Wenhua Liu

The Clp protease regulates antibiotic resistance, biofilm formation, stress response, and virulence in microorganisms through a post-transcriptional mechanism. Substrate recognition by the ClpS subunit is essential for the target protein degradation. However, the function of ClpS in Riemerella anatipestifer (R. anatipestifer) remains unclear. In this study, a clpS gene (B739_1609) in-frame deletion strain of R. anatipestifer was constructed using homologous recombination. The ΔclpS gene mutant exhibited no influence on the growth rate and shape of R. anatipestifer. However, the survival rate was significantly lower than that of the parent strain under different conditions, including 42 °C, hydrogen peroxide (10 mM), hydrochloric acid (20 mM), and iron deficiency (10 mM 2’2-dipyridyl). Moreover, R. anatipestifer can activate transcription of the clpS under heat and oxidative stress. The ΔclpS mutant strain exhibited defects in adhesion and invasion of RAW264.7 cells, as well as reduced pathogenicity in ducklings. Additionally, RNA-seq analysis revealed significant changes in the expression of 77 genes in the ΔclpS mutant strain, with 63 genes upregulated and 14 genes downregulated. The differentially expressed genes were primarily clustered in the following metabolic pathways: Global and Overview Maps, Energy Metabolism, Carbohydrate Metabolism, Metabolism of Cofactors and Vitamins, Glycan Biosynthesis and Metabolism, and Nucleotide Metabolism. These results demonstrate that clpS is involved in the stress response and virulence of R. anatipestifer. Moreover, these results can serve as a reference for understanding the molecular pathogenic mechanisms of R. anatipestifer.

Gastrodin (GAS), the principal bioactive component derived from Gastrodia elata Bl., has demonstrated efficacy in attenuating methamphetamine (MA) induced conditioned place preference (CPP) in animal models. However, the molecular mechanisms underlying its anti-addictive effects, particularly the role of miRNAs, remain insufficiently understood. A CPP model of MA dependence was established in rats. Hippocampal tissues were subjected to microarray analysis to identify differentially expressed miRNAs following GAS intervention. Key techniques including behavioral evaluation, stereotaxic injection of miR-128-3p antagomir, quantitative real time PCR, network pharmacology analysis, molecular docking, and dual-luciferase reporter assays were employed to elucidate the involvement of miR-128-3p in the anti-MA effects of GAS. GAS treatment significantly reversed MA induced CPP behaviors and mitigated the upregulation of hippocampal miR-128-3p. Inhibition of miR-128-3p via antagomir also attenuated MA induced CPP, mirroring the effect of GAS administration. Network pharmacology analysis identified 106 putative miR-128-3p targets enriched in learning, memory, and behavioral regulation pathways including PI3K Akt, MAPK, and FoxO signaling. Molecular docking revealed strong binding affinities between GAS, MA, and 6 key targets, with miR-128-3p interacting at multiple 3′ UTR sites. dual-luciferase reporter assays confirmed the suppressive effect of miR-128-3p on these targets. GAS exerts its inhibitory effect on MA dependence through modulation of miR-128-3p and its downstream targets. These findings suggest that miR-128-3p represents a promising therapeutic target, and GAS may serve as a novel miRNA based intervention strategy for MA addiction.

PACRG (Parkin co-regulated gene) shares a bi-directional promoter with the Parkinson’s disease-associated gene Parkin, but the physiological roles of PACRG have not yet been fully elucidated. Recombinant expression methods are indispensable for protein structural and functional studies. In this study, the coding region of PACRG was cloned to a conventional vector pQE80L, as well as two cold-shock vectors pCold II and pCold-GST, respectively. The constructs were transformed into Escherichia coli (DE3), and the target proteins were overexpressed. The results showed that the cold-shock vectors are more suitable for PACRG expression. The soluble recombinant proteins were purified with Ni²⁺ chelating column, glutathione S-transferase (GST) affinity chromatography and gel filtration. His₆ pull down assay and LC-MS/MS were carried out for identification of PACRG-binding proteins in HEK293T cell lysates, and a total number of 74 proteins were identified as potential interaction partners of PACRG. GO (Gene ontology) enrichment analysis (FunRich) of the 74 proteins revealed multiple molecular functions and biological processes. The highest proportion of the 74 proteins functioned as transcription regulator and transcription factor activity, suggesting that PACRG may play important roles in regulation of gene transcription.

