Podocyte contributes to the integrity of glomerular filtration barrier whose injury plays an important role in the initiation and progression of DN. Our study aimed to investigate the effect of Qiwei granules on podocyte lesion in diabetic KK-Ay mice were randomly divided in vehicle group and Qiwei granules group, while C57BL/6J mice were used as normal control. The mice were gavage with 1.37 g/kg/day Qiwei granules or water for 10 weeks. We measured water, food intake and body weight (BW) and fasting blood glucose (FBG) every 2 weeks, and urine protein every 4 weeks. At the end of the experiment, all surviving mice were sacrificed. The kidney weight and serum renal parameters were measured, and the renal morphology was observed. To search the underlying mechanism, we examined the podocyte positive marker, slit diaphragm protein expression and some involved cell signal pathway.Twelve-week-old male KK-Ay mice. In addition, the glomerular injuries and podocyte lesions were mitigated with Qiwei granules treatment. Furthermore, Qiwei granules increased expression of nephrin, CD2AP, and integrin alpha3beta1 in the podocytes of KK-Ay mice. Qiwei granules improved the phosphoration of Akt and inhibited cleaved caspase-3 protein expression.Qiwei granules treatment significantly improved the metabolic parameters, alleviated the urinary protein, and protected renal function in KK-Ay mice.These finding suggest that Qiwei granules protects the podocyte from the development of DN via improving slit diaphragm (SD) molecules expression and likely activating Akt signaling pathway in KK-A Diabetic nephropathy (DN), one of most common and serious complication of diabetes, is a major cause of end-stage renal disease (ESRD) . DiabetiChinese medicine comprised of all natural products is widespread used in the treatment of diabetes and its complications, which have been proved to alleviate the progression of diabetic nephropathy . Qiwei gy diabetes mice by TGF-β1/Smads signaling [It has been reported that Astragalus membranaceus, and Euonymus alatus both protects diabetic nephropathy kidney in animals ,16, Rehmignaling . But they mouse was chosen as a diabetic nephropathy model in our study. KK-Ay mouse is established by transfection of the yellow obese gene (Ay) into moderate KK mouse [y mouse is spontaneously developed and characterized of T2 Diabetes such as obesity, hyperglycemia, insulin resistance which is widely used as a T2D model [y mouse over 12 weeks age appears albuminuria, and diffuse mesangial matrix expansion, segmental glomerulosclerosis [y mice are considered as good diabetic nephropathy models renal lesions of which are similar with those of human.KK-AKK mouse . KK-Ay m2D model . Furtherclerosis . Therefoy diabetic mice kidneys.In the present study, we investigate whether Qiwei granules protect the podocyte and the underlying mechanism in the KK-AQiwei granules were prepared by Shanxi JinYu Pharmaceutical Company., Ltd. Qiwei granules consists of Astragalus membranaceus, Rehmannia glutinosa, Prunella vulgaris, Curcuma zedoaria, Euonymus alatus, Panax pseudoginseng, and Rheum officinale.The antibodies against integrin beta1 (integrin β1), pAkt (Ser473), Akt, cleaved caspase-3 and caspase-3 were purchased from Cell Signaling Technology, Inc. . Nephrin and integrin alpha3 (integrin α3) antibodies were purchased from R&D Systems, Inc. . CD2 associated protein antibody (CD2AP) was purchased from Abcam, Inc. . In-situ cell death detection kit POD was purchased from Roche Diagnositcs . Wilms tumor −1 antibody (WT-1) was purchased from Santa Cruz Biotechnology, Inc. .y mice and C57BL/6J mice were purchased from institute of Laboratory Animal Science of Chinese Academy of Medical Sciences . All mice were housed in same controlled conditions under temperature of 25 ± 1°C and humidity of 55% ± 5% on a regular 12 h light/dark cycle with free access to food and water. The KK-Ay mice were fed with high-fat diets , and C57BL/6J mice were fed with normal diets. After acclimating to new surroundings for 4 weeks, KK-Ay mice were randomly divided into two groups of 6 mice each, Qiwei granules group and vehicle group, while C57BL/6J mice (n = 6) were used as normal control group. The mice were gavage with 1.37 g/kg/day Qiwei granules (Qiwei granules group) or same volume water  as administration. Water, food intake and body weight (BW) of mice and fasting blood glucose (FBG) levels were measured every 2 weeks. All mice were placed into metabolic cages for urine collection at 0, 4, 8 weeks of treatment. After 10 weeks of treatment, all surviving mice were fasted for 12 h and sacrificed. The kidney were removed, cleaned for measurement of right kidney weight, and promptly frozen in liquid nitrogen. And portions were appropriated in 4% paraformaldehyde for histological analysis and in 2.5% glutaraldehyde solution for electron microscopic examination.Eight-week-old male KK-AThe study was approved by the Ethical Committee of Beijing University of Chinese Medicine, which was carried out in accordance with the Principles of Laboratory Animal Care published by National Institutes of Health.The blood was collected and sera were prepared by centrifugation, and measured by Beckman coulter. Serum urine nitrogen (BUN) and serum creatinine (Scr) were measured using the corresponding commercial kit . 24 h urinary samples were collected from mice in metabolic cages without restriction of water intake at the 0, 4, 8 weeks of treatment. After collection, urine volume was recorded and determined of 24 h urinary protein by Bradford method.3 samples of renal cortex were also fixed in 2.5% glutaraldehyde solution, rinsed in phosphate buffer, fixed in osmic acid and embedded in epon. Semithin 1 μm sections were cut and stained with uranyl acetate and then lead in nitrate solution. Representative glomeruli from 5 mice, each from untreated KK-Ay mice, KK-Ay mice treated with Qiwei granules, and normal control mice were examined. Images were acquired on a JEOL JEM-2100 transmission electron microscope .Parts of kidney tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. Under light microscopy, 2-3 μm sections were prepared and stained with hematoxylin and eosin (HE), Masson’s Trichrome, and periodic acid-Schiff (PAS). For PAS stain, the mesangial matrix expansion was evaluated in 10 randomly selected glomeruli in the cortex per animal under high magnification (×400) with Image-Pro Plus 6.0 . The area of fibrosis was investigated by measuring the collagen deposition staining by Masson’s Trichrome under × 200 magnification using Image-Pro Plus 6.0 software. 1 mmTUNEL was performed with the In-situ cell death detection kit POD to detect apoptotic positive cells. Staining for podocytes in renal tissues was performed using the protocol of polyclonal rabbit anti-mouse WT-1 antibody (1:200). Negative control for immunohistochemistry was run incubating with nonimmune serum instead of the primary antibody. For quantitative determination of podocyte numbers, the WT-1 positive cells were calculated in a total of 40 glomeruli area by Image J software. The averages of podocyte numbers per glomerulus were assessed from individual five mice each group.The kidney tissue was homogenized with ice-cold homogenized buffer containing 50 mM Tris–HCl (8.0), 150 mM NaCl, 0.025% NaN3, 0.1% SDS, 0.1 mg/ml PMSF, 0.01 mg/ml Aprotintin, 0.5% Na deoxycholate, and 0.1%Nonidet-P40. After incubation on ice for 20 min, lysates were centrifuged at 10000 rpm for 10 min and supernatants were isolated. Proteins were extracted by boiling in 0.5 mmol/ml Tris–HCl, PH 6.8, 10% SDS, 20% glycerol, 0.05% bromophenol blue, and 2-mercaptethanol. The proteins (40 μg/sample) were electrophoresed on a 7.5%-12.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) at 100 V for 2 h and transferred onto polyvinylidene fluoride (PVDF) membranes  at 30 mA for 2 h. The membrane was blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h, and incubated with the primary antibody nephrin (1:1000), CD2AP (1:2000), integrin α3 (1:1000), integrin β1 (1:500), pAkt (1:1000), Akt (1:1000), cleaved caspase-3 (1:1000) and caspase-3 (1:1000) overnight. After washing with TBST and incubated for 1 h with horseradish peroxidase-linked anti-rabbit and anti-mouse secondary antibodies . Detection was achieved using ECL kit . GAPDH was used as an internal control. The density of the bands was measured using IPP.Total RNA was extracted from the kidney using Trizol kit . Then RNA (1 μg) from each sample was reverse-transcribed to cDNA using the M-MLV RT Kit, according to the manufacturer’s instructions (Takara). THUNDERBIRD SYBR qPCR Mix was used for quantitative real-time RT-PCR analysis of each gene’s expression. The primers are listed as follow: nephrin forward, 5′- TCTGGCGGAGAAGACTGAG -3′; nephrin reverse, 5′- GTGCTAACCGTGGAGCTTCT -3′; integrin α3 forward, 5′- CTGGAGTGGCCCTATGAAGT -3′; integrin α3 reverse, 5′- TGACTCCAGGGTCAGAGAGA -3′; integrin β1 forward, 5′- CTTGCTGCTGATTTGGAAAC -3′; integrin β1 reverse, 5′- CTTCGGATTGACCACAGTTG -3′; GAPDH forward, 5′- GCAAGTTCAACGGCACAG -3′; GAPDH reverse, 5′- CGCCAGTAGACTCCACGAC -3′. Amplification was performed with a real-time PCR system (ABI Prism 7500). The amplification was performed as follows: 94°C for 15 min followed by 50 cycles of 94°C for 15 s, 60°C for 60 s, and 72°C for 10 min with a real-time PCR system (ABI Prism 7500). The data are expresses as a relative value after normalization