P < 0.01). Annexin V positive rate increased significantly when PBS was used as the buffer, compared to saline and water. These false negative results were also observed when we quantified some markers of MVs such as CD3 and CD19. A probable explanation for these findings is the production of insoluble Ca(H2PO4)2 or Ca3PO4 from calcium in the binding buffer and phosphate in PBS. Thus, considering the osmotic pressure of water, we suggest that saline is a more suitable buffer when counting MVs by flow cytometry.Microvesicles (MVs) are carriers of molecular and oncogenic signatures present in subsets of tumor cells and tumor-associated stroma, and a focus of cancer research. Although methods to detect MVs are mature, we were concerned that the buffer used could lead to false results when quantitating MVs by flow cytometry. In this work, we detected MVs by flow cytometry withthree different solutions: water, saline, and phosphate-buffered saline (PBS). The results demonstrated that PBS, when reacted with annexin V binding buffer, produced nano-sized vesicles even when there were no MVs in the sample. No similar events occurred in the saline and water groups ( Cellular microvesicles (MVs), 0.1–1 μm in size, are released by various cell types, especially cancer cells that are undergoing stress and activation . During Uniform methods to isolate and identify MVs are not yet defined. Electron microscopy, atomic force microscopy, nanoparticle tracking analysis, and flow cytometry have been reported in many studies . Among aK562 cells were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) Figure , 1B. ExtP < 0.0001 for K562 cells vs. all other groups)  Figure –2H.P < 0.0001). There was no significant difference between the other groups  , 165 ± 87 (water with annexin V binding buffer), 131 ± 50 , 93 ± 79 , 126 ± 76 (PBS), and 28,551 ± 5010 (PBS with annexin V binding buffer). The counts of nano-sized vesicles in tubes of PBS with annexin V binding buffer were significantly different , -allophycocyanin (APC), -phycoerythrin (PE), and annexin V antibodies. The results were similar with all antibodies when counting nano-sized vesicles in water  and the STAT3 inhibitor stattic (10 μM) were used to induce apoptosis in K562 cells. After 24 h in serum-free medium, the percentages of total, early, and late apoptosis of K562 cells were 47.2 ± 6.69, 48.9 ± 3.30, and 35.8 ± 3.32%, and 40.1 ± 3.81, 11.4 ± 10.0, and 9.17 ± 0.01% for elaiophylin and stattic, respectively (n = 3). The total, early, and late apoptosis percentages of control K562 cells (without elaiophylin and stattic) in serum-free medium for 24 h were 8.86 ± 2.05%, 13.7 ± 0.07%, 5.01 ± 0.85%, 8.55 ± 0.79%, 3.85 ± 1.20%, and 5.18 ± 0.72%, respectively (n = 3) Figure .P < 0.01)  Figure  (Supplem) Figure .For analysis of the positive rate and the positive nano-sized counts, the control, elaiophylin, and stattic groups were dissolved in saline or PBS with binding buffer and annexin V antibody. The positive percentages were 63.8 ± 5.83%, 68.5 ± 4.93%, 69.6 ± 4.32%, 70.7 ± 3.11%, 60.6 ± 8.06%, and 77.1 ± 4.12%, respectively Figure . The posIntercellular communication is essential for cancer cells to survive and thrive. Extracellular vesicles such as exosomes, MVs, and large oncosomes are involved in this communication process by shuttling reciprocal signals and other molecules between cancer and stromal cells, including fibroblast, endothelial, and immune cells –17. Extr2. Similar to our findings, they also found that the median fluorescence signal intensity increased with increasing CaCl2 concentrations when adding fluorescently labeled antibodies to the microprecipitates [MVs are defined as particles between 100 and 1000 nm in diameter that typically exhibit PS on the outer leaflet of their plasma membranes. Most studies have shown that MVs are typed and identified by fluorescence-activated cell sorting. Because exposure of PS is a typical marker for MVs, the standard procedure is to stain PS exposed on the external surface with annexin V –23. Apopipitates .2+ and, when added to PBS, generated microprecipitates that led to false positive results. Thus, PBS was not suitable for MV counting. Resuspending the MV pellet in 100 μL of saline could avoid artifacts from the calcium-phosphate microprecipitates, and false-positive results. Saline appears to be a better choice to improve the sensitivity of MV-based lipid biopsy and provides important information about optimal molecular monitoring schedules in cancer diagnosis and evaluating the efficacy of different treatments. Exosomes, with a diameter of 100 nm, could not be detected by flow cytometry because the lower limit of detection was 300 nm. Thus, we consider that PBS will not affect exosome counts.In our study, we confirmed that annexin V binding buffer contained Ca2. Before isolation of MVs, K562 cells were adjusted to 1 × 106/mL and cultured in serum-free RPMI 1640 for 4 h. Cells were pelleted at 100 × g for 5 min followed by centrifuging the supernatant at 3000 × g for 10 min to remove cellular debris. The resultant supernatant was centrifuged at 5000 × g for 30 min to remove smaller cellular debris, followed by centrifugation at 16,000 × g for 90 min to obtain MVs [2.The human chronic myeloid leukemia blast crisis cell line, K562, was purchased from the American Type Culture Collection . HUVECs were obtained from the cell bank of the Chinese Academy of Sciences. K562 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in 5% COtain MVs . HUVECs MVs were labeled with PKH26 red and PKH67 green fluorescent cell linker mini kits  following the manufacturer's instructions. MVs were visualized under a fluorescence microscope.Monoclonal antibodies  were purchased from BD Biosciences . Appropriate APC, PE, and FITC isotype antibodies were used as negative controls (BD Biosciences). The fluorescent nanoparticle size standard kit was purchased from Spherotech . Elaiophylin was provided by North China Pharmaceutical Group Corporation New Drug R&D center. Stattic was purchased from Sigma-Aldrich.6/mL of K562 cells, and diluted in 1 mL of double filtered saline or PBS. MVs were suspended by agitating for 5 s in a whirlpool oscillator, and a 100-μL sample was aspirated into different flow tubes.All analyses were performed on an LSR II flow cytometer (BD Biosciences) with DIVA (BD Biosciences) and FlowJo software . The MV Two blank groups were designed using fluorescent nanoparticles to confirm the MV gate, each with three subgroups: double filtered water, saline, and PBS without MVs. The volume of each tube was 100 μL. To determine whether PBS mixed with annexin V binding buffer generated nano-sized vesicles, the annexin V antibody was only added to tubes without the 10× annexin V binding buffer. In another blank group, the annexin V isotype-matched control IgG1-FITC, IgG1-APC, and IgG1-PE antibodies were added at 10, 6, 5, and 5 μL, respectively, with and without annexin V binding buffer. In the MV group, 6 μL of CD3, 5 μL of CD19, 6 μL of IgG1-FITC, 5 μL of IgG1-APC, 5 μL of IgG1-PE, and 10 μL of annexin V antibodies were added into the same solutions  with and without annexin V binding buffer. All antibodies added to the MV and blank groups were incubated for 30 min at room temperature in the dark. Event numbers of equal sample volumes were counted for 30 s.P < 0.05 was defined as being statistically significant.All statistical analyses were carried out using GraphPad Prism version 6.0  and SPSS for Windows, version 17.0 . Non-parametric and unpaired t-test comparisons were used to compare groups; the rates between groups were compared by the chi-square test. Two-sided


T, isolated from a facial abscess in a sugar glider, was characterized genetically and phenotypically. Cells of the strain were Gram-stain-positive, coccoid and catalase-negative. Morphological, physiological and phylogenetic data indicated that the isolate belongs to the genus Lactococcus. Strain 159469T was closely related to Lactococcus garvieae ATCC 43921T, showing 95.86 and 98.08 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Furthermore, a pairwise average nucleotide identity blast (ANIb) value of 93.54 % and in silico DNA–DNA hybridization value of 50.7  % were determined for the genome of strain 159469T, when compared with the genome of the type strain of Lactococcus garvieae. Based on the data presented here, the isolate represents a novel species of the genus Lactococcus, for which the name Lactococcus petauri sp. nov. is proposed. The type strain is 159469T (=LMG 30040T=DSM 104842T).A strain of lactic acid bacteria, designated 159469 Petaurus breviceps) are small marsupials native to Australia and New Guinea and commonly kept as pets. Due to a specialized dental structure that is designed for peeling bark and not for eating soft diets, they are prone to oral cavity disease as companion animals; abscesses due to facial trauma are also common [Pasteurella multocida and Toxoplasma gondii [Lactococcus.Sugar gliders . The strain was initially typed as a representative of L. garvieae based on biochemical profiles obtained from commercial diagnostic platforms and 16S rRNA gene Sanger sequencing. The pigmentation of the colonies, however, was inconsistent with this identification, and further characterization by whole-genome sequencing was performed. Phylogenetic and phenotypic analyses subsequently indicated that this strain represents a distinct species of the genus Lactococcus, sharing most of its sequence in common with L. garvieae and the pigmented phenotype of Lactococcuslactis. We propose to name 159469T as the type strain of Lactococcus petauri sp. nov.Strain 159469T using RNAmmer 1.2 [decipher [blast identified the 16S rRNA gene sequence from L. garvieae strain M14 as the closest match to the 16S rRNA gene sequence of isolate 159469T. Comparative phylogenetic analysis was carried out with 16S rRNA gene sequences of isolate 159469T and type strains of 16 species and subspecies of the genus Lactococcus with validly publsihed names. Strain 159469T clustered close to L. garvieae JCM 10343T (=ATCC 43921T) in a maximum-likelihood tree reconstructed on the basis of 16S rRNA gene sequences in RAxML v. 8 using the general time-reversible (GTRGAMMAI) substitution model and 1000 bootstrap repetitions  and maximum-parsimony trees in mega  [L. petauri sp. nov. 159469T with L. garvieae JCM 10343T, L. garvieae subsp. bovis BSN307T and Lactococcusformosensis NBRC 109475T.A 1537 bp 16S rRNA gene sequence was extracted from an assembled draft genome of isolate 159469decipher . NCBI bl. The 16 . All thrL. petauri sp. nov. 159469T was sequenced on an Illumina MiSeq platform with 2×250 bp paired-end reads, which were assessed for quality with FastQC version 0.11.2 and assembled de novo with SPAdes version 3.6.2 [quast version 3.2 [L. petauri sp. nov. 159469T and genomes of seven type strains of species and subspecies of the genus Lactococcus extracted from NCBI were analysed using kSNP version 2 with kmer