SETDB1 regulates short interspersed nuclear elements and chromatin loop organization in mouse neural precursor cells.

BACKGROUND: Transposable elements play a critical role in maintaining genome architecture during neurodevelopment. Short Interspersed Nuclear Elements (SINEs), a major subtype of transposable elements, are known to harbor binding sites for the CCCTC-binding factor (CTCF) and pivotal in orchestrating chromatin organization. However, the regulatory mechanisms controlling the activity of SINEs in the developing brain remains elusive. RESULTS: In our study, we conduct a comprehensive genome-wide epigenetic analysis in mouse neural precursor cells using ATAC-seq, ChIP-seq, whole genome bisulfite sequencing, in situ Hi-C, and RNA-seq. Our findings reveal that the SET domain bifurcated histone lysine methyltransferase 1 (SETDB1)-mediated H3K9me3, in conjunction with DNA methylation, restricts chromatin accessibility on a selective subset of SINEs in neural precursor cells. Mechanistically, loss of Setdb1 increases CTCF access to these SINE elements and contributes to chromatin loop reorganization. Moreover, de novo loop formation contributes to differential gene expression, including the dysregulation of genes enriched in mitotic pathways. This leads to the disruptions of cell proliferation in the embryonic brain after genetic ablation of Setdb1 both in vitro and in vivo. CONCLUSIONS: In summary, our study sheds light on the epigenetic regulation of SINEs in mouse neural precursor cells, suggesting their role in maintaining chromatin organization and cell proliferation during neurodevelopment.

Distinct epigenetic modulation of differentially expressed genes in the adult mouse brain following prenatal exposure to low-dose bisphenol A.

Bisphenol A (BPA) is a common component in the manufacture of daily plastic consumer goods. Recent studies have suggested that prenatal exposure to BPA can increase the susceptibility of offspring to mental illness, although the underlying mechanisms remain unclear. In this study, we performed transcriptomic and epigenomic profiling in the adult mouse brain following prenatal exposure to low-dose BPA. We observed a sex-specific transcriptional dysregulation in the cortex, with more significant differentially expressed genes was observed in adult cortex from male offspring. Moreover, the upregulated genes primarily influenced neuronal functions, while the downregulated genes were significantly associated with energy metabolism pathways. More evidence supporting impaired mitochondrial function included a decreased ATP level and a reduced number of mitochondria in the cortical neuron of the BPA group. We further investigated the higher-order chromatin regulatory patterns of DEGs by incorporating published Hi-C data. Interestingly, we found that upregulated genes exhibited more distal interactions with multiple enhancers, while downregulated genes displayed relatively short-range interactions among adjacent genes. Our data further revealed decreased H3K9me3 signal on the distal enhancers of upregulated genes, whereas increased DNA methylation and H3K27me3 signals on the promoters of downregulated genes. In summary, our study provides compelling evidence for the potential health risks associated with prenatal exposure to BPA, and uncovers sex-specific transcriptional changes with a complex interplay of multiple epigenetic mechanisms.

Interaction between Oligodendrocytes and Interneurons in Brain Development and Related Neuropsychiatric Disorders.

A variety of neurological and psychiatric disorders have recently been shown to be highly associated with the abnormal development and function of oligodendrocytes (OLs) and interneurons. OLs are the myelin-forming cells in the central nervous system (CNS), while interneurons are important neural types gating the function of excitatory neurons. These two types of cells are of great significance for the establishment and function of neural circuits, and they share similar developmental origins and transcriptional architectures, and interact with each other in multiple ways during development. In this review, we compare the similarities and differences in these two cell types, providing an important reference and further revealing the pathogenesis of related brain disorders.

ITPR2 Mediated Calcium Homeostasis in Oligodendrocytes is Essential for Myelination and Involved in Depressive-Like Behavior in Adolescent Mice.

