The dysbiosis signature of Fusobacterium nucleatum in colorectal cancer-cause or consequences? A systematic review.

Colorectal cancer (CRC) is the third most common cause of cancer globally and the fourth attributable cause of mortality and morbidity due to cancer. An emerging factor contributing to CRC is the gut microbiota and the cellular changes associated with it. Further insights on this may help in the prevention, diagnosis and new therapeutic approaches to colorectal cancer. In most cases of CRC, genetic factors appear to contribute less to its aetiology than environmental and epigenetic factors; therefore, it may be important to investigate these environmental factors, their effects, and the mechanisms that may contribute to this cancer. The gut microbiota has recently been highlighted as a potential risk factor that may affect the structural components of the tumor microenvironment, as well as free radical and enzymatic metabolites directly, or indirectly. Many studies have reported changes in the gut microbiota of patients with colorectal cancer. What is controversial is whether the cancer is the cause or consequence of the change in the microbiota. There is strong evidence supporting both possibilities. The presence of Fusobacterium nucleatum in human colorectal specimens has been demonstrated by RNA-sequencing. F. nucleatum has been shown to express high levels of virulence factors such as FadA, Fap2 and MORN2 proteins. Our review of the published data suggest that F. nucleatum may be a prognostic biomarker of CRC risk, and hence raises the potential of antibiotic treatment of F. nucleatum for the prevention of CRC.

Bacterial staphylokinase as a promising third-generation drug in the treatment for vascular occlusion.

Vascular occlusion is one of the major causes of mortality and morbidity. Blood vessel blockage can lead to thrombotic complications such as myocardial infarction, stroke, deep venous thrombosis, peripheral occlusive disease, and pulmonary embolism. Thrombolytic therapy currently aims to rectify this through the administration of recombinant tissue plasminogen activator. Research is underway to design an ideal thrombolytic drug with the lowest risk. Despite the potent clot lysis achievable using approved thrombolytic drugs such as alteplase, reteplase, streptokinase, tenecteplase, and some other fibrinolytic agents, there are some drawbacks, such as high production cost, systemic bleeding, intracranial hemorrhage, vessel re-occlusion by platelet-rich and retracted secondary clots, and non-fibrin specificity. In comparison, bacterial staphylokinase, is a new, small-size plasminogen activator, unlike bacterial streptokinase, it hinders the systemic degradation of fibrinogen and reduces the risk of severe hemorrhage. A fibrin-bound plasmin-staphylokinase complex shows high resistance to a2-antiplasmin-related inhibition. Staphylokinase has the potential to be considered as a promising thrombolytic agent with properties of cost-effective production and the least side effects.

Designing a multifunctional staphylokinase variant (SAK-2RGD-TTI) with appropriate thrombolytic activity in vitro.

OBJECTIVE: Thrombin, platelets, and plasmin are three key factors involved in hemostasis and thrombolysis. Thrombolytic therapy with clinically approved drugs is often followed by recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, new constructs were designed for the expression of recombinant staphylokinase (rSAK) and also a fusion protein composed of staphylokinase, 20 amino acids containing 2 RGD followed by tsetse thrombin Inhibitor (SAK-2RGD-TTI) in Pichia pastoris. RESULT: Modeling the tertiary structure of SAK-2RGD-TTI showed that the linker containing RGD and TTI did not interfere with proper folding of SAK. In laboratory testing, the purified SAK-2RGD-TTI (420 µg/mL) dissolved an average of 45% of the blood clot. The activity of the SAK-2RGD-TTI was also confirmed in various tests including human plasminogen activation assay, fibrin clot lysis assay, well diffusion method, activated partial thromboplastin time and platelet rich clot lysis assay. CONCLUSION: Our findings suggest that SAK-2RGD-TTI has improved therapeutic properties preventing reocclussion. It further confirms that it is practicable to assemble and produce a hybrid multifunctional protein that targets hemostatic process at various stages.

High-level expression of a biologically active staphylokinase in Pichia pastoris.

Staphylokinase (SAK) as the third generation thrombolytic molecule is a promising agent for the treatment of thrombosis. SAK variant of SAKфC was expressed in Pichia pastoris strains KM71H and GS115. The codon adaptation index of SAK was improved from 0.75 to 0.89. The expression of recombinant SAK (rSAK) reached to its maximum (310 mg/L of the culture medium) after 48-hr stimulation with 3% methanol and remained steady until day 5. The maximum activity of the enzyme was at pH 8.6 and 37°C. It was highly active at temperatures 20-37°C and pH ranges of 6.8-9 (relative residual activity more than 80%). It was determined that rSAK was 73.8% of the total proteins secreted by P. pastoris KM71H into the culture media. The specific activities of rSAK were measured as 9,002 and 21,042 U/mg for the nonpurified and purified proteins, respectively. The quantity of the purified protein (>99% purity) was 720 µg/mL with a purification factor of 2.34. Western blot analysis showed two bands of nearly 22 and 18.6 kDa. It was concluded that P. pastoris is a proper host for expression of biologically active and endotoxin-free rSAK due to its high expression and low protein impurity in culture supernatant.

