Antimicrobial and cytotoxic activities of flavonoid and phenolics extracted from Sepia pharaonis ink (Mollusca: Cephalopoda).

BACKGROUND: Several studies have been reported previously on the bioactivities of different extracts of marine molluscs. Therefore, we decided to evaluate the cytotoxic and antimicrobial activities of S. pharaonis ink as a highly populated species in the Red Sea. We extracted the flavonoids from the ink and analyzed their composition. Then we evaluated systematically the cytotoxic and antimicrobial properties of this extract. A pharmacokinetic study was also conducted using SwissADME to assess the potential of the identified flavonoids and phenolic compounds from the ink extract to be orally active drug candidates. RESULTS: Cytotoxic activity was evaluated against 5 cell lines (MCF7, Hep G2, A549, and Caco2) at different concentrations (0.4 µg/mL, 1.6 µg/mL, 6.3 µg/mL, 25 µg/mL, 100 µg/mL). The viability of examined cells was reduced by the extract in a concentration-dependent manner. The highest cytotoxic effect of the extract was recorded against A549 and Hep G2 cancer cell lines cells with IC50 = 2.873 and 7.1 µg/mL respectively. The mechanistic analysis by flow cytometry of this extract on cell cycle progression and apoptosis induction indicated that the extract arrests the cell cycle at the S phase in Hep G2 and MCF7, while in A549 cell arrest was recorded at G1 phase. However, it causes G1 and S phase arrest in Caco2 cancer cell line. Our data showed that the extract has significant antimicrobial activity against all tested human microbial pathogens. However, the best inhibitory effect was observed against Candida albicans ATCC 10,221 with a minimum inhibitory concentration (MIC) of 1.95 µg/mL. Pharmacokinetic analysis using SwissADME showed that most flavonoids and phenolics compounds have high drug similarity as they satisfy Lipinski's criteria and have WLOGP values below 5.88 and TPSA below 131.6 Å2. CONCLUSION: S. pharaonis ink ethanolic extract showed a promising cytotoxic potency against various cell lines and a remarkable antimicrobial action against different pathogenic microbial strains. S. pharaonis ink is a novel source of important flavonoids that could be used in the future in different applications as a naturally safe and feasible alternative of synthetic drugs.

The diagnostic utility of the triple markers Napsin A, TTF-1, and PAX8 in differentiating between primary and metastatic lung carcinomas.

Napsin A and thyroid transcription factor-1 (TTF-1) are useful biomarkers for differentiating lung adenocarcinoma from squamous cell carcinoma and also for differentiating primary lung adenocarcinoma from metastatic lung carcinoma. Pair-boxed 8 (PAX8) can help in distinguishing primary lung carcinoma from metastatic carcinomas and help to determine the primary sites of metastatic carcinomas. Immunohistochemistry for Napsin A, TTF-1, and PAX8 were performed on 193 cases of carcinoma: 50 primary lung carcinoma and 143 carcinomas from other sites. Napsin A and TTF-1 were positive in 54, 52 % of lung carcinomas cases, respectively. While in adenocarcinoma cases, their expressions were 86.7 and 83.3 %, respectively. PAX8 was negative in all lung carcinomas. TTF-1 and PAX8 were positive in 93.3 and 96.7 % of thyroid carcinoma cases and in 87.5 and 93.8 % of papillary carcinoma respectively, and both were positive in 100 % of follicular carcinoma. Napsin A was negative in all thyroid carcinomas. Napsin A and PAX8 were positive in 50 and 93.3 % of renal carcinoma cases and in 81.8 and 100 % of papillary carcinoma, 38.5 and 92.3 % of clear cell carcinoma, and 16.7 and 83.3 % of chromophobe carcinoma respectively. TTF-1 was negative in all renal carcinomas. PAX8 was positive in 80 % of ovarian carcinoma cases; 100 and 60 % of serous mucinous carcinomas, respectively. It was also positive in 100 % of endometrial carcinoma. Napsin A and TTF-1 were negative in both ovarian and endometrial carcinomas. Our data demonstrated that combined use of Napsin A, TTF-1, and PAX8 may help in differentiating between primary lung adenocarcinoma and metastatic lung carcinomas.

Toxocara canis: Prospective activity of Quercetin and venom of Cassiopea andromeda (Cnidaria: Cassiopeidae) against third-stage larvae in vitro.