Ca2+ signaling is essential for oligodendrocyte (OL) development and myelin formation. Inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) is an endoplasmic reticulum calcium channel and shows stage-dependent high levels in postmitotic oligodendrocyte precursor cells (OPCs). The role and potential mechanism of ITPR2 in OLs remain unclear. In this study, it is revealed that loss of Itpr2 in OLs disturbs Ca2+ homeostasis and inhibits myelination in adolescent mice. Animals with OL-specific deletion of Itpr2 exhibit anxiety/depressive-like behaviors and manifest with interrupted OPC proliferation, leading to fewer mature OLs in the brain. Detailed transcriptome profiling and signal pathway analysis suggest that MAPK/ERK-CDK6/cyclin D1 axis underlies the interfered cell cycle progression in Itpr2 ablated OPCs. Besides, blocking MAPK/ERK pathway significantly improves the delayed OPC differentiation and myelination in Itpr2 mutant. Notably, the resting [Ca2+]i is increased in Itpr2 ablated OPCs, with the elevation of several plasma calcium channels. Antagonists against these plasma calcium channels can normalize the resting [Ca2+]i level and enhance lineage progression in Itpr2-ablated OPCs. Together, the findings reveal novel insights for calcium homeostasis in manipulating developmental transition from OPCs to pre-OLs; additionally, the involvement of OLs-originated ITPR2 in depressive behaviors provides new therapeutic strategies to alleviate myelin-associated psychiatric disorders.

EIF4A3-mediated biogenesis of circSTX6 promotes bladder cancer metastasis and cisplatin resistance.

BACKGROUND: Cisplatin (CDDP)-based chemotherapy is a standard first-line treatment for metastatic bladder cancer (BCa) patients, and chemoresistance remains a major challenge in clinical practice. Circular RNAs (circRNAs) have emerged as essential regulators in carcinogenesis and cancer progression. However, the role of circRNAs in mediating CDDP chemosensitivity has yet to be well elucidated in BCa. METHODS: CircSTX6 (hsa_circ_0007905) was identified by mining the public circRNA datasets and verified by Sanger sequencing, agarose gel electrophoresis, RNase R treatment and qRT-PCR assays. Then, function experiments were performed to evaluate the effects of circSTX6 on BCa metastasis. Luciferase reporter assay, RNA pull-down, RNA immunoprecipitation (RIP), RNA stability assay, Fluorescence in situ hybridization (FISH) and Immunofluorescence (IF) were conducted to evaluate the interaction among circSTX6, miR-515-3p, PABPC1 and SUZ12. Animal experiments were performed to explore the function of circSTX6 in tumor metastasis and CDDP sensitivity. RESULTS: We identified that circSTX6 was significantly upregulated in clinical samples and cells of BCa. Functionally, circSTX6 promoted cell migration and invasion both in vitro and in vivo. Mechanistically, circSTX6 could act as a miR-515-3p sponge and abolish its effect on SUZ12. Moreover, circSTX6 was confirmed to increase the stability of SUZ12 mRNA by interacting with a mRNA stabilizer PABPC1 and subsequently promote the expression of SUZ12. Importantly, silencing of circSTX6 improved the chemosensitivity of CDDP-resistant bladder cancer cells to CDDP. Furthermore, in vivo analysis supported that knockdown of circSTX6 attenuated CDDP resistance in BCa tumors. CONCLUSION: These studies demonstrate that circSTX6 plays a pivotal role in BCa metastasis and chemoresistance, and has potential to serve as a therapeutic target for treatment of BCa.

A review on the hypoglycemic effect, mechanism and application development of natural dietary polysaccharides.

