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As a novel guanylate cyclase stimulator, SGC003F is being developed for the treatment of heart failure with a reduced ejection fraction (HFrEF). This study aimed to assess the effect of P-glycoprotein (P-gp) inhibition on SGC003F exposure in vivo, comparing plasma and tissue levels, and evaluating the role of P-gp in the small intestine, blood–brain barrier (BBB), and kidney in impacting the tissue exposure. Tariquidar, a P-gp inhibitor, was added to monolayer transport assays to observe the changes in the transmembrane characteristics of SGC003F. Rats were given SGC003F with tariquidar via various routes to measure plasma, tissue, urine, and fecal concentrations. The inclusion of tariquidar significantly altered the pharmacokinetics of SGC003F. In LLC-PK1-MDR1 cells, tariquidar reduced the efflux ratio of SGC003F from 6.56 to 1.28. In rats, it enhanced the plasma AUC by 3.05 or 1.61 times, increased the Cmax by 2.13 or 1.07 times, and notably improved bioavailability from 46.4% to 95%. Additionally, co-administration with tariquidar led to a decrease in fecal excretion and an increase in tissue exposure, with only a moderate effect on the partition ratios in the small intestine and brain. P-gp inhibition impacts SGC003F exposure, with plasma levels not fully reflecting tissue levels. P-gp in the small intestine and BBB affects SGC003F's pharmacokinetics, warranting further clinical drug–drug interaction (DDI) studies. [ABSTRACT FROM AUTHOR]

Here, we report the generation and comprehensive characterization of a knockin mouse model for the hotspot p.Arg87Cys variant of the cytoplasmic FMR1‐interacting protein 2 (CYFIP2) gene, which was recently identified in individuals diagnosed with West syndrome, a developmental and epileptic encephalopathy. The Cyfip2+/R87C mice recapitulated many neurological and neurobehavioral phenotypes of the patients, including spasmlike movements, microcephaly, and impaired social communication. Age‐progressive cytoarchitectural disorganization and gliosis were also identified in the hippocampus of Cyfip2+/R87C mice. Beyond identifying a decrease in CYFIP2 protein levels in the Cyfip2+/R87C brains, we demonstrated that the p.Arg87Cys variant enhances ubiquitination and proteasomal degradation of CYFIP2. ANN NEUROL 2023;93:155–163 [ABSTRACT FROM AUTHOR]

The two members of the cytoplasmic FMR1‐interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1‐2 × Myc and CYFIP2‐3 × Flag knock‐in mice and found that CYFIP1 and CYFIP2 are not significantly co‐immunoprecipitated with each other in the knock‐in brains compared with negative control wild‐type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size‐exclusion chromatography of WT mouse brains. Specifically, mass spectrometry‐based analysis of CYFIP1‐2 × Myc knock‐in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region‐ and age‐matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single‐cell RNA‐sequencing databases suggested non‐neuronal cell‐ and neuron‐enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co‐expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co‐localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1‐specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410 [ABSTRACT FROM AUTHOR]

Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1–12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13–16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ∼60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment. Functionally, Shank3-N overexpression altered the dendritic spine morphology in cultured neurons. Importantly, Shank3-N expression in Shank3B KO mice was not a compensatory response to a reduction of full-length Shank3 because expression was still detected in the brain after normalizing the level of full-length Shank3. Moreover, in another Shank3 KO line (Shank3 gKO) with a similar Shank3 exonal deletion as that in Shank3B KO mice but without a Neo cassette, the mRNA expression levels of Shank3 exons 1–12 were lower than those of wild-type mice and Shank3-N was not detected in the brain. In addition, the expression levels of genes neighboring Shank3 on chromosome 15 were altered in the striatum of Shank3B KO but not Shank3 gKO mice. These results suggest that the Neo cassette has potential off-target effects in Shank3B KO mice. [ABSTRACT FROM AUTHOR]

The cytoplasmic fragile X mental retardation 1 (FMR1)-interacting protein 2 (CYFIP2) gene is associated with epilepsy, intellectual disability (ID), and developmental delay, suggesting its critical role in proper neuronal development and function. CYFIP2 is involved in regulating cellular actin dynamics and also interacts with RNA-binding proteins. However, the adult brain function of CYFIP2 remains unclear because investigations thus far are limited to Cyfip2 heterozygous (Cyfip2+/−) mice owing to the perinatal lethality of Cyfip2 -null mice. Therefore, we generated Cyfip2 conditional knock-out (cKO) mice with reduced CYFIP2 expression in postnatal forebrain excitatory neurons (CaMKIIα-Cre). We found that in the medial prefrontal cortex (mPFC) of adult Cyfip2 cKO mice, CYFIP2 expression was decreased in both layer 2/3 (L2/3) and layer 5 (L5) neurons, unlike the L5-specific CYFIP2 reduction observed in adult Cyfip2+/− mice. Nevertheless, filamentous actin (F-actin) levels were increased only in L5 of Cyfip2 cKO mPFC possibly because of a compensatory increase in CYFIP1, the other member of CYFIP family, in L2/3 neurons. Abnormal dendritic spines on basal, but not on apical, dendrites were consistently observed in L5 neurons of Cyfip2 cKO mPFC. Meanwhile, neuronal excitability and activity were enhanced in both L2/3 and L5 neurons of Cyfip2 cKO mPFC, suggesting that CYFIP2 functions of regulating F-actin and excitability/activity may be mediated through independent mechanisms. Unexpectedly, adult Cyfip2 cKO mice did not display locomotor hyperactivity or reduced anxiety observed in Cyfip2+/− mice. Instead, both exhibited enhanced social dominance accessed by the tube test. Together, these results provide additional insights into the functions of CYFIP2 in the adult brain. [ABSTRACT FROM AUTHOR]