Toxocariasis is a zoonotic parasitic infection with worldwide distribution and high impact on human health. It has a limited clinical resolution with the available drugs, making it challenging to treat. Quercetin, which possesses biological and pharmacological qualities including antiparasitic, antioxidant, and anticancer activities, is a possible substitute for the current medications. Marine invertebrates can produce a vast array of different molecules, many of which are biologically active substances with distinct characteristics. In this study, we assessed the in vitro nematocidal effect of both quercetin and venom of Cassiopea andromeda (jellyfish) against third larvae of Toxocara canis. In microplates with Roswell Park Memorial Institute-1640 medium, larvae were incubated with ethanolic extract of quercetin (0.01, 0.02, 0.05, 0.08, 0.1, 0.25, and 0.5 mM/mL) and water extract of C. andromeda venom (15, 20, 25, 30, 35, 40, and 60 µg/mL) to evaluate their larvicidal effect. A scanning electron microscopy has investigated the possible effect of lethal concentration (LC90) of both extracts on the body wall of cultivated larvae, in comparison with those cultivated in albendazole. Our study revealed the effects of both quercetin and C. andromeda venom exposure on the mortality rate and the ultrastructure of T. canis third larva in comparison with control and albendazole-treated groups.

Pathological calcification in canine tendon-derived cells is modulated by extracellular ATP.

Tendon calcification is a commonly associated with degenerative tendinopathy of the Achilles tendons in dogs. It is characterised by the formation of calcific deposits and is refractory to treatment, often re-forming after surgical removal. Little is known about its pathogenesis and therefore the aims of this study were to develop an in vitro model of canine tendon calcification and use this model to investigate mechanisms driving calcification. Cells from the canine Achilles tendon were cultured with different calcifying media to establish which conditions were best able to induce specific, cell-mediated calcification. Once optimum calcification conditions had been established, the effect of ATP treatment on calcification was assessed. Results revealed that 2 mM di-sodium phosphate combined with 2 mM calcium chloride provided the optimum calcifying conditions, increasing calcium deposition and expression of osteogenic-related genes similar to those observed in tendon calcification in vivo. ATP treatment inhibited calcification in a dose-dependent manner, reducing calcium deposition and increasing cell viability, while osteogenic-related genes were no longer upregulated. In conclusion, the in vitro model of canine tendon calcification developed in this study provides the ability to study mechanisms driving tendon calcification, demonstrating that ATP plays a role in modulating tendon calcification that should be explored further in future studies.

Loading-related regulation of transcription factor EGR2/Krox-20 in bone cells is ERK1/2 protein-mediated and prostaglandin, Wnt signaling pathway-, and insulin-like growth factor-I axis-dependent.

Of the 1,328 genes revealed by microarray to be differentially regulated by disuse, or at 8 h following a single short period of osteogenic loading of the mouse tibia, analysis by predicting associated transcription factors from annotated affinities revealed the transcription factor EGR2/Krox-20 as being more closely associated with more pathways and functions than any other. Real time quantitative PCR confirmed up-regulation of Egr2 mRNA expression by loading of the tibia in vivo. In vitro studies where strain was applied to primary cultures of mouse tibia-derived osteoblastic cells and the osteoblast UMR106 cell line also showed up-regulation of Egr2 mRNA expression. In UMR106 cells, inhibition of ß1/ß3 integrin function had no effect on strain-related Egr2 expression, but it was inhibited by a COX2-selective antagonist and imitated by exogenous prostaglandin E2 (PGE2). This response to PGE(2) was mediated chiefly through the EP1 receptor and involved stimulation of PKC and attenuation by cAMP/PKA. Neither activators nor inhibitors of nitric oxide, estrogen signaling, or LiCl had any effect on Egr2 mRNA expression, but it was increased by both insulin-like growth factor-1 and high, but not low, dose parathyroid hormone and exogenous Wnt-3a. The increases by strain, PGE2, Wnt-3a, and phorbol 12-myristate 13-acetate were attenuated by inhibition of MEK-1. EGR2 appears to be involved in many of the signaling pathways that constitute early responses of bone cells to strain. These pathways all have multiple functions. Converting their strain-related responses into coherent "instructions" for adaptive (re)modeling is likely to depend upon their contextual activation, suppression, and interaction probably on more than one occasion.

Pro-Apoptotic Activity of Epi-Obtusane against Cervical Cancer: Nano Formulation, In Silico Molecular Docking, and Pharmacological Network Analysis.