The Safety of Schools Based on Heavy Metal Concentrations in Classrooms' Dust: A Systematic Review and Meta-Analysis.

BACKGROUND: Classroom is where children spend much of their time in; this study aimed to identify the concentration of heavy metals in the classroom dust based on the results of various studies in the world using the published data up to years 2018. METHODS: Fifteen studies were selected for the study according to the inclusion and exclusion criteria. The mean concentration of 11 heavy metals including arsenic, barium, cadmium, cobalt, chromium, copper, iron, lead, manganese, nickel, and zinc was extracted. RESULTS: The highest mean concentration of heavy metal (mg/kg) in classroom dust was related to iron (3904.7, 95%CI: 3657.1-8154.3), zinc (429.9, 95%CI: 182.8-677.1) and barium (419.2, 95%CI: 274.7-253.7), respectively. Subgroup analysis showed the maximum concentration (mg/kg) of iron in Iran (16945.5), zinc in Hong Kong (2293.5), barium in China (979.8), manganese in Iran (288.9), lead in Iran (258.8), chromium in Ghana (381.3), copper in Hong Kong (274.4), nickel in Iran (50.1), cobalt in China(43.4), arsenic in China(13.7) and cadmium in Hong Kong(8.7). CONCLUSION: Even safe and healthy classrooms can threaten children's health by heavy metals. These metals are important since they are naturally found throughout the earth's crust, accumulate in the food chain and contaminate drinking water as well as alloys in school equipment.

Protective effects of isatin and its synthetic derivatives against iron, copper and lead toxicity.

INTRODUCTION: While some metals are required for physiological functions in the form of essential trace elements, they can cause toxicity in the excessive concentrations. Chelation therapy was used to reduce the adverse effects of acute and chronic poisoning by metals. Isatin derivatives form complexes with copper ions indicating that they may have protective activity against metal overload. METHOD: In this study, four compounds (isatin and three isatin-derivatives Mj1, TR and Mk1) were evaluated for drug-likeliness. Then their potency inhibiting cell proliferation was determined in HEK293 cell culture assay. Finally, IC50 values for lead, copper, and iron was evaluated in the absence and also the presence of isatin and its derivatives. RESULTS: Isatin and its derivatives used in this study complied with the Lipinski criteria for drug-likeliness. The greatest difference between the IC50 values and the non-toxic dose was obtained for TR and Mj1, respectively. Pretreatment with the Mj1 increased the IC50 values for lead, iron, and copper, by 2.1, 1.7 and 1.7 times, respectively. At non-toxic dose, TR has only increased the IC50 values for lead and copper by 1.4 and 1.3 times without affecting iron cytotoxicity. Mk1 increased the IC50 values for lead, copper, and iron by 1.3, 1.8 and 1.7 times, respectively. CONCLUSIONS: Mj1 is suggested as a lead compound for developing therapeutic agents for lead (Pb) toxicity and Mk1 for copper and iron.

Comparison of expression optimization of new derivative of staphylokinase (SAK-2RGD-TTI) with the rSAK.

Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood-clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20-amino acid linker with 2 RGD (2 × arginine-glycine-aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut+ strain and KM71H as a Muts strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72-hr stimulation with 2.5% methanol and remained steady for 3-4 days. The highest expression was obtained at the range of 2-3% methanol. The SAK-2RGD-TT (relative activity >82%) was more active at 25-37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7-9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK-2RGD-TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK-2RGD-TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK-2RGD-TTI with high activity without requiring a complicated purification procedure.

Antioxidant protection of bulk fish oils by dispersed sugars and polyhydric alcohols.

Selected sugars (fructose, sucrose, or raffinose) and polyhydric alcohols (sorbitol or mannitol) were equilibrated directly with bulk fish oil (10% by weight, excess) and exposed to fluorescent lighting (2550 Lx) for 24 h at 5 degrees C. Data for room temperature-equilibrated samples revealed that polyols functioned as antioxidants in fish oil. Increased times and temperatures of equilibration (to 90-110 degrees C, 1-2 mmHg, to 2 h) greatly enhanced the antioxidant activity of polyols in fish oil exposed to light. Under accelerated oxidation conditions (60 degrees C) in the dark, dispersed sorbitol in bulk fish oil greatly suppressed the peroxide value, primarily by chelating transition metals, while fructose showed a limited antioxidant activity. Sugars with a lower molecular weight and smaller numbers of equatorial OH groups exhibited a higher rate of permeation of sugars into fish oil triacylglycerols and hence rendered greater antioxidant activities. The treatment of bulk fish oils with polyols and then using the oils in the preparation of emulsions greatly reduced their antioxidant activities as compared to those observed for treated bulk oils. The introduction of polyols dissolved in propylene glycol into bulk fish oils at 90 degrees C (0.025% polyol, 0.25 h of equilibration) provided a similar antioxidant activity to that imparted by the introduction of polyols into the oil by equilibrating excess polyols (10% by weight) with them at 90-110 degrees C for 2 h. However, regardless of the method of the introduction of polyols to bulk fish oil, an elevated temperature (90 degrees C) exposure during fish oil treatment was required to induce a notable antioxidant activity.

