First Report of Alternaria Leaf Spot Caused by Alternaria alternata on Ulmus parvifolia in Jiangsu, China.

Ulmus parvifolia Jacq. is an important tree with ornamental value, which is widely planted in Hebei and southern regions of China. In September 2022, a leaf spot symptom was observed on about approximately 20% U. parvifolia seedlings growing a tree farm (20000 m2) of Jiangsu Academy of Forestry (118°45'57.30″E, 31°51'27. 94″N). Gray to black spots appeared on leaves of seedlings. Five diseased leaves were collected from five different seedlings. The pieces were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in the darkness. Pure cultures were obtained by monosporic isolation. Six isolates with identical morphological features and the internal transcribed spacer (ITS) sequences were obtained (the isolate rate of 67%), and identified as Alternaria sp. A representative isolate, LY-1-1 was used for the further study. The colony of LY-1-1, growing on PDA was cotton-like and brown in color with gray-white aerial hyphae on their surfaces, and its reverse was dark grey. The conidia were ovate to pear-shaped, brown in color, with 1 to 4 transverse septa and 0 to 1 longitudinal septa, parietal cells extending into the beak, and measured 7.1 to 12.5×3.8 to 7.1 µm (n=35). These characteristics were consistent with the description of Alternaria sp. (Simmons 2007). The regions of ITS, large subunit ribosomal RNA (LSU), small subunit ribosomal RNA (SSU), anonymous region OPA10-2 genomic sequence (OPA10-2), Alternaria 1 major allergen (Alta1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1) genes (GenBank Accession No. OR047916, OR051904, OR047919, OR061065, OR061063, OR061064, and OR061062, respectively) were amplified (White et al. 1990; Woudenberg et al. 2015) and sequenced. These obtained sequences showed 99.86-100% similarity to the ITS (514/515 bp) of A. alternata isolate SPM-2 (OR378581), LSU (801/801 bp) of isolate B9 (OR366492), SSU (1019/1020 bp) of strain LSU0766 (MT000349), OPA10-2 (632/633 bp) of strain 19-1 (MN185000), Alta1 (470/470 bp) of strain CMML21-73 (OQ831518), GAPDH (177/177 bp) of isolate CS36-3 (KY814638), and TEF1 (240/240 bp) of isolate SY-6 (OP980553). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate LY-1-1 clustered in the A. alternata clade with 98% bootstrap support. Three 3-month-old U. parvifolia seedlings were wounded with a sterile needle and inoculated with 20 µL conidia suspension (1×106 spores/mL) on the left sides of leaves. Inoculation on the right side with 20 µL of sterile water was treated as a control. All inoculated plants were incubated in a greenhouse at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, typical gray to black spots were found on the left sides of all inoculated leaves, and the control did not have any leaf spot symptoms. Subsequently, the same fungus was reisolated and identified based on morphological and molecular traits, fulfilling Koch's postulates. The A. alternata has been reported to cause leaf spot on pecan (Wu et al. 2020), fruit spot on olive (Alam et al. 2019) and fruit rot on lychee (Alam et al. 2017). However, there are no other reports of A. alternata on U. parvifolia in the world. Thus, this study provides an important reference for the biology, epidemiology of A. alternata.

Molecular Mechanisms for Regulating Stomatal Formation across Diverse Plant Species.

Plant stomata play a crucial role in photosynthesis by regulating transpiration and gas exchange. Meanwhile, environmental cues can also affect the formation of stomata. Stomatal formation, therefore, is optimized for the survival and growth of the plant despite variable environmental conditions. To adapt to environmental conditions, plants open and close stomatal pores and even regulate the number of stomata that develop on the epidermis. There are great differences in the leaf structure and developmental origin of the cell in the leaf between Arabidopsis and grass plants. These differences affect the fine regulation of stomatal formation due to different plant species. In this paper, a comprehensive overview of stomatal formation and the molecular networks and genetic mechanisms regulating the polar division and cell fate of stomatal progenitor cells in dicotyledonous plants such as Arabidopsis and Poaceae plants such as Oryza sativa and Zea mays is provided. The processes of stomatal formation mediated by plant hormones and environmental factors are summarized, and a model of stomatal formation in plants based on the regulation of multiple signaling pathways is outlined. These results contribute to a better understanding of the mechanisms of stomatal formation and epidermal morphogenesis in plants and provide a valuable theoretical basis and gene resources for improving crop resilience and yield traits.