Diabetes mellitus (DM) as one chronic metabolic disease was greatly increased over recent decades. The major agents treating diabetes have noticeable side effects as well as the tolerability problems. The bioactive dietary polysaccharides from abundant natural resources exhibit good hypoglycemic effect with rare adverse effects, which might serve as a candidate to prevent and treat diabetes. However, the correlations between the hypoglycemic mechanism of polysaccharides and their structure were not mentioned in several studies, what's more, most of the current hypoglycemic studies on polysaccharides were based on in vitro and in vivo experiments, and there was a lack of knowledge about the effects in human clinical trials. The aim of this review is to discuss recent literature about the variety of dietary polysaccharides with hypoglycemic activity, as well the mechanism of action and the structure-function relationship are highlighted. Meanwhile, the application of dietary polysaccharides in functional foods and clinical medicine are realized with an in-depth understanding. So as to promote the exploration of dietary polysaccharides in low glycemic healthy foods or clinical medicine to prevent and treat diabetes.

A clinically applicable AI system for diagnosis of congenital heart diseases based on computed tomography images.

Congenital heart disease (CHD) is the most common type of birth defect. Without timely detection and treatment, approximately one-third of children with CHD would die in the infant period. However, due to the complicated heart structures, early diagnosis of CHD and its types is quite challenging, even for experienced radiologists. Here, we present an artificial intelligence (AI) system that achieves a comparable performance of human experts in the critical task of classifying 17 categories of CHD types. We collected the first-large CT dataset from three different CT machines, including more than 3750 CHD patients over 14 years. Experimental results demonstrate that it can achieve diagnosis accuracy (86.03%) comparable with junior cardiovascular radiologists (86.27%) in a World Health Organization appointed research and cooperation center in China on most types of CHD, and obtains a higher sensitivity (82.91%) than junior cardiovascular radiologists (76.18%). The accuracy of the combination of our AI system (97.20%) and senior radiologists achieves comparable results to that of junior radiologists and senior radiologists (97.16%) which is the current clinical routine. Our AI system can further provide 3D visualization of hearts to senior radiologists for interpretation and flexible review, surgeons for precise intuition of heart structures, and clinicians for more precise outcome prediction. We demonstrate the potential of our model to be integrated into current clinic practice to improve the diagnosis of CHD globally, especially in regions where experienced radiologists can be scarce.

TAGET: a toolkit for analyzing full-length transcripts from long-read sequencing.

Single-molecule Real-time Isoform Sequencing (Iso-seq) of transcriptomes by PacBio can generate very long and accurate reads, thus providing an ideal platform for full-length transcriptome analysis. We present an integrated computational toolkit named TAGET for Iso-seq full-length transcript data analyses, including transcript alignment, annotation, gene fusion detection, and quantification analyses such as differential expression gene analysis and differential isoform usage analysis. We evaluate the performance of TAGET using a public Iso-seq dataset and newly sequenced Iso-seq datasets from tumor patients. TAGET gives significantly more precise novel splice site prediction and enables more accurate novel isoform and gene fusion discoveries, as validated by experimental validations and comparisons with RNA-seq data. We identify and experimentally validate a differential isoform usage gene ECM1, and further show that its isoform ECM1b may be a tumor-suppressor in laryngocarcinoma. Our results demonstrate that TAGET provides a valuable computational toolkit and can be applied to many full-length transcriptome studies.

luxS contributes to intramacrophage survival of Streptococcus agalactiae by positively affecting the expression of fruRKI operon.

The LuxS quorum sensing system is a widespread system employed by many bacteria for cell-to-cell communication. The luxS gene has been demonstrated to play a crucial role in intramacrophage survival of piscine Streptococcus agalactiae, but the underlying mechanism remains largely unknown. In this study, transcriptome analysis, followed by the luxS gene deletion and subsequent functional studies, confirmed that impaired bacterial survival inside macrophages due to the inactivation of luxS was associated with reduced transcription of the fruRKI operon, encoding the fructose-specific phosphotransferase system. Further, luxS was determined not to enhance the transcription of fruRKI operon by binding its promoter, but to upregulate the expression of this operon via affecting the binding ability of catabolite control protein A (CcpA) to the catabolite responsive element (cre) in the promoter of fruRKI. Collectively, our study identifies a novel and previously unappreciated role for luxS in bacterial intracellular survival, which may give a more thorough understanding of the immune evasion mechanism in S. agalactiae.