to the GAPDH expression.Data are expressed as mean ± SEM and statistically analyzed by Dunnett test. Differences were considered significant for P < 0.05.y mice and Qiwei granules treated KK-Ay mice  at 12 weeks of age, and confirmed no significant differences between vehicle KK-Ay mice glomeruli were diffuse mesangial matrix expansion and capillary wall thickening  which contain a transmembrane region and intracellular region. So we investigated both the mRNA and protein expression of integrin α3β1 in KK-Ae Figure C, D. In e Figure A, C, D. y mice was significantly lower compared with normal mice, while the expression of total Akt was no difference between vehicle diabetic mice and normal mice. With Qiwei granules treatment, the expression of pAkt/Akt in KK-Ay mice was upregulated compared with vehicle KK-Ay mice. Further we examined the protein level of caspase-3 cleavage, the key downstream effector of apoptosis. The cleaved caspase-3 expression of vehicle diabetic mice was obviously higher than normal mice. Exposure to Qiwei granules resulted in decrease of cleaved caspase-3 expression in KK-Ay mice kindey. Thus, Qiwei granules improved the phosphorylation of Akt and inhibited cleavage of caspase-3.It has been shown that PI3K/AKT pathway related with SD molecules (SDs) is one key part of podocyte cell survival signaling to maintain podocyte functional integrity . Our stuy mice. In addition, the mitigation of podocyte lesion with Qiwei granules treatment was further demonstrated, following by the increases of SD molecules expression and likely activating Akt signaling pathway.Due to the constant increase in the incidence of type 2 diabetes mellitus, diabetic nephropathy has become the highly prevalent cause of chronic kidney disease worldwide. In China traditional Chinese medicines have been used for the diabetes mellitus and its complications for thousand of years. Chinese medicines have been attracted worldwide attention to become a new therapy for diabetic nephropathy. Qiwei granule, one of such traditional Chinese medicines, has been comprised of 6 Chinese herbs. In clinic study, Qiwei granule was shown to decrease blood glucose, improve hyperlipidemia, and reduce the proteinuria of DN patients . The prey mice. In addition, podocyte lesion demonstrated as foot process effacement and podocyte loss was found in KK-Ay mice. The treatment of Qiwei granules both reversed the glomerular and podocyte injuries.The classical hallmarks of diabetic nephropathy include glomerular hypertrophy, expansion of mesangial matrix and thickness of GBM . Recently mice compared with normal mice, whereas Qiwei granules treatment restored the SDs expressions. In combination, these results indicated that Qiwei granules treatment increased the expression levels of nephrin, CD2AP, and integrin α3β1 to protect from podocyte injury in diabetic KK-Ay mice.Slit diaphragm molecules between podocytes composed of multiple protein complexes contribute to the glomerular filtration barrier. Derangement of SDs results in proteinuria. Nephrin is a major transmembrane protein located in the glomerular SD region. The altered expression of nephrin has been observed in the initiation of proteinuria and linked to the development of proteinuria in DN ,32. CD2Ay mice. Administration of Qiwei granules improved the phosphorylation of Akt and increased the expression of SDs contributing to preservation of podocyte. Caspase-3 is a critical executioner of apoptosis and operates as the key effector enzyme in cell death. Activation of caspase-3 plays an important role in apoptosis. In accordance with previous studies [y mice. Qiwei granules reduced the overexpression of cleaved caspase-3 in diabetic mice possibly leading to inhibit apoptosis. These results indicated that Qiwei granules maybe affect podocyte apoptosis depending on Akt pathway and caspase-3.Apoptosis, one of the considered causes of podocyte loss results in the development of albuminuria in diabetic nephropathy. Akt is a serine/threonine protein kinase that plays a critical role in cell survival by inhibiting the pro-apoptotic signals. Previous studies show that nephrin and CD2AP stimulate Akt signaling pathway and thereby reduce podocyte apoptosis to protect podocyte in glomerular ,35. Our  studies ,37, our Previous studies are reported that high glucose induces podocyte apoptosis and reduction of SDs expressions contributing to podocyte injury . In the y mice. Qiwei granules treatment significantly improved the expression of SD molecules nephrin, CD2AP, integrin α3β1. Furthermore, it appears to activate Akt signaling pathway and inhibit the apoptosis in the kidney. It alleviated the podocyte lesion and glomerular pathologic changes, which resulted in the decrease of albuminuria and protection of renal function. Our study suggests that Qiwei granules ameliorates podocyte lesion to prevent progression of DN in KK-Ay mice.In conclusion, the Chinese formulation Qiwei granules protects podocyte in the kidney of diabetic KK-A


Complete nucleotide sequence showed that Φ1207.3 is 52,491 bp in length and contained 58 open reading frames (ORFs). A manual homology-based annotation with functional prediction of the hypothetical gene product was possible only for 34 out of 58 ORFs. Φ1207.3 codes for two different C-methylation systems, several phage structural genes, a lysis cassette (composed by a holin and a peptidoglycan hydrolase), and three site-specific resolvases of the serine recombinase family.Genetic element Φ1207.3 (formerly Tn Streptococcus pyogenes, the mef (A)/msr(D) pair of genes encoding eﬄux resistance to 14- and 15-membered macrolides is carried by a mobile genetic element originally described as a conjugative transposon which was called Tn1207.3 /msr(D) in S. pneumoniae  following essentially the protocol suggested by the manufacturer. Briefly, the 25-μl reaction mixture was in 1X LA PCR Buffer II Taq buffer and contained: (i) 2.5 mM MgCl2, (ii) 200 μM dNTPs, (iii) 10 pmol of each primer, (iv) 0.25 units of Takara LA Taq, (v) 1 μl of liquid bacterial culture. Thermal cycling profile was as follows: 1 cycle at 92∘C for 2 min, then 30 cycles at 50∘C for 10 s, 68∘C for 15 min, 92∘C for 10 s, and 1 cycle at 50∘C for 1 min and 68∘C for 20 min. A primer walking approach . Regions with different GC content can be could be identified in the DNA sequence of Φ1207.3: (i) the left end, containing ORFs 1–11, with 36.4% GC; (ii) ORFs 12–16 with 31.6% GC; (iii) ORFs 17–54 with 41,4% GC; (iv) the right end, corresponding to ORFs 55–58, with 37,8% GC . Comparison of Φ1207.3 with sequences present in public databases showed that: (i) the first 7,244 bp of the Φ1207.3 sequence are identical to the 7,244-bp element Tn1207.1 of S. pneumoniae , S. suis JS14 (GenBank no. CP002465), and S. agalactiae A909 (GenBank no. CP000114).The complete nucleotide sequence of Φ1207.3 was obtained by primer walking on four long PCR fragments spanning the whole element. Inverse PCR on genomic DNA of equences . Amplicoeumoniae , 2003;  Se Φm46.1 ; (iv) hoTable 1). Public protein databases and the Pfam protein family database were used for Blast searches of predicted gene products, taking into account significant homologies with functionally characterized proteins or good matches with Pfam domains. A putative ribosome binding sequence preceded all ORFs except orf36 and orf37. The alternative start codon TTG was present in orf34, orf36, orf43, and orf56, whereas orf37, orf38, and orf51 started with GTG. In the left arm of the element, the gene product of orf2 was predicted to be a site-specific resolvase of the serine recombinase family, mef(A) (orf4) and msr(D) (orf5) encoded respectively the transmembrane domains and the ATP-binding domains of the ABC transporter responsible for the M-type resistance to macrolides , orf30 (endonuclease), orf33 (cytosine-specific DNA methylase), while orf29 gene product presented an S-adenosylmethionine synthetase domain which may act as a methyl group donor for Orf33. In the central region, orf17, orf23, and orf25 encoded a DNA polymerase, a DNA primase, and a DNA helicase, possibly involved in phage DNA replication, whereas orf38 and orf39 encoded for the small and large subunit of the phage terminase, which, together with the portal protein encoded by orf40, could be involved in phage DNA packaging. orf42, orf44, orf45, and orf50 code for putative structural phage proteins. A putative lysis cassette, which is typically composed by a holin and an endolysin (hol (orf53) and skl (orf54). At the right end of the element, orf56 and orf57 gene products were homologous to resolvases of the serine recombinase family, possibly involved in excision, circularization, and site specific integration of Φ1207.3, whereas orf58 encoded a putative ADP-ribosyltransferase toxin.For 34 out of 58 ORFs it was possible a manual homology-based annotation with functional prediction of the hypothetical gene product  Φ10394.4 , integrated at the same chromosomal site of Φ1207.3  shows homology to two  Φ1207.3 ; (ii) Φmnsferase . The whoS. pyogenes prophages present in sequenced genomes have a tyrosine recombinase (integrase) as the recombination module (mec elements (types I to IV) of Staphylococcus aureus  by a mechanism which fits the operational definition of conjugation (Since Φ1207.3 can move among streptococcal species (jugation , this cojugation , could cThe 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.


