Isolation of a Virulent Clostridium perfringens Strain from Elaphurus davidianus and Characterization by Whole-Genome Sequence Analysis.

Clostridium perfringens (C. perfringens) is an important veterinary pathogen and a noteworthy threat to human and animal health. Recently, there has been a significant rise in the number of moose fatalities caused by this rare, endemic species in China. Currently, there is an increasing trend in conducting whole-genome analysis of C. perfringens strains originating from pigs and chickens, whereas fewer studies have been undertaken on Elaphurus davidianus-originating strains at the whole-genome level. Our laboratory has identified and isolated five C. perfringens type A from affected Elaphurus davidianus. The current study identified the most potent strain of C. perfringens, which originated from Elaphurus davidianus, and sequenced its genome to reveal virulence genes and pathogenicity. Our findings show that strain CX1-4 exhibits the highest levels of phospholipase activity, hemolytic activity, and mouse toxicity compared to the other four isolated C. perfringens type A strains. The chromosome sequence length of the CX1-4 strain was found to be 3,355,389 bp by complete genome sequencing. The current study unveils the genomic characteristics of C. perfringens type A originating from Elaphurus davidianus. It provides a core foundation for further investigation regarding the prevention and treatment of such infectious diseases in Elaphurus davidianus.

Simultaneous removal of antibiotic-resistant Escherichia coli and its resistance genes by dielectric barrier discharge plasma.

As emerging contaminants, antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have been widely detected in various aqueous environments. For antibiotic resistance to be inhibited in the environment, it is essential to control ARB and ARGs. In this study, dielectric barrier discharge (DBD) plasma was used to inactivate antibiotic resistant Escherichia coli (AR E. coli) and remove ARGs simultaneously. Within 15 s of plasma treatment, 108 CFU/mL of AR E. coli were inactivated by 97.9%. The rupture of the bacterial cell membrane and the increase of intracellular ROS are the main reasons for the rapid inactivation of bacteria. Intracellular ARGs (i-qnrB, i-blaCTX-M, i-sul2) and integron gene (i-int1) decreased by 2.01, 1.84, 2.40, and 2.73 log after 15 min of plasma treatment, respectively. In the first 5 min of discharge, extracellular ARGs (e-qnrB, e-blaCTX-M, e-sul2) and integron gene (e-int1) decreased by 1.99, 2.22, 2.66, and 2.80 log, respectively. The results of the ESR and quenching experiments demonstrated that ·OH and 1O2 played important roles in the removal of ARGs. This study shows that DBD plasma is an effective technique to control ARB and ARGs in waters.

Sodium butyrate ameliorates thiram-induced tibial dyschondroplasia and gut microbial dysbiosis in broiler chickens.

Thiram is a dithiocarbamate pesticide widely used in agriculture as a fungicide for storing grains to prevent fungal diseases. However, its residues have threatened the safety of human beings and the stability of the ecosystem by causing different disease conditions, e.g., tibial dyschondroplasia (TD), which results in a substantial economic loss for the poultry industry. So, the research on TD has a great concern for the industry and the overall GDP of a country. In current study, we investigated whether different concentrations (300, 500, and 700 mg/kg) of sodium butyrate alleviated TD induced under acute thiram exposure by regulating osteogenic gene expression, promoting chondrocyte differentiation, and altering the gut microbial community. According to the findings, sodium butyrate restored clinical symptoms in broilers, improved growth performance, bone density, angiogenesis, and chondrocyte morphology and arrangement. It could activate the signal transduction of the Wnt/ß-catenin pathway, regulate the expression of GSK-3ß and ß-catenin, and further promote the production of osteogenic transcription factors Runx2 and OPN for restoration of lameness. In addition, the 16S rRNA sequencing revealed a significantly different community composition among the groups. The TD group increased the abundance of the harmful bacteria Proteobacteria, Subdoligranulum, and Erysipelatoclostridium. The sodium butyrate enriched many beneficial bacteria, such as Bacteroidetes, Verrucomicrobia, Faecalibacterium, Barnesiella, Rikenella, and Butyricicoccus, etc., especially at the concentration of 500 mg/kg. The mentioned concentration significantly limited the intestinal disorders under thiram exposure, and restored bone metabolism.