Objective: Genetic variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) encoding an actin-regulatory protein are associated with brain disorders, including intellectual disability and epilepsy. However, specific in vivo neuronal defects and potential treatments for CYFIP2-associated brain disorders remain largely unknown. Here, we characterized Cyfip2 heterozygous (Cyfip2+/- ) mice to understand their neurobehavioral phenotypes and the underlying pathological mechanisms. Furthermore, we examined a potential treatment for such phenotypes of the Cyfip2+/- mice and specified a neuronal function mediating its efficacy.Methods: We performed behavioral analyses of Cyfip2+/- mice. We combined molecular, ultrastructural, and in vitro and in vivo electrophysiological analyses of Cyfip2+/- prefrontal neurons. We also selectively reduced CYFIP2 in the prefrontal cortex (PFC) of mice with virus injections.Results: Adult Cyfip2+/- mice exhibited lithium-responsive abnormal behaviors. We found increased filamentous actin, enlarged dendritic spines, and enhanced excitatory synaptic transmission and excitability in the adult Cyfip2+/- PFC that was restricted to layer 5 (L5) neurons. Consistently, adult Cyfip2+/- mice showed increased seizure susceptibility and auditory steady-state responses from the cortical electroencephalographic recordings. Among the identified prefrontal defects, lithium selectively normalized the hyperexcitability of Cyfip2+/- L5 neurons. RNA sequencing revealed reduced expression of potassium channel genes in the adult Cyfip2+/- PFC. Virus-mediated reduction of CYFIP2 in the PFC was sufficient to induce L5 hyperexcitability and lithium-responsive abnormal behavior.Interpretation: These results suggest that L5-specific prefrontal dysfunction, especially hyperexcitability, underlies both the pathophysiology and the lithium-mediated amelioration of neurobehavioral phenotypes in adult Cyfip2+/- mice, which can be implicated in CYFIP2-associated brain disorders. ANN NEUROL 2020;88:526-543. [ABSTRACT FROM AUTHOR]

Background: Infections caused by the carbapenem-resistant Enterobacter cloacae (CREC) bring great challenges to the clinical treatment and pose a serious threat to public health. In this study, we investigated the molecular characteristics of CREC in a tertiary hospital. Materials and Methods: A total of 12 non-duplicate CREC strains isolated during the period of November 2016 to July 2019 were subjected to automated microbial identification and antimicrobial susceptibility testing (AST) using the BD Phoenix-100 identification and antimicrobial susceptibility testing (ID/AST) system. The strains were also subjected to phenotypic screening for the detection of antibiotic resistance genes such as the carbapenemase and other β-lactamase genes, with the use of the polymerase chain reaction assay (PCR). Finally, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE)-based homology analysis were applied. Results: Four types of carbapenemases namely IMP-26, NDM-5, NDM-1, and KPC-2 were identified in 12 CREC strains. IMP-26 was the most prevalent type (6/12 strains, 50 %), followed by NDM-5 (3/12 strains, 25 %). The results of MLST revealed that these 12 strains could be divided into five sequence types (STs) among which ST544 was the dominant type (6/12 strains, 50 %). The PFGE results divided the 12 strains into four clusters. Conclusion: Our study indicated that the epidemics of the IMP-26-producing E. cloacae ST544 strain did occur in the intensive care unit (ICU) of a tertiary hospital. Therefore, early surveillance and strict implementation of control measures are crucial for the prevention of nosocomial infections and transmissions in hospitals. [ABSTRACT FROM AUTHOR]

Introduction: Carbapenem-resistant Enterobacteriaceae (CRE) pose a serious threat to clinical patient management and public health, as they are generally resistant to most antibiotics and cause infections with high mortality rates. Klebsiella pneumoniae ranks second among Enterobacteriaceae species that cause nosocomial infections. In this study, we investigated the epidemic characteristics of carbapenem-resistant K. pneumoniae (CRKP) in the pediatric intensive care unit (PICU) of Yanbian University Hospital. Materials and Methods: A total of 14 non-duplicate CRKP strains, collected from March  2015 to November 2019, were subjected to automated microbial identification and antimicrobial susceptibility tests using the Phoenix-100 ID/AST system. The strains were also subjected to genotypic resistance testing, polymerase chain reaction assays to detect genes encoding carbapenemases and other β-lactamases, multi-locus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE)-based homology analysis. Results: Two carbapenemase genes, KPC-2 and NDM-1 (in eight and six strains, respectively), were detected. MLST enabled the division of the strains into two sequence types, ST11 and ST1224 (containing eight and six strains, respectively). PFGE results classified the 14 strains into clonotypes A–D, of which clonotypes A and B belonged to ST11, while clonotypes C and D belonged to ST1224. Conclusion: Our study reveals that epidemics of the KPC-2-ST11 and NDM-1-ST1224 strains occurred in the PICU of Yanbian University Hospital. Surveillance and strict implementation of prevention and control measures are crucial to prevent the occurrence and rapid spread of nosocomial infections. [ABSTRACT FROM AUTHOR]