Cancer is a major disease that threatens human health all over the world. Intervention and prevention in premalignant processes are successful ways to prevent cancer from striking. On the other hand, the marine ecosystem is a treasure storehouse of promising bioactive metabolites. The use of such marine products can be optimized by selecting a suitable nanocarrier. Therefore, epi-obtusane, previously isolated from Aplysia oculifera, was investigated for its potential anticancer effects toward cervical cancer through a series of in vitro assays in HeLa cells using the MTT assay method. Additionally, the sesquiterpene was encapsulated within a liposomal formulation (size = 130.8 ± 50.3, PDI = 0.462, zeta potential -12.3 ± 2.3), and the antiproliferative potential of epi-obtusane was investigated against the human cervical cancer cell line HeLa before and after encapsulation with liposomes. Epi-obtusane exhibited a potent effect against the HeLa cell line, while the formulated molecule with liposomes increased the in vitro antiproliferative activity. Additionally, cell cycle arrest analysis, as well as the apoptosis assay, performed via FITC-Annexin-V/propidium iodide double staining (flow cytofluorimetry), were carried out. The pharmacological network enabled us to deliver further insights into the mechanism of epi-obtusane, suggesting that STAT3 might be targeted by the compound. Moreover, molecular docking showed a comparable binding score of the isolated compound towards the STAT3 SH2 domain. The targets possess an anticancer effect through the endometrial cancer pathway, regulation of DNA templated transcription, and nitric oxide synthase, as mentioned by the KEGG and ShinyGo 7.1 databases.

Abdominal Hernia in Equine: Animal Level Risk Factors and Repair Using Polypropylene Mesh.

There is a paucity of information regarding the frequency and risk factors associated with the occurrence of an abdominal hernia in different equine species especially donkeys, and mules. In addition, the effectiveness of using polypropylene mesh for the surgical management of abdominal hernia in donkeys and mules is still unknown. The purpose of the present study was to evaluate the efficacy of using polypropylene mesh for the surgical treatment of abdominal hernia in horses, donkeys, and mules and to address the occurrence and risk factors associated with an abdominal hernia in the selected animal species. Based on clinical and ultrasound proof of abdominal hernia, 48 animals were included in the current study. A questionnaire was created to imply the assumed risk factors associated with occurence and clinical findings of abdominal hernia. Hernioplasty using polypropylene mesh was used for the surgical treatment. The abdominal hernia was prevalent (P < .05) in foals followed by adult horses, donkeys, and mules (25, 15, 6, and 2), respectively. Ages from 3 to 5 years exhibited more abdominal hernias in donkeys, mules, and horses (12.5%, 4.2%, and 31.3%, P < .05), respectively. In all studied animals, females were more prevalent than males (70.8%, n = 34 vs. 29.2%, n = 14) respectively. The complication of abdominal wall hernia was positively affected by the time to repair admitted cases (P = .000). The majority of the animals (36 cases) recovered without complications, however, 10 cases had mild difficulties such as suture abscess, wound infection, serous fluid accumulation, and hematoma postsurgery. In addition, two cases of postoperative recurrence were documented. The results herein indicated that polypropylene mesh hernioplasty may be an alternative to the treatment of equine abdominal hernia. Recognizing the potential risk factors associated with an abdominal hernia in different equine species may be helpful to construct the best preventive measures.

Mechanical loading enhances the anabolic effects of intermittent parathyroid hormone (1-34) on trabecular and cortical bone in mice.

The separate and combined effects of intermittent parathyroid hormone (iPTH) (1-34) and mechanical loading were assessed at trabecular and cortical sites of mouse long bones. Female C57BL/6 mice from 13 to 19 weeks of age were given daily injections of vehicle or PTH (1-34) at low (20 microg/kg/day), medium (40 microg/kg/day) or high (80 microg/kg/day) dose. For three alternate days per week during the last two weeks of this treatment, the tibiae and ulnae on one side were subjected to a single period of non-invasive, dynamic axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle). Two levels of peak load were used; one sufficient to engender an osteogenic response, and the other insufficient to do so. The whole tibiae and ulnae were analyzed post-mortem by micro-computed tomography with a resolution of 5 microm. Treatment with iPTH (1-34) modified bone structure in a dose- and time-dependent manner, which was particularly evident in the trabecular region of the proximal tibia. In the tibia, loading at a level sufficient by itself to stimulate osteogenesis produced an osteogenic response in the low-dose iPTH (1-34)-treated trabecular bone and in the proximal and middle cortical bone treated with all doses of iPTH (1-34). In the ulna, loading at a level that did not by itself stimulate osteogenesis was osteogenic at the distal site when combined with high-dose iPTH (1-34). At both levels of loading, there were synergistic effects in cortical bone volume of the proximal tibia and distal ulna between loading and high-dose iPTH (1-34). Images of fluorescently labelled bones confirmed that such synergism resulted from increases in both endosteal and periosteal bone formation. No woven bone was induced by iPTH (1-34) or either level of loading alone, whereas the combination of iPTH (1-34) and the "sufficient" level of loading stimulated woven bone formation on endosteal and periosteal surfaces of the proximal cortex in the tibiae. Together, these data suggest that in female C57BL/6 mice, under some but not all circumstances, mechanical loading exerts an osteogenic response with iPTH (1-34) in trabecular and cortical bone.