Excess reactive oxygen species can cause cellular damage, and are involved in many pathological processes such as inflammation, atherosclerosis and cancer.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac111 gene is highly conserved in lepidopteran-specific baculoviruses, and its function in the AcMNPV life cycle is still unknown. To investigate the function of ac111, an ac111-knockout AcMNPV (vAc111KO) was constructed through homologous recombination in Escherichia coli. Viral growth curve analysis and plaque assays showed that the deletion of ac111 had no effect on infectious budded virion production. Quantitative real-time polymerase chain reaction analysis confirmed that viral DNA replication was unaffected in the absence of ac111. Electron microscopy revealed that the ac111 deletion did not affect nucleocapsid assembly, occlusion-derived virion formation, or the embedding of occlusion-derived virions into the occlusion bodies. However, in vivo bioassays showed that although the deletion of ac111 did not affect the per os infectivity of AcMNPV in Spodoptera exigua larvae, it led to an approximately five-fold reduction in infectivity of AcMNPV in Trichoplusia ni larvae, and vAc111KO took approximately 21 h longer to kill Trichoplusia ni larvae than the wild-type viruses. Taken together, our results demonstrated that although ac111 is not essential for virus replication in vitro, it plays an important role in the per os infectivity of AcMNPV in a host-dependent manner.

Soft corals of the genus Sinularia are widely distributed in the South China Sea. Two new lipid analogues (Z)-N-(3,4-dihydroxyphenethyl)-3-methyldo-dec-2-enamide (1) and (E)-1-(2,5-dihydroxy-4-methylphenyl)-6-acetoxy-2,3,7-trihydroxy-3,7,11,15-tetramethylhexadeca-11,14-dien (2), together with three known compounds (3–5) were isolated from the soft coral Sinularia flexibilis. Their structures were elucidated on the basis of extensive spectroscopic analysis, including one and two-dimensional NMR, MS, IR, UV, as well as by comparison with published data.

Key points: The hD4.7 variant has been linked to attention‐deficit/hyperactivity disorder (ADHD); however, the underlying mechanism is unknown. We found that activation of hD4.7 induced over‐suppression of glutamatergic excitatory network bursts and under‐suppression of GABAergic inhibitory network bursts in the prefrontal cortex (PFC) circuitry. Methylphenidate, a psychostimulant drug used to treat ADHD, normalized the effects of hD4.7 on synchronous network bursts in PFC pyramidal neurons. The findings of the present study suggest that the aberrant regulation of PFC synchronous network activity by hD4.7 may underlie its involvement in ADHD. A unique feature of the human D₄ receptor (hD₄R) gene is the existence of a large number of polymorphisms in exon 3 coding for the third intracellular loop, which consists of a variable number of tandem repeats. The hD₄R variants with long repeats have been linked to attention‐deficit/hyperactivity disorder (ADHD); however, the underlying mechanism is unknown. Emerging evidence suggests that selective attention is controlled by the rhythmic synchronization in the prefrontal cortex (PFC) and its connected networks. In the present study, we examined the role of hD₄R variants in regulating PFC synchronous network activity. D₄R knockout mice with viral infection of hD4.4 or hD4.7 in the medial PFC were used. Whole‐cell patch‐clamp recordings were performed to examine the effects of activating hD4.x on the spontaneous large scale correlated activity in PFC pyramidal neurons. We found that, compared to the normal four‐repeat variant hD4.4, the ADHD‐linked variant hD4.7 induces more suppression of glutamatergic excitatory network bursts and less suppression of GABAergic inhibitory network bursts in the PFC circuitry. Methylphenidate, a psychostimulant drug used to treat ADHD, normalized the effects of hD4.7 on synchronous network bursts in PFC pyramidal neurons. These results reveal the differential effects of hD₄R variants on the integrated excitability of PFC circuits. It is suggested that the aberrant regulation of PFC network activity by hD4.7 may underlie its involvement in ADHD. The methylphenidate‐induced normalization of synaptic circuitry regulation may contribute to its effectiveness in ADHD treatment.

Converging evidence suggests that females and males show different responses to stress; however, little is known about the mechanism underlying the sexually dimorphic effects of stress. In this study, we found that young female rats exposed to 1 week of repeated restraint stress show no negative effects on temporal order recognition memory (TORM), a cognitive process controlled by the prefrontal cortex (PFC), which was contrary to the impairment in TORM observed in stressed males. Concomitantly, normal glutamatergic transmission and glutamate receptor surface expression in PFC pyramidal neurons were found in repeatedly stressed females, in contrast to the significant reduction seen in stressed males. The detrimental effects of repeated stress on TORM and glutamate receptors were unmasked in stressed females when estrogen receptors were inhibited or knocked down in PFC, and were prevented in stressed males with the administration of estradiol. Blocking aromatase, the enzyme for the biosynthesis of estrogen, revealed the stress-induced glutamatergic deficits and memory impairment in females, and the level of aromatase was significantly higher in the PFC of females than in males. These results suggest that estrogen protects against the detrimental effects of repeated stress on glutamatergic transmission and PFC-dependent cognition, which may underlie the stress resilience of females.