Design and Synthesis of a Triazine-Based sp2 Carbon-Conjugated Covalent Organic Framework for Blue Light Photocatalysis.

Fully conjugated covalent organic frameworks (COFs) can exhibit great potential in semiconductor photocatalysis. But their syntheses remain elusive due to the low reversibility of vinylene linkage. Herein, by tuning the amount of base and temperature, a novel triazine-based sp2  carbon-conjugated COF (TA-sp2 c-COF) is successfully constructed over Cs2 CO3 . Besides, the influence of modulating factors on the chemical and optoelectronic properties of TA-sp2 c-COF is thoroughly investigated. TA-sp2 c-COF adopts an eclipsed AA stacking structure with uniform micropores (1.4 nm). The blue light photocatalysis of the highly crystalline TA-sp2 c-COF is established for the selective oxidative coupling of amines with oxygen, and the predominant role of superoxide is identified in forming imines. This work foretells that meticulous modulation of reaction conditions is the key to constructing sp2  carbon-conjugated COFs toward solar photocatalysis.

Strophiofimbrins A and B: Two Rearranged Norditerpenoids with Novel Tricyclic Carbon Skeletons from Strophioblachia fimbricalyx.

Two rearranged norditerpenoids with novel tricyclic carbon skeletons, strophiofimbrin A (1) and strophiofimbrin B (2), were isolated from Strophioblachia fimbricalyx. Their structures were established by 1D/2D NMR spectroscopy, HRESIMS, quantum chemistry calculations, and X-ray diffraction analyses. 1 and 2 represented the first examples of diterpenoids with unprecedented 5/6/7-fused ring systems. In the proposed biosynthetic pathway, they were suspected to derive from cleistanthane norditerpenoids via ring opening, expansion, cyclization, and rearrangement based on the existence of phenanthrenone and cleistanthane diterpenoids from Strophioblachia and Trigonostemon, two closely related genera of the Euphorbiaceae family. Furthermore, compounds 1 and 2 exhibited significant proliferation inhibition and obvious neuroprotective effects.

Analysis of the Analgesic Effect, Emotion, and Safety of Esketamine in Cesarean Section Analgesia for Puerperae.

Objective: This retrospective study aimed to evaluate the effectiveness and safety of esketamine as an analgesic during cesarean section procedures. Methods: 102 puerperae undergoing cesarean section were divided into a control group and an esketamine (SK) group. Various parameters, including HR, MAP, and postoperative pain, were analyzed. Blood gas analysis and Apgar scores were assessed in neonates. Postoperative depression and satisfaction were evaluated in puerperae. Drug concentrations were measured using liquid-phase tandem mass spectrometry. Results: No significant differences in dimension levels were observed between the two groups (P > .05). However, the SK group showed better HR and MAP indicators at various time points, less postoperative pain, and better mental well-being on postpartum days 1, 3, and 7 (P < .05). Adverse reaction rates were similar between groups (P > .05), but postoperative satisfaction was significantly different (P = .027). Neonatal outcomes did not differ significantly (P > .05). In the SK group, SK2 and SK3 groups had better results compared to SK1 (P < .05). Conclusion: Esketamine during cesarean section stabilized vital signs, reduced pain, and improved well-being in puerperae without affecting newborns. Optimal dosage: 30 µg/kg/h esketamine, 15 ng/kg/h sufentanil.

[Clinical and molecular genetic analysis of a child with Schmid type metaphyseal chondrodysplasia].