size 19 [N=109) were used to reconstruct a maximum-likelihood phylogeny with 1000 bootstrap repetitions in RaxML  . An ANIb pairwise similarity matrix was used to plot the dendrogram in R 3.3.2 [L. garvieae JCM 10343T was shown to have the most similar genome (93.54 %) to L. petauri sp. nov. 159469T as suggested by ANIb. The pairwise ANIb values <95 % compared with representatives of other species in the genus Lactococcus confirmed strain 159469T as a representative of a novel species [L. petauri sp. nov. 159469T and another strain, currently classified as a representative of L. garvieae in the NCBI database  was 98.51 %. This indicates the existence of another strain of the novel species described here as L. petauri sp. nov. Strain PAQ102015-99 was described as a putative pathogen of salmonid fish used for vaccine development.The same eight genomes were used to compute pairwise average nucleotide identity  R 3.3.2  using th species . InteresIn silico DNA–DNA hybridization (DDH) analysis was performed on the aforementioned eight genomes using GGDC 2.1 method 2, which is recommended for draft genomes . The highest DDH value (DDH=50.7 %) was obtained for isolates L. petauri sp. nov. 159469T and L. garvieae JCM 10343T. Considering DDH of 70 % as a species threshold, in silico DDH further confirmed isolate 159469T as a representative of a novel species . The Biolog and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) platforms both ranked L. garvieae as the closest match with scores of 0.734 and 2.1. The strain was propagated on trypticase soy agar (TSA) with 5 % Sheep Blood (BD) at 33–37 °C and demonstrated growth typical of a facultative anaerobe.Strain 159469L. petauri sp. nov. 159469T using L. garvieae ATCC 43921T as a control. M17 medium (BD Difco) was used for all tests except where indicated differently. Strains were incubated at 30 °C except where indicated differently. Colonies grown on M17 agar were small, round and cream-coloured when incubated aerobically for 24 h at 30 °C. However, colonies appeared larger and orange-coloured when incubated anaerobically for 24 h at 30 °C on M17 agar. L. petauri sp. nov. 159469T was oxidase-negative (Hardy Diagnostics) and produced acid and no gas from glucose (BD BBL).Further phenotypic characterization was performed for L. petauri sp. nov. 159469T was able to grow at temperatures of between 6 and 40 °C, but not at 45 °C. Growth at various pH levels was assessed by inoculating pH-adjusted M17 broth and incubating at 30 °C for 14 days. The novel species was able to grow from pH 4.0 to 10.0. Tolerance to various sodium concentrations was determined by inoculating M17 broth containing 3, 4, 5, 6, 7 and 8 % (w/v) NaCl and incubating at 30 °C for 14 days. The novel species was able to grow with 3 to 7 % (w/v) NaCl and no growth was seen at 8 % (w/v) NaCl. Results of the temperature, pH and NaCl tests can be seen in Lactococcus.Growth at various temperatures was determined by plating overnight cultures on M17 agar plates and incubating at 4, 6, 10 and 14 °C for 21 days, 20 and 25 °C for 14 days, 30, 35, 37 and 40 °C for 7 days, and 45 and 55 °C for 3 days. Production of acid from carbohydrates was determined by using API 50 CH kits (bioMérieux). The kits were used according to the manufacturer’s instructions and incubated for 48 h at 30 °C. Enzymic activity was assessed using the API ZYM kit (bioMérieux). The kit was used according to the manufacturer’s instructions. Strains were initially grown aerobically at 30 °C for 24 h on M17 agar. Once the strip was inoculated, incubation occurred for 4 to 4.5 h at 37 °C. The results of the API tests can be seen in L. petauri sp. nov. 159469T were C16 : 0 (40.93 %) and C14 : 0 (14.43 %). The complete fatty acid profile of the novel species is shown in Lactococcus cannot be made due to the use of different incubation conditions by other authors for fatty acid analysis. Fatty acid profiles of other species in the genus can also be seen in Analysis of fatty acid methyl esters was performed by Microbial ID according to the instructions of the Microbial Identification System. The organism was cultured on TSA for 24 h at 28 °C before harvesting. The major fatty acids of Lactococcus petauri (pe.tau′ri. N.L. gen. n. petauri of Petaurus pertaining to the sugar glider Petaurus breviceps).d-ribose, d-galactose, d-glucose, d-fructose, d-mannose, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, sucrose, trehalose, gentobiose and d-tagatose; to some degree also from d-mannitol and potassium gluconate. Possesses active esterase, esterase lipase, leucine arylamidase, α-chymotrypsin, acid phosphatase, α-glucosidase and β-glucosidase, and weakly active valine arylamidase and naphthol-AS-BI-phosphohydrolase. The major fatty acid is C16 : 0.Cells are Gram-stain-positive cocci, catalase-negative, oxidase-negative, beta-haemolytic, mucoid and facultatively anaerobic. The type strain is orange-pigmented when grown aerobically on TSA with 5 % sheep blood or when grown anaerobically on M17 agar; cream coloured when grown aerobically on M17 agar. It grows at 6–40 °C, but not at 4 or 45 °C; it grows with 7 % (w/v) NaCl and at pH 4.0–10.0, but not at pH 3.0. The organism grows optimally at 20–40 °C, between pH 6.0 and 7.0, and at NaCl concentrations of 3 % NaCl or lower. Produces acid from T (=LMG 30040T=DSM 104842T). The genomic DNA G+C content of the type strain, determined on the basis of the whole-genome sequence, is 37.7 mol%.The type strain, isolated from a sugar glider in the USA, is 159469


Escherichia coli O157:H7 in non-intact beef and veal. Coarse ground beef and veal patties  inoculated with E. coli O157:H7, aerobically stored before double pan-broiling for 0–360 s without rest or to 55, 62.5, 71.1, and 76 °C  with 0.5- or 3.5-min rest. Microbial population and qualities including color, cooking losses, pH, water activity, fat, and moisture content, were tested. After cooking the beef and veal patties, the weight losses were 17.83–29%, the pH increased from 5.53–5.60 to 5.74–6.09, the moisture content decreased from 70.53–76.02% to 62.60–67.07%, and the fat content increased (p < 0.05) from 2.19–6.46% to 2.92–9.45%. Cooking beef and veal samples with increasing internal temperatures decreased a* and b* values and increased the L* value. Escherichia coli O157:H7 was more sensitive to heat in veal compared to beef with shorter D-value and “shoulder” time. Cooking to 71.1 and 76 °C reduced E. coli O157:H7 by >6 log CFU/g regardless of rest time. Cooking to 55 °C and 62.5 °C with a 3.5-min rest achieved an additional 1–3 log CFU/g reduction compared to the 0.5-min rest. Results should be useful for developing risk assessment of non-intact beef and veal products.This study compared the quality variation and thermal inactivation of  Escherichia coli O157:H7 can generate shiga toxins that can cause, with as few as 10 cells, severe hemolytic uremic syndrome in infected humans [E. coli O157:H7 has been considered an adulterant of raw, non-intact beef products since 1999 [E. coli O157:H7 outbreaks in the United States since 2000 [E. coli O157:H7, on the meat surface may be translocated and trapped in sterile internal tissues, thus protected from thermal destruction if the meat is undercooked. A recent survey showed that 40–58% of US consumers ordered beefsteaks at medium rare (60–62.8 °C) to rare (54.4–57.2 °C), which could potentially put consumers at a high risk from E. coli O157:H7 contaminated non-intact veal meat if consumers order the same way as beefsteaks [nce 1999 . The Unince 1999 . These nnce 2000 . During E. coli O157:H7 contaminated non-intact beef has been documented in numerous studies [Thermal processing includes using high temperature to inactive spoilage and foodborne pathogens is one of the most effective and widely used technologies for meat products preservation . The eff studies ,7,8 indiE. coli outbreaks in the US, since 2009 there have been multiple recalls of veal products amounting to 14,600 lb (ca. 6649 kg) due to possible E. coli O157:H7 and STEC contamination [E. coli contamination [E. coli O157:H7 and three (0.24%) of 1232 samples to be positive for STEC in beef; in contrast, in veal, three (3.49%) of 86 samples were positive for E. coli O157:H7 and 4 (4.00%) of 100 samples were positive for STEC [E. coli O157:H7 strains in non-intact veal products [Veal, which originated from Europe, is the meat from 16–18-week-old calves. In the past 10 years, 25% of American households purchased veal products in restaurants or retail stores at least once every three months . Differemination . Accordimination . Accordifor STEC . The difproducts ,14. The safety of beef and veal products is important to the industry and to consumers, but consumers tend to identify the quality of products based on appearance. Cornforth and Jayasingh  stated tE. coli O157:H7 in coarse ground beef and veal patties. We hypothesize that (1) beef and veal patties have similar tendencies in quality change throughout storage and cooking and (2) a higher internal temperature with a longer rest time will increase the inactivation of E. coli O157:H7 in beef and veal patties. The novelty of this study are (1) a detailed side-by-side comparison study of quality attributes and thermal inactivation activity of E. coli O157:H7 between beef and veal and (2) the thermal kinetics study was conducted in a commercial size patties cooked on a griller instead of using small amount of meat heated in water bath.The objective of this study is to investigate the quality variances, including color variation in non-intact coarse ground beef and veal patties during aerobic storage and cooking and to evaluate the thermal inactivation of Escherichia coli O157:H7 strains ATCC 43895, ATCC 43888, and ATCC 43889  were cultured and sub-cultured individually in 10 mL of tryptic soy broth (TSB) at 35 °C for 24 h. The three cultures were then mixed and centrifuged  at 4629× g for 15 min at 4 °C. The harvested cells were washed twice with 10 mL of phosphate-buffered saline (PBS), centrifuged as described above, and re-suspended in 30 mL of fresh PBS. The washed pathogen cells were 10-fold diluted in PBS to obtain an initial inoculum level of 8 log CFU/mL, and then 40 mL of this prepared inoculum was added into 2 kg of coarse ground beef or veal to reach the inoculation level of ~6 log CFU/g. E. coli O157:H7 inoculum cocktail in a bowl-lift stand mixer  at medium speed for two minutes to ensure an even distribution of the inoculum into the sample, which simulates E. coli O157:H7 contamination during the preparation of non-intact beef or veal products. A manual hamburger patty maker  was then used to make beef or veal patties with 180 g of grounded meat. The beef/veal patties (2.1 cm thick and 12.4 cm diameter) were packaged aerobically in foam trays  with the absorbent pads, covered using air-permeable plastic film  and stored at 4.0 °C for four days.Fresh beef knuckles and veal round top were purchased from a local meat retailer for each