Characterization of the antioxidant activity of sugars and polyhydric alcohols in fish oil emulsions.

Polyols have been incorporated into fish oil emulsions as a means for the inhibition of lipid oxidation and suppression of fishy flavor. However, the role of sugars and polyhydric alcohols as antioxidants has not been clearly established. Selected polyols were evaluated for their performance as antioxidants and modifiers of oxidation pathways in a model system. Oil/water (O/W) emulsions were prepared with freshly steam-deodorized menhaden oil. A layer of emulsion in aluminum pans held at 5 degrees C was exposed to 2550 lx fluorescent lights for 24 h before peroxide values and volatile flavor compounds were analyzed by GC headspace entrainment procedure. Antioxidant activity was confirmed for fructose, sucrose, raffinose, sorbitol, or mannitol when incorporated at 16% of the aqueous phase into model fish oil-in-water emulsions. Peroxide values were suppressed 10-18% in treated samples compared to control samples. Viscosity data did not exclude possible contributions from a restricted oxygen diffusion mechanism in the antioxidant activity, but revealed that emulsion viscosity did not govern fish oil oxidation rates. Combining polyols with phenolic antioxidants (alpha-tocopherol, BHT, or TBHQ) frequently diminished the antioxidant activity compared to that for individual phenolic antioxidants, which was interpreted as indicating that the H-donating activity of phenolic antioxidants was hindered by the H-bonding activity of polyols. A viscosity-based inhibition of the retroaldol conversion of (E,Z)-2,6-nonadienal to (Z)-4-heptenal with a high fructose concentration (67%) was attributed to a restriction of molecular mobility of reactants, but the conversion was only slightly inhibited by the concentration of fructose (16%) used in experimental emulsions. The data supported a hypothesis that either or both free radical scavenging and transition state metal chelation activities were provided by polyols in fish oil emulsions. Also, polyols retarded the water-requiring retroaldol decomposition of (E,Z)-2,6-nonadienal to (Z)-4-heptenal in the model systems and the reaction may be involved in some suppression of fishy flavors in emulsions.

Role of continuous phase protein on the oxidative stability of fish oil-in-water emulsions.

Whey protein isolate (WPI), soy protein isolate (SPI), and sodium caseinate (CAS) can inhibit lipid oxidation when they produce a positive charge at the interface of emulsion droplets. However, when proteins are used to stabilize oil-in-water emulsions, only a fraction of them actually absorb to the emulsion droplets, with the rest remaining in the continuous phase. The impact of these continuous phase proteins on the oxidative stability of protein-stabilized emulsions is not well understood. WPI-stabilized menhaden oil-in-water emulsions were prepared by high-pressure homogenization. In some experiments WPI was removed from the continuous phase of the emulsions through repeated centrifugation and resuspension of the emulsion droplets (washed emulsion). Unwashed emulsions were more oxidatively stable than washed emulsions at pH 7.0, suggesting that continuous phase proteins were antioxidative. The oxidative stability of emulsions containing different kinds of protein in the continuous phase decreased in the order SPI > CAS > WPI, as determined by both hydroperoxide and headspace propanal formation. Iron-binding studies showed that the chelating ability of the proteins decreased in the order CAS > SPI > WPI. The free sulfhydryls of both WPI and SPI were involved in their antioxidant activity. This research shows that continuous phase proteins could be an effective means of protecting omega-3 fatty acids from oxidative deterioration.

Chemical and sensory analysis of strawberry flavoured yogurt supplemented with an algae oil emulsion.

A yogurt mix (2 g fat and 17g solids/100 g) was supplemented with an algae oil emulsion to provide 500 mg omega-3 fatty acids per 272 g serving of yogurt white mass. The emulsion was added to the yogurt mix either before or after the homogenization step and prior to pasteurization. It was then flavoured with a strawberry fruit base and fermented and stored for up to three weeks. The oxidative deterioration of the products was determined by hydroperoxide measurements and by trained and consumer sensory evaluations. The hydroperoxide content of the supplemented yogurts increased over the storage treatment and was unaffected by the stage of addition. The trained panel could distinguish a stronger fishy flavour in both of the supplemented yogurts after 22 days storage, but the consumer panel rated both control and supplemented samples similarly, as 'moderately liked'.