OBJECTIVE: To analyze the clinical features and genotype of a child with Schmid type metaphyseal chondrodysplasia. METHODS: Clinical data of the child and her parents was collected. The child was subjected to high-throughput sequencing, and candidate variant was verified by Sanger sequencing of her family members. RESULTS: Whole exome sequencing revealed that the child has harbored a heterozygous c.1772G>A (p.C591Y) variant of the COL10A1 gene, which was not found in either of her parents. The variant was not found in the HGMD and ClinVar databases, and was rated as likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). CONCLUSION: The heterozygous c.1772G>A (p.C591Y) variant of the COL10A1 gene probably underlay the Schmid type metaphyseal chondrodysplasia in this child. Genetic testing has facilitated the diagnosis and provided a basis for genetic counselling and prenatal diagnosis for this family. Above finding has also enriched the mutational spectrum of the COL10A1 gene.

Diagnosis of asthmatic pneumonia in children by lung ultrasound vs. chest X-ray: an updated systematic review and meta-analysis.

Introduction: Chest X-ray (CXR) is used as the standard diagnostic method in lung diseases, especially in pneumonia, but unfortunately, despite the high risk of receiving radiation, it also has a high false negative rate. Therefore, some researchers recommend ultrasound to diagnose pneumonia. Aim: To investigate the accuracy of lung ultrasound compared to CXR for the diagnosis of pneumonia children by meta-analysis method. Material and methods: Original articles which evaluated accuracy of lung ultrasound compared to chest X-ray for the diagnosis of pneumonia in children, published between 1 January 2010 and 20 March 2021, were identified in the PubMed, Web of Science, Embase, Scopus and Cochrane Library databases. Data synthesis and statistical analysis were carried out using STATA software. Odds ratios with 95% confidence interval (CI), fixed effect model and mean difference with 95% CI, random effect model (REM) were calculated. Results: At the first step, 1016 potentially important research abstracts and titles were discovered in our electronic searches, 8 papers were in agreement with our inclusion criteria. The statistical analysis showed sensitivity of 95.5% (95% CI: 93.6-97.1%) and specificity of 96.3% (95% CI: 92.1-98.4%) for the lung ultrasound, and CXR sensitivity and specificity were 87.4% (95% CI: 84.3-90.0%) and 98.6% (95% CI: 95.8-99.6%), respectively. Conclusions: The present study showed that ultrasound can be useful in diagnosing pneumonia in children, and due to the lack of risk of receiving radiation, this method is even more preferred.

Integrated methylome and transcriptome analysis unravel the cold tolerance mechanism in winter rapeseed(Brassica napus L.).

BACKGROUND: Cytosine methylation, the main type of DNA methylation, regulates gene expression in plant response to environmental stress. The winter rapeseed has high economic and ecological value in China's Northwest, but the DNA methylation pattern of winter rapeseed during freezing stress remains unclear. RESULT: This study integrated the methylome and transcriptome to explore the genome-scale DNA methylation pattern and its regulated pathway of winter rapeseed, using freezing-sensitive (NF) and freezing-resistant (NS) cultivars.The average methylation level decreased under freezing stress, and the decline in NF was stronger than NS after freezing stress. The CG methylation level was the highest among the three contexts of CG, CHG, and CHH. At the same time, the CHH proportion was high, and the methylation levels were highest 2 kb up/downstream, followed by the intron region. The C sub-genomes methylation level was higher than the A sub-genomes. The methylation levels of chloroplast and mitochondrial DNA were much lower than the B. napus nuclear DNA, the SINE methylation level was highest among four types of transposable elements (TEs), and the preferred sequence of DNA methylation did not change after freezing stress. A total of 1732 differentially expressed genes associated with differentially methylated genes (DMEGs) were identified in two cultivars under 12 h and 24 h in three contexts by combining whole-genome bisulfite sequencing( and RNA-Seq data. Function enrichment analysis showed that most DMEGs participated in linoleic acid metabolism, alpha-linolenic acid metabolism, carbon fixation in photosynthetic organisms, flavonoid biosynthesis, and plant hormone signal transduction pathways. Meanwhile, some DMEGs encode core transcription factors in plant response to stress. CONCLUSION: Based on the findings of DNA methylation, the freezing tolerance of winter rapeseed is achieved by enhanced signal transduction, lower lipid peroxidation, stronger cell stability, increased osmolytes, and greater reactive oxygen species (ROS) scavenging. These results provide novel insights into better knowledge of the methylation regulation of tolerance mechanism in winter rapeseed under freezing stress.