Qualitative and Quantitative Determination of Decoquinate in Chicken Tissues by Gas Chromatography Tandem Mass Spectrometry.

A novel precolumn derivatization-GC-MS/MS method was developed for the determination of decoquinate residues in chicken tissues (muscle, liver, and kidney). The samples were extracted and purified by liquid-liquid extraction combined with solid-phase extraction and derivatized with acetic anhydride and pyridine. The recovery rates for decoquinate were 77.38~89.65%, and the intra-day and inter-day RSDs were 1.63~5.74% and 2.27~8.06%, respectively. The technique parameters meet the necessities for veterinary drug residue detection in China, the US, and the EU. Finally, the method was applied to analyze tissues of 60 chickens bought from a neighborhood supermarket, and solely one sample of chicken muscle contained 15.6 µg/kg decoquinate residue.

Transcriptomic profiling of the developing brain revealed cell-type and brain-region specificity in a mouse model of prenatal stress.

BACKGROUND: Prenatal stress (PS) is considered as a risk factor for many mental disorders. PS-induced transcriptomic alterations may contribute to the functional dysregulation during brain development. Here, we used RNA-seq to explore changes of gene expression in the mouse fetal brain after prenatal exposure to chronic unpredictable mild stress (CUMS). RESULTS: We compared the stressed brains to the controls and identified groups of significantly differentially expressed genes (DEGs). GO analysis on up-regulated DEGs revealed enrichment for the cell cycle pathways, while down-regulated DEGs were mostly enriched in the neuronal pathways related to synaptic transmission. We further performed cell-type enrichment analysis using published scRNA-seq data from the fetal mouse brain and revealed cell-type-specificity for up- and down-regulated DEGs, respectively. The up-regulated DEGs were highly enriched in the radial glia, while down-regulated DEGs were enriched in different types of neurons. Cell deconvolution analysis further showed altered cell fractions in the stressed brain, indicating accumulation of neuroblast and impaired neurogenesis. Moreover, we also observed distinct brain-region expression pattern when mapping DEGs onto the developing Allen brain atlas. The up-regulated DEGs were primarily enriched in the dorsal forebrain regions including the cortical plate and hippocampal formation. Surprisingly, down-regulated DEGs were found excluded from the cortical region, but highly expressed on various regions in the ventral forebrain, midbrain and hindbrain. CONCLUSION: Taken together, we provided an unbiased data source for transcriptomic alterations of the whole fetal brain after chronic PS, and reported differential cell-type and brain-region vulnerability of the developing brain in response to environmental insults during the pregnancy.

Predictive performance of the perivascular fat attenuation index for interventional antegrade percutaneous coronary intervention for chronic total occlusion.

OBJECTIVES: This study aimed to investigate the association between the perivascular fat attenuation index (FAI) and the success of the antegrade percutaneous coronary intervention (PCI) for chronic total occlusion (CTO). METHODS: This study evaluated patients with only one CTO lesion observed on conventional coronary angiography (CAG) who underwent coronary computed tomography angiography (CCTA) < 1 month before CAG, from 2018 to 2019. The clinical data, CCTA-based CTO lesion morphologic characteristics, and perivascular FAI of CTO lesions were recorded and analysed. RESULTS: In total, 156 patients with CTOs were enrolled in this study. Successful antegrade PCI (A-PCI) was achieved in 105 CTO lesions (67.3%). The perivascular FAI of the failed A-PCI group was significantly lower than the successful A-PCI group (-84.76 ± 10.44 Hounsfield unit (HU) vs. -67.54 ± 9.94 HU; p < 0.001), and the cut-off value determined by the receiver operating characteristic (ROC) curve was -77.50 HU. Multivariable analysis revealed no statistical significance in the clinical data, FAI ≤ -77.50 HU (odds ratio (OR): 33.96), negative remodeling (OR: 4.36), severe calcification degree (OR: 4.43) and occlusion length ≥ 20.25 mm (OR: 3.89) were independent predictors of A-PCI failure. The prediction performance of combining the three morphologic characteristics (severe calcification, occlusion length ≥ 20.25 mm, and negative remodeling) with FAI ≤ -77.50 HU was better than that of the three morphologic characteristics alone (0.93 versus 0.77, p < 0.001). CONCLUSIONS: As a non-invasive index for detecting coronary inflammation, FAI complements indicators based on coronary CTA well and may be helpful for choosing appropriate interventional strategies. KEY POINTS: • Perivascular FAI of CTO was significantly higher in the failed A-PCI group. • The combination of FAI with other morphological predictors showed higher predictive performance of failed A-PCI for CTOs. • FAI is a good complement to indicators based on coronary CTA.