Drosophila, ventral nerve cord (VNC) occupies most of the larval central nervous system (CNS). However, there is little literature elaborating upon the specific types and growth of neurites as defined by their structural appearance in Drosophila larval VNC neuropil. Here we report the ultrastructural development of different types VNC neurites in ten selected time points in embryonic and larval stages utilizing transmission electron microscopy. There are four types of axonal neurites as classified by the type of vesicular content: clear vesicle (CV) neurites have clear vesicles and some T-bar structures; Dense-core vesicle (DV) neurites have dense-core vesicles and without T-bar structures; Mixed vesicle (MV) neurites have mixed vesicles and some T-bar structures; Large vesicle (LV) neurites are dominated by large, translucent spherical vesicles but rarely display T-bar structures. We found dramatic remodeling in CV neurites which can be divided into five developmental phases. The neurite is vacuolated in primary (P) phase, they have mitochondria, microtubules or big dark vesicles in the second (S) phase, and they contain immature synaptic features in the third (T) phase. The subsequent bifurcate (B) phase appears to undergo major remodeling with the appearance of the bifurcation or dendritic growth. In the final mature (M) phase, high density of commensurate synaptic vesicles are distributed around T-bar structures. There are four kinds of morphological elaboration of the CVI neurite sub-types. First, new neurite produces at the end of axon. Second, new neurite bubbles along the axon. Third, the preexisting neurite buds and develops into several neurites. The last, the bundled axons form irregularly shape neurites. Most CVI neurites in M phase have about 1.5–3 µm diameter, they could be suitable to analyze their morphology and subcellular localization of specific proteins by light microscopy, and they could serve as a potential model in CNS in vivo development.In  Neurites are swollen projections that form from neuronal somata, dendrites or axons. Axonal neurites are often characterized with synapses, synaptic vesicles, microtubules, and mitochondria in TEM. Synapses, located in the free surface of an axonal neurite, are communication connections between axonal neurites and target cells, the main site of nerve signal processing and neural information transmission, and a bridge the neural network circuit. Neurites undergo several dynamic processes, such as neurite growth, extension, retraction, and branching, which are regulated by many molecules Drosophila NMJ, type I boutons are repeatedly wrapped by the subsynaptic reticulum (SSR) that is formed by the muscle cell membrane There are, however, notable differences between mammalian and insect neurite morphology. In the Drosophila. Adult Drosophila NMJs are classified by two distinct types of synaptic boutons (types I and II). Type II boutons are small (0.5–1.5 µm in diameter) and contain octopamine. Type I boutons are densely packed with clear synaptic vesicles, are comparatively larger (the diameters 0.8–5.5 µm), and contain glutamate Drosophila NMJs are classified by three types synaptic boutons  according to the size, characteristics of SSR, and compositions of synaptic vesicles. Larval type I boutons are divided into type-Ib and type-Is. The type-Ib boutons, characterized by a thick SSR, are the largest, with diameters of 3–5 µm Depending on different classification criteria, the NMJ boutons and brain boutons can be divided into different types in Drosophila mushroom body calyx, there are three morphological types of PN boutons according to the synaptic vesicle composition . CCV-PNs (clear-core vesicle-projection neurons) have exclusively clear-core synaptic vesicles, while DCVPNs (dense-core vesicle-projection neurons) have mixed clear- and dense-core vesicles. DB-PNs (dark bouton-projection neurons) have a dark cytoplasm, with both clear- and dense-core vesicles Drosophila first instar larval neuropils of brain and VNC: they are termed globular, varicose, axiform, and dendritiform, according to characteristics of their appearance and synapse  DrosophilaDrosophila VNC plays an important physiological role.In adult Drosophila development, the number of boutons increases 10-fold With Drosophila CNS. Here, we analyzed the morphological classification and growth of Drosophila VNC axon neurite in ten periods from embryo to larvae, and identified four types of axon neurites, five developmental phases of the CV neurite, four kinds of growth manners of CVI neurites in TEM.However, little literature has reported the fine ultrastructure and development of the complex, seemingly disorderly neurites in Drosophila, VNC occupied most of CNS in larvae stages  neurite, dense-core vesicles (DV) neurite, mixed vesicles (MV) neurite and large vesicles (LV) neurite, according to the size and inclusions of vesicles.Neurites were divided into dendrite neurites and axon neurites in I and CVII, according to color and appearance of neurite. CVI neurites were numerous and widely distributed throughout the neuropil. They were light-colored and had synaptic vesicles with a diameter of 32.87 nm±3.9 , but DVII neurite vesicles were bigger, measuring 97.29 nm±24.0  in L3w period . LV neurites formed in L1–2 period, and we could observe the huge vesicles characteristic of LV neurites during the L1–12 period  dramatically changed in size , 70.2±19.9 at L1–12 period , and 65.1±15.5 at L1–22 period . However, there were no statistically significant differences among the big dark synaptic vesicles in S phase , 56.8±11.8 at L1–12 period , and 61.2±11.8 at L1–22 period . However, the big dark synaptic vesicles had a statistically significant difference between the L1–2 period and L1–12 period, but no statistically significant difference between L1–12 period and L1–22 period in T phase , 55.8 nm±8.2 at L1–12 period , 55.8 nm±7.6 at L1–22 period , 53.8 nm±7.3 at L2–2 period , 53.0 nm±7.3 at L2–12 period , 55.0 nm±6.8 at L2–22 period , 55.7 nm±8.4 at L3–2 period , 55.2 nm±9.8 at L3–12 period  and 56.3 nm±8.0 at L3w period . However, there were no statistically significant differences among the contiguous developmental periods  in the whole instar larval stage  in S phase, 60.1 nm±14.3  in T phase, and 54.8 nm±8.3  in B phase. Moreover, the big dark synaptic vesicles had no statistically significant differences between S phase and T phase, but had statistically significant differences between S phase and B phase, or T phase and B phase in the first instar larval stage , and decreased sharply during L1–12 period (9.8±3.1/mm2), then remained at a low level during L1–22 period (5.9±2.9/mm2) and the following periods (Data not shown). The density of CV neurites in S phase (2) and L1–12 period (286.1±8.3/mm2) to L1–22 period (179.3±12.8/mm2). The density of CV neurites in T phase (2) to L1–12 period (172.9±10.8/mm2), but decreased from L1–12 period to L1–22 period (134.1±10.3/mm2). The density in B phase (2) and L1–12 period (122.1±9.8/mm2) to L1–22 period (184.6±8.2/mm2). The density of total CV neurites including P, S, T, B phase (2) and L1–12 period (589.9±19.0/mm2) to L1–22 period (505.0±13.0/mm2).The amount of CV neurites in P, S, T and B phases varied dynamically during larval development. The appearance and length were not different in the first instar larvae , and the S phase  decrease T phase  increase B phase  increase B phase  decreaseThe frequency  and densI neurites were produced constantly throughout the larval stage. We could find the P phase at L1–2 period  Drosophila larval NMJs contain three types of synaptic boutons  0 boutons, DV boutons, and MV boutons According to various criteria, the neurites in Drosophila VNC neuropil according to their vesicle characteristics: clear vesicle (CV) neurite, dense-core vesicle (DV) neurite, mixed vesicle (MV) neurite, and large vesicle (LV) neurite. The morphological characteristics of the CV neurite, the DV neurite, and the MV neurite in VNC are very similar to the CV bouton, DV bouton, and MV bouton in NMJ. During the L3w period, the CV neurites  that are an important basis for the classification of type I, II, and III boutons in the NMJ, so we used the classification criteria described by Jia neurites ; Fig. 7Jneurites  and NMJ neurites  looks vew period . In compendritic , and are boutons , but witI neurites  and size neurites  and DVIIneurites  accordinI neurites  have a lot of small commensurate synaptic vesicles that cluster around the T-bar structure. The shape of CVII neurites remained bifurcated or dendritic in M phase  cycle. For the embryonic analyses, the flies of 6–9 days post-eclosion were gathered on the yeasted apple juice agar plates in dark, 20 minutes later, the eggs were collected, and the embryonic stages were given according to Campos-Ortega and Hartenstein The wile-type flies were 2O, and stained in 2% aqueous uranyl acetate for 2 hr. The specimens were dehydrated in an ethanol series, passed through propylene oxide two times, and embedded into a sheet about 1–2 mm in Epon812 (SPI Science) to allow the image of VNC to be captured with the Olympus microscope. Ultra-thin sections (90 nm thick) that were cut from the VNC cross-section of the abdominal segments 2–4 with Leica UC6 using a diamond knife. Grids were post-stained with 2% saturated uranyl acetate in 50% ethanol and 1% lead citrate (PH 12), examined with H-7650 electron microscope and recorded with a Ganton 830 digital CCD. More than 3 animals for each developmental period were analyzed. The electron microscope procedure of NMJ sample: See accompanying article by Atwood et al. Embryos were prepared for TEM using standard techniques I neurite were collected from L1–2 period, L1–12 period, and L1–22 period. The big dark synaptic vesicles in B phase were collected throughout the larval stages. The counted clear vesicles were less than 45 nm in L3w period, and the counted big dark vesicles were more than 45 nm in S phase, T phase and B phase. For statistic analysis of CV neurites, the images were collected by electron microscope at 12000× magnification in the first instar larvae. The data were measured using Image J (NIH), and the data were analysized using GraphPad Prism.The synaptic vesicles for measuring the diameter of CV neurite, DV neurite, MV neurite, and LV neurite were collected from L3w period. The big dark synaptic vesicles in S phase and T phase for measuring the diameter of the CV


This work investigated the effects of Vitamin E (VE) on aberrant crypt foci (ACF) incidence, oxidative stress parameters , and malondialdehyde (MDA) levels), and expression of both cyclooxygenase-2 (COX2) and proliferating cellular nuclear antigen (PCNA) in experimental colorectal carcinogenesis. Male Wistar rats received subcutaneous injections of 1,2-dimethylhydrazine (DMH) twice a week, for two weeks (40 mg/kg), except for the Control group. Animals were separated into groups that received different amounts of VE in the diet: 0 IU (0×), 75 IU , 225 IU (3× RDI), or 1500 IU (20× RDI), during (dDMH) or after (aDMH) administration of carcinogen. The 0×dDMH and 3×dDMH groups showed decreased serum VE levels. Hepatic VE concentration was higher in 3×aDMH as compared with the other groups. All the groups, except the Control and the 0×aDMH groups, had reduced GSH levels. The 0×dDMH, 0×aDMH, and 20×aDMH groups exhibited increased MDA levels. The aDMH groups had higher ACF incidence and PCNA expression. The 0×aDMH group presented higher ACF rate, followed by 20×aDMH. Moreover, the 3×aDMH group displayed reduced ACF incidence and COX2 expression. Multivariate analysis revealed that GSH modulated homocysteine levels and COX2. These results suggested that 1500 IU of VE is hazardous, whereas 225 IU of VE has beneficial effects on chemical colorectal carcinogenesis. Colorectal cancer (CRC) is the third most common cancer worldwide. It is estimated that over 1.2 million new CRC cases emerged and 608,700 deaths occurred due to this condition in 2008. Australia, New Zealand, Europe, and North America have the highest incidence rates [Diet has been the most studied etiological factor in colorectal carcinogenesis and may account for approximately 50% of the attributable risk in countries with high susceptibility, such as the USA, England, and Australia .Vitamins and minerals have been suggested to influence the risk of developing CRC. Approximately 12% of colon cancer cases have been attributed to the Western-style diet . On the CRC development is a multistage process that involves a number of pathological alterations, from discrete microscopic lesions in the mucosa, such as aberrant crypt foci (ACF), to malignant tumors . ACF areIncreased cell proliferation is one of the mechanisms that contribute to malignant transformation, so it may serve as a pre-malignant lesion marker . Rapid cCyclooxygenases (COX) are key enzymes in prostaglandin synthesis. Overproduction of the isoform type 2, COX2, occurs in multiple stages of colon carcinogenesis . COX2 exReactive oxygen species production occurs under basal conditions, which requires continuous deactivation of these compounds. Increased prooxidant synthesis causes oxidative stress and induces damage in biological molecules. Indeed, these processes have been implicated in a variety of degenerative pathologies, including cancer .dl-Alpha-Tocopheryl Acetate (ATA) supplementation  reduces oxidative stress in healthy adults [VE is a potent inhibitor of human cancer COX2 activity, cell cycle progression, and proliferation ; it may y adults . Hence, This study was approved by the Ethics Committee for Animal Experimentation of Ribeirão Preto Medical School, University of São Paulo, in May 2007, under protocol number 051/2007.The 80 male Wistar rats used in this work weighed approximately 300 g. The Central Animal Facility of the Ribeirão Preto Medical School, University of São Paulo supplied the animals. The rats were housed in individual stainless steel cages, at 25 °C, in a 12 h light/dark cycle, with ad libitum water intake. The chow was replaced three times a week, and consumption was measured throughout the experiment.After acclimation for one week, the rats were randomly separated into eight groups of 10 rats each. All the groups received four doses of DMH. The exception was the Control group, which received saline injections see .