Induced Near-Infrared Emission and Controlled Photooxidation based on Sulfonated Crown Ether in Water.

Regulation of physio-chemical properties and reaction activities via noncovalent methodology has become one of increasingly significant topics in supramolecular chemistry and showed inventive applications in miscellaneous fields. Herein, we demonstrate that sulfonated crown ether can form very stable host-guest complexes with a series of push-pull-type photosensitizers, eventually leading to the dramatic fluorescence enhancement in visible and near-infrared regions. Meanwhile, severe suppression in singlet oxygen (1 O2 ) production is found, mainly due to the higher energy barriers between the excited single and triple states upon host-guest complexation. Moreover, such complexation-induced tuneable 1 O2 generation systems has been utilized in adjusting the photochemical oxidation reactions of polycyclic aromatic hydrocarbons (anthracene) and sulfides ((methylthio)benzene) in water. This supramolecularly controlled photooxidation based on the selective molecular binding of crown ether with photosensitizers may provide a feasible and applicable strategy for monitoring and modulating many photocatalysis processes in aqueous phase.

[Clinical characteristics and genetic analysis of a pedigree affected with neonatal central core disease].

OBJECTIVE: To investigate the possible causative factors of central core disease(CCD), the clinical features of a neonatal case with CCD and five patients in the pedigree line were analyzed for RYR1 gene variant. METHODS: Medical and family history inquiries and detailed clinical examinations were performed in the proband. High-throughput sequencing technology was applied to analyze the gene variant of the proband, and Sanger sequencing was applied to verify the pedigree distribution of the variant. RESULTS: The whole exon sequencing results showed that the proband has a missense variant of c. 14591A>C (p.Tyr4864Ser) in the RYR1 gene which was unreported previously; Sanger sequencing results showed that the father, grandfather, the eldest aunt and second aunt of the proband all carried the same variant. The c.14591 A>C variant of RYR1 gene was predicted to be a likely pathogenic (PM2+PM5+PP1+PP3) according to the American College of Medical Genetics and Genomics standards and guidelines. CONCLUSION: The RYR1 gene c.14591A>C (p.Tyr4864Ser) variant may be the genetic cause of the pedigree and genetic testing helps to clarify the diagnosis. Identification of this variant has enriched the variant spectrum of the RYR1 gene.

Generation of Tunable Ultrastrong White-Light Emission by Activation of a Solid Supramolecule through Bromonaphthylpyridinium Polymerization.

Herein, we reported solid supramolecular bromonaphthylpyridinium polymers (P-BrNp), which exhibit tunable phosphorescence emission in the amorphous state enabled by sulfobutylether-ß-cyclodextrin (SBE-ß-CD) and diarylethene derivatives. The monomer BrNp gave single fluorescence emission at 490 nm, while an apparent room-temperature phosphorescence (RTP) at 550 nm emerged for P-BrNp copolymers with various feed ratios. Through fluorescence-phosphorescence dual emission, P-BrNp-0.1 displayed an ultrahigh white-light emission quantum yield of 83.9 %. Moreover, the subsequent assembly with SBE-ß-CD further enhanced the phosphorescent quantum yield of P-BrNp-0.1 from 64.1 % to 71.3 %, accompanied by the conversion of photoluminescence emission from white to yellow. Diarylethene monomers were introduced as photoswitches to realize reversible RTP emission, which can be used in switchable data encryption and multifunctional writing ink.

Metal-Organic Framework-Encapsulated CoCu Nanoparticles for the Selective Transfer Hydrogenation of Nitrobenzaldehydes: Engineering Active Armor by the Half-Way Injection Method.