Histone Methyltransferase SETDB1 Regulates the Development of Cortical Htr3a-Positive Interneurons and Mood Behaviors.

BACKGROUND: GABAergic (gamma-aminobutyric acidergic) interneurons (INs) are highly heterogeneous, and Htr3a labels a subpopulation of cortical INs originating from the embryonic caudal ganglionic eminence. SETDB1 is one of the histone H3K9 methyltransferases and plays an essential role in the excitatory neurons, but its role in regulating cortical inhibitory INs remains largely unknown. METHODS: In this study, we generated transgenic mice with conditional knockout of Setdb1 in neural progenitor cells (Setdb1-NS-cKO) and GABAergic neurons (Setdb1-Gad2-cKO). In addition, we performed RNA sequencing, ATAC-seq (assay for transposase-accessible chromatin with sequencing), chromatin immunoprecipitation sequencing, luciferase assay, chromatin conformation capture, and CRISPR (clustered regularly interspaced short palindromic repeats)/dCas9 to study the epigenetic mechanism underlying SETDB1-mediated transcriptional regulation of Htr3a. We also performed in situ hybridization and whole-cell recording to evaluate the functional properties of cortical Htr3a+ INs and behavioral tests for mood. RESULTS: We detected significant upregulation of Htr3a expression in the embryonic ganglionic eminence of Setdb1-NS-cKO and identified the endogenous retroviral sequence RMER21B as a new target of SETDB1. RMER21B showed enhancer activity and formed distal chromatin interaction with the promoter of Htr3a. In addition, we observed an increased number and enhanced excitability of Htr3a+ INs in the knockout cortex. Moreover, Setdb1-Gad2-cKO mice exhibited anxiety- and depressive-like behaviors, which were partially reversed by a 5-HT3 receptor antagonist. CONCLUSIONS: These findings suggest that SETDB1 represses Htr3a transcription via RMER21B-mediated distal chromatin interaction in the embryonic ganglionic eminence and regulates the development of cortical Htr3a+ INs and mood behaviors.

TonB systems are required for Aeromonas hydrophila motility by controlling the secretion of flagellin.

The TonB system is required for the active transport of iron compounds across the outer membrane in Gram-negative bacteria. Our previous data indicated that three TonB systems act coordinately to contribute to the motility of Aeromonas hydrophila NJ-35. In this study, we found that flagellum biogenesis was defective in the ΔtonB123 mutant. Subcellular localization indicated that the flagellin subunits FlaA and FlaB were trapped in the cytoplasm of ΔtonB123 mutant with reduced molecular mass. Overexpression of FlaA or FlaB in the ΔtonB123 mutant was unable to restore the secretion of flagellin subunits. Further investigation demonstrated that flagellins in the ΔtonB123 mutant showed a weak affinity for the flagellin-specific chaperone FliS, which is necessary for the export of flagellins. Deglycosylation analysis indicated that flagellins in the cytoplasm of the ΔtonB123 mutant were almost nonglycosylated. Our data suggested that disruption of tonB123 impairs the formation of flagella by inhibiting flagellin glycosylation and decreasing the binding affinity of flagellin for the chaperone FliS. Taken together, our findings indicate a new role of the TonB system in flagellar biogenesis in A. hydrophila.