®  tubes and centrifuged at 2500 rpm for 10 min; serum samples were stored at −70 °C for later analysis. The large bowel was longitudinally excised in the mesenteric border throughout its full extension. The distal colon and rectum were fixed in 10% buffered formalin and embedded in paraffin using methods appropriate for the analysis. For histological (H & E stain) examination of the ACF, samples were embedded in face and sliced (5-μm sections), providing a view of the transversally cut of colonic crypts [Animals were euthanized by decapitation 30 days after the fourth DMH injection. Blood was collected in Vacutainerc crypts . For immSymmetrical dimethylhydrazine dihydrochloride  was dissolved in 1 mM EDTA (pH 7.0) immediately before use at a dose of 40 mg/kg of body weight. All the exposed groups received subcutaneous DMH injections twice a week, for two weeks.dl-Alpha-Tocopheryl Acetate (ATA) 50% powder  was included in the vitamin mix  [The diet was prepared according to AIN-93M recommendations  Table 1Table 1.  Brazil) . All the Brazil) ), 59.3 m Brazil) . Table 2control group: Standard AIN-93M diet (75 IU of VE) up to the end of the experiment (six weeks) and subcutaneous saline injection .Carcinogen group: Standard AIN-93M diet (75 IU of VE) up to the end of the experiment (six weeks) and subcutaneous DMH injection .0×dDMH group: AIN-93M diet, VE-free , during DMH injection.0×aDMH group: AIN-93M diet, VE-free , after DMH injection.3×dDMH group: AIN-93M diet, VE 3× RDI , during DMH injection.3×aDMH group: AIN-93M diet, VE 3× RDI , after DMH injection.20×dDMH group: AIN-93M diet, VE 20× RDI , during DMH injection.20×aDMH group: AIN-93M diet, VE 20× RDI , after DMH injection.Hepatic and serum levels of VE were measured by high-performance liquid chromatography . Briefly® . Briefly, 15 μL of plasma and 300 μL of working solution were homogenized in a tube. Each tube was covered and incubated for 30 min at 37 °C in a water bath. Then, the treated sample was transferred to a cuvette and analyzed.Plasma homocysteine concentrations were obtained by immunoassay using Immulite2000 kit2) was calculated [The colonic mucosa was screened to identify, quantify, and analyze the frequency and distribution of ACF, considered early biomarkers of carcinogenesis . After slculated  Figure  was calcTo estimate colonic cyclooxygenase-2 (COX2) expression, 4-μm slices of the paraffin-embedded colon were immunostained with an antibody against COX2, clone 4H12, dilution 1:200 . One hundred colonic crypts and surrounding connective tissue were observed in each colon. The results for COX2 index (iCOX2) were expressed as positive cell number per colonic crypt .To estimate epithelial cell proliferation, colonic mucosa was immunostained with an antibody against proliferating cell nuclear antigen, clone PC10, dilution 1:100 . One hundred colonic crypts nuclei were observed in each colon, and the PCNA index (iPCNA) was estimated as the ration positively marked nucleus/total nucleus ratio per crypt .p < 0.05 for all the analyses. The software programs SAS® 9  and Graphpad Prism 4.0  were used.Data are expressed as mean ± SEM. ANOVA test was used to determine significant differences among the groups, regardless of the Tukey’s Multiple Comparison Test for parametric analysis  or Kruskall-Wallis Test with Dunn for non-parametric analysis (PCNA-Li). A multivariate analysis was performed to evaluate the effect of: (1) MDA, GSH, hepatic VE, homocysteine, iPCNA, and COX2 on ACF; (2) MDA, GSH, hepatic VE, and COX2 on homocysteine; (3) MDA, GSH, and hepatic VE on COX2; and (4) MDA, hepatic VE, and homocysteine on GSH. The significance level was set at p < 0.05), 0×dDMH (p < 0.001), 0×aDMH (p < 0.01), and 3×aDMH (p < 0.001) groups. The 20×dDMH group gained less weight as compared with the 0×dDMH (p < 0.01) and 3×aDMH (p < 0.01) groups. The 3×aDMH group gained more weight as compared with the Carcinogen group (p < 0.05)  . Necropsp < 0.05). The 20×dDMH group presented increased VE concentration as compared with the 0×aDMH and 3×dDMH groups (p < 0.01) (p < 0.01) (Groups receiving VE-free (0×aDMH and 0×dDMH) diet and the 3×dDMH group presented reduced serum VE concentration. The 3×aDMH group had lower serum levels of VE than the Carcinogen group ( < 0.01) . Hepatic < 0.01) C.p < 0.05). Groups receiving 20×RDI VE  showed decreased GSH levels as compared with the Control group (p < 0.05)  .p < 0.05). The 0×dDMH group had lower serum homocysteine concentration than the 20×aDMH group (p < 0.05)  .p < 0.001), followed by the 20×aDMH group (p < 0.001). The 3×aDMH group presented the lowest incidence of ACF (p < 0.001) (Diet modification after exposure to carcinogen resulted in higher incidence of ACF. The 0×aDMH group had the highest number of ACF (< 0.001) D.p < 0.05). Compared with the Control group, supplementation in the 20×dDMH group elevated iPCNA (p < 0.001) (Administration of the carcinogen and diet modification after exposure to the carcinogen increased iPCNA expression (< 0.001) A.p < 0.01)  B.Multivariate analysis showed that increased hepatic GSH content reduced plasma homocysteine levels . The rise of one unit in GSH levels increased iCOX2 by 0.11 units . Increased hepatic MDA levels slightly raised GSH levels .The present study has been the first to investigate how VE (ATA) intake  during or after exposure to DMH affects chemical colorectal carcinogenesis, as analyzed by indirect  and direct (incidence of ACF) parameters. Because tissue and serum levels of alpha-tocopherol are higher than the levels of other tocopherols, we decided to use this isoform in the study. Another reason for our choice was the fact that alpha-tocopherol is the isoform that usually occurs in commercial supplements consumed by the general population without regular medical or nutritional monitoring, which could cause serious health impacts.This study showed that exposure to the carcinogen alone did not affect the serum and hepatic levels of VE. VE (ATA) supplementation at 3× RDI (225 IU) increased hepatic VE storage and reduced the serum values of this vitamin, suggesting the existence of tissue, serum, and peripheral control of VE. These findings agreed with previous studies, suggesting that VE needs protection from other antioxidant systems to prevent its depletion and loss of its regulatory properties. Lack of oxidative mechanisms compensation culminates in VE waste, and VE oxidation products arise .On the other hand, exposure to the carcinogen decreased the hepatic GSH content. GSH is the major regulator of oxidative stress; low GSH level has been associated with cancer pathogenesis . Becaused-alpha tocopherol supplementation in healthy adults does not benefit lipid peroxidation [in vitro studies have suggested that high alpha-tocopherol levels have a paradoxical role in LDL autoxidation, causing VE-mediated peroxidation [VE (ATA) depletion and 20× RDI supplementation (1500 IU) after exposure to carcinogen augmented hepatic MDA levels. Advanced lipid peroxidation end products, such as MDA, emerged as a consequence of VE insufficiency. These end products may impact cell homeostasis and modify gene expression . A recenxidation , which cxidation ,33.et al. (1980) [et al. (1985) [Diet modification after exposure to carcinogen greatly impacts the incidence of ACF. Here, the group receiving VE-free diet after DMH exposure (0×aDMH group) presented the highest number of ACF, followed by the 20×aDMH group. The 3×aDMH group had decreased incidence of ACF. Literature works have reported both inhibition of  and incr. (1980)  demonstr. (1985)  describeMost studies on colorectal adenoma have shown that VE exerts a protective effect, suggesting that it acts early in tumorigenesis . The preThe present work also found that administration of carcinogen and diet modification after exposure to carcinogen resulted in higher iPCNA expression. Tumorigenesis is a multistep process; one of the controversial mechanisms contributing to the malignant transformation is increased cell proliferation, considered a marker of pre-malignant lesions . Rapid cThe present study showed that the group receiving VE (ATA) supplementation at 3× RDI displayed reduced iCOX2 expression. COX2 overexpression occurs in a variety of malignant diseases and induces cell growth by stimulating proliferation and angiogenesis . In the Although the 3×dDMH group did not display lower incidence of ACF, our findings suggested that 225 IU of VE might have an anti-initiation and/or anti-promotion role in chemical colorectal carcinogenesis.The multivariate statistical analysis showed that elevated hepatic GSH levels increased iCOX2 and decreased plasma homocysteine and hepatic MDA levels. These findings indicated that oxidative stress has an important role in chemical colorectal carcinogenesis—it interferes with early carcinogenesis biomarkers, like COX2, which reinforces the need to maintain oxidative balance.2O2) [S-transferases, which are enzymes that aid detoxification of drugs and endogenous toxins underlying oxidative stress [The formation of reactive oxygen species is a normal consequence of a variety of essential biochemical reactions, and excess oxygen radicals can originate in chronic diseases of the gastrointestinal tract. With the progression of the disease, the production of these radicals increases, to culminate in enhanced lipid peroxidation as a result of cell membrane degeneration and DNA damage. Autoxidation can take place in the presence of high homocysteine concentrations, to produce highly detrimental and reactive hydrogen peroxide (H2O2) . Therefo2O2) . In turn2O2) , and its2O2) . As for 2O2) . In the e stress .This study showed that VE (ATA) acts in different pathways of colorectal carcinogenesis: it affects cell growth, inflammation, oxidative stress, and cell death. Gene expression regulation, cellular stress, and protein kinase C (PKC) activity through VE could be the mechanisms involved in this process. Some authors have shown that VE  can inhibit PKC activity ,49, moduIn the current study, we have employed different doses of DL-Alpha-Tocopheryl Acetate (ATA) at different times of exposure to carcinogen to investigate how Vitamin E affects the early stages of colon carcinogenesis. We have shown that oxidative stress participates in the process of cancer initiation and promotion. Diet modification after exposure to carcinogen increased PCNA and COX2 expression. Supplementation at 20× the recommended daily intake (1500IU) was as hazardous as the absence of Vitamin E (ATA), as judged from the increase in the incidence of ACF. Supplementation at three times the recommended daily intake lowered the incidence of ACF, cell proliferation, and COX2 expression, showing that supplementation with 225 IU has a protective role during the early stages of colorectal chemical carcinogenesis in rats.