replicate. The meat was manually cut into trimmings and then coarse ground in a meat grinder  with a kidney plate (0.95 cm diameter). The ground meat was then mixed with 40 mL of the aforementioned After four days of storage, the beef or veal patties were removed from their packages, weighed, and double pan-broiled in a Farberware grill  with a set-up temperature of 177 °C (or 350 °F) (1) for 0, 30, 60, 90, 120, 180, 240, and 360 s with 0-min rest to determine the thermal dynamic parameters  (2) to an internal geometric target temperature of 55, 62.5, 71.1, or 76 °C, followed by a 0.5- or 3.5-min rest. The cooked patties were allowed to rest on the tray after cooking without any cover. Double broiling, also known as contact grilling, is when the food  is cooked on both sides simultaneously by applying two cooking surfaces, from both the bottom and the top, greatly reducing the cooking time. A type-K thermocouple was attached to the geometric center of the patty to monitor the internal temperature throughout cooking using PicoLog , a real-time data-recording software [The objective color of non-intact beef or veal patties was measured on each day of storage and after cooking to 55, 62.5, 71.1, or 76 °C  using a portable spectrophotometer , with full spectral data being obtained as L* (lightness), a* (redness), and b* (yellowness), along with reflectance data . For theE. coli O157:H7, respectively. Colonies were counted manually after incubation at 35 °C for 48 h. The samples below the detect limit of spread-plating were enriched at 35 °C for 48 h and streak-plated onto MacConkey agar to enrich any cells that were not recovered. Cooking losses were determined by measuring the difference in patty weight before cooking and after cooking when the samples had cooled to room temperature. The pH of the meat homogenate was measured after microbial analysis using a digital pH meter . The water activity (aw) indicates the availability of water for bacterial growth. The water activity of the uncooked and cooked samples was measured using an AquaLab water activity meter . All of the samples were tested for fat and moisture content at the Meat Science Lab of the University of Illinois at Urbana– Champaign. For microbiological analysis, the individual uncooked or cooked beef or veal samples were transferred to a Whirl-Pak filter bag  with a 1:1 ratio of nutrient broth by weight and homogenized  for 2 min. Serial 10-fold dilutions of each sample in PBS were surface-plated onto tryptic soy agar  supplemented with 0.1% sodium pyruvate  and MacConkey agar  for the enumeration of total bacterial populations and p = 0.05. Microbial populations (log CFU/g) were analyzed using the PROC MIXED procedure of Statistical Analysis System , with independent variables including beef or veal, cooked internal temperatures, rest time, and interactions between two or three variables. USDA-Integrated-Predictive-Modeling-Program software [The experiment was repeated twice, with three samples in each replicate in quality and microbial thermal inactivation studies. The quality parameters of beef and veal samples, including cooling losses, pH, water activity, fat and moisture content, were analyzed with a one-way ANOVA of SAS. All of the comparisons were performed with software , provideThe initial geometric center temperature of uncooked beef and veal patties ranged from 3.6 °C to 4.8 °C and from 4.1 °C to 8.9 °C, respectively. The cooking of beef samples by double pan-broiling required 330, 360, 430 and 460 s to reach the internal center temperatures of 55, 62.5, 71.1 and 76 °C, respectively A. In veap < 0.05) cooking losses (24.16–29%) compared to those from cooking to 55 °C (17.83%) (p < 0.05) cooking losses (28.25–29%) (Cooking caused weight losses ranging from 17.83% to 29% in non-intact beef samples  and from(17.83%) . In veal.25–29%) . Higher p < 0.05) in the pH of beef and veal patties, resulting in pH values ranging from 5.98 to 6.09  as the cooked internal temperature increased from 55 °C to 76 °C (p < 0.05) the moisture content to approximately 63% (beef) and 67–68%  compared to the 66% (beef) and 71%  in samples that were cooked to 55 °C  increased fat content of 8.62% to 9.45%, irrespective of the cooked internal temperatures  increased fat content ranging from 2.79% to 3.02% was found in cooked veal samples compared to that in the uncooked samples.The fat content of fresh beef and veal patties was 6.46% and 2.19% , respecteratures . Previoup < 0.05) a* and b* values of 26.98 and 22.63 and a higher L* value of 59.83 were detected in fresh veal patties (p < 0.05) from 34.75 to 15.27, from 25.86 to 13.93, and from 46.32 to 39.85 in beef patties and decreased (p < 0.05) from 26.98 to 12.77, from 22.63 to 16.53, and from 59.82 to 57.87 in veal samples by the end of storage. These results agree with those of a previous study [The color index a*, b*, and L* values of freshly prepared beef patties were 34.75, 25.89, and 44.94, respectively . Compare patties . The lesus study , which fp < 0.05) the L* value from 46.67 to 47.08 compared to the value that was obtained at 55 °C (50.32). In veal samples, the L* value decreased (p < 0.05) from 68.93 to 66.45 when the cooked temperature increased from 55 to 71.1 °C, while the L* value returned to 68.45 after cooking to 76 °C. There results suggest that the doneness of cooked beef and veal patties cannot be determined by external color change.After cooking to 55–76 °C, the external color values of a* and b* ranged from 11.86 to 13.46 and from 16.96 to 18.45 in beef samples (p < 0.05) value of 53.66 to 54.19 compared to the value of 50.24 in samples that were cooked to 55 °C  (p < 0.05) a* value of 11.21 to 12.2 and a lower b* value of 15.56 to 16.25 than those of the samples that were cooked to 55 °C or 62.5 °C, with an a* value of 16.25 to 17.19 and a b* value of 18.49 to 18.84 (p > 0.05) in L* values, ranging from 70.09 to 72.96, among the veal samples that were cooked from 55 °C to 76 °C  with various heating times. As expected, the pathogen population in beef and veal samples decreased with the increasing of heating time. After cooking at 177 °C for 360 s, a reduction of 5.67 and 6.42 log CFU/g was observed in ground beef and veal samples, respectively . As shown in In this study, three survival models in the USDA-IPMP software were used to evaluate the fitness of the model to predict the thermal inactivation kinetics of E. coli O157:H7 in ground beef, turkey, lamb, and pork meat, with cooked temperatures from 55 °C to 65 °C in water bath settings, and no studies have reported thermal dynamic parameters of veal products yet. Results of this study showed that the D-value of E. coli O157:H7 in ground veal samples cooked at 177 °C on a commercial double pan-broiling griller were 63.27, 189.5, and 29.41 s calculated from Linear, Mafart-Weibull, and Buchanan Two-Phase Linear models, respectively, which were significantly lower than those from the beef samples  sensitive to heat in veal samples than in beef samples. Very limited studies have reported the thermal inactivation of d others  recentlyd others . In thisE. coli O157:H7 to 5.1–7.0 log CFU/g. To reduce the possibility of food-borne outbreaks due to E. coli O157:H7 contamination, the USDA-FSIS recommends cooking non-intact veal products to an internal temperature of 62.5 °C (145 °F) with at least a three-minute rest time [E. coli O157:H7 on non-intact beef and veal products. By double pan-broiling to 55 °C and 62.5 °C with a 0.5-min rest, 1.95 to 4.79 log CFU/g and 1.97 to >6.0 log CFU/g reductions of E. coli O157:H7 were achieved in beef and veal samples, respectively (p < 0.05) 0.98 to 1.14 log CFU/g reduction was reached when the rest time extended from 0.5 min to 3.5 min after cooking from 55 °C to 62.5 °C (p < 0.05) reduction of 2.68 log CFU/g was achieved compared to the 0.5-min rest (E. coli O157:H7 was below the detect limit (>6.0 log CFU/g reduction) when veal samples were cooked to 62.5 °C with a 0.5- or 3.5-min rest (p < 0.05) lower than the same amount in beef samples on MacConkey agar. This result might be explained by the higher moisture content (slightly less fat) in combination with less mature and less thick collagen in veal muscle tissue, allowing for more efficient heat transfer. The compaction density of the physiologically less mature veal tissue versus beef tissue in the patty could also be a factor affecting heat transfer, which produces a greater steaming effect inside the veal patties, resulting greater pathogen reduction. Over all, the present study demonstrates that cooking coarse ground beef and veal patties to an internal end-point temperature of ≥62.5 °C with at least a 3-min rest achieves a >5.0 log reduction of E. coli O157:H7 cells. As expected, double pan-broiling beef and veal samples to 71.1 °C (well done doneness) and 76 °C (beyond well-done doneness) decreased the overall pathogen populations to below the detection limit (>6 log CFU/g reduction), regardless of the rest time , in agreest time . To the ectively . For bee 62.5 °C . This enmin rest . It is imin rest , signifiE. coli O157:H7 is more vulnerable in veal compared to the beef during thermal processing. A higher internal temperature and longer rest time cause an increased inactivation of E. coli O157:H7, and veal and beef patties present similar tendencies in terms of quality change throughout storage and cooking, supporting our hypothesis. The results of this study cover various aspects of beef and veal quality changes during storage and cooking that will be beneficial for intact and non-intact beef and veal preparation at multiple points, including retail, foodservices, and at home. It was also verified that cooking coarse ground beef or veal to an internal end-point temperature of 62.5 °C with a 3.5-min rest will not generate a great food safety risk. This information will be useful for the USDA-FSIS in developing risk assessments of E. coli O157:H7 in non-intact and intact beef and veal products.In conclusion,


