Photodynamic therapy: a new antimicrobial approach to infectious disease?

Photodynamic therapy (PDT) employs a non-toxic dye, termed a photosensitizer (PS), and low intensity visible light which, in the presence of oxygen, combine to produce cytotoxic species. PDT has the advantage of dual selectivity, in that the PS can be targeted to its destination cell or tissue and, in addition, the illumination can be spatially directed to the lesion. PDT has previously been used to kill pathogenic microorganisms in vitro, but its use to treat infections in animal models or patients has not, as yet, been much developed. It is known that Gram-(-) bacteria are resistant to PDT with many commonly used PS that will readily lead to phototoxicity in Gram-(+) species, and that PS bearing a cationic charge or the use of agents that increase the permeability of the outer membrane will increase the efficacy of killing Gram-(-) organisms. All the available evidence suggests that multi-antibiotic resistant strains are as easily killed by PDT as naive strains, and that bacteria will not readily develop resistance to PDT. Treatment of localized infections with PDT requires selectivity of the PS for microbes over host cells, delivery of the PS into the infected area and the ability to effectively illuminate the lesion. Recently, there have been reports of PDT used to treat infections in selected animal models and some clinical trials: mainly for viral lesions, but also for acne, gastric infection by Helicobacter pylori and brain abcesses. Possible future clinical applications include infections in wounds and burns, rapidly spreading and intractable soft-tissue infections and abscesses, infections in body cavities such as the mouth, ear, nasal sinus, bladder and stomach, and surface infections of the cornea and skin.     

Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism

L-Ascorbate catabolism involves reversible oxidation to DHA (dehydroascorbic acid), then irreversible oxidation or hydrolysis. The precursor-product relationships and the identity of several major DHA breakdown products remained unclear. In the presence of added H2O2, DHA underwent little hydrolysis to DKG (2,3-dioxo-L-gulonate). Instead, it yielded OxT (oxalyl L-threonate), cOxT (cyclic oxalyl L-threonate) and free oxalate (~6:1:1), essentially simultaneously, suggesting that all three product classes independently arose from one reactive intermediate, proposed to be cyclic-2,3-O-oxalyl-L-threonolactone. Only with plant apoplastic esterases present were the esters significant precursors of free oxalate. Without added H2O2, DHA was slowly hydrolysed to DKG. Downstream of DKG was a singly ionized dicarboxy compound (suggested to be 2-carboxy-L-xylonolactone plus 2-carboxy-L-lyxonolactone), which reversibly de-lactonized to a dianionic carboxypentonate. Formation of these lactones and acid was minimized by the presence of residual unreacted ascorbate. In vivo, the putative 2-carboxy-L-pentonolactones were relatively stable. We propose that DHA is a branch-point in ascorbate catabolism, being either oxidized to oxalate and its esters or hydrolysed to DKG and downstream carboxypentonates. The oxidation/hydrolysis ratio is governed by reactive oxygen species status. In vivo, oxalyl esters are enzymatically hydrolysed, but the carboxypentonates are stable. The biological roles of these ascorbate metabolites invite future exploration.     

Study on impact of habitat degradation on proximate composition and amino acid profile of Indian major carps from different habitats