N′-[(Z)-(phenyl)methyl]benzohydrazide (HL1), which was characterized by NMR spectroscopy and single-crystal X-ray structure study. X-ray diffraction analyses of the crystals revealed a nonplanar molecule, existing in the keto-amine form, with intermolecular hydrogen bonding forming a seven-membered ring system. The reaction of HL1 with Co(II), Ni(II), and Cu(II) halides gave the corresponding complexes, which were characterized by elemental analysis, molar conductance, magnetic measurements, and infrared and electronic spectral studies. The compounds were screened for their in vitro cytotoxic activity against HL-60 human promyelocytic leukemia cells and antimicrobial activity against some bacteria and yeasts. Results showed that the compounds are potent against HL-60 cells with the IC50 value ≤5 μM, while some of the compounds were active against few studied Gram-positive bacteria.Reaction of 1-phenyl-3-methyl-4-benzoyl-pyrazol-5-one and benzoyl hydrazide in refluxing ethanol gave  N′-[(Z)-(phenyl)methyl]benzohydrazide and its Co(II), Ni(II) and Cu(II) complexes, the in vitro antimicrobial activity against some bacteria and yeasts, and their antiproliferative effect on human promyelocytic leukemia cells.Compounds derived from 4-acylpyrazol-5-one have continued to receive considerable attention due to their pharmaceutical importance and high biological activity –3. They 1H and 13C NMR spectra were obtained from a Bruker AV 500 MHz for 1H and 125 MHz for 13C using a 5 mm quadra nuclei probe (QNP). The X-ray crystallographic data were obtained at the University of Auckland on a Bruker SMART APEX II diffraction equipped with a CCD area detector, using MoKα radiation (λ = 0.71073 Å). The software SMART was used for the collection of data frames, for indexing reflections, and to determine lattice parameters; SAINT was used for the integration of the intensity of the reflections and for scaling benzohydrazide , ethanolic solution (10 mL) of the metal chloride (0.001 mol) was added with constant stirring. The coloured mixture was then refluxed for 4 h. The resulting metal complex was filtered hot, washed with boiling mixture of 1 : 1 water/ethanol, dried under suction, and kept in vacuum over CaCl2.To an ethanolic solution (20 mL) of Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Proteus sp., Serratia marcescens, and Enterobacter sp. and the Gram-positive (G+) were Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Bacillus cereus ATCC 11778, Bacillus subtilis ATCC 6633, Bacillus thuringiensis, Enterococcus faecalis ATCC 29212, Enterococcus faecalis ATCC 51299, Streptococcus pneumoniae ATCC 49617, and Listeria monocytogenes. Also, yeast strains such as Candida utilis, Candida albicans ATCC 90028, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae ATCC 9763 were used. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Salmonella typhimurium ATCC 14028, Listeria monocytogenes, Proteus sp., Serratia marcescens, and Enterobacter sp. were cultured in nutrient broth (NB) (Merck) at 37°C; Streptococcus pneumoniae ATCC 49617, Enterococcus faecalis ATCC 29212, and Enterococcus faecalis ATCC 51299 were cultured in brain heart infusion broth (BHIB) (Merck) at 37°C for 24 h.; Micrococcus luteus ATCC 9341, Bacillus cereus ATCC 11778, Bacillus subtilis ATCC 6633, and Bacillus thuringiensis were cultured in nutrient broth (NB) (Merck) at 30°C; Candida utilis, Candida albicans ATCC 90028, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae ATCC 9763 were cultured in malt extract broth (MEB)  at 30°C for 24 h.Fifteen bacteria and five yeast cultures were used in the study. Ten bacterial strains and two yeast strains were obtained from the American Type Culture Collection . Other microorganism strains were obtained from Adnan Menderes University Faculty of Medicine. The Gram-negative (G−) wereThe antimicrobial activities of all the synthesized compounds were determined by the disc diffusion method , 25, andμg·mL−1) of all the synthesized compounds were prepared in DMSO. The inoculum suspensions of the tested bacteria and yeasts were prepared from the broth cultures (18–24 h) and the turbidity equivalent adjusted to 0.5 McFarland standard tube to give a concentration of 1 × 108 bacterial cells and 1 × 106 yeast cells/mL. To test the antimicrobial activity of all the synthesized compounds, a Mueller Hinton Agar (MHA) plate was inoculated with 0.1 mL broth culture of bacteria or yeast. Then a hole of 6 mm in diameter and depth was made on top with a sterile stick and filled with 50 μL of synthesized compounds. Plates inoculated with E. coli ATCC 25922, S. typhimurium ATCC 14028, S. aureus ATCC 25923, S. epidermidis ATCC 12228, E. faecalis ATCC 29212, Enterococcus faecalis ATCC 51299, S. pneumoniae ATCC 49617, Listeria monocytogenes, Proteus sp., S. marcescens, and Enterobacter sp. were incubated at 37°C for 24 h, while those with M. luteus ATCC 9341, Bacillus subtilis ATCC 6633, B. cereus ATCC 11778, B. thuringiensis, S. cerevisiae ATCC 9763, C. albicans ATCC 90028, C. glabrata, C. utilis, and C. tropicalis were incubated at 30°C for 24 h. At the end of incubation time, the diameters of the inhibition zones formed on the MHA were evaluated in millimeters. Disc of chloramphenicol (C30), gentamicin (CN10), tetracycline (TE30), erythromycin (E15), ampicillin (AMP10), and nystatin (NS100) were used as positive controls. The measured inhibition zones of the study compounds were compared with those of the reference discs.The standard method of Antimicrobial Disc Susceptibility Tests reported by the National Committee for Clinical Laboratory Standards  was usedμL of Mueller Hinton Broth (MHB) was placed in each well. After, the compounds were dissolved in DMSO (2 mg mL−1) and transferred into the first well. Two fold serial dilution of the compounds were carried out to determine the MIC, within the concentration range 256 to 0.125 μg mL−1. Cultures were grown at 30–37°C (18–20 h) and the final inoculum was approximately 106 cfu mL−1. The lowest concentration of the study compounds that resulted in complete inhibition of the microorganisms was represented as MIC (μg mL−1). Similar procedures were done on positive controls, streptomycin (I. E. Ulagay). In each case, the test was performed in triplicate and the results were expressed as means.Minimum inhibitory concentration (MIC) was determined by reported method . The stu2. All media and supplements were obtained from Life Technologies. HL 60 cells were seeded in T-25 tissue culture flasks at a concentration of 1 × 105/mL and incubated with increasing concentrations  of the study compounds. Cell counts and IC50 values were determined at 24 and 72 h using a Thoma slide. Experiments were done in triplicate. The percentage of cell divisions compared to the untreated control was calculated as follows:HL-60 promyelocytic leukaemia cells were purchased from ATCC. Cells were grown in RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum, 1% L-glutamine, and 1% penicillin/CO1 has been reported in literature [1 was solved by direct method. The nitrogen and oxygen atoms were located followed by other atoms in subsequent refinements. All nonhydrogen atoms were refined as anisotropic. The X-ray crystal structures and atom numbering schemes of HL1 are as shown in The crystal structure of HLterature  but was terature , and con−1 cm2 mol−1 [All the metal complexes are colored and soluble in polar solvents  but insoluble in solvents such as hexane, pentane, tetrahydrofuran and carbon tetrachloride. Analytical and physical data of all the study compounds are shown in m2 mol−1 –32.−1 in the spectral of HL1 and the metal complexes were assigned to NH stretching vibrations. Two strong v(C=O) bands are observable in the keto-amine form of HL1, one assignable to v(C=O) of the lateral chain comprising the hydrazide moiety and the other to the v(C=O) of pyrazolone ring [−1) were observed at 1645 and 1596, respectively. The v(C=O) band assigned to the hydrazide moiety shifted to lower frequencies in the metal complexes, suggesting that the carbonyl oxygen was involved in coordination to the metal ions [−1 have been assigned to imino v(C=N) in all the metal complexes. The bands in the region of 1437–1440 cm−1 in the metal complexes have been assigned to v(C–O) [The relevant stretching frequencies are tabulated in one ring –35. Thestal ions –38. Bando v(C–O) , 39 resu−1 in the ligand and the metal complexes have been assigned to v(N–N). Bands in the range ~510–520 cm−1 in the metal complexes were assigned to v(M–O) while bands in the range ~420–480 cm−1 were assigned to v(M–N) [ vO–H of water of crystallization was observed in the region ~3370–3470 cm−1 in the metal complexes. HL1 reacted as the enol-imino form (5)), when coordinated to metal ions in solution, coordinating via pyrazolone ring enolic (C–O), azomethine (C=N), and hydrazide side chain (C=O).The bands in the region ~1025–1120 cmo v(M–N) –43. vO–H1 are as shown in 4). The conjugated system that extended from the pyrazolone ring confers extra stability on form (4). In the 1H NMR spectrum the 3-methyl protons signal was observed as singlet upfield at 2.17 ppm. The phenyl protons appeared as multiplets in the range 7.26–8.07 ppm while the signal due to –NH was observed at 8.83 ppm. The 13C-NMR spectrum of the ligand exhibited 17 carbon signals comprising 16 sp2 and one sp3 (–CH3) carbons; the signal at δ16.06 has been assigned to carbon of the pyrazolone core side 3-methyl. Carbons of benzene rings are present at δ 118.47–165.18. The most deshielded carbons have been assigned relevant signals downfield. The assignments are as follows: C-1 = 127.55, C-2 = 127.42, C-3 = 129.70, C-4 = 127.42, C-5 = 127.55, C-6 = 131.43, C-7 = 138.75, C-8 = 163.64, C-9 = 130.69, C-10 = 148.27, C-11 = 132.78, C-12 = 130.04, C-13 = 128.83, C-14 = 130.04, C-15 = 15.44, C-16 = 149.28, C-17 = 120.34, C-18 = 154.55, C-19 = 138.22, C-20 = 119.85, C-21 = 129.70, C-22 = 125.26, C-23 = 129.70 and C-24 = 119.85 ppm (see (4) in The possible keto forms of HL−1 which were assigned to n → π* and π → π* transitions [9 octahedral Cu2+ complex the 2D free-ion term is split into two levels by the Oh symmetry and further split on distortion to D4h or C4v symmetry [−1 which have been assigned to 2B1g→2B2g,2B1g→2A1g and 2B1g→2Eg transitions respectively [8 configuration in an octahedral field has a ground state of 3F, which is split into 3A2g, 3T2g, and 3T1g(F) states and the excited state designated as 3T1g(P). Three bands can be observed in the spectra. The Ni(II) complex displays three bands at 12,545, 14,174, and 