Genetic Diversity and Population Structure of the Rice False Smut Pathogen Ustilaginoidea virens in the Sichuan-Chongqing Region.

Rice false smut caused by Ustilaginoidea virens is one of the most devastating fungal diseases of rice panicles worldwide. In this study, two novel molecular markers derived from single nucleotide polymorphism-rich genomic DNA fragments and a previously reported molecular marker were used for analyzing the genetic diversity and population structure of 167 U. virens isolates collected from nine areas in the Sichuan-Chongqing region, China. A total of 62 haplotypes were identified, and a few haplotypes with high frequency were found and distributed in two to three areas, suggesting gene flow among different geographical populations. All isolates were divided into six genetic groups. Groups I and VI were the largest, with 61 and 48 isolates, respectively. The pairwise FST values showed significant genetic differentiation among all compared geographical populations. Analysis of molecular variance showed that intergroup genetic variation accounted for 40.17% of the total genetic variation, while 59.83% of genetic variation came from intragroup genetic variation. The unweighted pair-group method with arithmetic means dendrogram and population structure revealed that the genetic composition of isolates collected from Santai, Nanchong, Yongchuan, and Wansheng dominated by the same genetic subgroup was different from those collected from other areas. In addition, genetic recombination was found in a few isolates. These findings will help to improve the strategies for rice false smut management and resistance breeding, such as evaluating breeding lines with different isolates or haplotypes at different elevations and landforms.

Effective degradation of Direct Red 81 using FeS-activated persulfate process.

As a burgeoning advanced oxidation process (AOP), heterogeneous activation of persulfate (PS) for synthetic refractory contaminants decontamination has recently received much attention. In this study, FeS was selected as a heterogeneous PS activator to facilitate the degradation of a typical recalcitrant contaminant of diazo dye Direct Red 81 (DR 81). The results showed that approximately 95% of 0.03 mM DR 81 was removed within 60 min with FeS and PS doses of 1.5 × 10-3 M. The efficient decomposition of DR 81 by the FeS/PS system was assumed to be mainly attributed to the highly reactive SO4-• and •OH, which was related to PS cleavage by both dissolved Fe2+ leached from FeS and Fe2+ bound on the FeS surface. Except for strongly alkaline conditions, DR 81 decolorations by FeS/PS were insignificantly affected by operational parameters such as temperature, initial solution pH, and rotate speed. Meanwhile, the presence of five inorganic anions being studied had distinct impacts on DR 81 degradation and followed a strict order of NO3- < Cl- < SO42- < CO32- < PO43-. However, FeS/PS system was highly adaptable, and FeS, which is used as a PS activator was more stable. GC/MS and TOC data revealed that thorough mineralization of DR 81 by PS/FeS in an initial fast reaction phase to transform DR 81 to aromatic intermediates, followed by a slow reaction phase that mineralized these organic intermediates into carboxylic acids and carbon dioxide through further oxidation.

Sharper photonic nanojets generated by microspheres under higher-order radially polarized beam illumination.

Photonic nanojets (PNJs) generated from a single microsphere illuminated by higher-order radially polarized (RP) beams are investigated. The effects of the size parameters of higher-order RP beams, the refractive index, and radius of the dielectric microsphere on the full width at half-maximum and peak intensity of the PNJ are numerically discussed and qualitatively interpreted. The results show that the minimal width of the PNJ can be obtained by optimally adjusting the size parameter. The PNJ beam waist becomes gradually narrower with increasing the radial mode number. As compared to the case of plane wave illumination, sharper PNJs are more easily generated when irradiated by a higher-order RP beam, even for microspheres with lower refractive indices or larger radii. Our findings can promote potential applications of PNJs in a variety of fields including super-resolution microscopy, nanolithography, and optical data storage.

Directional control of the off-normal scattering from a single nanodisk by superposed linearly and radially polarized beams.

A scheme to dynamically control the off-axis directional scattering from a silicon nanodisk is proposed, which is based on focused fields formed by the coherent superposition of radially and linearly polarized beams. When the phase condition of the generalized Kerker conditions is satisfied at a specified wavelength, the amplitude requirement for the off-axis directional scattering along a required direction can be fulfilled by tuning the magnitude ratio of the two focused beams. Therefore, directional control of the off-axis scattering in the meridional plane is achieved without the manipulation of the working wavelength. Our findings provide new possibilities of future potential applications of all-dielectric nanoantennas.