SEM micrographs indicate a mean particle size of 9.873 ± 0.034 µm and particles that are moderately dispersed in the matrix. The XRD patterns indicate Al2O3 is of the β-phase polytype which possesses high ionic conductivity. The storage modulus (G′) of the composites, or the rigidity, increases by nearly two orders of magnitude, with variations in particle volume fraction and electrical field strength. The increase in the storage modulus is caused the ionic polarization of the alumina particles and the induced dipole moments set up in the natural rubber matrix.The electrorheological properties (ER) of natural rubber (XL)/alumina (Al The role of inorganic particles has been evaluated using the tube model theory [Combining a dielectric ceramic and a polymer host to form a flexible dielectric composite is interesting in view of the greater flexibility in tailoring these materials towards particular mechanical, electrical, and thermal properties, and/or a coupling between these properties ,2. Carpinsducers . López-Ml theory . Molecull theory . The backbone of natural rubber (NR), polyisoprene, is derived from the polyacetylene backbone through the saturation of every other double bond. Polyisoprene is a potential candidate for materials used in various devices: solar cells, LEDs, and FETs. Natural rubbers combine high tensile strength, flexibility, and tear strength with outstanding resistance to fatigue ,6. Exteret al. reported that β- and β"-alumina are high temperature solid electrolytes that exhibit high ionic conductivity. These materials have been used in energy storage, alkali-metal thermal-to-electric conversion (AMTEC) cells, and gas sensors [2O3) are a low thermal-expansion, a good thermal-shock fracture resistance, a low density, high creep resistance, good chemical and thermal stability, a high melting point , and excellent toughness and strength. Vinod et al. and Jakubowickz reported that alumina can also reduce heat, humidity, light, ozone and gamma radiation, flame resistance, and the crack growth in natural rubber [2O3) is a potential candidate as the dispersed phase in the natural rubber matrix [Alumina is a linear dielectric material with ionic polarization with potential uses as electrolytic capacitors, optical and photoluminescent materials . The mos sensors . Many of sensors ,9. β-alu 1,828 ±  °C, and l rubber . Aluminar matrix ,13. Factr matrix ,19,20,21r matrix ,19,20,21r matrix ,23,24,25The aim of this work was to study the electrical and electrorheological properties of composites based on alumina particles (the dispersed phase) embedded in a dried natural rubber type XL (matrix or the continuous phase). In addition, detailed characterizations by SEM, FTIR, and XRF of composites, the matrix, and the dispersed phase shall be reported. 2O3), natural rubber (XL) and the XL/Al2O3 composites are shown in The mean diameter of alumina particles is 9.873 ± 0.034 µm, as determined by the particle size analyzer, and consistent with that found by SEM. The chemical composition of alumina powder, as measured by the X-ray fluorescence is 99.74% . The FTIR spectra of alumina , 3,000–2,900 cm-1 ν(C-H) and (=CH), 2,900–2,800 cm-1 ν(CH2), 1,800–1,600 cm-1 ν(C=C), 1,500–1,300 cm-1 (CH3 asymmetric deformation), 1,300–1,100 cm-1 ν(C-O-C), 1,100–740 cm-1 (=CH bending), and 640–580 cm-1 ν(Al-O). Our FTIR result is consistent with those reported by Ismail et al. [et al. [2O3, and XL/Al2O3, are shown in The characteristic peaks of XL/All et al.  and Kans [et al. . The XRD2O3 resemble those recorded at the International Center for Diffraction Data . This observation indicates a partial phase transformation from the corundum phase  to β- and β"-alumina forms . The structures of β- and β"-alumina are the closely related polytypes; both have the same general stacking pattern of Al-O spinel-like blocks separated by Na-O planes [+, Ca2+, and Na+) as traced by XRF can migrate through a surplus of cationic sites in the crystal lattice. The microstructures of pure natural rubber (XL), XL/Al_6, and XL/Al_7, and alumina particles are shown in SEM micrographs of 2O3 composites are: 0.9200 and 0.9428 g/cm3 (XL/Al_0); 0.9216 and 0.9449 g/cm3 (XL/Al_1); 0.9274 and 0.9527 g/cm3 (XL/Al_2); 0.9333 and 0.9607 g/cm3 (XL/Al_3); 0.9383 and 0.9674 g/cm3 (XL/Al_4); 0.9424 and 0.9730 g/cm3 (XL/Al_5); 0.9460 and 0.9780 g/cm3 (XL/Al_6); and 0.9489 and 0.9818 g/cm3 (XL/Al_7). The XRD patterns of alumina powder and XL/Al2O3), and XL/Al2O3 composites were measured by the impedance analyzer; the data are tabulated in 2O3, and XL/Al_7, respectively. The electrical conductivity values of XL, Al2O3, and XL/Al_7 are 6.517 × 10-9, 1.502 × 10-7, and 2.683 × 10-8 (Ω.m)-1, respectively. The electrical conductivity of XL/Al_7 composite is approximately two orders of magnitude higher than that of the natural rubber XL. The polyisoprene can be swollen by the hexane, and the alumina is intercalated in the natural rubber XL matrix. The alumina has a partial phase transformation from rhombohedral to the hexagonal β- and β"- phase formations as shown by the XRD pattern. The β- and β"- alumina phases have high ionic conductivity [2O3 acts as Al-O spinel-like blocks in Na-O planes under an applied AC electrical field.The electrical properties of the non-conducting natural rubber (XL), the insulating material  exhibits the highest electrorheological responses, the storage modulus G′(ω) and the loss modulus G″(ω), under the stimulation of an external electrical field. For the XL/Al_0 system, the G′ (ω = 1 rad/s) increases from ~1.16 × 104 Pa to 1.25 × 104 Pa as the electrical field is varied from 0 to 2 kV/mm; ΔG' = 0.09 x 104 Pa. For the XL/Al_7 system, the G′ (ω = 1 rad/s) increases from ~2.00 × 104 Pa to 2.44 × 104 Pa as the electrical field is varied from 0 to 2 kV/mm; ΔG′ = 0.44 × 104 Pa. The storage modulus of the composite without alumina (XL/Al_0) increases significantly upon electrical fields from 0 to 2 kV/mm because natural rubber (XL) is an elastic elastomer. The XL elastic behavior is caused by bond distortions. In its relaxed state rubber consists of long, coiled-up polymer chains that are interlinked and rotate freely about its neighbor. As an electrical field is applied, bond lengths deviate from the minimum energy equilibrium and the strain energy is stored electrostatically. vs. particle volume fraction at the electrical field strengths of 2 kV/mm. In this figure, we also show the storage modulus, G′ο(ω =1 rad/s), without an electrical field versus particle volume fraction. Both ΔG′and G′ο appear to increase monotonically with increasing particle volume fraction. For the storage modulus G′ο(ω = 1 rad/s) without an electrical field, the storage modulus increases from 11,590 Pa to 20,011 Pa as the volume fraction is varied from 0.00000 to 0.00968, as also tabulated in ο, vs. particle volume fraction. The storage modulus sensitivity values at an electrical field strength of 2 kV/mm are 7.8854% (XL/Al_0), 23.6483% (XL/Al_1), 24.6102% (XL/Al_2), 24.1534% (XL/Al_3), 22.3179% (XL/Al_4), 23.9230% (XL/Al_5), 22.8915% (XL/Al_6), and 21.8755% (XL/Al_7). The storage modulus sensitivity of our composites is thus nearly independent of particle volume fractions; but it is higher than that of pure natural rubber (XL/Al_0). vs. electrical field of the three composites  at various electrical field strengths are shown in The storage moduli G′(ω =1 rad/s) 2O3) was purchased from Sigma-Aldrich Chemical Co., Ltd. (USA). The specific gravity of the alumina powder was 3.90 g/cm3. Hexane (HPLC grade) was obtained from Lab-Scan Co., Ltd. The starting natural rubber  was supplied by Venus Technology Co., Ltd. . STR XL is a dried rubber type in an extra light color slab and with a low dirt content of less than 0.04%. The specific gravity, impurity content, Mooney viscosity, initial plasticity, and plasticity retention of natural rubber (STR XL) are 0.92 g/cm3, 0.02% by weight, 62.8, 32.0, and 81.3, respectively. This natural rubber (STR XL) exhibits a high compounded gum tensile strength, good hot tensile properties, and possesses low level non-polymer constituents [Aluminum oxide , diperseV is the volume of the dispersed phase (Al2O3), and totalV is the total volume of the dispersed phase (Al2O3) and the matrix (STR XL) phase. Fourier transform infrared spectra (FTIR) were recorded on a Perkin Elmer, model Spectrum One spectrometer with a spectral resolution of 4 cm-1. The samples were mixed with the single-crystal potassium bromide (KBr).. X-ray diffraction (XRD) data were taken and analyzed using a Bruker, D8 Discover analyzer with a VANTEC-1 detector and a double-crystal wide-angle goniometer. Scans were obtained from 10° to 80° 2θ at a scan speed of 5° 2θ/min in 0.05° or 0.03° 2θ increments using CuKα radiation (λ = 0.154 nm). Peak positions were compared with standard JCPDS files to identify the crystalline phases. SEM micrographs were obtained using a scanning electron microscope  equipped with EDS for X-ray microanalysis. The samples of natural rubber XL, XL/Al2O3, and alumina powder (Al2O3) were mounted on a stub using carbon paste and were sputter-coated to ~0.1 µm of gold to improve conductivity. An acceleration voltage of 20 kV with magnifications of 1000 and 3200 times were used. The electrical properties were measured and obtained using an impedance analyzer  equipped with an LCR meter . The samples were prepared according to the ASTM B263-94 standard for the electrical property measurement. Composite samples were prepared as thin discs having a diameter of 38 mm and a thickness of 0.50 mm. In our experiment, the electrical properties were measured at frequencies from 500 to 106 Hz with an AC current of 2 A for samples at various volume fractions: Φ =effρ is the effective resistivity (Ω.m), R is the resistance (Ω), A is the cross-section discs (m2), and L is the thickness (m).The measured electrical resistivity of the composite materials was converted to electrical conductivity as follows :ρeff=RAA controlled-strain rheometer  was used to measure the dynamic rheological properties of the composites under controlled strain with a copper parallel plate geometry (25 mm in diameter). The typical sample thickness of the parallel plate gap was 1.0 ± 0.1 mm. An electrical field for the ER measurement was applied using a high voltage power supply . Strain sweep tests were first carried out to determine the suitable strains to measure as functions of frequency at various DC electrical field strengths. The samples were pre-sheared at a low frequency (0.04 rad/s) with the electrical field on for 9 min in order to attain the equilibrium polarization. Each measurement was carried out at 27 °C and was repeated at least two or three times. Cumulative and fractional distributions were measured by using a particle size analyzer . The samples were dispersed in a water medium and vibrated in an ultrasonic cleaner for 20 min. Cρ = pρ Φ m+ ρ (1-Φ), where ρc,pρ and mρ are the densities (g/cm3) of the composites, the alumina, and the elastomer, respectively. Chemical compositions were obtained using an Oxford, model ED2000 X-ray fluorescence spectrometer (XRF), with a tube current of 1000 µA and an acquisition lifetime of 30 s. Bulk densities of the alumina particles, the elastomer, and the composites were determined using a pynconometer by weighing the samples of known volumes with an accurate 4-digit balance. The composite densities were calculated from3, a relative permittivity of 2.0, and it is colorless. The alumina particles were dispersed using a magnetic bar stirrer at room temperature for 24 h. Each suspension was then poured into a Petri dish glass and allowed to dry at room temperature overnight. A thin disc with the thickness less than 0.5 mm and of light yellow in color was obtained. ER suspensions, with alumina particulate volume fractions of 0.00000 (XL/Al_0), 0.00054 (XL/Al_1), 0.00248 (XL/Al_2), 0.00448 (XL/Al_3), 0.00613 (XL/Al_4), 0.00750 (XL/Al_5), 0.00873 (XL/Al_6), 0.00968 (XL/Al_7), were prepared. The non-polar molecules tend to be soluble in non-polar solvents such as hexane, carbon tetrachloride, diethyl ether, and benzene. The dried natural rubber (XL) samples were swollen in a 20% by volume hexane solution. The hexane was HPLC grade with a density of 0.655 g/cm2O3 particle volume fraction of 0.00968 has the highest electrical and electrorheological responses. The electrical properties, namely dielectric constant and electrical conductivity, of XL/Al_7 are 2.123 and 2.683 × 10-8 (Ω.m)-1, respectively. With and without an electrical field, the dynamic moduli G′ο, G′2kV/mm, G″ο, G″2kV/mm, and ∆G′/G′ο of the XL/Al_7 composite are 20011 Pa, 24388 Pa, 2827 Pa, 3639 Pa, and 21.88%, respectively. Our results suggest that small amounts of alumina (Al2O3) particles can be used as a filler to absorb loss and store additional elastic energy within the natural rubber matrix under electrical field. The XL/Al2O3 composites studied are soft and flexible materials and potential candidates for biomimetric actuators and/or artificial muscles.The experimental data show that the XL/Al_7 composite with an Al


