Purification and Characterization of Antibacterial Activity against Phytopathogenic Bacteria in Culture Fluids from Ganoderma lucidum

Previous studies of Ganoderma lucidum have focused on its medicinal applications. Limited information is available about its antibacterial activity against plant pathogens. Thus, the goal of this study was to purify and characterize the antibacterial activity against plant pathogenic bacteria from culture fluids of G. lucidum. The nature of the bioactive components was determined using heat boiling, organic solvents, dialysis tubing, gel exclusion chromatography (GEC), proteinase sensitivity, HPLC, HPLC-APCI-MS, and GC-MS. The bioactive compounds were neither lipid, based on their solubility, nor proteic in nature, based on proteinase digestion and heat stability. The putative-bioactive polysaccharides have molecular weights that range from 3500 to 4500 Daltons as determined by dialysis tubing, GEC and APCI-MS analysis. The composition of the antibacterial compounds was determined by GC-MS. This is the first report of small polysaccharides produced by G. lucidum with activity against bacterial plant pathogens.

A reassessment of several erstwhile methods for isolating DNA fragments from agarose gels

Molecular biology research often requires extraction of DNA fragments from agarose gels. In the past decades, there have been many methods developed for this purpose. Currently most researchers, especially novices, use commercial kits for this extraction, although these kits cost money and the procedures involved are not necessarily easier than some erstwhile methods. We herein reintroduce and reassess several simple and cost-free older methods. One method involves excising a slice of the gel containing the DNA fragment, followed by a thaw-and-freeze procedure to release the DNA from the gel slice into the gel-making buffer. The second method involves a dialysis tubing and requires electroelution of the DNA from the gel slice in the tubing. The third one is to centrifuge the gel slice to release the DNA. The fourth method requires electro-transfer of the DNA from the gel into a filter paper, while the fifth one includes either allowing the DNA in the slice to be dissolved into a buffer or dissolving the DNA-containing gel slice, followed by DNA precipitation with ethanol or isopropanol. The strengths and weaknesses of these methods are discussed to assist researchers in making their choice. We also point out that some of the end uses of the DNA fragment in the agarose gel may not actually require extraction of the DNA. For instance, a tiny DNA-containing gel block or filter paper can be directly used as the template in a nested or semi-nested polymerase chain reaction to preliminarily determine the identity of the DNA fragment.

Possibilities of microemulsion application in rectal administration of indomethacin

Rectal administration is a suitable route of administration for drugs that are either very irritating to the intestine (e.g., indomethacin) or are more effective when the first-pass effect in the liver is circumvented. Microemulsions are a tool for the improvement of penetration of sparingly soluble drugs. They are mainly used in topical and transdermal drug delivery. However, they find application also in other routes of administration, mainly due to their ability to solubilize sparingly soluble drugs. The selection of a suppository base depends on the physical properties of the drug. The study focused on evaluating the effect of the microemulsion as the solubilizer of sparingly soluble indomethacin in hydrophilic and lipophilic suppository bases compared with Polysorbate 80 as the excipient contained in the microemulsion. The reference suppositories were prepared by the traditional moulding technique from Adeps solidus or Macrogol suppository base without the previous drug solubilization. The microemulsion-based suppositories were prepared after the initial solubilization of the drug in the microemulsion or Polysorbate 80, followed by the addition of suppository base to maintain the same drug/solubilizer ratio. The suppositories were tested for softening time, hardness, and uniformity of mass. The dissolution test was performed using the dialysis tubing method in the basket apparatus. The amount of indomethacin released into the dissolution medium was determined spectrophotometrically at 320 nm. The results indicate that solubilization of indomethacin in the microemulsion had a positive effect on in vitro drug release but not as significant as in the case of Polysorbate 80 used alone. The enhancement ratio for Polysorbate 80 in Adeps suppositories was 2.9, for the microemulsion in Adeps suppositories was 1.1, and for Polysorbate 80 in Macrogol suppositories was 7.4 after 3 hours. The test of uniformity of mass had shown that all suppositories (reference, solubilizer-containing) are within the permitted limits. The softening time was reduced by adding the solubilizer to each type of suppository base.

Synthesis and Characterization of a Fullerenol Derivative for Potential Biological Applications

Several biological barriers are generally responsible for the limited delivery of cargoes at the cellular level. Fullerenols have unique structural features and possess suitable properties for interaction with the cells. This study aimed to synthesize and characterize a fullerenol derivative with desirable characteristics (size, charge, functionality) to develop cell penetration vehicles. Fullerenol was synthesized from fullerene (C60) solubilized in toluene, followed by hydroxylation with hydrogen peroxide and tetra-n-butylammonium hydroxide (TBAH) as a phase transfer catalyst. The obtained product was purified by a Florisil chromatography column (water as the eluent), followed by dialysis (cellulose membrane dialysis tubing) and freeze-drying (yield 66%). Subsequently, a silane coupling agent was conjugated on the fullerenol surface to render free amine functional groups for further covalent functionalization with other molecules. Characterization via UV–VIS, FTIR-ATR, Raman, DLS, and SEM techniques was conducted to evaluate the composition, size, morphology, surface functionality, and structural properties. We are currently working on the conjugation of the potent cell-penetrating agents Buforin II (BUFII) and the Outer Membrane Protein A (OmpA) on the surface of the fullerenol to estimate whether cell penetration and endosome escape are improved concerning conventional polymeric vehicles and our previous developments with iron oxide nanoparticles.