Ursolic acid (UA), a plant-derived compound, has many properties beneficial to health. In the present study, we synthesised three series of novel UA derivatives and evaluated their anti-Toxoplasma gondii activity both in vitro and in vivo. Most derivatives exhibited an improved anti-T. gondii activity in vitro when compared with UA (parent compound), whereas compound 3d exhibited the most potent anti-T. gondii activity in vivo. Spiramycin served as the positive control. Additionally, determination of biochemical parameters, including the liver and spleen indexes, indicated compound 3d to effectively reduce hepatotoxicity and significantly enhance anti-oxidative effects, as compared with UA. Furthermore, our molecular docking study indicated compound 3d to possess a strong binding affinity for T. gondii calcium-dependent protein kinase 1 (TgCDPK1). Based on these findings, we conclude that compound 3d, a derivative of UA, could act as a potential inhibitor of TgCDPK1. [ABSTRACT FROM AUTHOR]

Genetic variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, which encodes excitatory postsynaptic core scaffolds cause numerous brain disorders. Several lines of Shank3 knock-out (KO) mice with deletions of different Shank3 exons have previously been generated and characterized. The different Shank3 KO mouse lines have both common and line-specific phenotypes. Shank3 isoform diversity is considered a mechanism underlying phenotypic heterogeneity, and compensatory changes through regulation of Shank3 expression may contribute to this heterogeneity. However, whether such compensatory changes occur in Shank3 KO mouse lines has not been investigated in detail. Using previously reported RNA-sequencing analyses, we identified an unexpected increase in Shank3 transcripts in two different Shank3 mutant mouse lines (Shank3B and Shank3ΔC) having partial deletions of Shank3 exons. We validated an increase in Shank3 transcripts in the hippocampus, cortex, and striatum, but not in the cerebellum, of Shank3B heterozygous (HET) and KO mice, using qRT-PCR analyses. In particular, expression of the N-terminal exons 1–12, but not the more C-terminal exons 19–22, was observed to increase in Shank3B mice with deletion of exons 13–16. This suggests a selective compensatory activation of upstream Shank3 promoters. Furthermore, using domain-specific Shank3 antibodies, we confirmed that the increased Shank3 transcripts in Shank3B KO mice produced a small Shank3 isoform that was not detected in wild-type mice. Taken together, our results illustrate another layer of complexity in the regulation of Shank3 expression in the brain, which may also contribute to the phenotypic heterogeneity of different Shank3 KO mouse lines. [ABSTRACT FROM AUTHOR]

The SH3 and multiple ankyrin repeat domains 3 (Shank3) proteins are core organizers of the postsynaptic density in neuronal excitatory synapses, and their defects cause various neurodevelopmental and neuropsychiatric disorders. Mechanistically, Shank3 directly and indirectly interacts with hundreds of synaptic proteins with diverse functions and potentially exerts its regulatory roles in synaptic development and function via these interactors. However, Shank3‐dependent regulation of synaptic abundance has been validated in vivo for only a few Shank3 interactors. Here, using a quantitative proteomic analysis, we identified 136 proteins with altered synaptic abundance in the striatum of Shank3‐overexpressing transgenic (TG) mice. By comparing these proteins with those found in a previous analysis of the postsynaptic density of Shank3 knock‐out (KO) striatum, we identified and confirmed that cylindromatosis‐associated deubiquitinase (Cyld), a deubiquitinase specific for Lys63‐linked polyubiquitin chains, was up‐ and down‐regulated in Shank3 TG and KO striatal synapses, respectively. Consistently, we found that the synaptic levels of Lys63‐linked polyubiquitin chains were down‐ and up‐regulated in the Shank3 TG and KO striata, respectively. Furthermore, by isolating and analyzing the synaptic Cyld complex, we generated a Cyld interactome consisting of 103 proteins, which may include Cyld substrates. Bioinformatic analyses suggested associations of the Cyld interactome with a few brain disorders and synaptic functions. Taken together, these results suggest that Shank3 regulates the synaptic abundance of Cyld in the mouse striatum and, thereby, potentially modulates the Lys63‐linked polyubiquitination of striatal synaptic proteins. [ABSTRACT FROM AUTHOR]

Both deletions and duplications of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, encoding excitatory postsynaptic scaffolds, are causally associated with various brain disorders, suggesting that proper Shank3 dosage is critical for normal brain development and function. In addition to its well-established synaptic functions, recent studies have suggested that Shank3 can also affect gene expression in the nucleus. However, it has not been investigated whether there are a group of genes whose directional expression is regulated in a Shank3 dosage-dependent manner (i.e. showing opposite changes in expression following Shank3 reduction and overexpression). This is an important issue to be examined for better understanding why neuronal development and function are sensitive to Shank3 dosage, and how much transcriptional changes contribute to neuronal phenotypes affected by Shank3 dosage. To examine this, we performed transcriptome analyses on the striatum of Shank3 heterozygous and knock-out mice, which identified three and 17 differentially expressed genes, respectively. We then compared the results to those of our previous striatal transcriptome analysis of Shank3 overexpressing mice and identified 31 candidate genes showing directional expression changes in a Shank3 dosage-dependent manner. However, overall, their Shank3 dosage-dependent fold changes were very subtle (average of absolute log2(fold change) was 0.139). Meanwhile, the gene set enrichment analyses of the striatal transcriptome suggested that Shank3 dosage may affect anchoring junction-related functions. Taken together, these results suggest that Shank3 dosage minimally affects directional gene expression changes in the mouse striatum. [ABSTRACT FROM AUTHOR]

FIGURE: Expression and stability of wild-type (WT) or mutant CYFIP2 in HEK293T cells or cultured neurons. Second, we examined protein levels of wild-type or mutant CYFIP2 by transfecting the constructs into HEK293T cells or cultured rat cortical neurons (Fig). [Extracted from the article]