Zanthoxylum acanthopodium has insecticidal effect in Chinese traditional medicine. In this study, the essential oil from the dried Zanthoxylum plant was used as a larvicidal compound against the malaria mosquitoes, Anopheles anthropophagus and Anopheles sinensis.Zanthoxylum essential oil were investigated by gas chromatography and mass spectroscopy (GC–MS). The larvicidal bioassays of the whole oil, as well as the main compounds in the oil  were performed using WHO method.Compounds in the cis-linalool oxide (3.76%), cis-limonene oxide (3.06%). A dose-dependent effect on mortality was recorded with increasing concentrations of essential oil and compounds increasing mortality of the larvae. Larvicidal bioassays revealed that 24 h LC50 of the whole essential oil was 36.00 mg/L and LC90 was 101.49 mg/L against An. anthropophagus, while LC50 was 49.02 mg/L and LC90 was 125.18 mg/L against An. sinensis. Additionally, 24 h LC50 of estragole were 38.56 and 41.67 mg/L against An. anthropophagus and An. sinensis, respectively, while the related LC90 were 95.90 and 107.89 mg/L. LC50 of eucalyptol were 42.41 and 45.49 mg/L against An. anthropophagus and An. sinensis, while the related LC90 were 114.45 and 124.95 mg/L.In total, 63 main compounds (99.32%) were found in the oils, including estragole (15.46%), eucalyptol (10.94%), β-caryophyllene (5.52%), Z. acanthopodium and its several major compounds may have potential for use in the control of malaria mosquitoes.The essential oil of  Malaria, a widespread disease in tropical and subtropical regions , can cause symptoms that include fever, headache, vomiting, and even progress to coma or death . As the Anopheles is the only vector of human malaria [Plasmodium [Anopheles anthropophagus and Anopheles sinensis have been chosen as the test target mosquito species in this research, because they are rife and harmful in China. The mosquito An. sinensis is a member of the Anopheles hyrcanus group, distinguished from other series by the presence of pale bands  on the palpi and a tuft of dark scales on the clypeus on each side. Anopheles anthropophagus is a major vector in central China, able to spread malaria 18–20 times better than An. sinensis [An. anthropophagus and An. sinensis live on the surface of water. Compared to adults, the ability of larvae to escape the insecticide is limited, so the time from larvae to adult is an important period to control mosquitoes [Among mosquitoes,  malaria  and, of asmodium . Anophelsinensis , 7. The Generally, mosquito larval control is carried out by conventional synthetic insecticides, which have high efficiency and low cost. Their efficacy may decay gradually because mosquitoes develop resistance against insecticides . By contZanthoxylum acanthopodium is a shrub or small tree in the Zanthoxylum genus of Citrus family, native to southern China, Bangladesh, Bhutan, northern India, Nepal, Laos, Burma, northern Thailand Vietnam, Indonesia, and Peninsular Malaysia. It has 5–13 alternate leaves with serrated edge (3–8 cm long and 1–2 cm wide) and axillary inflorescences with densely globose clusters of unisexual flowers [Zanthoxylum piperitum, it has similar tongue-numbing characteristic seed pericarps, which are often used as spices in cooking, and traditional Chinese medicine reported great effect on contraception, relieving pain and killing parasites [Zanthoxylum acanthopodium, with many active ingredients, is a novel agent with special effect. In the present study, the chemical composition of the essential oil from Zanthoxylum acanthopodium was determined and its larvicidal activity was evaluated against An. anthropophagus and An. sinensis.Zanthoxylum acanthopodium were collected from 33 families of plants in the Ailao Mountains, Yunnan Province, China, in May 2013. Plant specimens were identified by Dr. Yin-zhang Zhou. The plants were dried under shade (at room temperature). The voucher specimen (No. IBSC/0387990) was deposited in the South China Botanical Garden, Chinese Academy of Sciences.The aerial parts of g) of Zanthoxylum acanthopodium were chopped and subjected to hydrodistillation for 6 h using a Clevenger type apparatus. The obtained oils were dried over hydrous sodium sulfate for 24 h, filtered, and then stored at 4 °C in brown sealed glass vials until tested.The air-dried plant materials  with a HP-5 5% phenylmethylsiloxane capillary column  and an FID detector was used for GC–FID analysis. Helium gas (1 mL/min) was used as the carrier gas. Injector and mass transfer line temperatures were set at 250 and 280 °C, respectively. The EO solution (1 μL) in hexane was injected and analysed under the following column conditions: initial column temperature of 40 °C for 1 min, increased to 250 °C at a 3 °C/min, and maintained at 250 °C for 20 min.n-alkanes (C8–C25) in an HP-5 MS column. The major oil components were identified by co-injection with standards (whenever possible) and confirmed using the Wiley (V.7.0) and National Institute of Standards and Technology V.2.0 GC–MS library on the basis of Kovats indices. The relative concentration of each compound in the oil was quantified on the basis of the peak area integrated in the analysis program.The oils were quantitatively and qualitatively analysed using a GC–MS 6890-5975 system  equipped with a HP-5 MS fused silica capillary column . For GC–MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas (1 mL/min) was used as carrier gas. Injector and mass transfer line temperatures were set at 250 and 280 °C, respectively. EO solution (1 μL) in hexane was injected and analysed under the following column conditions: initial column temperature at 40 °C for 1 min, increased to 250 °C at a 3 °C/min heating ramp, and maintained at 250 °C for 20 min. Kovats indices were calculated for all of the volatile components by using a homologous series of An. sinensis and An. anthropophagus were received from Centre for Disease Control and Prevention of Guangdong Province, China. Twenty-five larvae were reared in 250 mL glass beaker and fed with Brewer’s yeast/dog biscuit (1:3) [Zanthoxylum acanthopodium were evaluated according to the standard procedures suggested by the WHO [The eggs of it (1:3) , 15. The the WHO . The ess50, LC90 and other statistics at 95% fiducial limits of upper confidence limit and lower confidence limit, and Chi square values were calculated by using the software using Statistical Package of Social Sciences (SPSS) 13.0 for windows, the significance level was set at P < 0.05.The average larval mortality data were subjected to probit analysis for calculating LCg of dried plant material yielded 7.2 mL  of a light- yellow oil with a distinct smell. The GC–MS analysis of Zanthoxylum acanthopodium essential oil revealed 63 components representing 99.32% of the oil , cis-limonene oxide (3.06%).The steam distillation of 2000il Table . The comZanthoxylum acanthopodium essential oil, main compounds were estragole, eucalyptol and β-caryophyllene. They were often reported in the essential oil extracted from different kinds of Zanthoxylum plants. Estragole is a component isolated from various trees and plants and its extraction from Zanthoxylum plants has been described but is relatively rare. Wang et al. [Zanthoxylum schinifolium, estragole was the major compound (69.52%). Eucalyptol was common in many plants including Zanthoxylum plants in related literature. Liu et al. [Zanthoxylum avicennae leaves and stems had high content of monoterpenoids including 53.05% eucalyptol. Prieto et al. [Zanthoxylum monophyllum oil were eucalyptol (9.19%); Eiter et al. [Zanthoxylum clava-herculis and found the content of eucalyptol ranged from 16 to 43%. Similar research was also carried out on the essential oil from the fruits of Zanthoxylum bungeanum [Zanthoxylum rhetsoides and Zanthoxylum myriacanthum [Zanthoxylum essential oils. It reported to be found in the essential oils of Zanthoxylum newbouldia leaves (36%) [Zanthoxylum syncarpum (9.2–9.3%) [Zanthoxylum acanthopodium leaves (3.0%) [Zanthoxylum rubescens leaf (22.1%) [Zanthoxylum setulosum (13.7%) [Zanthoxylum ekmanii (11.5%) [Zanthoxylum procerum leaves (7.0%) [Z. acanthopodium essential oil shares some similar main constituents with other species of Zanthoxylum.In the g et al.  showed tu et al.  showed to et al.  determinr et al.  analysedungeanum ; Zanthoxacanthum , the cones (36%) ; Zanthox.2–9.3%) ; Zanthoxs (3.0%) ; Zanthox (22.1%) ; Zanthox (13.7%) ; Zanthox (11.5%) ; Zanthoxs (7.0%) . The resAn. sinensis and An. anthropophagus with increase in essential oil and test compounds concentration. The mortality of An. sinensis was 17, 54, 68, 83, 94 and 100% when the oil at concentration of 25.0, 50.0, 100.0, 125, and 150 mg/L. The mortality of An. anthropophagus was 36, 62, 78, 90, 97 and 100% when the oil at concentration of 25.0, 50.0, 100.0, 125, and 150 mg/L. The LC50 and LC90 values against An. sinensis larvae were 49.02 and 125.18 mg/L and for An. anthropophagus were 36.00 and 101.49 mg/L, respectively. Between two compounds tested for 24 h, estragole exhibited a stronger mosquito larvicidal activity than the oil with LC50 41.67, 38.56 mg/L and LC90 107.89, 95.90 mg/L for An. sinensis and An. anthropophagus. Eucalyptol had less LC50 values (45.49 and 42.41 mg/L) and LC90 values (124.95 and 111.45 mg/L).Tables An. sinensis and An. anthropophagus used in these experiments, act as vectors for many disease-causing viruses and cause serious health problems worldwide in both humans and animals. Although Zanthoxylum is a known potential source of anthelmintic agents for traditional Chinese medicine, only a few studies have assessed the anti-mosquito activity of Zanthoxylum species. Trongtokit et al. [Zanthoxylum limonella had repellent effect against Aedes aegypti, Culex quinquefasciatus, and Anopheles dirus. Effects of Zanthoxylum armatum essential oil against Aedes aegypti (LC50 = 54 