23,809, cm−1 assignable to 3A2g→3T2g(F), 3A2g→3T1g(F), and 3A2g→3T1g(P) transitions, respectively [−1 for Co(II) complexes have been assigned to 4T1g(F)→4T1g(P), 4T1g(F)→4A2g(F) and 4T1g(F)→4T2g(F) transitions, respectively [The significant electronic absorption bands in the spectra of the ligand and the metal complexes recorded in DMSO solution are presented in nsitions , 50. Forsymmetry . Three sectively –54. The ectively . The magectively . The magectively .On the basis of microanalytical data, magnetic moments, conductivity measurements, and spectral analysis, http://dx.doi.org/10.1155/2014/718175. The MIC values which suggested that some of the studied compounds exhibited appreciable antimicrobial activity are showed in 1) showed significant antimicrobial activity against four Gram- positive bacteria Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778, and Bacillus subtilis ATCC 6633; the Co complex showed antimicrobial activity against two Gram-positive bacteria Bacillus cereus ATCC 11778 and Bacillus subtilis ATCC 6633. Cu(II) complex exhibited antimicrobial activity against three Gram-positive bacteria Micrococcus luteus ATCC 9341, Staphylococcus aureus ATCC 25923, and Bacillus cereus ATCC 11778. However, none of the study compounds exhibited antimicrobial activity against tested Gram-negative bacteria and yeasts. Gram-negative bacteria are noted for the protective lipopolysaccharide (LPS) in the outer membrane of their cell walls, which protects the sensitive inner membrane and the cell wall from drugs and dyes [The results of antimicrobial activities of the study compounds reported as inhibition zone diameter (mm) are showed in and dyes . This prand dyes , 59. Theand dyes . On coorμg mL−1. The lowest MICs were observed for HL1 (4–8 μg mL−1) against the four tested microorganisms. Low MICs were also recorded for Cu complex (Staphylococcus aureus ATCC 25923) and Co complex (Bacillus cereus ATCC 11778). The results suggest that the compounds showed good antimicrobial activity against these organisms.MICs were measured for the compounds that showed appreciable growth inhibition zones; the study was restricted to microorganisms that were affected by the compounds. From the results of MIC tests shown in human promyelocytic leukemia cells) cell line is a leukemic cell line that has been used for laboratory research. The HL-60 cultured cell line provides a continuous source of human cells for studying the molecular events of myeloid differentiation and the effects of physiologic, pharmacologic, and virologic processes. The results of antiproliferative activities of investigated compounds against human cancer (HL-60) cell line are presented in 1 and its Cu(II) complex exhibit cytotoxic effects at the lowest tested concentration with IC50 value between ~3 and 5 μM while the Co(II) and Ni(II) complexes exhibit better cytotoxic effects with IC50 value ~2-3 μM. μM) were able to inhibit the proliferation of more than 50% of the HL-60 cell. The tested compounds can be described as potent anticancer agents due to their considerably high antiproliferative effects. This is consistent with documented facts by the US NCI screening program, that if the IC50 value (following the incubation of a compound and cell lines between 48 and 72 h) is less than 10 μM, the compound is considered to have in vitro cytotoxic activity [HL-60 , Cu(II), and Co(II) complexes. The electronic data and magnetic moments are in favour of octahedral geometry for Ni(II), Cu(II), and Co(II) complexes. The compounds exhibited better antimicrobial activity against Gram-positive bacterial strains studied. The cytotoxicity test results showed that the compounds are potential anticancer agents.1 has been deposited with CCDC, deposition number 931219, and is freely available from http://www.ccdc.cam.ac.uk/data_request/cif.A CIF file containing complete information on the structure of HL


Staphylococcus aureus, the disease impact of chromosomally integrated prophages on virulence is well described. However, the existence of extra-chromosomal prophages, both plasmidial and episomal, remains obscure. Despite the recent explosion in bacterial and bacteriophage genomic sequencing, studies have failed to specifically focus on extra-chromosomal elements. We selectively enriched and sequenced extra-chromosomal DNA from S. aureus isolates using Roche-454 technology and uncovered evidence for the widespread distribution of multiple extra-chromosomal prophages (ExPΦs) throughout both antibiotic-sensitive and -resistant strains. We completely sequenced one such element comprised of a 43.8 kbp, circular ExPΦ (designated ФBU01) from a vancomycin-intermediate S. aureus (VISA) strain. Assembly and annotation of ФBU01 revealed a number of putative virulence determinants encoded within a bacteriophage immune evasion cluster (IEC). Our identification of several potential ExPΦs and mobile genetic elements (MGEs) also revealed numerous putative virulence factors and antibiotic resistance genes. We describe here a previously unidentified level of genetic diversity of stealth extra-chromosomal elements in S. aureus, including phages with a larger presence outside the chromosome that likely play a prominent role in pathogenesis and strain diversity driven by horizontal gene transfer (HGT).In  S. aureus. Integrated prophages encode S. aureus-specific toxins such as Panton-Valentine leukotoxin (PVL) and staphylococcal enterotoxin A Escherichia, Bacillus, Halomonas, Chlamydia, and Borrelia species S. aureus.Chromosomally integrated prophages are known to play key roles in the pathogenicity and virulence of Borrelia burgdorferi, B31, contained 12 different plasmidial prophages, , while Chlamydia pneumoniae contained only one B. burgdorferi and C. pneumoniae is attributed to evolutionary pressure on their smaller genomes to remove all non-essential DNA B. burgdorferi because of its higher copy number prophages combined with its small chromosome, allowing for the identification and sequencing of these additional, smaller, genetic elements. Thus, plasmidial prophages might not be as rare as this study indicated, but rather difficult to identify. Additional evidence also suggests that lysogeny in the environment favors the plasmidial form. B. anthracis strains isolated worldwide have nearly identical chromosomal prophage sequences, yet up to 20% of isolates encode a diverse array of additional inducible phages B. anthracis phages  with significant roles in the adaptive behavior of the pathogen A report by Casjens in 2003 showed the existence of only two plasmidial prophages in over 80 published bacterial DNA sequences among diverse species S. aureusS. aureus phages Φs80b and Φs84b could integrate into the chromosome of S. aureus strain s64c but remained plasmidial or solely extra-chromosomal in S. aureus strain 8325–4. Thus, the S. aureus host cell determined the extra-chromosomal state of these prophages: episomal in S. aureus s64c, and plasmidial in 8325–4. In strain s64c, this extra-chromosomal state was reported as ‘transient’, where the integrated and extra-chromosomal states could switch during serial passages σH, was found to directly affect integration and excision of S. aureus phages; sigH deletion from the S. aureus genome caused an increase in the episomal state of the phage −5) in the bacterial population that they cannot be detected using current sequencing techniques.In this study we term transient phages as ‘episomal’, and those existing solely in the cytoplasm as ‘plasmidial’. Two previous reports have examined extra-chromosomal prophages in Streptococcus pyogenes chromosomal island in an M1 serotype (SpyCIM1), can integrate at a specific site in the chromosome during stationary phase of growth, blocking transcription of a mismatch repair gene, mutL, thereby increasing the mutation rate. During exponential growth however, SpyCIM1 excises and replicates in the extra-chromosomal compartment of the cell, enabling mutL transcription E. coliBacillus subtilisClostridium difficileEnterococcus faecalis V583 S. aureus ExPΦ is unclear, these studies suggest it will likely have an important role in cellular processes.MGEs in both the integrated and circularized states can have major effects on the bacterial cell. For example, a phage-like MGE in S. aureus has been biased toward sequencing the bacterial chromosome; not surprisingly, smaller, low-copy, extra-chromosomal elements (like ExPΦ) would have been overlooked unless specifically isolated S. aureus may be a transient event and therefore potentially difficult to detect due to extremely low-copy numbers. In this study, we developed a method to effectively enrich extra-chromosomal DNA that includes low-copy number episomal and/or plasmidial ExPΦs and other extra-chromosomal elements from S. aureus. In addition, a NGS approach was used to achieve extremely high coverage and delineate genetic diversity of MGEs. Our results revealed naturally present and potentially widespread ExPΦs in virulent and antibiotic-resistant strains of S. aureus. ExPΦs were shown to be either episomal or plasmidial. The complete genomic sequence of one extra-chromosomal S. aureus phage ФBU01 from a VISA strain, was assembled and annotated, and found to contain scn, chp, sak, and sep IEC virulence genes associated with β-hemolysin containing phages S. aureus pathogenesis.Next-generation sequencing (NGS) in Staphylococcus aureus strains were cultured in Bacto Brain Heart Infusion  and incubated at 37°C. S. aureus from freezer stocks were revived on BHI agar plates and incubated for 16 to 24 hrs. One to two-day-old single colonies were inoculated into BHI broth and grown overnight (12–15 hrs). Cultures were then diluted 1∶100 in 150 mL BHI and grown to an OD600 of 0.6 to 0.9. Cultures were then centrifuged at 4,000 RPM for 20 minutes at 4°C. Cell pellets were used immediately or stored overnight at −20°C.Bacterial strains used in this study are described in Table S1 in S. aureus cell pellets were resuspended in 12 mL of the QIAGEN  Resuspension Buffer  supplemented with 200 µg lysostaphin from Staphylococcus simulans , 800 ng of PlySs2 lysin 2O at 65°C.g for 30 min or as needed at 4°C. The centrifugation force was kept below 5000×g to prevent shearing of large plasmids . When needed, samples under a total volume of 100 µl were concentrated further by Speedvac (Thermo Scientific Savant) set at low drying rate. Samples were stored at −20°C and shipped overnight on ice to the sequencing facility. Samples treated with Plasmid-Safe linear DNase were incubated for 2–4 hrs at 37°C with 1 µl 25 mM ATP, 2.5 µl 10× Reaction Buffer and 1 µl (10 U) Plasmid-Safe Linear DNase (25 µl reaction volume). Heating at 70°C for 30 min then inactivated the linear DNase.DNA was separated by electrophoresis at 50 V for 60 min on a 0.7 % Agarose, 1X, TAE gel. DNA was stained with ethidium bromide, and visualized under ultraviolet transillumination on an Alpha Imager HP . Total DNA was estimated by measuring final concentrations of extra-chromosomal DNA preparations using Thermo Scientific  NanoDrop 1000, software version 3.1.0. If needed, samples were concentrated using Amicon Ultra 0.5 mL 30,000 MWCO centrifugal units ; centrifuged at 2000×et al. 2005 De novo assembly of sequences was performed using Roche gsAssembler Newbler at default settings. Final assemblies were submitted to Genbank with the following accession numbers: ΦBU01, KF831354; pBU108a, KF831355; pBU108b, KF831356; and pBU108c, KF831357.