Molecular and Phenotypic Analysis of Hemolytic Aeromonas Strains Isolated from Food in Egypt Revealed Clinically Important Multidrug Resistance and Virulence Profiles.

The aim of this study was to determine the public health significance of hemolytic Aeromonas species isolated from 213 food samples in Egypt, based on their virulence and antimicrobial-resistance potential. We recovered 63 strains, isolated from fish, raw milk, karish cheeses, and ras cheese in 29 (31.18%) of 93, 10 (25.00%) of 40, 13 (32.50%) of 40, and 11 (27.50%) of 40 samples, respectively. The most prevalent virulence gene was alt (50.79%), followed by aerA (34.92%), asa1 (39.68%), ahh1 (20.63%), act (11.11%), and ast (3.17%). Thirteen strains screened in this study carried no hemolysin gene, but only the alt gene, and another eight hemolytic strains screened, carried no virulence gene. The virulence signatures " ahh1+ aerA" and " alt+ act," in which the genes interact synergistically to induce severe diarrhea, were detected in two and four strains, respectively. Most showed resistance to third-generation cephalosporins, aztreonam, and imipenem, which indicates the complexity of the ß-lactamase production in our hemolytic Aeromonas strains. Fourteen (22.22%) of 63 strains carried one or more antimicrobial-resistance markers, including the blaCTX-M, blaTEM, tet(A), tet(E), and intI1 genes, which were detected in 6.34, 3.17, 3.17, 4.76, and 14.28% of isolates, respectively. In conclusion, the majority of hemolytic Aeromonas strains isolated from the intestinal contents of healthy fish and naturally contaminated milk and cheeses were not commensal but had developed multidrug-resistance and virulence profiles, indicating an emerging potential health risk. Importantly, screening for certain hemolysin genes may not be reliable in predicting the pathogenic potential of Aeromonas species and, thereby, the safety of analyzed foods. Our findings indicate that specific criteria are required for the phenotypic and molecular analysis of Aeromonas species in food items, particularly those eaten without further treatment, to ensure their safety.

Team Approach Helps Patient Survive High-voltage Electric Burn.

A 20-year-old man was subjected to high-voltage electric burn, which was occupational. The patient was admitted to AlGalaa Military Medical Complex almost 2 weeks after the accident had happened. According to Lund and Browder's chart, the patient had a 40% total body surface area burn involving the upper limbs, anterior and posterior trunks, and the left thigh (third- and fourth-degree burns). The aim of this study was to stabilize the patient by conducting lifesaving operations in multiple scheduled sessions, bilateral below-elbow amputations, escharotomies, and excision of affected ribs and cartilages. A left latissimus dorsi flap used to cover the left side of the anterior chest wall. Skin grafting (split thickness, meshed 1:3) was done to cover the raw areas. Multiple aggressive operations by a multidisciplinary team saved the patient's life. The victim suffered a major injury and was handicapped, but he survived. It was not necessary to replace the excised ribs with prosthesis because of the preserved sternum. An electric burn poses a burden on many people in addition to the patients themselves.

Parathyroid hormone's enhancement of bones' osteogenic response to loading is affected by ageing in a dose- and time-dependent manner.

Decreased effectiveness of bones' adaptive response to mechanical loading contributes to age-related bone loss. In young mice, intermittent administration of parathyroid hormone (iPTH) at 20-80µg/kg/day interacts synergistically with artificially applied loading to increase bone mass. Here we report investigations on the effect of different doses and duration of iPTH treatment on mice whose osteogenic response to artificial loading is impaired by age. One group of aged, 19-month-old female C57BL/6 mice was given 0, 25, 50 or 100µg/kg/day iPTH for 4weeks. Histological and µCT analysis of their tibiae revealed potent iPTH dose-related increases in periosteally-enclosed area, cortical area and porosity with decreased cortical thickness. There was practically no effect on trabecular bone. Another group was given a submaximal dose of 50µg/kg/day iPTH or vehicle for 2 or 6weeks with loading of their right tibia three times per week for the final 2weeks. In the trabecular bone of these mice the loading-related increase in BV/TV was abrogated by iPTH primarily by reduction of the increase in trabecular number. In their cortical bone, iPTH treatment time-dependently increased cortical porosity. Loading partially reduced this effect. The osteogenic effects of iPTH and loading on periosteally-enclosed area and cortical area were additive but not synergistic. Thus in aged, unlike young mice, iPTH and loading appear to have separate effects. iPTH alone causes a marked increase in cortical porosity which loading reduces. Both iPTH and loading have positive effects on cortical periosteal bone formation but these are additive rather than synergistic.