ppm), Anopheles stephensi (LC50 = 58 ppm) and Culex quinquefasciatus (LC50 = 49 ppm) was analysed by Tiwary et al. [Zanthoxylum articulatum was examined with respect to its larvicidal properties against the larvae of Aedes aegypti and showed that LC50 was 77.62 ppm [Zanthoxylum piperitum had insecticidal effect against Aedes gardnerii, Anopheles barbirostris, Armigeres subalbatus, Culex tritaeniorhynchus, Culex gelidus, Culex vishnui group, and Mansonia uniformis [Zanthoxylum beecheyanum was also reported, 24 h LC50 was 6.895 mg/mL against Culex pipiens adults and 119.020 mg/mL against 4 instar larvae [Mosquitoes, including t et al. , 31 showy et al. . The ess7.62 ppm . Essentiniformis . The relr larvae .Zanthoxylum essential oil, it can be attributed to some compounds in the oil that can lead to alteration in the membrane structure of larval cells [Lasioderma serricorne adults with 24 h LD50 value of 15.58 mg/adult, and in the fumigant toxicity test, the 24 h LD50 was 5.18 mg/L air. When against Maize weevils, 24 h LC50 values of estragole was 14.10 ppm [50 of eucalyptol was inactive at concentrations even as high as 100 mg/L against early fourth instar mosquito larvae of Culex pipiens. Against the 3rd instars of the Culex pipiens 24 h LC50 values of eucalyptol was 91.45 mg/L [Zanthoxylum acanthopodium essential oil against two anopheline mosquito species. Yang et al. [Clove Bud oils with reference to repellent activity against the bean bugs Riptortus clavatus. You et al. [Lasioderma serricorne. Wang et al. [50 = 6.21 mg/L for Tribolium castaneum and 14.07 mg/L air for L. serricorne.When comes to the larvicidal mechanism of the al cells , 37, espal cells  showed e4.10 ppm . Kimbari4.10 ppm  showed t.45 mg/L . The curg et al.  showed tu et al.  showed tg et al.  showed tTo sum up, a series of investigations for chemical compounds from natural products have revealed that some essential oil with adequate active ingredients is essential to the development of new insecticidal drugs, especially in view of the vast worldwide flora. There is also some evidence indicating that essential oils often prove to be more effective than their components, indicating synergy .Zanthoxylum acanthopodium essential oil, including estragole (15.46%), eucalyptol (10.94%), β-caryophyllene (5.52%), cis-linalool oxide (3.76%), cis-limonene oxide (3.06%). Both whole essential oil and its main compounds  showed significant larvicidal activity. The results revealed that Zanthoxylum acanthopodium essential oil could be further developed as a potential agent to control the larvae of malaria mosquito.In total, 63 main compounds (99.32%) were found in


Confocal imaging of mouse small intestines from animals expressing GCaMP3 in ICC were used to investigate inhibitory neural regulation of ICC in the deep muscular plexus (ICC-DMP). We hypothesized that Ca2+ signaling in ICC-DMP can be modulated by inhibitory enteric neural input. ICC-DMP lie in close proximity to the varicosities of motor neurons and generate ongoing Ca2+ transients that underlie activation of Ca2+-dependent Cl− channels and regulate the excitability of SMCs in the SIP syncytium. Electrical field stimulation (EFS) caused inhibition of Ca2+ for the first 2–3 s of stimulation, and then Ca2+ transients escaped from inhibition. The NO donor (DEA-NONOate) inhibited Ca2+ transients and Nω-Nitro-L-arginine (L-NNA) or a guanylate cyclase inhibitor (ODQ) blocked inhibition induced by EFS. Purinergic neurotransmission did not affect Ca2+ transients in ICC-DMP. Purinergic neurotransmission elicits hyperpolarization of the SIP syncytium by activation of K+ channels in PDGFRα+ cells. Generalized hyperpolarization of SIP cells by pinacidil (KATP agonist) or MRS2365 (P2Y1 agonist) also had no effect on Ca2+ transients in ICC-DMP. Peptidergic transmitter receptors (VIP and PACAP) are expressed in ICC and can modulate ICC-DMP Ca2+ transients. In summary Ca2+ transients in ICC-DMP are blocked by enteric inhibitory neurotransmission. ICC-DMP lack a voltage-dependent mechanism for regulating Ca2+ release, and this protects Ca2+ handling in ICC-DMP from membrane potential changes in other SIP cells.Gastrointestinal motility is coordinated by enteric neurons. Both inhibitory and excitatory motor neurons innervate the syncytium consisting of smooth muscle cells (SMCs) interstitial cells of Cajal (ICC) and PDGFRα In fact this level of motor control is due not only to the functions of smooth muscle cells (SMCs), because the behavior of SMCs is modulated by interstitial cells . ICC and PDGFRα+ cells are electrically coupled to SMCs  generate pacemaker activity that develops into electrical slow waves  expressed specifically in murine ICC to investigate the responses of ICC-DMP to enteric inhibitory neurotransmission. Our results show that enteric inhibitory neurotransmitters exert powerful inhibitory effects on Ca2+ release, which would be expected to reduce activation of Ano1 and development of spontaneous transient inward currents (STICs) and reduce the excitatory drive exerted upon the SIP syncytium by ICC-DMP.The precise mechanisms through which ICC transduce inputs from motor neurons are poorly understood, largely because past studies have relied upon Gt(ROSA)26Sortm38(CAG−GCaMP3)Hze/J) and their corresponding wild-type siblings (C57BL/6) were purchased from Jackson Laboratories  and subsequently crossed with Kit-Cre mice (c-Kit+/Cre−ERT2) provided by Dr. Dieter Saur . Kit-Cre-GCaMP3 mice underwent treatment with tamoxifen at 6–8 weeks of age (2 mg for 3 consecutive days), as previously described . The jejunal segments opened along the mesenteric border, and intra-luminal contents were removed by washing with KRB. Mucosal and sub-mucosal layers were removed by sharp dissection, and the remaining 3, 15.5; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.5; and glucose, 11.5. The KRB was bubbled with a mixture of 97% O2–3% CO2 and warmed to 37 ± 0.2°C. All drugs were purchased from Tocris Bioscience  and dissolved in solvents recommended by the manufacturer to create appropriate stock solutions. Final concentration used for experiments were obtained by diluting with KRB. All work was performed according to biosafety level II regulations.Tissues were maintained by perfusing with KRB containing (mmol/L): NaCl, 120.35; KCl, 5.9; NaHCONeural responses were elicited by electrical field stimulation  generated by a Grass stimulator Grass S48 stimulator  and delivered via two platinum electrodes placed on either side of muscle strips. Responses evoked by EFS were completely abolished by tetrodotoxin .+/copGFP mice as previously described . The PCR primers used and their GenBank accession numbers are listed in Table Jejunal ICC were dispersed from Kit2+ imaging experiments were performed in the presence of nicardipine (100 nM) to minimize movement artifacts resulting from contractions.For imaging studies, the muscles were equilibrated with continuous perfusion of warmed KRB solution at 37°C for 1 h. Imaging was performed with a spinning-disk confocal microscope  mounted to an upright Nikon Eclipse FN1 microscope equipped with a 60x 1.0 NA CFI Fluor lens . GCaMP3, expressed in ICC within the jejunal muscles, was excited at 488 nm using a laser coupled to a Borealis system . Emitted fluorescence (>515 nm) was captured using a high-speed EMCCD Camera . Image sequences were acquired at 33 fps using MetaMorph software . In some experiments images were acquired with an Eclipse E600FN microscope  equipped with a 60x 1.0 CFI Fluor lens . In this system, GCaMP3 was excited at 488 nm , as previously described  images and imported into custom software  for initial pre-processing analysis. Tissue movement was stabilized to ensure accurate measurement of Ca2+ transients from ICC-DMP. Whole cell ROIs were used to generate spatio-temporal (ST) maps of Ca2+ activity in individual ICC-DMP recorded in situ. ST maps were then imported as TIFF files into Image J  for post-hoc quantification analysis of Ca2+ events.Analysis of Ca2+ event frequency in ICC-DMP was expressed as the number of events fired per cell per second (s−1). Ca2+ event amplitude was expressed as ΔF/F0, the duration of Ca2+ events was expressed as full duration at half maximum amplitude (FDHM), and Ca2+ event spatial spread was expressed as μm of cell propagated per Ca2+ event. Unless otherwise stated, data is represented as mean ± standard error (S.E.M.). Statistical analysis was performed using either a student's t-test or with an ANOVA test where appropriate . In all statistical analyses, P < 0.05 was taken as significant. P < 0.05 are represented by a single asterisk (*), P < 0.01 are represented by two asterisks (**), P < 0.001 are represented by three asterisks (***) and P < 0.0001 are represented by four asterisks (****). When describing data throughout the text, n refers to the number of animals used in that dataset while c refers to the numbers of cells used in that same data set.Ca2+ transients in ICC-DMP were ongoing and stochastic in nature, as previously reported  resulted in multiphasic responses (inhibition of Ca2+ transients followed by enhancement of these events), as shown in a representative spatiotemporal map (ST map) and traces of Ca2+ transients . During EFS (5 s), an initial inhibitory period was observed in which Ca2+ transients ceased for ~2 s. The firing of Ca2+ transients escaped from inhibition during the final 3 s of EFS, and a robust increase in Ca2+ transient firing was apparent during this period and after cessation of EFS . The amplitude of Ca2+ transients decreased from 0.8 ± 0.07 ΔF/F0 prior to EFS to 0.14 ± 0.05 ΔF/F0 during the inhibitory phase , duration decreased from 189 ± 7 ms before EFS to 27 ± 9.7 ms , and the spatial spread of Ca2+ transients decreased from 11.1 ± 0.7 to 2 ± 0.9 μm . The number of active firing sites also decreased during the inhibitory phase of EFS from 5.2 ± 0.4 to 0.3 ± 0.09 . Lastly, the propagation velocity of Ca2+ signals decreased from 71.4 ± 5.6 μm/s prior EFS to 11.4 ± 4.4 μm/s during the first 2 s of EFS .In this study we focused our analysis on the inhibitory responses imposed on ICC-DMP by nerve stimulation, so Ca Figures . During 2+ firing sites in ICC-DMP, from a single site to 13 sites . The nature of Ca2+ transients during sustained EFS (5 s) was examined in more detail by the analysis described in Figure 2+ transients ceased during the initial 2 s of EFS, as shown in the ST map . The delay periods describing the escape from inhibition are plotted as a summary histogram in Figure n = 19, c = 48).In this and a previous study we noted a range in the number of Cap Figure . During s Figure . Ca2+ tre Figure . A greate Figure . The inh2+ transients in these cells are displayed in ST maps in Figures 2+ firing site active in cell 2 before anything occurred in cell 1  incubated for 15 min, increased the firing frequency of Ca2+ transients in ICC-DMP from 1.5 ± 0.3 s−1 in control to 2.5 ± 0.5 s−1, as shown in Figures P = 0.0042, n = 