‘Whole-genome sequencing’ (WGS) on extra-chromosomal DNA samples was performed using the Roche-  454 Sequencing Genome Sequencer FLX as described in Margulies http://www.nibi.nih.gov/BLAST). Bacteriophage gene identification was performed using BLASTP analysis. Homologies found in the GenBank database were based on greater than 50 percent identity Contigs generated by Roche gsAssembler Newbler resulting from the Roche-454 sequencing runs were identified as plasmid, bacteriophage, chromosome, or miscellaneous MGE by BLASTN analysis comparing to GenBank database 5′-CCTGTTGCTTGGGTAACTGTATC-3′ and 5′-AATGGCAGAAAGTGGCTGG-3′ for identified bacteriophage in the NRS19 strain and primers 5′TGCCATTGTGATGAGGAGGG-3′ and 5′-GCAACGCAGATTGTTTGAGTG-3′ for the identified bacteriophage of the NRS26 strain. These PCR products were purified with QIAquick PCR Purification Kit # 28106 and labeled for use as a Southern blot probe using Biotin DecaLabel DNA Labeling Kit . DNA transferred to nylon membrane was pre-hybridized and hybridized based on standard Southern blot procedures Aliquots of 200–400 ng extra-chromosomal DNA were separated (50 volts for 60 min) on a 0.7 % agarose, 1X TAE gel. Invitrogen  SYBR Safe DNA gel stain (# S33102) was added to the gel at a 1∶10,000 dilution. DNA was treated and transferred from gel to Positively Charged Nylon Transfer Membrane  using standard Southern blot procedures et al. 2000 S. aureus cultures were grown overnight, then diluted 1∶100 in BHI, and 10 mL samples were taken at 6, 8, and 24hrs. Samples of φBU01 were also taken at an OD600 of 0.12, 0.65, 1.3, 2.0 and 2.5 (24 hrs). Samples were concentrated to an adjusted OD600 of 5.0–10.0. Cultures were added to Lonza  SeaPlaque GTG low melting agarose (# 50111) and allowed to solidify. Samples were treated in 0.75 % agarose disks with 100 µg/mL RNase A, 50 µg/mL lysostaphin, 1 mg/mL lysozyme, and 25 µg/mL PlySs2 for 5 hrs. Agarose disks were then treated with 1 mg/mL proteinase K for 17 hours and overnight with SmaI. DNA was separated by pulse-field electrophoresis for 23 hours at 6.0 V/cm. Gel was stained with ethidium bromide for 30 min and destained in dH20 for 30 min.Pulsed field gel electrophoresis was performed with a modified procedure from Chung De novo assembly of the sequence contigs was performed using MacVector assembly (without template) with the PHRAP assembly algorithm, default settings Primers were designed based on preliminary mapping of the contigs to the reference genome  was spotted on BHI soft-agar overlays  of S. aureus reporter strains NRS77, RN4220, and Newman , 0.002% (w/v) gelatin). Prior to verification of bacteriophage DNA by PCR, bacteriophage samples were treated with 2 µl DNase  at 37°C for 30 min. DNase in the bacteriophage samples was inactivated by incubation at 75°C for 10 min.Phage induction with mitomycin C was performed following a modified protocol described by Cao 2O, and stained with 2% 0.2 µm filtered uranyl acetate. Excess solution was wicked away and grids were allowed to dry at least 30 min before visualization. Grids were examined on the JEOL 100CX transmission electron microscope using AMT V600 software (Advanced Microscopy Techniques) and images captured at 33,000x and 50,000x magnification.PEG-precipitated phage samples as described above were visualized at The Rockefeller University Electron Microscopy Resource Center. Briefly, 10 µL of sample was spotted on glow discharged 200-mesh copper grids with carbon film  and incubated for 2.5 min. Grids were then washed 2x in ddHPCR amplicons for Southern analysis, primer walking for sequence gap closure, and for verification of the presence of free phage particles in culture supernatants were all generated using NRS19 extra-chromosomal DNA preparation as template. Two DNA polymerases were used: (1) Taq PCR Master Mix  and (2) Phusion DNA Polymerase . The reaction condition for the Taq polymerase were as follows: (1) 95°C for two min; (2) 95°C for 30 sec; (3) appropriate annealing temperature for 30 sec; (4) 72°C for one min per kb; (5) repeat steps two through five 29 times; (6) 72°C for 10 min; and (7) 4°C indefinitely. The reaction conditions used for the Phusion polymerase were as follows: 1) 98°C for 30 sec; (2) 98°C for 10 sec; (3) appropriate annealing temperature for 30 sec; (4) 72°C for 30 sec per kb; (5) repeat steps two through five 29 times; (6) 72°C for 10 min; and (7) 4°C indefinitely. Following PCR, DNA was separated by electrophoresis at 100 V for 20 min on a 1.5 % agarose gel, run in 0.5 X TAE buffer. DNA was stained with EtBr, and visualized under ultraviolet transillumination on a Cell Biosciences  Alpha Image HP.Staphylococcus NARSA strains (Table S1 in S. aureus phage genomes S. aureus (VISA), three of seven methicillin-resistant S. aureus (MRSA), and three of five methicillin-sensitive S. aureus (MSSA) extra-chromosomal DNA samples possessed phage genes (S. aureus (VRSA) strains (VRS2 and VRS3a) and the one vancomycin-intermediate S. epidermidis (VISE) strain (NRS53). Thus, Roche-454 based sequencing of S. aureus extra-chromosomal DNA revealed evidence of the possible widespread existence of ExPΦs for the first time in natural isolates of these human pathogens.Extra-chromosomal DNA from 24 ge genes . Phage DS. aureus strains in more detail to categorize the MGEs found in our sequencing data. From BLASTN sequence analysis, all S. aureus extra-chromosomal contigs could be categorized as either plasmid, phage, or in some cases chromosomal DNA , in that the fewer the number of contigs, the lower the score. Assemblies with large numbers of contigs, such as that of NRS26 (1993 contigs) and NRS145 (500 contigs), generally had higher Q39 scores , possibly indicating that either the depth of coverage was relatively low, or the presence of more than one genomic species. This could occur with either low-level chromosomal contamination or the presence of multiple phages.Each extra-chromosomal DNA fraction was prepared as described in S. aureus ФNM3 phage genome (Siphoviridae family of phages. The assembled circular sequence of ФBU01 and Southern blot identification of a Plasmid-Safe (a DNase specific for linear DNA) treated sample of NRS19 , was determined by primer walking on PCR-amplified extra-chromosomal phage DNA . Using ae genome . The pree genome  were usee genome ; PCR#1. of NRS19  providedBacillus anthracis) S. aureus.  or linear  and NRS26 . These eThe efficacy of the linear DNase is shown in a control study that removed sheared, contaminating chromosomal DNA from an extra-chromosomal DNA sample , and NRS26 by pulsed-field gel electrophoresis (PFGE)  . Primerss (PFGE) . After 1s (PFGE) . Howevers (PFGE) , suggestS. aureus strains NRS19 and NRS26 were treated with mitomycin C S. aureus reporter strains; controls without mitomycin C treatment were negative. Polyethylene glycol-precipitated phage preparations were first treated with DNase to remove any host chromosomal DNA not contained within the phage particles. Phage particles were then disrupted, and the released DNA was used as template in PCR. DNA extracted from mitomycin C-induced culture supernatants of NRS19 (which includes ΦBU01) and NRS26 was subjected to PCR analysis. Primers bdu211, bdu212 and bdu215, bdu216 described above were used to determine if ExPΦ DNA could be detected in the induced phage fraction. The primers specific for the ExPΦs of NRS19 and NRS26 detected the presence of phages from both strains , then ФBU01 DNA should represent approximately 0.0078% of the extra-chromosomal DNA sample. Thus, ФBU01 is represented in our extra-chromosomal sample at a nearly 100-fold higher percentage  and NRS109 (MSSA) represent two such samples. Since these strains did not appear to contain ExPΦs, we were curious if they contained integrated prophage. Upon treatment with mitomycin C, we could induce phage from NRS24, which plaqued on S. aureus strain RN4220 . The lack of any extra-chromosomal phage contigs in the prophage-positive strains NRS24 and NRS109 indicates that the extra-chromosomal phage DNA of strains NRS19 and NRS26 are not the result of spontaneous excision but rather more stable, potentially plasmidial forms.In addition, some of the extra-chromosomal S. aureus strains identified several putative, phage-encoded virulence factors with the potential to be horizontally transferred to other bacteria , and Bacillus cereus plamidial-prophage pE33L54 contains multiple integrases Vibrio parahaemolyticus is 92% identical to integrated VHML phage. The replication of linear plasmidial-phages requires a replication protein (RepA), an origin of replication (ori), a protelomerase (Tel), and a telomere resolution site. Both Vp58.5 and VHML phage contain these elements. Therefore the presence of single genes such as integrases, is not a reliable indicator of whether a bacteriophage can integrate or is solely excised S. aureus.However, an integrase does not always determine the fate of plasmidial and integrated bacteriophages. g to reduce potential shearing of large DNA elements. Furthermore, Roche-454 sequencing was selected as it generated DNA sequencing read lengths longer than other NGS platforms de novo assembly of contig sequences of low-copy DNA elements.Extra-chromosomal DNA from nine VISA, seven MRSA, two VRSA, one VISE and five virulent MSSA strains were isolated and screened for ExPΦs using Roche-454 sequencing  containing a region of 98% percent identity with a query coverage of 83%. ФNM3 is a defective β-haemolysin (hlb) converting phage essential to the virulence of S. aureus Newman strain sea, sak, chp, and scn), the CHIPS gene responsible for reduction in neutrophil recruitment, phage structural