Separation and Detection of Abamectin, Ivermectin, Albendazole and Three Metabolites in Eggs Using Reversed-Phase HPLC Coupled with a Photo Diode Array Detector.

An innovative and sensitive approach using high-performance liquid chromatography-photo diode array detection (HPLC-PDAD) was developed and optimized for the simultaneous determination of abamectin (ABM), ivermectin (IVM), albendazole (ABZ) and three metabolites in eggs. The samples were extracted with acetonitrile (MeCN)/water (90:10, v/v), and the extracts containing the targets were cleaned up and concentrated by a series of liquid-liquid extraction (LLE) steps. A reversed-phase C18 column and a mobile phase consisting of 0.1% trifluoroacetic acid (TFA) aqueous solution and methanol (MeOH) were utilized to perform optimal chromatographic separation. The developed method was validated on the basis of international guidelines. The limits of detection (LODs) and quantitation (LOQs) were 2.1-10.5 µg/kg and 7.8-28.4 µg/kg, respectively. Satisfactory linear relationships were observed for the targets in their corresponding concentration ranges. The mean recoveries ranged from 85.7% to 97.21% at 4 addition levels, with intraday and interday relative standard deviations (RSDs) in the ranges of 1.68-4.77% and 1.74-5.31%, respectively. The presented protocol was demonstrated to be applicable and reliable by being applied for the detection of target residues in locally sourced egg samples.

Microglia-specific transcriptional repression of interferon-regulated genes after prolonged stress in mice.

Stress-induced neuroinflammation is considered an important mechanism in the pathogenesis of depression. As immune effector cells in the brain, microglia play an essential role in neuroinflammation under stress, but the underlying mechanism remains controversial. Here, we performed RNA-seq and ATAC-seq to study microglia-specific epigenomic changes in mice after 12 weeks of exposure to mild stress. Our study revealed that chronic stress induced pronounced anxiety and depressive-like behavioral changes. However, microglia did not manifest a state of neuroinflammatory activation; instead, they displayed morphological changes characterized by hyper-ramification. Furthermore, we revealed large-scale transcriptional repression in microglia isolated from the stressed brain, including many interferon (IFN)-regulated genes (IRGs) and some encompassing DNA repeats. GSEA showed that the down-regulated genes were enriched in the IFN-mediated neuroimmune signaling pathways. In addition, integrative analysis with a published scRNA-seq dataset revealed that these down-regulated genes were enriched in a distinct subpopulation of "Interferon microglia". ATAC-seq analysis further showed that differential gene expression was positively correlated with the changes in chromatin accessibility, and the IFN-stimulated response element (ISRE) was enriched in the down-regulated ATAC-seq loci. Interestingly, this phenotype was not associated with the production of IFNs. Instead, the gene encoding Activating Transcription Factor 3 (ATF3) was significantly increased in the stressed microglia, which might contribute to the transcriptional repression of IRGs. Our study reported microglia-specific transcriptional repression of IRGs independent of the production of IFNs, providing some new insights into neuroimmune dysregulation under prolonged stress.

Concurrent Determination of Tigecycline, Tetracyclines and Their 4-Epimer Derivatives in Chicken Muscle Isolated from a Reversed-Phase Chromatography System Using Tandem Mass Spectrometry.