Variants of the SH3 and multiple ankyrin repeat domain 3 (SHANK3) gene, encoding excitatory postsynaptic core scaffolding proteins, are causally associated with numerous neurodevelopmental and neuropsychiatric disorders, including autism spectrum disorder (ASD), bipolar disorder, intellectual disability, and schizophrenia (SCZ). Although detailed synaptic changes of various Shank3 mutant mice have been well characterized, broader downstream molecular changes, including direct and indirect changes, remain largely unknown. To address this issue, we performed a transcriptome analysis of the medial prefrontal cortex (mPFC) of adult Shank3 -overexpressing transgenic (TG) mice, using an RNA-sequencing approach. We also re-analyzed previously reported RNA-sequencing results of the striatum of adult Shank3 TG mice and of the prefrontal cortex of juvenile Shank3+/ΔC mice with a 50–70% reduction of Shank3 proteins. We found that several myelin-related genes were significantly downregulated specifically in the mPFC, but not in the striatum or hippocampus, of adult Shank3 TG mice by comparing the differentially expressed genes (DEGs) of the analyses side by side. Moreover, we also found nine common DEGs between the mPFC and striatum of Shank3 TG mice, among which we further characterized ASD- and SCZ-associated G protein-coupled receptor 85 (Gpr85), encoding an orphan Gpr interacting with PSD-95. Unlike the mPFC-specific decrease of myelin-related genes, we found that the mRNA levels of Gpr85 increased in multiple brain regions of adult Shank3 TG mice, whereas the mRNA levels of its family members, Gpr27 and Gpr173 , decreased in the cortex and striatum. Intriguingly, in cultured neurons, the mRNA levels of Gpr27 , Gpr85 , and Gpr173 were modulated by the neuronal activity. Furthermore, exogenously expressed GPR85 was co-localized with PSD-95 and Shank3 in cultured neurons and negatively regulated the number of excitatory synapses, suggesting its potential role in homeostatic regulation of excitatory synapses in Shank3 TG neurons. Finally, we performed a gene set enrichment analysis of the RNA-sequencing results, which suggested that Shank3 could affect the directional expression pattern of numerous ribosome-related genes in a dosage-dependent manner. To sum up, these results reveal previously unidentified brain region-specific and broad molecular changes in Shank3 -overexpressing mice, further elucidating the complexity of the molecular pathophysiology of SHANK3 -associated brain disorders. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Enterobacteriaceae (CRE) are usually resistant to most of antibiotics. Infections caused by such bacteria have a high mortality and pose a serious threat to clinical management and public health. Enterobacter cloacae ranks third among Enterobacteriaceae that cause nosocomial infections. In this study, the molecular characteristics of carbapenem-resistant E. cloacae in China were investigated. From November 2012 to August 2016, 55 non-repetitive strains of carbapenem-resistant E. cloacae were collected from 12 hospitals in 11 Chinese cities. The bacteria were identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry. Antimicrobial susceptibility tests were determined by agar dilution method. Carbapenemase and other β-lactamase genes were detected with PCR and sequencing. Multilocus sequence typing and plasmid conjugation tests were performed. Among the 55 E. cloacae strains, 50 strains were detected to produce 8 types of carbapenemase including NDM-1, NDM-5, IMP-4, IMP-26, IMP-1, KPC-2, and VIM-1. NDM-1 accounted for 68.0% (34/50) among the carbapenemase-producing E. cloacae. A total of 24 sequence types were identified and ST418 was the most common, accounting for 20% (11/55). For further investigation, a pulsed-field gel electrophoresis (PFGE) assay was conducted to identify the PFGE patterns of the strains. These 23 isolates yielded 13 PFGE patterns, which were designated as type A–M. Eight isolates obtained from Shenzhen had the same PFGE pattern (type A) and the remaining 15 isolates belonged to the other 12 PFGE patterns (type B–M). The observation that 8 of the 15 blaNDM−1-positive E. cloacae isolates obtained from Shenzhen with the same PFGE pattern (type A) suggested a transmission outbreak of a common strain. S1-nuclease PFGE and Southern blotting were also conducted to estimate the size of plasmids harbored by blaNDM−1-positive strains. The results showed that the plasmids harboring the blaNDM−1 gene ranged in size from approximately 52–58 kilobases. Our study indicates that carbapenem-resistant E. cloacae strains that produce NDM carbapenemase have strong resistance. Early detection and monitoring of the prevalence of these strains are urgent. [ABSTRACT FROM AUTHOR]