This investigation is aimed to study an impact of habitat degradation on proximate composition and amino acid (AAs) profile of Catla catla, Labeo rohita and Cirrhinus mrigala collected from polluted, non-polluted area (upstream) and a commercial fish farm. The amino acid profile was estimated by the amino acid analyzer. C. catla collected from the polluted environment had highest lipid, protein and ash contents (12.04?±?0.01, 13.45?±?0.01 and 0.93?±?0.03%, respectively). The high protein content (14.73?±?0.01 and 14.12?±?0. 01%) was recorded in C. catla procured from non-polluted (upstream) wild habitat of River Chenab and controlled commercial fish farm. Farmed fish species showed comparatively higher moisture contents followed by upstream and polluted area fishes. C. mrigala showed significant differences in amino acid and proximate composition collected from a polluted site of the river Chenab. C. catla collected from non-polluted site of the river showed an excellent nutrient profile, followed by L. rohita (wild and farmed) and C. mrigala (polluted area), respectively. All fishes from the polluted areas of the River Chenab indicated a significant decrease in the concentration of some AAs when compared to farmed and wild (upstream) major carps. Omitting of some important AAs was also observed in the meat of fish harvested from polluted habitat of this river. C. mrigala and L. rohita exhibited a significant increase in the concentration of some of non-essential amino acids such as cysteine in their meat. The results indicated that wild fish (upstream) and farmed fish species had highest protein contents and amino acid profile and hence appeared to be the best for human consumption. The proximate composition and AAs profiles of fish harvested from the polluted area of the river clearly indicated that efforts shall be made for the restoration of habitat to continue the requirement of high quality fish meat at a low cost to the human population.     

Peptide-induced inflammatory increase in vascular permeability improves photosensitizer delivery and intersubject photodynamic treatment efficacy

Photodynamic therapy (PDT) treatment can exhibit high intersubject variability due to the inherent differences in drug delivery within the tissue to be treated. In this study, the increased perfusion of the lipid-associated photosensitizer verteporfin was studied using substance P, a peptide known to increase vascular permeability. The transvascular permeability coefficient was quantified before and after administration of substance P, and the mean value increased from 0.026 to 0.043 microm/s with the induced inflammation. Correspondingly, there was a 40-50% increase in uptake of verteporfin in the tumor parenchyma in tumors injected with substance P compared to those without. This increased drug uptake resulted in a modest increase in tumor doubling time from 4 days with regular PDT to 6.2 days with substance P and PDT. There was also a significant reduction in the interindividual variability in with substance P plus PDT from 64% to 13%. The resulting treatment was therefore more effective and there was less variability in dose between subjects.     

Photodynamic therapy for cutaneous leishmaniasis: the effectiveness of topical phenothiaziniums in parasite eradication and Th1 immune response stimulation.

Photodynamic therapy (PDT) is emerging as a therapeutic modality in the clinical management of cutaneous leishmaniasis (CL). The efficacy of PDT against CL has been demonstrated previously with aminolevulinic acid, although the prolonged terms of therapy were less than ideal, and the search for new photosensitizers (PS) is ongoing. However, phenothiaziniums have demonstrated high parasiticidal effects in vitro. The subject of our investigation is the in vivo activity of two PS, 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride (EtNBSe) and (3,7-Bis(N,N-dibutylamino) phenothiazinium bromide (PPA904). The results of our comparative analysis of the efficacy of these two phenothiazinium analogues demonstrated a high antiparasitic activity of EtNBSe in vitro, and the higher efficacy of PPA904 in a mouse model of CL. The kinetics of photodestruction are different in parasite and mammalian cells, and with both dyes, the macrophages are more susceptible to photodynamic effects than L. major parasites. As the number of parasites in the lesions undergoes a biphasic change, temporarily increasing on days 2-4 and decreasing on days 5-7, more than one treatment is required within an interval of 5 to 7 days. We have also shown that PPA904-PDT can provide an immunomodulating, dose-dependent efflux on IL-12p70 production. This mechanism could be responsible for promoting a more rapid healing in PPA904-PDT treated mice. Our initial data indicate that phenothiaziniums exhibit a high parasiticidal effect in vivo against CL; this finding may be of use in establishing curative PDT regimens for future clinical trials.     

Real-time fluorescence monitoring of phenothiazinium photosensitizers and their anti-mycobacterial photodynamic activity against Mycobacterium bovis BCG in in vitro and in vivo models of localized infection.