Rapid Quantification of Fungicide Effectiveness on Inhibiting Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici).

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important and devastating diseases of wheat; therefore, it is necessary to rapidly and accurately quantify fungicide effectiveness to monitor Pst sensitivity and manage the disease. In this study, a rapid method of quantifying the fungicide effectiveness with detached leaves was developed. The results showed that 0.5% water agar containing 75 µg/ml of 6-benzylaminopurine and filter paper worked the best for maintaining wheat leaves. The disease incidences of different concentrations of spore suspension were compared. When the spore concentrations were 5 and 10 mg/ml, the disease incidences had no significant differences at 12 and 15 days after inoculation (P < 0.05). Fungicide treatment tests revealed that there were no significant differences in the efficacies of triadimefon on rust suppression between detached leaves in the culture dishes and direct spray on seedlings. We also developed a Photoshop software method that can replace the current classification method and accurately measure the proportion of sporulation area on infected leaves. The sensitivity baseline of Pst to triadimefon was estimated as 0.1453 ± 0.0081 µg/ml, and all the values of EC50 were tested for normal distribution using the Shapiro-Wilk test (W = 0.204). The baseline can be used to test the sensitivity of different Pst isolates to triadimefon.

Novel 2, 8-bit derivatives of quinolines attenuate Pseudomonas aeruginosa virulence and biofilm formation.

Signal molecules are stimulators of multiple quroum-sensing virulence and biofilm formation. Small molecule analogues have been suspected as a potent inhibitor in therapeutic strategy. Herein, we synthesized a series of small molecule compounds from the 2, 8-bit derivatives of quinoline by Suzuki coupling reaction. We found that these compounds have the biofilm inhibitory effect in normal condition instead of phosphate limitation state. Furthermore, lacZ reporter strain assay and rhamnolipids as well as pyocyanin experiments showed that these compounds did not affect las and pqs system but reduced the expression of rhl. All these results suggest that quinoline derivatives can be treated as potent inhibitors against biofilm and reduce virulence through the rhl system. This research will be useful in designing new quorum sensing inhibitors to attenuate the infection of bacteria.

Ellipsoidal Abstract and Illustrative Representations of Molecular Surfaces.

Molecular visualization is often challenged with rendering of large molecular structures in real time. The key to LOD (level-of-detail), a classical technology, lies in designing a series of hierarchical abstractions of protein. In the paper, we improved the smoothness of transition for these abstractions by constructing a complete binary tree of a protein. In order to reduce the degree of expansion of the geometric model corresponding to the high level of abstraction, we introduced minimum ellipsoidal enveloping and some post-processing techniques. At the same time, a simple, ellipsoid drawing method based on graphics processing unit (GPU) is used that can guarantee that the drawing speed is not lower than the existing sphere-drawing method. Finally, we evaluated the rendering performance and effect on series of molecules with different scales. The post-processing techniques applied, diffuse shading and contours, further conceal the expansion problem and highlight the surface details.

Prediction of Enzyme Function Based on Three Parallel Deep CNN and Amino Acid Mutation.

During the past decade, due to the number of proteins in PDB database being increased gradually, traditional methods cannot better understand the function of newly discovered enzymes in chemical reactions. Computational models and protein feature representation for predicting enzymatic function are more important. Most of existing methods for predicting enzymatic function have used protein geometric structure or protein sequence alone. In this paper, the functions of enzymes are predicted from many-sided biological information including sequence information and structure information. Firstly, we extract the mutation information from amino acids sequence by the position scoring matrix and express structure information with amino acids distance and angle. Then, we use histogram to show the extracted sequence and structural features respectively. Meanwhile, we establish a network model of three parallel Deep Convolutional Neural Networks (DCNN) to learn three features of enzyme for function prediction simultaneously, and the outputs are fused through two different architectures. Finally, The proposed model was investigated on a large dataset of 43,843 enzymes from the PDB and achieved 92.34% correct classification when sequence information is considered, demonstrating an improvement compared with the previous result.

Antibacterial Biphenanthrenes from the Fibrous Roots of Bletilla striata.