Neosporosis is an infectious disease caused by Neospora caninum , and it primarily affects cattle and dogs. An infection by N. caninum causes fetal abortion and neonatal mortality. Previous proteomics and immunoscreening analyses revealed that N. caninum dense granule antigen 2 (NcGRA2) has potential for serodiagnosis of N. caninum. Consequently, we expressed the truncated NcGRA2 (NcGRA2t), which lacks a signal peptide. We compared the serodiagnostic performances of recombinant NcGRA2t with that of truncated surface antigen 1 of N. caninum (NcSAG1t). Specificity testing using sera from mice infected with Toxoplasma gondii indicated that the NcGRA2t recombinant protein does not cross-react with T. gondii . In addition, we detected anti-NcGRA2t antibody at the acute stage in experimentally infected dogs, while detecting anti-NcSAG1t antibody during both the acute and chronic stages. Our results suggest that the levels of anti-NcGRA2 antibody reflect parasite activation in dogs. In conclusion, antibodies against NcGRA2t and NcSAG1t are suitable indicators to distinguish the acute and chronic stages of N. caninum infection. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: The concentration of tyrosine and the ratio of branch-amino acid to the aromatic amino acid in phenylketonuria (PKU) patients are much lower than that of normal people, which reveal that PKU patients have amino acid metabolism disorder. The aim of the present study was to investigate the arginine level in blood, the expression of argininosuccinate synthetase (ASS), the rate-limiting enzyme in arginine synthesis pathway, and the methylation of ASS in patients with PKU. Design and Methods: Twenty-five children with PKU and 65 healthy controls were investigated in this study. Blood concentration of arginine was analyzed by automatic amino acid analyzer. The methylation of ASS gene promoter was evaluated by using methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing PCR (BSP) methods, and the mRNA level of ASS was evaluated by semi-quantitative RT-PCR. Results: Blood concentration of arginine in PKU patients without dietary control was 0.017±0.009mmol/L while in normal persons was 0.129±0.007mmol/L, which is statistically significant (P<0.001). The promoter of ASS was methylated in PKU (15/15, 100%) but not in normal persons (0/15). The mRNA level of ASS in PKU patients was lower than that of normal people, which was well correlated with its methylation status. Conclusions: The silencing of ASS due to aberrant promoter CpG methylation may be an important mechanism for arginine biosynthesis disorders in PKU. High levels of phenylalanine and low levels of arginine are common characteristics in PKU patients. These findings would extend the current understanding of arginine, ASS in the development of PKU disease. [Copyright &y& Elsevier]

Abstract: Toxoplasma gondii is the etiologic agent of toxoplasmosis. Although the combination of sulfadiazine and pyrimethamine is used as therapy for this disease, these drugs can have serious side effects and its use is limited in pregnancy. Therefore there is a need for new anti-T. gondii drugs in the clinic. Some systems for T. gondii drug screening have been described, but these have limitations and can be difficult. In order to solve these problems, we established a system to screen drugs in vitro that involved using cell viability methods to calculate drug selectivities, which are Trypan blue, [3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuzolium, inner salt] (MTS) method and lactate dehydrogenase (LDH) assay. These assays were simple to establish and perform. The IC50 values calculated from the morphological assay were not significantly different from the EC50 values calculated using the other three methods. In particular, the results of the morphological assay showed a distinct association with the MTS assay (R =0.9841). These assays could be used for a wide range of applications in the screening of new drugs and may provide an alternative to the techniques currently used to screen for candidate anti-T. gondii compounds in vitro. In this study, we also tested many compounds and identified some that had a good anti-T. gondii effect in vitro based on the MTS assay. This simple and fast system allowed us to determine which compounds to investigate further using in vivo experiments. [Copyright &y& Elsevier]

• Four series of DHA derivatives were evaluated for anti- T. gondii activity in vitro. • Compound A2 had the better anti- T. gondii activity than DHA in in vitro and in vivo. • Compound A2 had better inhibitory effects on T. gondii in vivo than spiramycin. In this study, four series of dihydroartemisinin derivatives were designed, synthesized, and evaluated for anti-toxoplasma gondii activity, and calculated the selectivity index (SI). It was the higher the SI, the better the effect of this compound against Toxoplasma gondii. Our goal was to filter out compounds that were bigger SI than the lead compound. The compound with the highest SI was selected for the anti-toxoplasmosis test in mice in vivo. Among the synthesized compounds, the (3R,5aS,6R,8aS,9R,12R,12aR)-3,6,9-trimethyl-decahydro-12 H -3,12-epoxy[1,2]di-oxepino[4,3 - i ]isochromen-10-yl-(te-rt- butoxycarbonyl)- l -alaninate (A2) exhibited the most potent anti- T. gondii activity and low cytotoxicity (SI: 6.44), yielding better results than the lead compound DHA (SI: 1.00) and the clinically used positive-control drug spiramycin (SI: 0.72) in vitro. Furthermore, compound A2 had better growth inhibitory effects on T. gondii in vivo than spiramycin did and significantly reduced the number of tachyzoites in the peritoneal cavity of mice (P < 0.01). The evaluation of the data generated in the T. gondii mouse infection model indicates that compound A2 treatment was a good inhibitor of T. gondii in vivo and that it was effective in relieving the liver damage induced by T. gondii. In addition, the results of a docking study revealed that A2 could become a better T. gondii calcium-dependent protein kinase1 (Tg CDPK1) inhibitor. For this reason, compound A2 has potential as an anti-parasitic drug. Further studies are required to elucidate the mechanism of the action of compound A2, as well as to develop drug delivery systems for patients. [ABSTRACT FROM AUTHOR]

Variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) gene are associated with early-onset epileptic encephalopathy, intellectual disability, and developmental delay. However, the current understanding of the molecular functions of CYFIP2 is limited to those related to actin dynamics, and thus, the detailed mechanisms of CYFIP2 -associated brain disorders remain largely unknown. Here, we isolated the neonatal forebrain CYFIP2 complex using newly generated Cyfip2-3×Flag knock-in mice, and performed mass spectrometry-based analyses to identify proteins in the complex. The CYFIP2 interactome, consisting of 140 proteins, contained not only the expected actin regulators but also 25 RNA-binding proteins (RBPs) including Argonaute proteins. Functionally, overexpression of brain disorder-associated CYFIP2 R87 variants, but not wild-type, inhibited stress granule formation in HeLa cells. Mechanistically, the CYFIP2 R87 variants formed intracellular clusters with Argonaute proteins under both basal and stress conditions, and thereby possibly preventing their assembly into stress granules. Beyond identifying CYFIP2 interactors in vivo , these results may provide novel insights for better understanding the molecular mechanisms of CYFIP2 -associated brain disorders. • The forebrain CYFIP2 complex was isolated from neonatal Cyfip2-3×Flag KI mice. • Mass spectrometry-based analyses identified 140 proteins in the CYFIP2 complex. • The complex included actin regulators and RNA-binding proteins such as Argonaute. • Disease-associated CYFIP2 R87 variants inhibited stress granule formation in cells. • The CYFIP2 R87 variants formed intracellular clusters with Argonaute. [ABSTRACT FROM AUTHOR]