5, c = 11). The duration of Ca2+ transients was also increased by L-NNA from 232 ± 11 to 250 ± 6.5 ms . However, neither the amplitude  nor the spatial spread  of Ca2+ transients was affected significantly by L-NNA.The nitric oxide synthase (NOS) inhibitor 2+ transients were dramatically inhibited by this compound Figure 2+ transients during control conditions was 0.9 ± 0.2 s−1, and was reduced to 0.14 ± 0.09 s−1 by DEA-NONOate . The amplitude of Ca2+ transients was reduced from 0.7 ± 0.14 ΔF/F0 to 0.2 ± 0.13 ΔF/F0 in DEA-NONOate , duration was decreased by DEA-NONOate from 242 ± 21.2 ms to 50 ± 33.6 ms , and spatial spread was reduced from 8.9 ± 8 μm to 1.8 ± 1.2 μm . These results suggest that NO was released tonically from enteric nerves, however at lower levels than was achieved by addition of DEA-NONOate (10 μM), and NO modulates the basal level of firing of Ca2+ transients in ICC-DMP.The NO donor, DEA-NONOate (10 μM) caused effects opposite of L-NNA and Ca2+ transients for about ~2 s after the onset of stimulation . L-NNA also increased the amplitude of Ca2+ transients from 0.2 ± 0.1 ΔF/F0 to 1 ± 0.1 ΔF/F0 , the duration from 42 ± 14 ms to 216 ± 14.9 ms , and the spatial spread from 1.3 ± 0.7 μm to 11.3 ± 1.3 μm .As above, EFS evoked inhibition of Can Figure . L-NNA , amplitude (P = 0.91), duration (P = 0.48), or spatial spread (P = 0.86) . MRS 2500 (1 μM) also failed to affect Ca2+ transients in ICC-DMP during EFS, as shown in Figures 2+ transients were also unaffected by MRS 2500 during the initial 2s of EFS .As several studies have reported, enteric inhibitory signaling by purines is mediated by P2Y1 receptors in post-junctional cells . The amplitude of Ca2+ transients increased during this period from 0.1 ± 0.07 ΔF/F0 to 0.7 ± 0.08 ΔF/F0 , Ca2+ transient duration increased from 30 ± 16.2 ms to 201 ± 17.83 ms , and the spatial spread of Ca2+ transients increased from 0.9 ± 0.5 μm to 10.5 ± 1.3 μm .While the P2Y1 receptor antagonist had no resolvable effect on the inhibition of Ca2+-activated K+ channels  in GI muscles cause significant hyperpolarization due to opening of apamin-sensitive, small conductance Ca2+ transients in ICC-DMP. In the presence of TTX, ATP (100 μM) also had no significant effects on Ca2+ transient frequency (P = 0.85), amplitude (P = 0.31), duration (P = 0.1) or spatial spread . Similarly, the selective P2Y1 receptor agonist MRS 2365 (1 μM) had no effect on ICC-DMP Ca2+ transient frequency (P = 0.25), amplitude (P = 0.62), duration (P = 0.62), or spatial spread . Hyperpolarization of the SIP syncytium with the KATP channel agonist, pinacidil , amplitude (P = 0.36), duration (P = 0.26), or spatial spread . These data suggest that membrane potential transients have no effect on Ca2+ tranisents in ICC-DMP, and purines which exert their hyperpolarizing effects on SIP cells other than ICC-DMP do not affect the Ca2+ transients in ICC-DMP.Application of purinergic agonists had no significant effects on CaGucy1a1 and Gucy1b1, protein kinase cGMP-dependent type 1: Prkg1, and inositol-1,4,5 triphosphate receptor I-associated G kinase substrate (IRAG) (aka murine retrovirus integration site 1 homolog): Mrvi1 transcripts. We noted elevated expression in all of these downstream mediators of nitrergic responses in ICC relative to unsorted cells . Gucy1a1 transcripts were higher in ICC in comparison to unsorted cells , duration (P = 0.33), and spatial spread (P = 0.5) were not significantly different after ODQ . The role of sGC in mediating nitrergic responses in ICC-DMP was also shown by an activator of sGC. Bay 58-2667 (1 μM), reduced firing frequency of Ca2+ transients , amplitude from 0.3 ± 0.06 ΔF/F0 to 0.1 ± 0.04 ΔF/F0 , duration from 215 ± 8.2 ms to 72 ± 26.9 ms , and spatial spread from 7.6 ± 0.5 μm to 2.7 ± 1.2 μm .Similar to L-NNA, ODQ (10 μM), an inhibitor of sGC, significantly increased basal Ca Figures  from 1.62+ transients during the first 2 s of EFS was 0.6 ± 0.3 s−1 in control and increased to 4 ± 0.5 s−1 in the presence of ODQ . ODQ also increased Ca2+ transient amplitude during EFS from 0.4 ± 0.15 ΔF/F0 to 1.1 ± 0.2 ΔF/F0 , duration from 99 ± 37.2 ms to 252 ± 7.8 ms , and spatial spread from 2.8 ± 1.3 μm to 11.3 ± 1.4 μm .ODQ (10 μM) also blocked the inhibitory effects of EFS on ICC-DMP during the initial 2 s of stimulation Figures . In thes2+ transients in ICC-DMP. We tested the effects of two PKG inhibitors, KT 5823 (1 μM) and Rp-8-pCPT-cGMPS (10 μM). However, Neither of these compounds had any effect on the basal activity of Ca2+ transients in ICC-DMP , amplitude , duration  or spatial spread , n = 4, c = 11. Rp-8-pCPT-cGMPS also failed to exert any significant changes in Ca2+ transient frequency , amplitude , duration  or spatial spread , n = 8, c = 21. We next tested if inhibition of PKG relieved the EFS evoked inhibitory response in ICC-DMP. KT 5823 and Rp-8-pCPT-cGMPS both had no significant effects on Ca2+ transients during the initial 2 s period of EFS. Across all cells tested, there was no significant change in the firing frequency (P = 0.57), amplitude (P = 0.49), duration (0.96) or spatial spread (0.58) of Ca2+ transients during the initial 2 s period of EFS in the presence of KT 5823 . Also in the presence of Rp-8-pCPT-cGMPS (10 μM) there was no significant change in the firing frequency (P = 0.67), amplitude (P = 0.37), duration (0.92) or spatial spread (0.81) of Ca2+ transients during the initial 2 s period of EFS in the presence of the drug .We next sought to evaluate the role of cGMP dependent protein kinase (PKG) on basal firing of Ca Figures . KT 5823We also tested the PKG inhibitors seeking a positive control for their poor performance against nitrergic effects in ICC. Contractile experiments were performed on muscles of the colon, aorta and corpus cavernosum. Neither KT 5823 nor Rp-8-pCPT-cGMPS reduced the inhibitory effects of sodium nitroprusside  or DEA NONOate (10 μM) on muscles pre-contracted with norepinephrine  or carbachol  (data not shown). Taken together, the lack of effects of commercial PKG inhibitors on smooth muscles, in general, might be due to poor penetration of the drugs into cells in intact tissues or targets besides PKG contributing to inhibitory responses. This is a question in need of further evaluation.Vipr1 and Vipr2) and PACAP receptor (Adcyap1r1). We noted elevated expression in all of peptidergic receptors in ICC relative to unsorted cells . Vipr1 transcripts were higher in ICC in comparison to unsorted cells  are also released from nerve terminals and might modulate ICC-DMP activity. Therefore, we examined the expression profile of peptide receptors in sorted ICC from small intestinal muscles and characterized expression of VIP receptors . Similarly, the amplitude of Ca2+ transients was reduced from 0.5 ± 0.06 ΔF/F0 to 0.2 ± 0.08 ΔF/F0 by VIP . The duration of Ca2+ transients was decreased from 224 ± 9.8 ms to 66 ± 28 ms by VIP . Finally, VIP also significantly decreased the spatial spread of Ca2+ transients from 9.7 ± 1.4 μm to 5 ± 2.3 μm . VIP 6-28 (10 μM) caused a significant increase in basal Ca2+ transient firing in a similar manner to blocking nitrergic input with application of L-NNA . The amplitude (P = 0.38), duration (P = 0.67) and spatial spread (P = 0.24) of Ca2+ transients was not significantly affected by VIP 6-28, n = 5, c = 23 , amplitude (P = 0.16), or spatial spread (P = 0.58) of Ca2+ transients was significantly affected during the initial 2 s period of EFS VIP 6–28 (10 μM) . However, the duration of Ca2+ transients in the initial 2 s period of EFS was significantly increased from 59 ± 26.9 ms in control to 110 ± 29.5 ms in the presence of VIP 6-28 . It is possible that multiple receptors mediate responses to inhibitory peptides in ICC.While VIP 6-28 increased the basal level of CaA Figure , VIP 6-2A Figure . Neither2+ transients in ICC-DMP, the intramuscular class of ICC in the small intestine. Ca2+ handling mechanisms in ICC are of interest because Ca2+ release from stores couples to activation of Ano1 channels and the generation of STICs  in which ongoing Ca2+ transients in ICC-DMP were largely abolished. Shutting off of STICs during the initial phase of EFS may explain a portion of the hyperpolarization responses to nerve stimulation seen in electrophysiological recordings  leading to non-linear changes in voltage-dependent conductances, membrane potential and excitation-contraction coupling. At present there are no organ-specific, unified models of the many responses in GI muscles that might be initiated or repressed by stimulation of enteric neurons.It is difficult to reconcile generalized tissue level responses to EFS  with events occurring in a single type of ICC. Neurotransmission may affect conductances and Ca2+ firing sites within ICC-DMP escaped from inhibition. In our experiments this occurred within about 2 s from the onset of stimulation. However, the Ca2+ release sites escaped inhibition with different temporal characteristics. The heterogeneity in the periods before escape from inhibition occurred is further demonstration of the independence of Ca2+ release sites, even within individual cells, and supports the conclusion that there is no cellular or multi-cellular correlation between Ca2+ release sites within the ICC-DMP network . We showed that Ca2+ release events were virtually blocked if neurokinin receptor antagonists were present . Thus, ICC have the receptor and the molecular apparatus to transduce nitrergic signals and produce cGMP. Others have found that knock down of Gucy1b1 in ICC, using Cre-LoxP technology, abolished nitrergic inhibitory junction potentials (IJP) in gastric fundus and reduced IJPs in the colon, but these authors also reported that knockdown of Gucy1b1 in SMCs also either reduced the amplitude or shortened IJPs  is expressed in ICC and is the main receptor/transducer of the inhibitory effects of NO in the GI tract  is the principal downstream signaling molecule mediating nitrergic responses, and Prkg1 is expressed in ICC in the small intestine -associated cGMP-kinase substrate , and this gene is also expressed in ICC of the small intestine. IRAG co-precipitates with IP3R and was found to be indispensable for cGMP regulation of Ca2+ release in model cells or cultured human colonic SMCs  responsible for these effects are complex. Ca2+ transients initiate inward currents in ICC-DMP that are conducted to other cells in the SIP syncytium. Thus, suppression of Ca2+ transients in ICC-DMP by inhibitory neural inputs would tend to reduce SMC excitability and reduce contractile force in the small intestine.In summary, CaSB, BD, and KS: Conception and design of the experiments; SB, BD, CC, KK, and KS: Collection, analysis, and interpretation of data; SB, BD, and KS: Drafting the article and revising it critically for intellectual content. All authors read and approved the manuscript before submission.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. The reviewer MC and handling Editor declared their shared affiliation.