Four new 9',10'-dihydro-biphenanthrenes, including an unprecedented 1,2'-linked biphenanthrene, 4,7,3',5'-tetramethoxy-9',10'-dihydro(1,2'-biphenanthrene)-2,7'-diol (1), a new 1,3'-linked biphenanthrene, 4,7,7'-trimethoxy-9',10'-dihydro(1,3'-biphenanthrene)-2,2',5'-triol (2), and two new 1,1'-linked biphenanthrenes, 4,7,4'-trimethoxy-9',10'-dihydro(1,1'-biphenanthrene)-2,2',7'-triol (3) and 4,7,3',5'-tetramethoxy-9',10'-dihydro(1,1'-biphenanthrene)-2,2',7'-triol (4), as well as two known biphenanthrenes (5, 6), were isolated from a 95% ethanol extract of the fibrous roots of Bletilla striata. Their structures were determined by spectroscopic and spectrometric methods. Atropisomerism of these compounds was considered based on their chiral optical properties and potential energy surface scans at the ab initio HF/3-21G level, which revealed their racemic mixture form. Compounds 2-6 showed potent antibacterial activities against six Gram-positive bacterial strains.

Lasiodiplodia sp. ME4-2, an endophytic fungus from the floral parts of Viscum coloratum, produces indole-3-carboxylic acid and other aromatic metabolites.

BACKGROUND: Studies on endophytes, a relatively under-explored group of microorganisms, are currently popular amongst biologists and natural product researchers. A fungal strain (ME4-2) was isolated from flower samples of mistletoe (Viscum coloratum) during a screening program for endophytes. As limited information on floral endophytes is available, the aim of the present study is to characterise fungal endophytes using their secondary metabolites. RESULTS: ME4-2 grew well in both natural and basic synthetic media but produced no conidia. Sequence analysis of its internal transcribed spacer rDNA demonstrated that ME4-2 forms a distinct branch within the genus Lasiodiplodia and is closely related to L. pseudotheobromae. This floral endophyte was thus identified as Lasiodiplodia sp. based on its molecular biological characteristics. Five aromatic compounds, including cyclo-(Trp-Ala), indole-3-carboxylic acid (ICA), indole-3-carbaldehyde, mellein and 2-phenylethanol, were found in the culture. The structures of these compounds were determined using spectroscopic methods combined with gas chromatography. To the best of our knowledge, our work is the first to report isolation of these aromatic metabolites from a floral endophyte. Interestingly, ICA, a major secondary metabolite produced by ME4-2, seemed to be biosynthesized via an unusual pathway. Furthermore, our results indicate that the fungus ME4-2 is a potent producer of 2-phenylethanol, which is a common component of floral essential oils. CONCLUSIONS: This study introduces a fungal strain producing several important aromatic metabolites with pharmaceutical or food applications and suggests that endophytic fungi isolated from plant flowers are promising natural sources of aromatic compounds.

A sensitive enzyme-free electrochemical sensor based on ZnWO4@co-MNPC@MIP for specific recognition and determination of chloramphenicol in milk sample.

We have carefully built a new chloramphenicol (CAP) electrochemical sensor, which takes the zinc tungstate @ cobalt magnetic nanoporous carbon @ molecularly imprinted polymer (ZnWO4@Co-MNPC@MIP) as the core. First, we successfully prepared Co-MNPC nanomaterials using an efficient one-step hydrothermal method and a direct carbonization method. Next, we recombined ZnWO4 with Co-MNPC and synthesized the completely new ZnWO4@Co-MNPC complex by using the hydrothermal method. To further improve its performance, we combined ZnWO4@Co-MNPC with a molecular imprinted polymer and coated a molecular imprinted (MIP) shell on the surface of ZnWO4@Co-MNPC by precipitation polymerization. This shell not only gives the sensor a new performance but also gives it a stronger peak current, resulting in a more accurate detection of CAP. Under optimal conditions, the ZnWO4@Co-MNPC@MIP (MMIP) electrode has a stronger CAP detection peak current than the one-component electrode, with a fairly wide linear range: 0.007-200 µM and 200-1400 µM. Even more surprisingly, the detection limit is as low as 0.0027 µM, which allows the sensor to maintain excellent selectivity and stability in the face of various interferences, making it an excellent electrochemically modified electrode. Compared to magnetic non-molecular imprint sensors (MNIPs), MMIP sensors have higher detection efficiency. After practical application, we found that the ZnWO4@Co-MNPC@MIP modified electrode was satisfactory in milk samples.