Variants of the SHANK3 gene, which encodes a core scaffold protein of the postsynaptic density of excitatory synapses, have been causally associated with numerous brain disorders. Shank3 proteins directly bind zinc ions through their C-terminal sterile α motif domain, which enhances the multimerization and synaptic localization of Shank3, to regulate excitatory synaptic strength. However, no studies have explored whether zinc affects the protein interactions of Shank3, which might contribute to the synaptic changes observed after zinc application. To examine this, we first purified Shank3 protein complexes from mouse brain synaptosomal lysates that were incubated with different concentrations of ZnCl 2 , and analyzed them with mass spectrometry. We used strict criteria to identify 71 proteins that specifically interacted with Shank3 when extra ZnCl 2 was added to the lysate. To characterize the zinc-induced Shank3 interactome, we performed various bioinformatic analyses that revealed significant associations of the interactome with subcellular compartments, including mitochondria, and brain disorders, such as bipolar disorder and schizophrenia. Together, our results showing that zinc affected the Shank3 protein interactions of in vitro mouse synaptosomes provided an additional link between zinc and core synaptic proteins that have been implicated in multiple brain disorders. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii ( T . gondii ) is an important pathogen which can causes serious public health problems. Since the current therapeutic drugs for toxoplasmosis present serious host toxicity, research on effective and new substances of relatively low toxicity is urgently needed. This study was carried out to evaluate the anti-parasitic effect of oxymatrine (OM) and matrine (ME) against T . gondii in vitro and in vivo . In our study, the anti- T . gondii activities of ME and OM were evaluated in vitro using cell counting kit-8 assay, morphological observation and trypan blue exclusion assay. In vivo , mice were sacrificed four days post-infection and ascites were drawn out to determine the extent of tachyzoite proliferation. Viscera indexes and liver biochemical parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione (GSH) and malondialdehyde (MDA), were examined to evaluate the toxicity of compounds to mice. As a result, OM and ME showed anti- T . gondii activity but low selectivity toxicity to HeLa cells. Both compounds also significantly decreased the number of tachyzoites in peritoneal cavity and recovered the levels of ALT, AST, GSH and MDA in liver. Moreover, the mice treated with OM or ME achieved better results in viscera index and survival rate than that of spiramycin. These results suggest that OM and ME are likely the sources of new drugs for toxoplasmosis, and further studies will be necessary to compare the efficacy of drug combination, as well as identify its action of mechanism. [ABSTRACT FROM AUTHOR]

Abstract: A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection. [Copyright &y& Elsevier]

Toxoplasma gondii is an important pathogen that causes serious public health problems. Currently, therapeutic drugs for toxoplasmosis cause serious side effects, and more effective and novel substances with relatively low toxicity are urgently needed. Ursolic acid (UA) has many properties that can be beneficial to healthcare. In this study, we synthesized eight series of UA derivatives bearing a tetrazole moiety and evaluated their anti- T. gondii activity in vitro using spiramycin as a positive control. Most of the synthesized derivatives exhibited better anti- T. gondii activity in vitro than UA , among which compound 12a exhibited the most potent anti- T. gondii activity. Furthermore, the results of biochemical parameter determination indicated that 12a effectively restored the normal body weight of mice infected with T. gondii , reduced hepatotoxicity, and exerted significant anti-oxidative effects compared with the findings for spiramycin. Additionally, our molecular docking study indicated that the synthesized compounds could act as potential inhibitors of T. gondii calcium-dependent protein kinase 1 (TgCDPK1), with 12a possessing strong affinity for TgCDPK1 via binding to the key amino acids GLU129 and TYR131. Image 1 • The synthesized ursolic acid derivatives bearing tetrazole moieties were evaluated for their anti- T. gondii activities. • Compound 12a is a promising hit for the development of new anti- T. gondii agents. • The investigation of the mechanism in vivo and binding mode of action of 12a to TgCDPK1 was performed. [ABSTRACT FROM AUTHOR]

New anti- Toxoplasma gondii agents are in demand due to the emergence of high toxicity. Ginseng polysaccharides and saponins can be used to treat the replication of Toxoplasma gondii in an attempt to determine whether the medicinal uses of ginseng are supported by pharmacological effects. Anti- Toxoplasma gondii activities of ginseng polysaccharides and saponins were examined in vitro and in vivo . The findings are the survival time and rate of Toxoplasma gondii infected mice after the intake of the total polysaccharides and saponins increased compared to untreated control mice. The survival rate of mice treated with ginseng saponins was the highest at 83.3%, the phenomenon of splenomegaly of mice was decreased especially ( p < 0.05) treated with ginseng polysaccharides. Accordingly, ginseng polysaccharides and saponins have a potential application in anti- Toxoplasma gondii treatments.