Pleurotus tuber-regium on carbon tetrachloride (CCl4) deleterious effects on the reproductive system of male rats. Thirty six rats were divided into six groups of six animals each. Group I (negative control) received 10 ml/kg olive oil intraperitoneal weekly in addition to feed and water ad libitum. Group II (positive control) received CCl4 10 ml/kg (30% in Olive oil) weekly. Group III, IV, and V received 100 mg, 20 0mg, and 500 mg P. tuber-regium (33.3% in feed) daily in addition to 10 ml/kg CCl4 weekly. Group VI received 500 mg P. tuber-regium (33.3% in feed) daily. After 4 weeks, sperm motility, epididymal count and vitality were determined. Serum malondialdehyde (MDA), testosterone, Luteinizing hormone (LH), Follicle stimulating hormone (FSH), prolactin and oestradiol were estimated using enzyme-linked immunosorbent assay (ELISA) kits. Histopathologic examinations of the testis were carried out. Carbon tetrachloride significantly reduced the sperm motility (54.33 ± 3.79%), epididymal count , LH (0.88 ± 0.14), FSH (2.04 ± 0.33), and Testosterone (2.02 ± 0.24) when compared with control (89.33 ± 9.01), 91.91 ± 1.92 × 106/ml, 13.12 ± 0.19, 2.74 ± 0.32, 3.64 ± 0.62, and 4.16 ± 0.23, respectively, which were reversed by P. tuber-regium administration. Co-administration of P. tuber-regium plus CCl4 significantly reduced MDA level. P. tuber-regium showed dose dependent ameliorative activity against CCl4 deleterious action on the testis and may be beneficial in the management of male infertility.The high rate of male infertility and the meager resources to manage same in sub Saharan Africa have necessitated the search for cost effective and available alternatives. Mushrooms have been used traditionally in folk medicine and as nutraceuticals. This study has investigated the effect of the wild mushroom  Infertility affects at least 20% of married couples and growing evidence suggest an increasing incidence of male reproductive problems . In AfriPleurotus tuber-regium is an edible mushroom found in Nigeria  in Olive oil  was usedSprague-Dawley rats with body weights 180–200 g acclimatized for 2 weeks were maintained under controlled conditions of temperature (23 ± 2°C) and humidity (50 ± 5%) and a 12-h light–dark cycle, were used for the experiment. The animals were housed in sanitized polypropylene cages containing sterile paddy husk as bedding. The bedding of the cages was changed daily and the cages were cleaned as well. They had free access to standard rat pellet diet and water ad libitum. The procedures were performed according to the guidelines on the use of animals and approved by the Institutional Animal Ethical Committee.Thirty six male P. tuber-regium (50 – 5000 mg/kg b.w.) were administered orally to male rats. These animals were observed daily for toxicological manifestations like behavioral changes, neural and autonomic toxicities, feeding pattern changes, etc. There was no mortality recorded during this period even up to the dose of 5000 mg/kg  at a dose of 10 ml/kg weekly in addition to standard feed and water.Group II – Positive control received CClP. tuber-regium in feed (33.3% w/w) along with 10 ml/kg CCl4 (30% in olive oil) weekly.Group III – rats were treated orally with 100 mg/kg b.w. of P. tuber-regium in feed (33.3% w/w) along with 10 ml/kg CCl4 (30% in olive oil) weekly.Group IV – rats were treated orally with 200 mg/kg b.w. of P. tuber-regium in feed (33.3% w/w) along with 10 ml/kg CCl4 (30% in olive oil) weekly.Group V – rats were treated orally with 500 mg/kg b.w. of P. tuber-regium in feed (33.3% w/w) only with standard feed and water.Group VI – rats were treated orally with 500 mg/kg b.w. of Treatments continued for 4 weeks. Blood was collected by retero orbital sinus puncture and serum was separated by centrifugation at 3000 r.p.m. Rats were sacrificed under ether anesthesia; the testis was excised, weighed, rinsed clean in saline, weighed and preserved in 10% formalin for histopathological study. Sperm was harvested from the caudal epididymis and mounted on a slide to determine sperm motility at 40× magnification. The motility assessment was expressed as percentage motile forms. The epididymal filtrate was then mixed in equal volume with eosin-nigrosin stain and a smear made of it was used for epididymal sperm vitality . The cauSerum lipid peroxidation was quantified as malondialdehyde (MDA) according to the method described by Serum levels of testosterone, Luteinizing hormone (LH), Follicle stimulating hormone (FSH), prolactin and oestradiol were estimated using enzyme-linked immunosorbent assay (ELISA) kits , according to the manufacturer’s instruction.p < 0.05 were considered significant. Graph pad prism 5.0 software was used for statistical analysis.Data were expressed as mean ± SD of the number of animals used in each group of the experiment. One way analysis of variance (ANOVA) was used to analyze the difference among the groups followed by Bonferroni’s posttest. Values of Table 1 shows the effect of P. tuber-regium on the testis and body weight CCl4 treated rats administration and treatment on the body weight of rats. Carbon tetrachloride significantly decreased the body weight [from 221 ± 2.24 (control) to 193 ± 1.87 g (CCl4 treated groups)] and increased the absolute (from 1.73 ± 0.40 (control) to 2.74 ± 0.12 g CCl4 group and relative weight of the testis [0.91 ± 0.20 (control) to 1.40 ± 0.02 CCl4 treated group p < 0.01]. Treatment with 100, 200, and 500 mg P. tuber-regium gave significant and dose-dependent reversal in the body weight and testis weight in CCl4 only treated group.P. tuber-regium on sperm motility, epididymal count, vitality and (MDA of CCl4 treated rats is shown on Table 2. Carbon tetrachloride significantly reduced the sperm motility (54.33 ± 3.79%), epididymal count  when compared with control (89.33 ± 9.01), 91.91 ± 1.92 × 106/ml and 13.12 ± 0.19, respectively. There was significant increase in the sperm motility, epididymal count and vitality following P. tuber-regium administration. The reversal effect of P. tuber-regium on these seminal parameters were dose-dependent. There was significant difference between the MDA level (14.00 ± 3.50) in the CCl4 treated rats and the untreated control (1.40 ± 0.32 μg/mg protein). Following the co-administration of 100, 200, and 500 mg P. tuber-regium plus CCl4 there was significant reduction in the level of the MDA  in dose-dependent manner.The effect of Table 3 shows the effect of P. tuber-regium on LH, FSH, Testosterone, Prolactin, and Estradiol of CCl4 treated rats. Administration of carbon tetrachloride significantly decreased the levels of LH (0.88 ± 0.14), FSH (2.04 ± 0.33), and Testosterone (2.02 ± 0.24) compared to control 2.74 ± 0.32, 3.64 ± 0.62, and 4.16 ± 0.23, respectively. The CCl4, however, increased significantly the levels of prolactin from 1.06 ± 0.21 to 3.58 ± 0.48 and oestradiol from 0.81 ± 0.10 (control) to 2.89 ± 0.18 in the CCl4 treated group. There was dose-dependent reversal of the effects of CCl4 on these hormonal parameters in the P. tuber-regium treated groups.Figure 1 present the histological changes in the CCl4 exposed animals. Administration of CCl4 to the animals lead to degeneration of the seminiferous tubules with different degrees of sperm cell arrest when compared to control. However, treatments with P. tuber-regium lead to improvement in sperm cell population while the 500 mg P. tuber-regium only group had there seminiferous tubules and sperm cell population similar to control.P. tuber-regium on carbon tetrachloride induced testicular injury in animal model. After hepatic handling CCl4 generates chloride and trichloromethyl radicals CCl3, which reacts with oxygen to produce CCl3 O2. CCl3 bind to fatty acids to generate alkoxy and peroxy radicals , which will phosphorylate proteins. The phosphorylated proteins will further phosphorylate other proteins or induce new protein synthesis, i.e., steroidogenic acute regulatory protein . The funn levels , prolactn levels , or by rn levels  and has Histological examination suggests the consequences of oxidative stress in spermatogenesis with sperm cell maturation arrest. The result conform with the sperm parameter result of decreased sperm count since maturation arrest will ultimately decrease the number of sperm reaching the epididymis .P. tuber-regium may protect the testis from CCl4 induced damage as evidenced by the by the ameliorative effects on the epididymal sperm count, motility, viability, sex hormones, MDA, and testicular histomorphology. Since biochemical hypogonadism is considered a main feature of male infertility, dietary supplementation with P. tuber-regium with its significant reversal of all the deleterious testicular effects in CCl4 treated rats may hold some promise in the management of involuntary childlessness amongst married couples.In this study has shown that KO: Designed study, carried out the bench work and analysed data, IS: Design, and OO: Designed the study, analysed data and write up.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.