Immunotoxicity of skin acid secretion produced by the sea slug Berthellina citrina in mice spleen: Histological and Immunohistochemical study.

Acid secretion containing sulfuric and hydrochloric acids is a fascinating defensive phenomenon within many groups of marine organisms. This study aimed to investigate the mice spleen histology and immunotoxicity using skin acid secretion (SAS) of the sea slug Berthellina citrina after oral administration. The spleen showed atrophy in the white pulp, decrease in the splenocytes density, megakaryocytes cytoplasmic degeneration as well as inflammatory cells infiltrations. The white and red pulp splenocytes number decreased time-dependently in the treated spleens. Additionally, the size of the megakaryocytes increased as compared with the control. The administration with SAS increased the number of the IgA(+) cells aggregation in the splenic red pulp. Furthermore, after 7days of the administration, large number of dispersed IgA(+) cells were distributed in splenic parenchyma. The IgA(+) cells numbers increased time-dependently as compared with those in the control. The aggregation sizes and number of the F4/80(+) cell in the splenic red pulp were increased. Furthermore the F4/80(+) cells numbers increased time-dependently as compared with those in the control. The UEAI(+) cells were found as free cells but not in aggregations in the control splenic red pulp. Contradictory to the number of IgA(+) cells and F4/80(+) cells the number of the UEAI(+) cells decreased time-dependently after administration with SAS. Hematologically, abnormal numbers of WBCs different cells were observed after administration with SAS. This study provides new insight about the toxicity of a marine extract may be used in natural products industry or medical applications.

Cytotoxic cembranoids from the Red Sea soft coral Sarcophyton glaucum.

One new cembrane diterpene, 2R,7R,8R-dihydroxydeepoxysarcophine (1), together with three known compounds, 7alpha,8beta-dihydroxydeepoxysarcophine (2), 7beta-acetoxy-8alpha-hydroxydeepoxysarcophine (3), and sarcophine (4), have been isolated from the Red Sea soft coral Sarcophyton glaucum. Their structures were determined using 1D and 2D NMR spectroscopy. 7beta-Acetoxy-8alpha-hydroxydeepoxysarcophine (3) exhibits cytotoxic activity against HepG2, HCT-116, and HeLa cells with IC50 values of 3.6, 2.3, and 6.7 microg/mL, respectively.

The mouse fibula as a suitable bone for the study of functional adaptation to mechanical loading.

Bones' functionally adaptive responses to mechanical loading can usefully be studied in the tibia by the application of loads between the knee and ankle in normal and genetically modified mice. Such loading also deforms the fibula. Our present study was designed to ascertain whether the fibula adapts to loading in a similar way to the tibia and could thus provide an additional bone in which to study functional adaptation. The right tibiae/fibulae in C57BL/6 mice were subjected to a single period of axial loading (40 cycles at 10 Hz with 10-second intervals between each cycle; approximately 7 min/day, 3 alternate days/week, 2 weeks). The left tibiae/fibulae were used as non-loaded, internal controls. Both left and right fibulae and tibiae were analyzed by micro-computed tomography at the levels of the mid-shaft of the fibula and 25% from its proximal and distal ends. We also investigated the effects of intermittent parathyroid hormone (iPTH) on the (re)modelling response to 2-weeks of loading and the effect of 2-consecutive days of loading on osteocytes' sclerostin expression. These in vivo experiments confirmed that the fibula showed similar loading-related (re)modelling responses to those previously documented in the tibia and similar synergistic increases in osteogenesis between loading and iPTH. The numbers of sclerostin-positive osteocytes at the proximal and middle fibulae were markedly decreased by loading. Collectively, these data suggest that the mouse fibula, as well as the tibia and ulna, is a useful bone in which to assess bone cells' early responses to mechanical loading and the adaptive (re)modelling that this engenders.