A novel armor-type composite of metal-organic framework (MOF)-encapsulated CoCu nanoparticles with a Fe3 O4 core (Fe3 O4 @SiO2 -NH2 -CoCu@UiO-66) has been designed and synthesized by the half-way injection method, which successfully serves as an efficient and recyclable catalyst for the selective transfer hydrogenation. In this half-way injection approach, the pre-synthetic Fe3 O4 @SiO2 -NH2 -CoCu was injected into the UiO-66 precursor solution halfway through the MOF budding period. The formed MOF armor could play a role of providing significant additional catalytic sites besides CoCu nanoparticles, protecting CoCu nanoparticles, and improving the catalyst stability, thus facilitating the selective transfer hydrogenation of nitrobenzaldehydes into corresponding nitrobenzyl alcohols in high selectivity (99 %) and conversion (99 %) rather than nitro group reduction products. Notably, this method achieves the precise assembly of a MOF-encapsulated composite, and the ingenious combination of MOF and nanoparticles exhibits excellent catalytic performance in the selective hydrogen transfer reaction, implementing a "1+1>2" strategy in catalysis.

[Clinical characteristics and molecular genetics of seven neonates with congenital nephrogenic diabetes insipidus].

OBJECTIVE: To explore the clinical characteristics, genetic basis and clinical treatment of seven neonates with congenital nephrogenic diabetes insipidus (NDI). METHODS: Clinical data of the patients were collected. High-throughput sequencing was carried out to detect potential variants. Sanger sequencing was used to verify the results. RESULTS: The patients were all males, with the age of onset being 10 to 21 days. All patients were admitted to the hospital for intermittent fever as the first symptom during the neonatal period. Additional symptoms had included polydipsia and polyuria. After the treatment, 5 patients had recovered, the remainders still had NDI symptoms and developmental retardation. Five children were found to harbor pathogenic variants of the AVPR2/AQP2 gene, which included one in-frame mutation of c.645_646insGCACCTACCCTGGGTATCGCC, two missense mutations of c.541C>T and c.419C>A, and two hemizygous deletions of the AVPR2/AQP2 gene. Among these, two were unreported previously. Cases 6 and 7 were a pair of twins. Both had carried homozygous missense variants of c.538G>A of the AVPR2/AQP2 gene, which was known to be pathogenic. CONCLUSION: AVPR2/AQP2 is the main pathogenic gene for congenital NDI, for which two novel pathogenic variants have been discovered in this study. Above results have provided a basis for clinical diagnosis and genetic counseling for the affected pedigrees.

Controllable Singlet Oxygen Generation in Water Based on Cyclodextrin Secondary Assembly for Targeted Photodynamic Therapy.

The construction of supramolecular assembly whose singlet oxygen (1O2) generation capability can be controllably regulated in water still remains challenging. Herein, a novel cyclodextrin secondary assembly was fabricated from the photochromic-switch moiety diarylethene-bridged dicyclodextrin, the adamantane-polypyridyl ruthenium photosensitizer, and the cancer-cell-targeting ligand ß-cyclodextrin-grafted hyaluronic acid, which not only possessed cancer-cell-targeting ability but also served as cell imaging and photodynamic therapy agents with noninvasive controllability. In virtue of the multivalent interactions between the three components, they could self-assemble in two stages to form uniform spherical nanoparticles (OF-NPs) with average diameters of about 80 nm, as indicated by scanning electron microscopy, high-resolution transmission electron microscopy, atomic force microscopy, and dynamic light scattering. Significantly, the prepared OF-NPs exhibited excellent photochromic performance and can transform into their ring-closed form (CF-NPs), accompanied by the efficient energy transfer from donor 2 to CF-1 and gradual quenching of 1O2 generation. Cellular imaging experiments showed that OF-NPs could specifically target the mitochondria of A549 cancer cells, while CF-NPs displayed a negligible red fluorescence signal in A549 cells due to the energy-transfer process. Furthermore, in vitro cytotoxicity tests revealed that upon irradiation with 450 nm light, OF-NPs with 10 µM concentration displayed a remarkable higher cytotoxicity with the cell death rate of up to 88% toward A549 cancer cells, which was approximately 4.4 times higher than that of CF-NPs. Additionally, the apoptosis rate of A549 cells induced by OF-NPs under light irradiation was 4.68 times higher than that of CF-NPs. These well-designed cyclodextrin secondary assemblies successfully achieve noninvasive control over the generation of 1O2 both in water and in cancer cells by irradiation at distinct wavelengths and are further applied in targeted PDT, which avoid the inadvertent photosensitizer activation and provide a new approach for cancer therapy with more safety and high efficiency.