Shank3 is a member of the Shank family proteins (Shank1-3), which are abundantly present in the postsynaptic density (PSD) of neuronal excitatory synapses. As a core scaffold in the PSD, Shank3 plays a critical role in organizing the macromolecular complex, ensuring proper synaptic development and function. Clinically, various mutations of the SHANK3 gene are causally associated with brain disorders such as autism spectrum disorders and schizophrenia. However, recent in vitro and in vivo functional studies and expression profiling in various tissues and cell types suggest that Shank3 also plays a role in cardiac function and dysfunction. For example, Shank3 interacts with phospholipase Cβ1b (PLCβ1b) in cardiomyocytes, regulating its localization to the sarcolemma and its role in mediating Gq-induced signaling. In addition, changes in cardiac morphology and function associated with myocardial infarction and aging have been investigated in a few Shank3 mutant mouse models. This review highlights these results and potential underlying mechanisms, and predicts additional molecular functions of Shank3 based on its protein interactors in the PSD, which are also highly expressed and function in the heart. Finally, we provide perspectives and possible directions for future studies to better understand the roles of Shank3 in the heart., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kim, Ko, Jin, Zhang, Kang, Ma, Li, Choi and Han.)

Six series of (+)-usnic acid derivatives were synthesized. The IC 50 values of these compounds were determined in T. gondii infected HeLa cells (μM) and in HeLa cells (μM), and their selectivity indexes (SI) were calculated. In vitro, most of the derivatives tested in this study exhibited more anti activity than that of the parent compound (+)-usnic acid and the positive control drugs. Among these derivatives, methyl ( E )-(1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[ b , d ]furan-2(1 H )-ylidene)ethyl)phenylalaninate ( D3 ) showed the most effective anti- T. gondii activity (selectivity >2.77). In comparison with the clinically used positive control drugs sulfadiazine (selectivity 1.15), pyrimethamine (selectivity 0.89), spiramycin (selectivity 0.72), and the lead compound (+)-usnic acid (selectivity 0.96), D3 showed better results in vitro. Furthermore, D3 and ( E )-6-acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-(quinolin-6-ylamino)ethylidene)dibenzo[ b , d ]furan-1,3(2 H ,9b H )-dione ( F3 ) had greater inhibitory effects on T. gondii (inhibition rates 76.0% and 64.6%) in vivo in comparison to spiramycin (inhibition rate 55.2%); in the peritoneal cavity of mice, the number of tachyzoites was significantly reduced ( p < 0.001) in vivo. Additionally, some biochemical parameters were measured and spleen indexes were comprehensively evaluated, and the results indicated that mice treated with both compound D3 and compound F3 showed reduced hepatotoxicity and significantly enhanced antioxidative effects in comparison to the normal group. Granuloma and cyst formation were effected by the inhibition of compound D3 and compound F3 in liver sections. Overall, these results indicated that D3 and F3 for use as anti- T. gondii agents are promising lead compounds.

Cytoplasmic FMR1-interacting protein 2 (CYFIP2) is a key component of the WAVE regulatory complex (WRC) which regulates actin polymerization and branching in diverse cellular compartments. Recent whole exome sequencing studies identified de novo hotspot variants in CYFIP2 from patients with early-onset epileptic encephalopathy and microcephaly, suggesting that CYFIP2 may have some functions in embryonic brain development. Although perinatal lethality of Cyfip2 -null ( Cyfip2 -/- ) mice was reported, the exact developmental time point and cause of lethality, and whether Cyfip2 -/- embryonic mice have brain abnormalities remain unknown. We found that endogenous Cyfip2 is mainly expressed in the brain, spinal cord, and thymus of mice at late embryonic stages. Cyfip2 -/- embryos did not show lethality at embryonic day 18.5 (E18.5), but their body size was smaller than that of wild-type (WT) or Cyfip2 +/- littermates. Meanwhile, at postnatal day 0, all identified Cyfip2 -/- mice were found dead, suggesting early postnatal lethality of the mice. Nevertheless, the brain size and cortical cytoarchitecture were comparable among WT, Cyfip2 +/- , and Cyfip2 -/- mice at E18.5. Using RNA-sequencing analyses, we identified 98 and 72 differentially expressed genes (DEGs) from the E18.5 cortex of Cyfip2 +/- and Cyfip2 -/- mice, respectively. Further bioinformatic analyses suggested that extracellular matrix (ECM)-related gene expression changes in Cyfip2 -/- embryonic cortex. Together, our results suggest that CYFIP2 is critical for embryonic body growth and for early postnatal survival, and that loss of its expression leads to ECM-related gene expression changes in the embryonic cortex without severe gross morphological defects.