A quantitative and qualitative method using a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) detection approach was developed and validated for the analysis of tigecycline, four tetracyclines and their three 4-epimer derivatives in chicken muscle. Samples were extracted repeatedly with 0.1 mol/L Na2EDTA-McIlvaine buffer solution. After vortexing, centrifugation, solid-phase extraction, evaporation and reconstitution, the aliquots were separated using a C8 reversed-phase column (50 mm × 2.1 mm, 5 µm) with a binary solvent system consisting of methanol and 0.01 mol/L trichloroacetic acid aqueous solution. The typical validation parameters were evaluated in accordance with the acceptance criteria detailed in the guidelines of the EU Commission Decision 2002/657/EC and the U.S. Food and Drug Administration Bioanalytical Method Validation 05/24/18. The matrix-matched calibration curve was linear over the concentration range from the limit of quantitation (LOQ) to 400 µg/kg for doxycycline, and the calibration graphs for tetracycline, chlortetracycline, oxytetracycline, their 4-epimer derivatives and tigecycline showed a good linear relationship within the concentration range from the LOQ to 200 µg/kg. The limits of detection (LODs) for the eight targets were in the range of 0.06 to 0.09 µg/kg, and the recoveries from the fortified blank samples were in the range of 89% to 98%. The within-run precision and between-run precision, which were expressed as the relative standard deviations, were less than 5.0% and 6.9%, respectively. The applicability was successfully demonstrated through the determination of residues in 72 commercial chicken samples purchased from different sources. This approach provides a novel option for the detection of residues in animal-derived food safety monitoring.

Comparative transcriptome combined with morphophysiological analyses revealed the molecular mechanism underlying Tetrahymena thermophila predation-induced antiphage defense in Aeromonas hydrophila.

Protozoan predation has been demonstrated to be a strong driving force for bacterial defence strategies in the environment. Our previous study demonstrated that Aeromonas hydrophila NJ-35, which evolved small-colony variants (SCVs), displayed various adaptive traits in response to Tetrahymena thermophila predation, such as enhanced phage resistance. However, the evolutionary mechanisms are largely unknown. In this study, we performed a genome- and transcriptome-wide analysis of the SCV1, representing one strain of the SCVs, for identification of the genes of mutation and altered expression underlying this phage resistance phenotype. Our study demonstrated that phage resistance caused by T. thermophila predation was due to the downregulation of a flagellar biosynthesis regulator, flhF, in SCV1. Interestingly, we confirmed that phage resistance in SCV1 was not straightforwardly attributable to the absence of flagella but to FlhF-mediated secretion of extracellular protein that hinders phage adsorption. This finding improves our understanding of the mechanisms by which A. hydrophila lowers the susceptibility to phage infection under predation pressure, and highlights an important contribution of bacterium-protozoan interactions in driving the adaptive evolution of pathogens in complex environments.

CRISPR Contributes to Adhesion, Invasion, and Biofilm Formation in Streptococcus agalactiae by Repressing Capsular Polysaccharide Production.

The clustered regularly interspaced palindromic repeat (CRISPR)-associated (Cas) system functions classically as a prokaryotic defense system against invading mobile genetic elements, such as phages, plasmids, and viruses. Our previous study revealed that CRISPR deletion caused increased transcription of capsular polysaccharide (CPS) synthesis-related genes and severely attenuated virulence in the hypervirulent piscine Streptococcus agalactiae strain GD201008-001. Here, we found that CRISPR deficiency resulted in reduced adhesion, invasion, and biofilm formation abilities in this strain by upregulating the production of CPS. However, enhanced CPS production was not responsible for the attenuated phenotype of the ΔCRISPR mutant. RNA degradation assays indicated that inhibited transcription of the cps operon by CRISPR RNA (crRNA) was not due to the base pairing of the crRNA with the cps mRNA but to the repression of the promoter activity of cpsA, which is a putative transcriptional regulator of the capsule locus. IMPORTANCE Beyond protection from invading nucleic acids, CRISPR-Cas systems have been shown to have an important role in regulating bacterial endogenous genes. In this study, we demonstrate that crRNA inhibits the transcription of the cps operon by repressing the activity of promoter PcpsA, leading to increases in the abilities of adhesion, invasion, and biofilm formation in S. agalactiae. This study highlights the regulatory role of crRNA in bacterial physiology and provides a new explanation for the mechanism of crRNA-mediated endogenous gene regulation in S. agalactiae.

Erratum: Metformin inhibits the proliferation of A431 cells by modulating the PI3K/Akt signaling pathway.

[This corrects the article DOI: 10.3892/etm.2015.2220.].