Novel hapten design, antibody recognition mechanism study, and a highly sensitive immunoassay for diethylstilbestrol in shrimp.

Over the past few years, there has been a lack of progress in the quality of diethylstilbestrol (DES) antibodies used in immunoassay. In this study, a new immunizing hapten was designed for remarkably sensitive and specific antibody generation against diethylstilbestrol. By introducing a benzene ring instead of the traditional linear chain alkane as the hapten spacer, a more specific immune reaction was induced in the process of immunization. The developed polyclonal antibodies were characterized using the indirect competitive enzyme-linked immunosorbent assay (icELISA). Under optimized conditions, the half maximal inhibitory concentration (IC50) of the best polyclonal antibody was 0.14 ng/mL and it displayed low cross-reactions (CRs) with the structural analogs such as hexestrol (HEX) and dienestrol (DI). The molecular modeling and quantum chemical computation revealed that the lowest CR of the DES antibody to DI was mainly due to the huge three-dimensional conformational difference between DES and DI. Finally, a highly sensitive icELISA method based on the polyclonal antibody was developed for the determination of DES in shrimp tissue. The limit of detection (LOD) was as low as 0.2 µg/kg in shrimp and the recoveries in the spiked samples ranged from 83.4 to 90.8% with the coefficient of variation less than 13.8%. These results indicated that the use of an aromatic ring as the immunizing hapten spacer arm could be a potential strategy for the enhancement of anti-DES antibody sensitivity, and the established icELISA was applicable to the trace detection of DES in shrimp. Graphical abstract.

Docosahexaenoic acid phospholipid differentially modulates the conformation of G90V and N55K rhodopsin mutants associated with retinitis pigmentosa.

Rhodopsin is the visual photoreceptor of the retinal rod cells that mediates dim light vision and a prototypical member of the G protein-coupled receptor superfamily. The structural stability and functional performance of rhodopsin are modulated by membrane lipids. Docosahexaenoic acid has been shown to interact with native rhodopsin but no direct evidence has been established on the effect of such lipid on the stability and regeneration of rhodopsin mutants associated with retinal diseases. The stability and regeneration of two thermosensitive mutants G90V and N55K, associated with the retinal degenerative disease retinitis pigmentosa, have been analyzed in docosohexaenoic phospholipid (1,2-didocosa-hexaenoyl-sn-glycero-3-phosphocholine; DDHA-PC) liposomes. G90V mutant reconstituted in DDHA-PC liposomes significantly increased its thermal stability, but N55K mutant showed similar thermal sensitivity both in dodecyl maltoside detergent solution and in DDHA-PC liposomes. The retinal release process, measured by fluorescence spectroscopy, became faster in the lipid system for the two mutants. The opsin conformation was stabilized for the G90V mutant allowing improved retinal uptake whereas no chromophore binding could be detected for N55K opsin after photoactivation. The results emphasize the distinct role of DHA on different phenotypic rhodopsin mutations associated with classical (G90V) and sector (N55K) retinitis pigmentosa.

Aloe-emodin inhibits Staphylococcus aureus biofilms and extracellular protein production at the initial adhesion stage of biofilm development.