Effect of umbilical cord blood-mononuclear cells on knee osteoarthritis in rabbits.

BACKGROUND: To investigate the effect and underlying mechanism of umbilical cord blood-mononuclear cells (UCB-MNCs) in treating knee osteoarthritis (KOA) in rabbits. METHODS: A rabbit KOA model was prepared by anterior cruciate ligament transection (ACLT). Fifty New Zealand white rabbits were randomly divided into the control group, model group, sodium hyaluronate (SH) group, platelet-rich plasma (PRP) group and UCB-MNC group. Knee injections were performed once a week for five consecutive weeks. The gross view of the knee joint, morphology of knee cartilage and structural changes in the knee joint were observed on CT scans, and graded by the Lequesne MG behavioral score and the Mankin score. TNF-α and IL-1ß levels in the synovial fluid of the knee were measured by the enzyme-linked immunosorbent assay (ELISA). Expression levels of MMP-13 and COL-II in the knee cartilage were detected by Western blotting and qRT-PCR. RESULTS: The Lequesne MG behavioral score and the Mankin score were significantly higher in the model group than those in the control group (P < 0.05). Rabbits in the SH, PRP and UCB-MNC groups had sequentially lower scores than those in the model group. Imaging features of KOA were more pronounced in the model group than in the remaining groups. CB-MNC significantly relieved KOA, compared to SH and PRP. Significantly higher levels of TNF-α and IL-1ß in the synovial fluid of the knee, and up-regulated MMP-13 and down-regulated COL-II in the knee cartilage were detected in the model group than in the control group. These changes were significantly reversed by the treatment with SH, PRP and UCB-MNCs, especially UCB-MNCs. CONCLUSION: Injections of UCB-MNCs into knees protect the articular cartilage and hinder the progression of KOA in rabbits by improving the local microenvironment at knee joints.

Case report of a giant lipoma in the inguinal-perineal area.

INTRODUCTION: Lipomas are benign tumors that occur in the back, shoulder, neck, and extremities. Giant lipomas of the inguinal-perineal area are extremely rare. CASE REPORT: We present a case of a 63-year-old man with a giant lipoma in the inguinal-perineal area. Ultrasound examination showed a heterogeneous hyperechoic mass (14.6 × 8.3 cm) located in the inguinal area, presumably an inguinal hernia. Computed tomography (CT) results showed fat tissue radiographic patterns, and lack of enhancement of the left inguinal area extending to the lateral scrotum. The patient underwent an operation and a radical resection was performed. Histology results revealed a lipoma. At the 1-month follow-up, the patient did not show any evidence of recurrence. CONCLUSION: Giant lipomas occurring in the inguinal-perineal area are extremely rare and are easily confused with other lesions in the groin area. We recommend a detailed preoperative adjunctive examination such as CT. Open surgical complete excision is the ideal treatment.

Promotion of Wound Healing Using Nanoporous Silk Fibroin Sponges.

Wound dressings are important for wound repair. The morphology of the biomaterials used in these dressings, and in particular, the pore structure affects tissue regeneration by facilitating attachment and proliferation of cells due to the hierarchical multiscale, water absorbance, and nutrient transport. In the present study, silk fibroin (SF) sponges with walls containing nanopores (SFNS) were prepared from SF nanoparticles generated during the autoclaving of SF solutions, followed by leaching the SF nanoparticles from the freeze-dried sponges of SF. The nano/microporous structure, biofluid absorbance, and porosity of the SF sponges with and without nanopores were characterized. In vitro cell proliferation, in vivo biocompatibility, and wound healing were evaluated with the sponges. The results demonstrated that SFNS had significantly increased porosity and water permeability, as well as cell attachment and proliferation when compared with SF sponges without the nanopores (SFS). Wound dressings were assessed in a rat skin wound model, and SFNS was superior to SFS in accelerating wound healing, supported by vascularization, deposition of collagen, and increased epidermal thickness over 21 days. Hence, such a dressing material with a hierarchical multiscale pore structure could promote cell migration, vascularization, and tissue regeneration independently without adding any growth factor, which would offer a new strategy to design and engineer better-performed wound dressing.