Heparin-free hemodialysis

Hemodialysis is the most widely applied renal replacement therapy. Due to the fact that the hemocyte - dialyzer contact leads to the activation of the coagulation pathway, adequate anticoagulation to provide fluent blood flow is crucial. Since the standard parenteral use of heparin is not free from complications and may increase already raised bleeding risk in renal patients, the alternative methods of performing hemodialysis, including heparin-free procedures, are being investigated. These include the usage of anticoagulants regionally in the extracorporeal circuit or repeated saline flushes or other substituting compounds. Citrate module has already become the standard anticoagulant in intensive care for patients on continuous hemofiltration. Its usage in intermittent dialysis program requires some protocol modifications, but it is a valuable input in the development of heparin-free strategies. The other approach that allows reduced heparin usage is the use of an airless dialysis tubing system. Amongst coated dialyzer membranes, the one with heparinized hydrogel polyacrylonitrile was perceived as a significant step forward. Despite the fact that innovative strategies may turn out to be time and resource consuming and not always free of side-effects, they are worth investigating.

Mechanical Properties of Arterial Elastin With Water Loss

Elastin is a peculiar elastomer in that it requires water to maintain resilience, and its mechanical properties are closely associated with the immediate aqueous environment. The bulk, extra- and intrafibrillar water plays important roles in both elastic and viscoelastic properties of elastin. In this study, a two-stage liquid–vapor method was developed to investigate the effects of water loss on the mechanical properties of porcine aortic elastin. The tissue samples started in a phosphate-buffered saline (PBS) solution at their fully hydrated condition, with a gravimetric water content of 370±36%. The hydration level was reduced by enclosing the tissue in dialysis tubing and submerging it in polyethylene glycol (PEG) solution at concentrations of 10%, 20%, 30%, and 45% w/v, which reduced the water content of the samples to 258±34%, 224±20%, 109±9%, and 58±3%, respectively. The samples were then transferred to a humidity chamber to maintain the hydration level while the samples underwent equi-biaxial tensile and stress relaxation tests. The concentration of 10% PEG treatment induced insignificant changes in tissue dimensions and stiffness, indicating that the removal of bulk water has less effect on elastin. Significant increases in tangent modulus were observed after 20% and 30% PEG treatment due to the decreased presence of extrafibrillar water. Elastin treated with 45% PEG shows a very rigid behavior as most of the extrafibrillar water is eliminated. These results suggest that extrafibrillar water is crucial for elastin to maintain its elastic behavior. It was also observed that the anisotropy of elastin tends to decrease with water loss. An increase in stress relaxation was observed for elastin treated with 30% PEG, indicating a more viscous behavior of elastin when the amount of extrafibrillar water is significantly reduced. Results from this study shed light on the close association between the bulk, extra- and intrafibrillar water pools and the mechanics of elastin.

Cortical Blindness and Altered Mental Status following Routine Hemodialysis, a Case of Iatrogenic Cerebral Air Embolism

Cerebral air embolism is a known complication from a myriad of iatrogenic causes. This case describes a 60-year-old female presenting from hemodialysis with altered mental status, bilateral homonymous hemianopia, and repetitive speech. A noncontrast head CT revealed air in the vein of Galen and the deep cerebral veins of the left thalamus and occipital sulcus, a complication from air being introduced into the patient via improper flushing of dialysis tubing. The retrograde flow of air bubbles in the upright patient during dialysis was likely responsible for the air embolus lodging in the cerebral vasculature. This patient was transferred to receive hyperbaric therapy, whereupon the patient survived with residual attention and spatial deficits.

(1→3)-β–d-Glucan and Galactomannan for Differentiating Chemical “Black Particles” and Fungal Particles Inside Peritoneal Dialysis Tubing

BackgroundAseptic, sheet-like foreign bodies observed inside Tenckhoff (TK) catheter lumens (referred to as “black particles”) are, on gross morphology, hardly distinguishable from fungal colonization because these contaminants adhere tightly to the catheter. Detection of fungal cell wall components using (1→3)-β–d-glucan (BG) and galactomannan index (GMI) might be an alternative method for differentiating the particles.MethodsForeign particles retrieved from TK catheters in 19 peritoneal dialysis patients were examined microscopically and cultured for fungi and bacteria. Simultaneously, a Fungitell test (Associates of Cape Cod, Falmouth, MA, USA) and a Platelia Aspergillus ELISA assay (Bio-Rad Laboratories, Marnes-La-Coquette, France) were used to test the spent dialysate for BG and GMI respectively.ResultsOf the 19 patients, 9 had aseptic black particles and 10 had fungal particles in their tubing. The fungal particles looked grainy, were tightly bound to the catheter, and appeared more “colorful” than the black particles, which looked sheet-like and could easily be removed by milking the tubing. Compared with effluent from patients having aseptic particles, effluent from patients with fungal particles had significantly higher levels of BG (501 ± 70 pg/mL vs. 46 ± 10 pg/mL) and GMI (10.98 ± 2.17 vs. 0.25 ± 0.05). Most of the fungi that formed colonies inside the catheter lumen were molds not usually found in clinical practice, but likely from water or soil, suggesting environmental contamination. Interestingly, in all 10 patients with fungal colonization, visualization of black particles preceded a peritonitis episode and TK catheter removal by approximately 1–3 weeks; in patients with aseptic particles, a 17-week onset to peritonitis was observed.ConclusionsIn all patients with particle-coated peritoneal dialysis tubing, spent dialysate should be screened for BG and GMI. Manipulation of the TK catheter by squeezing, hard flushing, or even brushing to dislodge black particles should be avoided. Replacement of the TK catheter should be suspended until a cause for the particles is determined.