concentration of HAase in human serum samples. The results showed no obvious difference between this method and enzyme-linked immunosorbent assay, indicating the good accuracy and usability of the suggested method.

Miniature Two-Photon Microscopic Imaging Using Dielectric Metalens.

Miniature two-photon microscopy has emerged as a powerful technique for investigating brain activity in freely moving animals. Ongoing research objectives include reducing probe weight and minimizing animal behavior constraints caused by probe attachment. Employing dielectric metalenses, which enable the use of sizable optical components in flat device structures while maintaining imaging resolution, is a promising solution for addressing these challenges. In this study, we designed and fabricated a titanium dioxide metalens with a wavelength of 920 nm and a high aspect ratio. Furthermore, a meta-optic two-photon microscope weighing 1.36 g was developed. This meta-optic probe has a lateral resolution of 0.92 µm and an axial resolution of 18.08 µm. Experimentally, two-photon imaging of mouse brain structures in vivo was also demonstrated. The flat dielectric metalens technique holds promising opportunities for high-performance integrated miniature nonlinear microscopy and endomicroscopy platforms in the biomedical field.

In vivo evaluation of T-Scan in quantifying occlusal contact.

BACKGROUND: The T-scan system has been used previously to analyse occlusion, but the quantitative analysis of occlusal contact by T-Scan system has yet to be reported. OBJECTIVES: To evaluate the reliability and validity of T-Scan system for quantitatively measuring occlusal contact area and occlusal contact number. METHODS: Twenty-two individuals with normal occlusion, 11 men and 11 women, were recruited for the study. Two occlusal analysis methods, including silicone transmission analysis method (STA) and T-Scan occlusion analysis method (TSO), were used to make quantitative analysis to measure occlusal contact area (OCA) and occlusal contact number (OCN). A test-retest check was performed with an interval of 2 weeks. The values of intraclass correlation coefficients (ICC) between test-retest of each method were calculated for reliability evaluation. Pearson correlations analysis, paired t-tests, regression analysis and Bland-Altman analysis were performed for validity evaluation. RESULTS: The ICC values of STA were greater than those of TSO for OCA while for OCN, ICC values of TSO were greater than STA. The higher OCA and OCN values were found in TSO compared with STA. Pearson's correlation coefficient indicated strong relations between TSO and STA (0.730-0.812) for OCA, while good relations between then (0.569-0.583) for OCN. Paired t-test showed a significant difference between the OCA and OCN values between TSO and STA. Bland-Altman analysis showed good agreement between OCA and OCN values of TSO and STA both in men and women. Regression analysis identified a linear correlation between OCA values obtained from these two methods. CONCLUSIONS: T-Scan method showed strong reliability for measuring OCA and OCN quantitatively. Strong correlations were found between OCA values from TSO and STA method, but the validity of TSO for measuring OCN needs to be promoted. CLINICAL SIGNIFICANCE: T-Scan system demonstrates good potential in quantitative analysis of occlusion, which will expand its clinical application.

Genome-Wide Identification, Expression and Response to Estrogen of Vitellogenin Gene Family in Sichuan Bream (Sinibrama taeniatus).

To enhance our understanding of teleost reproductive physiology, we identified six Sichuan bream (Sinibrama taeniatus) vitellogenin genes (vtg1-6) and characterized their sequence structures. We categorized them into type Ⅰ (vtg1,4,5 and 6), type Ⅱ (vtg2) and type Ⅲ (vtg3) based on differences in their subdomain structure. The promoter sequence of vtgs has multiple estrogen response elements, and their abundance appears to correlate with the responsiveness of vtg gene expression to estrogen. Gene expression analyses revealed that the vitellogenesis of Sichuan bream involves both heterosynthesis and autosynthesis pathways, with the dominant pathway originating from the liver. The drug treatment experiments revealed that 17ß-estradiol (E2) tightly regulated the level of vtg mRNA in the liver. Feeding fish with a diet containing 100 µg/g E2 for three weeks significantly induced vtg gene expression and ovarian development, leading to an earlier onset of vitellogenesis. Additionally, it was observed that the initiation of vtg transcription required E2 binding to its receptor, a process primarily mediated by estrogen receptor alpha in Sichuan bream. The findings of this study provide novel insights into the molecular information of the vitellogenin gene family in teleosts, thereby contributing to the regulation of gonadal development in farmed fish.

[Identification of a novel variant in a patient with Calsequestrin 1 related myopathy].

OBJECTIVE: To explore the genetic basis of a myopathic patient with pathological characteristics including tubular aggregates and vacuoles. METHODS: Next generation sequencing was carried out for the patient, and candidate variant was verified by Sanger sequencing. RESULTS: Genetic testing revealed that the patient has harbored a heterozygous c.730G>C (p.D244H) variant of Calsequestrin 1 (CASQ1) gene. The same variant was not found in his unaffected parents. Based on guidelines from the American College of Medical Genetics and Genomics, the variant was rated as pathogenic (PS1+PM2+PP3). CONCLUSION: The novel c.730G>C (p.D244H) variant of the CASQ1 gene probably underlay the myopathy in this patient. Above finding has enriched the mutational spectrum of the CASQ1 gene.