Data for a total of 164 bloodstream infection cases due to carbapenem-resistant Enterobacteriaceae (CRE) from 2013 to 2017 were retrospectively collected from 36 tertiary hospitals in 19 provinces in China to evaluate the outcomes and risk factors for mortality by univariable and multivariable analysis. The most frequent infecting species was Klebsiella pneumoniae (69.5%, 114/164). The overall in-hospital and 14-day mortality rates were 32.9% (54/164) and 31.1% (42/135), respectively. Multivariable analysis revealed that septic shock (adjusted odds ratio [aOR], 6.339; 95% confidence interval [CI], 1.586 to 25.332; P  = 0.009), the Pitt bacteremia score (aOR, 1.300; 95% CI, 1.009 to 1.676; P  = 0.042), and the Charlson comorbidity index (aOR, 1.392; 95% CI, 1.104 to 1.755; P  = 0.005) were independently associated with a hazard effect on mortality. Combination therapy, especially tigecycline-based combination therapy, resulted in relatively low rates of in-hospital mortality and failure in clearance of CRE infection. Survival analysis revealed that appropriate therapy was associated with a lower 14-day mortality rate than inappropriate therapy (including nonactive therapy; P  = 0.022), that combination therapy was superior to monotherapy ( P  = 0.036), that metallo-β-lactamase producers were associated with a lower 14-day mortality than strains without carbapenemases or KPC-2 producers ( P  = 0.009), and that strains with MICs of >8 mg/liter for meropenem were associated with a higher 14-day mortality rate than those with MICs of ≤8 mg/liter ( P  = 0.037). Collectively, the severity of illness, meropenem MICs of >8 mg/liter, and carbapenemase-producing types were associated with the clinical outcome. Early detection of the carbapenemase type and initiation of appropriate combination therapy within 96 h might be helpful for improving survival., (Copyright © 2018 American Society for Microbiology.)

Carbapenem-resistant Enterobacteriaceae (CRE) are usually resistant to most of antibiotics. Infections caused by such bacteria have a high mortality and pose a serious threat to clinical management and public health. Enterobacter cloacae ranks third among Enterobacteriaceae that cause nosocomial infections. In this study, the molecular characteristics of carbapenem-resistant E. cloacae in China were investigated. From November 2012 to August 2016, 55 non-repetitive strains of carbapenem-resistant E. cloacae were collected from 12 hospitals in 11 Chinese cities. The bacteria were identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry. Antimicrobial susceptibility tests were determined by agar dilution method. Carbapenemase and other β-lactamase genes were detected with PCR and sequencing. Multilocus sequence typing and plasmid conjugation tests were performed. Among the 55 E. cloacae strains, 50 strains were detected to produce 8 types of carbapenemase including NDM-1, NDM-5, IMP-4, IMP-26, IMP-1, KPC-2, and VIM-1. NDM-1 accounted for 68.0% (34/50) among the carbapenemase-producing E. cloacae . A total of 24 sequence types were identified and ST418 was the most common, accounting for 20% (11/55). For further investigation, a pulsed-field gel electrophoresis (PFGE) assay was conducted to identify the PFGE patterns of the strains. These 23 isolates yielded 13 PFGE patterns, which were designated as type A-M. Eight isolates obtained from Shenzhen had the same PFGE pattern (type A) and the remaining 15 isolates belonged to the other 12 PFGE patterns (type B-M). The observation that 8 of the 15 bla NDM-1 -positive E. cloacae isolates obtained from Shenzhen with the same PFGE pattern (type A) suggested a transmission outbreak of a common strain. S1-nuclease PFGE and Southern blotting were also conducted to estimate the size of plasmids harbored by bla NDM-1 -positive strains. The results showed that the plasmids harboring the bla NDM-1 gene ranged in size from approximately 52-58 kilobases. Our study indicates that carbapenem-resistant E. cloacae strains that produce NDM carbapenemase have strong resistance. Early detection and monitoring of the prevalence of these strains are urgent.

Toxoplasma gondii is an important opportunistic pathogen that causes toxoplasmosis, which has very few therapeutic treatment options. The most effective therapy is a combination of pyrimethamine and sulfadiazine; however, their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound, (Z)-1-[(5-nitrofuran-2-yl)methyleneamino]-imidazolidine-2,4-dione (nitrofurantoin), showed anti-T. gondii effects in vitro and in vivo. In HeLa cells, the selectivity of nitrofurantoin was 2.3, which was greater than that of pyrimethamine (0.9). In T. gondii-infected female ICR mice, the inhibition rate of T. gondii growth in the peritoneal cavity was 44.7% compared to the negative control group after 4-day treatment with 100 mg/kg of nitrofurantoin. In addition, hematology indicators showed that T. gondii infection-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, biochemical parameters involved in liver injury, were reduced by nitrofurantoin significantly. Moreover, nitrofurantoin exerted significant effects on the index of antioxidant status, i.e., malondialdehyde (MDA) and glutathione (GSH). The nitrofurantoin-treated group inhibited the T. gondii-induced MDA levels while alleviating the decrease in GSH levels. Thus, nitrofurantoin is a potential anti-T. gondii candidate for clinical application.

Introduction: A lot of in vitro technologies have been developed to screen drugs for toxoplasmosis, which is caused by Toxoplasma gondii and is one of the most serious infectious diseases in the world. However, developed screening methods still have limitation such as inaccuracy, labor-intensive and time-consuming procedure. Therefore, the development of simpler, more efficient and accurate high-throughput screening assay is needed., Areas Covered: The present review gives the overview of in vitro screening technologies described in literatures so far including morphological assay, incorporation of [(3)H]uracil assay, enzyme-linked immunosorbent assay (ELISA), colorimetric microtiter assay (β-galactosidase assay), flow cytometric quantification assay, yellow fluorescent protein assay and cell viability assay. The authors discuss how these methods are efficient and/or limited for screening anti-T. gondii drugs. The authors further suggest brand-new technologies which are faster, simpler, more effective and available for high-throughput screening., Expert Opinion: Options for clinical treatment of toxoplasmosis are currently very limited. Thus, more accurate in vitro screening methods must be established to identify the most effective anti-T. gondii drugs from random screening of compounds. At the same time, based on genome information, combination of an appropriate screening technology, combinatorial chemistry and computational biology may increase the efficiency of target-based drug discovery against T. gondii.

An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.