An objective was to explore the photodynamic activity of two cationic photosensitizers (PS) (benzo[a]phenothiazinium chloride and benzo[a]phenoselenazinium chloride) against Mycobacterium bovis BCG both in vitro and in a murine model of BCG-granuloma. The hypothesis being tested in this study was that cationic molecules could best interact with the negatively charged membrane of BCG as a model for mycobacterial infection. Cells in culture were incubated with various concentrations of PS and subsequently illuminated using a 635 nm diode laser. Dark- and light-induced killing profiles were generated as a function of fluence and dye concentration. In vivo, local injection of the PS into subcutaneous Mycobacterium-induced granuloma sites in murine model was followed by red light illumination of the same area. A special microscope was fabricated for real-time in vivo fluorescent microscopy to monitor EtNBS delivery to subcutaneous murine granulomata. Both PS demonstrated good in vitro antimycobacterial photodynamic activity with varying degrees of toxicity under dark conditions. Real time in vivo monitoring of benzophenothiazine chloride in the mouse model indicated that this fluorescent photosensitizer was delivered rapidly to the subcutaneous granuloma site. In vivo, photosensitizer specific dark- and photo-toxicities depended on the structure, concentration of the photosensitizer and the light dose utilized. Cationic phenothiazine photosensitizers are promising candidates for use in anti-mycobacterial PDT for localized diseases such as cutaneous and pulmonary granulomata.     

Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea,Prokaryotes and Eukaryotes

Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.     

Effects of noradrenaline and metaraminol on isolated aortic strips and auricles obtained from untreated and reserpine-treated rabbits

The isolated aortic strips of the untreated rabbits responded to noradrenaline in concentrations ranging from 20 to 160 ng/mL, whereas similar preparations from reserpine-treated animals responded to lower concentrations (from 2.5 to 10 ng/mL) and larger effects were produced by concentrations ranging from 20 to 160 ng/mL. Comparatively stronger concentrations (from 0.4 to 1.6 microgram/mL) of metaraminol were required to produce suitable contractile responses, which were not modified by reserpine pretreatment. On the isolated auricles obtained from untreated rabbits, noradrenaline was used in concentrations of 20 and 80 ng/mL, but these concentrations were reduced to 2.5 and 10 ngmL in the case of tissues obtained from reserpinized rabbits. Comparatively stronger (0.2 and 0.8 microgram/mL) concentrations of metaraminol were required to produce a stimulant effect on this preparation, and the sensitivity was not altered by reserpine pretreatment. These observations lead to the conclusions that though reserpine pretreatment produces a well-marked increase in the sensitivity of isolated rabbit aortic strips and auricles to noradrenaline, it does not modify the sensitivity of these preparations to metaraminol. Reserpine was used in 0.3 mg/kg doses injected subcutaneously for 3 successive days.     

Molecular Modeling and docking of Wheat Hydroquinone Glucosyl transferase by using Hydroquinone,Phenyl phosphorodiamate and n-(n butyl) Phosphorothiocic Triamide as Inhibitors

In agriculture high urease activity during urea fertilization causes substantial environmental and economical problems by releasing abnormally large amount of ammonia into the atmosphere which leads to plant damage as well as ammonia toxicity. All over the world, urea is the most widely applied nitrogen fertilizer. Due to the action of enzyme urease; urea nitrogen is lost as volatile ammonia. For efficient use of nitrogen fertilizer, urease inhibitor along with the urea fertilizer is one of the best promising strategies. Urease inhibitors also provide an insight in understanding the mechanism of enzyme catalyzed reaction, the role of various amino acids in catalytic activity present at the active site of enzyme and the importance of nickel to this metallo enzyme. By keeping it in view, the present study was designed to dock three urease inhibitors namely Hydroquinone (HQ), Phenyl Phosphorodiamate (PPD) and N-(n-butyl) Phosphorothiocic triamide (NBPT) against Hydroquinone glucosyltransferase using molecular docking approach. The 3D structure of Hydroquinone glucosyltransferase was predicted using homology modeling approach and quality of the structure was assured using Ramachandran plot. This study revealed important interactions among the urease inhibitors and Hydroquinone glucosyltransferase. Thus, it can be inferred that these inhibitors may serve as future anti toxic constituent against plant toxins.