Case report: Submucosal cavernous lymphangioma causing jejuno-jejunal intussusception in an adult.

Cavernous lymphangioma often occurs in the head, neck, trunk, and extremities of infants and children, and it is rare to cause a small intestine intussusception in adults. In this case, a 32-year-old woman presented with abdominal pain, vomiting, and a 5 cm × 5 cm abdominal mass on the left side of the abdomen. Laboratory tests showed anemia and CT showed small intestinal intussusception. After conservative treatments, her symptoms disappeared. However, 18F-FDG PET/CT suggested malignancy and her symptoms reappeared after eating something. Segmental jejunal resection was performed and pathology showed submucosal cavernous lymphangioma. At the 1-year follow-up, the patient was asymptomatic. Then this paper reviewed the literature on small intestinal cavernous lymphangioma in adults and found that this is the first English case report of intussusception caused by a jejunal submucosal cavernous lymphangioma in an adult. Current problem is that adult intussusception and intestinal lymphangioma are difficult to diagnose preoperatively. Imaging techniques such as tomography and PET/CT aid in the diagnosis of these benign lesions. Surgical resection was considered to be the required treatment and seems to have had no recurrence in adults according to the literature.

Gastroprotective effect of an active ingredients group of Lindera reflexa Hemsl. On Ethanol-Induced gastric ulcers in Rats: Involvement of VEGFR2/ERK and TLR-2/Myd88 signaling pathway.

Lindera reflexa Hemsl. (LR) has been used for the treatment of gastrointestinal disorders. The present study was carried out to investigate the gastroprotective effect of an active ingredients group of Lindera reflexa Hemsl. (LRG) on ethanol-induced gastric ulcer in rats and its possible mechanisms. The ulcer area was measured, and samples of gastric tissue were taken for histochemical, pathological, and biochemical analyses. Pretreatment with LRG protected the gastric mucosa as seen by reduction the GUI and gastric juice volume, regulated gastric acid secretion. LRG counteracted the ethanol-induced oxidative stress by increasing the levels of depleted SOD and CAT as well as significantly attenuating the lipid peroxidation by reducing the levels of MDA and MPO. LRG also reduced release of inflammatory mediator TNF-α, increased the content of PGE2 and inhibited MTL secretion. Immunofluorescence and Western blot analyses confirmed that the co-localization of TLR-2 and MyD88 protein in the gastric mucosa of LRG-treated rats was significantly lower than that of rats with gastric ulcers. Furthermore, LRG also modulated the expression of Ki-67 antigens. LRG markedly increased the expression of phosphorylated form of extracellular signal-regulated kinaseVEGFR2, ERK1/2, AKT and p38, thereby protecting the gastric mucosa. These findings indicated that the gastroprotective effect of LRG is attributable to its antioxidant, anti-inflammatory, and antisecretory properties. In addition, LRG can ameliorate ethanol-induced gastric ulcers in rats by regulating the VEGFR2/ERK and TLR-2/MyD88 signaling pathways.

Six undescribed derivatives of stilbene isolated from Lindera reflexa Hemsl. and their anti-tumor and anti-inflammatory activities.

Six undescribed stilbene derivatives Reflexanbene DH (1-4, 6) and Reflexanbene J (5), as well as one known stilbene 3,5-dimethoxystilbene (7), were isolated from the dried roots of Lindera reflexa Hemsl. Their structures and absolute configurations were elucidated using spectroscopy and electronic circular dichroism (ECD) analysis. In cytotoxic assays, moderately inhibitory activities of Reflexanbene F (3) against MGC80-3 and A549 cell lines were observed, with IC50 values of 15.42 and 5.09 µM, respectively. The IC50 value of Reflexanbene E (2) on A549 cell lines was 19.78 µM. The isolated compounds were also tested for their inhibitory effect against LPS-induced NO and IL-6 production in RAW 264.7 cells. In particular, Reflexanbene J (5) and Reflexanbene H (6) showed significant inhibition of NO production in LPS-stimulated macrophage RAW 264.7 cells at the concentration of 20 µM. Furthermore, the expression of IL-6 protein in the LPS-induced RAW 264.7 cells can also be significantly inhibited by different concentrations (5, 10 and 20 µM, p < 0.05 or p < 0.01) of compounds 1-7.