genes, and an integrase gene (tyrosine recombinase family XerC). ФNM3 is unique in that it does not encode an excisionase, and primarily remains integrated within the chromosome ФBU01 shares sequence homology to several phages, including S. aureus MGEs that may be identified in this analysis include phages, plasmids, transposons, S. aureus pathogenicity islands (SaPIs), and staphylococcal cassette chromosomes (SCCs), all of which may be transferred horizontally between staphylococci Extra-chromosomal isolations separated by electrophoresis contained multiple bands  and 4 in−4, while φ11 in the same host has an excision rate closer to 10−2. In the same study, ΦSA2mw had excision rates over 10−2 in NCTC8325, yet under 10−3 in host strain MW2 −4 are within the label of “spontaneous excision”, with a range of 10−4 to 10−5. However, rates of 10−2 represent a two-log increase and potentially occur in 1% of the population. Based on our calculations, we predict that at a minimum, the episomal phages identified by our technique would excise at a rate of 10−2 or greater  into S. aureus recipient strains mec elements in the extra-chromosomal sequencing results. Therefore, it is plausible that MGEs detected in this study, including ExPΦs, could be transferred by phage transduction.Integrated phages can be vertically transferred from mother to daughter cells, passing on the phage's genetic information, associated virulence factors, and transcriptional regulators that can have important effects on the bacterial cell. The extra-chromosomal form provides multiple DNA copies of the phage, yet its relevance in vertical and HGT is currently overlooked. While the rate at which ExPΦs are transferred is unclear, it is possible that they could mediate their own transfer or be transferred by helper phages or other elements. Transduction by phage plays a pivotal role in S. aureus are needed before categorizing these genetic elements as true plasmidial prophages. The mechanism for the replication of ΦBU01 is presently unclear, however, a putative DnaD replication protein is encoded on ΦBU01. DnaD replication proteins have been found in S. aureus phages B. subtilisS. aureus phage DNA replication modules Further studies on the replication of ExPΦs in S. aureus. These elements may play a major role in bacterial virulence and human disease, as they encode multiple S. aureus virulence determinants. Perhaps their presence as multiple ExPΦ is beneficial by allowing for increases in gene dosage as a result of accumulative higher expression levels. Importantly, ExPΦs could potentially facilitate the dissemination of these virulence factors through transduction or in tandem with another MGE. Understanding the biology of these elements and their contribution to pathogenesis will pave the way for better epidemiology, diagnostics, and therapeutics for S. aureus. Furthermore, the approach described here can also easily be extended to reveal the cytoplasmic DNA elements of other bacterial pathogens.In conclusion, it is clear that advances in NGS have allowed for cost effective whole genomic sequencing of a number of organisms, leading to the sequencing of more 10,000 bacterial genomes and several thousand phage genomes to date File S1Supplementary Figures and Tables.(DOCX)Click here for additional data file.File S2Preliminary alignments of 454 sequencing contigs identified as bacteriophage DNA.(DOCX)Click here for additional data file.


P ≤ 0.05 and P ≤ 0.01, resp.). While ouabain significantly increased the atrial beating rate in control group (P ≤ 0.05), same treatment did not show similar effect in clofibrate-treated group (P > 0.05). Injection of ouabain significantly increased the atrial and systemic levels of all studied inflammatory cytokines (P ≤ 0.05). Pretreatment with clofibrate could attenuate the ouabain-induced elevation of IL-6 and TNF-α in atria , as well as ouabain-induced increase in IL-6 in plasma (P ≤ 0.05). Based on our findings, clofibrate may possess antiarrhythmic properties through mitigating the local and systemic inflammatory factors including IL-6 and TNF-α.Considering the cardioprotective and anti-inflammatory properties of clofibrate, the aim of the present experiment was to investigate the involvement of local and systemic inflammatory cytokines in possible antiarrhythmic effects of clofibrate in ouabain-induced arrhythmia in rats. Rats were orally treated with clofibrate (300 mg/kg), and ouabain (0.56 mg/kg) was administered to animals intraperitoneally. After induction of anesthesia, the atria were isolated and the onset of arrhythmia and asystole was recorded. The levels of inflammatory cytokines in atria were also measured. Clofibrate significantly postponed the onset of arrhythmia and asystole when compared to control group ( PPAR-α possesses an important role in lipid metabolism [α is predominantly expressed in tissues with high fatty acid oxidation rate including heart, liver, and kidney [α knockout mice, indicating that PPAR-α plays a critical role in cardioprotective effects of fibrates [α agonists have anti-inflammatory properties [The fibrate class of hypolipidemic drugs is used extensively in treatment of metabolic syndrome in which hyperlipidemia and hypertension are most prominent manifestations of this disorder. Fibrates are ligands of the peroxisome proliferator-activated receptors (PPARs) , 2. Thestabolism . PPAR-α d kidney . It has d kidney , 8. Intefibrates , 8. Sincoperties –12, and operties –15. Ther ad libitum access to food and water. All experiments were conducted in Tehran University of Medical Sciences in accordance with the recommendations of the ethics committee on animal experimentation of the medical school.Male Wistar albino rats (body weight 250–280 g) were obtained from the Department of Pharmacology and Comparative Biology Unit . Animals were housed under the standard laboratory conditions, temperature 22 ± 2°C, humidity 70%–80%, and 12 h light-dark cycle, and haveα were provided from Sigma-Aldrich . ELISA kit for measurement of IL-1β was purchased from Abcam, UK.All materials were purchased from Merck (Germany), unless noted otherwise. Clofibrate was a gift from Zahravi Pharmaceutical Company, Iran. Ouabain and enzyme-linked immunosorbent assay (ELISA) kit for measurement of IL-6 and TNF-In order to study the isolated atria, 12 rats were randomly divided into 2 equal groups. First group (treatment) received clofibrate  once daily for 14 days, while the second group (control) received only olive oil  once daily for 14 days. For measuring the inflammatory cytokines, 12 rats were divided into three equal groups. First 2 groups received only olive oil  once daily for 14 days, while third group was given clofibrate  once daily for 14 days. From the days 12th to 14th, first group received normal saline, 1 mL/kg, intraperitoneally (i.p.) once daily, whereas the second and third groups were injected by ouabain dissolved in normal saline at the dose of 0.56 mg/kg  once dai2 and 5% CO2) modified Krebs solution at 37°C and pH 7.4. The composition of the solution was as follows (mM): NaCl 118.0, KCl 4.7, CaCl2 2.6, MgCl2 1.2, NaH2PO4 1.0, NaHCO3 25.0, glucose 11.1, EDTA 0.004, and ascorbic acid 0.11. A preload tension of 1000 mg was applied to the atria and tissues were allowed to equilibrate for 30 min [After induction of anesthesia with ketamine  and diazepam  (i.p.), heart was rapidly removed and atria were carefully dissected from the ventricles, attached to a tissue holder, and then were immersed in a tissue bath containing 25 mL of carbogenated , and the contents of IL-1β, IL-6, and TNF-α in atria and plasma were calculated and reported as ng/mL.The levels of inflammatory cytokines in atria and plasma were measured by specific ELISA kits. Briefly, each atrium was homogenized in 1 mL of phosphate buffered saline  and then centrifuged at 10000 g for 15 min at 4°C. For measurement of plasma levels of cytokines, blood samples were taken and centrifuged at 4000 g for 15 min at 4°C. IL-1t-test was carried out to compare the time of onset of arrhythmia or asystole as well as atrial beating rate and contractile force between treatment and control groups. Paired Student's t-test was used to detect the effects of ouabain on atrial beating rate and contractile force within groups. Comparison of inflammatory cytokines between three groups was done by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. P values less than 0.05 were considered statistically significant.Statistical analyses were carried out using GraphPad Prism 5 software . The results are presented as mean ± SEM. Unpaired Student's P ≤ 0.05). Also, we detected a significant increase in the onset time of asystole in the treatment group (66.19 ± 12.33 min), while this time for control group was 22.77 ± 7.17 min  rather than control group , while such effect was not observed in treatment group . Pretreatment with clofibrate attenuated the ouabain-induced elevation of IL-6 and TNF-α in atria  possess arrhythmogenic properties. In this context, TNF-α and IL-1β have been reported to boost susceptibility to arrhythmia in rat ventricular myocytes through increase of calcium leakage from the sarcoplasmic reticulum [α in transgenic animals causes various atrial and ventricular arrhythmias [α, contribute to pathophysiology of ventricular arrhythmias [α agonist, clofibrate. It seems that the role of IL-6 in mediating the antiarrhythmic properties of clofibrate is more prominent than TNF-α, because clofibrate reduced IL-6, locally and systemically, while it only decreased the local elevation of TNF-α. On the other hand, the local antiarrhythmic effects of clofibrate appear superior to systemic antiarrhythmic properties.Inflammatory cytokines play a key role in mediating the inflammatory responses under pathologic conditions. TNF- as IL-6 . Evidenc as IL-6 , 24. Sim as IL-6 , 14. Witeticulum . Moreovehythmias –28. Recehythmias . These fα agonist, clofibrate, can reduce the experimental myocardial infarct size up to 43% in rats [α agonists protect the heart against the ischemia-reperfusion injury [Several lines of research have indicated that there is a high rate of atrial fibrillation and other kinds of cardiac arrhythmias in metabolic syndrome and lipid metabolism disorders , 31. Evi in rats , and it n injury , 8. Sincα.It is concluded that clofibrate might possess antiarrhythmic properties in reducing the cardiac arrhythmias. It seems that clofibrate shows this beneficial effect through the modulation of some local and systemic inflammatory cytokines including IL-6 and TNF-