Staphylococcus aureus (S. aureus) biofilms are clinically serious and play a critical role in the persistence of chronic infections due to their ability to resist antibiotics. The inhibition of biofilm formation is viewed as a new strategy for the prevention of S. aureus infections. Here, we demonstrated that minimum inhibitory concentrations (MICs) of aloe-emodin exhibited no bactericidal activity against S. aureus but affected S. aureus biofilm development in a dose-dependent manner. Further studies indicated that aloe-emodin specifically inhibits the initial adhesion and proliferation stages of S. aureus biofilm development. Scanning electron microscopy (SEM) indicated that the S. aureus ATCC29213 biofilm extracellular matrix is mainly composed of protein. Laser scanning confocal microscope assays revealed that aloe-emodin treatment primarily inhibited extracellular protein production. Moreover, the Congo red assay showed that aloe-emodin also reduced the accumulation of polysaccharide intercellular adhesin (PIA) on the cell surface. These findings will provide new insights into the mode of action of aloe-emodin in the treatment of infections by S. aureus biofilms.

Acacetin Protects Mice from Staphylococcus aureus Bloodstream Infection by Inhibiting the Activity of Sortase A.

Staphylococcus aureus (S. aureus) is a major cause of infection in hospitals and communities. Widespread dissemination of multi-drug resistant S. aureus is a serious threat to the health of humans and animals. An anti-virulence strategy has been widely considered as an alternative therapeutic approach. Inhibitors of virulence factors are able to treat S. aureus infections without influencing the growth or viability of bacteria and rarely lead to bacterial resistance. Sortase A (SrtA) is a membrane-associated cysteine transpeptidase that catalyzes up to 25 surface proteins that covalently bind to cell wall peptidoglycans. In S. aureus, most of these surface proteins have been identified as important virulence factors that are vital in bacterial pathogenesis. In the present study, we show that acacetin, a natural flavonoid compound, inhibits the activity of SrtA in S. aureus (IC50 = 36.46 ± 4.69 µg/mL, 128 µM) which affects the assembly of protein A (SpA) to cell walls and reduces the binding of S. aureus to fibrinogen (Fg). The mechanism of the interaction between acacetin and SrtA were preliminarily discussed using molecular dynamics simulations. The results suggested that acacetin adopted a compact conformation binding at the pocket of the SrtA via residues Arg-139 and Lys-140. By performing an animal infection model, we demonstrated that acacetin was able to protect mice from renal abscess formation induced by S. aureus and significantly increased survival rates. Taken together, these findings suggest that acacetin may be a promising candidate for the development of anti-S. aureus drugs.

Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa.

Mutations in the visual photoreceptor rhodopsin are the cause of the retinal degenerative disease retinitis pigmentosa. Some naturally occurring mutations can lead to protein conformational instability. Two such mutations, N55K and G90V, in the first and second transmembrane helices of the receptor, have been associated with sector and classical retinitis pigmentosa, respectively, and showed enhanced thermal sensitivity. We have carefully analyzed the effect of phospholipid bicelles on the stability and ligand binding properties of these two mutants and compared it with those of the detergent-solubilized samples. We have used a phospholipid bilayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). We find that DMPC/DHPC bicelles dramatically increase the thermal stability of the rhodopsin mutants G90V and N55K. The chromophore stability and regeneration of the mutants were also increased in bicelles when compared to their behavior in a dodecyl maltoside detergent solution. The retinal release process was slowed in bicelles, and chromophore entry, after illumination, was improved for the G90V mutant but not for N55K. Furthermore, fluorescence spectroscopy measurements showed that bicelles allowed more exogenous retinal binding to the photoactivated G90V mutant than in a detergent solution. In contrast, N55K could not reposition any chromophore either in the detergent or in bicelles. The results demonstrate that DMPC/DHPC bicelles can counteract the destabilizing effect of the disease-causing mutations and can modulate the structural changes in the ensuing receptor photoactivation in a distinct specific manner for different retinitis pigmentosa mutant phenotypes.

Differential light-induced responses in sectorial inherited retinal degeneration.

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking. M39R showed a faster rate for transducin activation than WT rhodopsin with a faster metarhodopsinII decay, whereas N55K presented a reduced activation rate and an altered photobleaching pattern. N55K also showed an altered retinal release from the opsin binding pocket upon light exposure, affecting its optimal functional response. Our data suggest that these sector RP mutations cause different protein phenotypes that may be related to their different clinical progression. Overall, these findings illuminate the molecular mechanisms of sector RP associated with rhodopsin mutations.