Tulbaghia violacea (Harv) Exerts its Antifungal Activity by Reducing Ergosterol Production in Aspergillus flavus.

Opportunistic infections in immunosuppressed patients have led to an increase in fungal infections, with Aspergillus being one of the main causative agents. Medicinal plants exhibiting antifungal activity have the potential to be used as chemotherapeutic agents. However, often their mechanisms of action are not fully researched. Tulbaghia violacea exhibits antifungal activity towards Candida, Aspergillus flavus and Aspergillus parasiticus but its mode of action has only recently begun to be investigated. This study aimed to ascertain the effect of T. violacea rhizome extracts on ergosterol production in A. flavus and the mechanism of inhibition. The MIC of a T. violacea rhizome extract against A. flavus was first determined, using a broth dilution assay, to be 15 mg/ml. Thereafter, the culture was subjected to sub-inhibitory concentrations of the extract before sterol intermediates of the ergosterol biosynthetic pathway were isolated and analysed for dose-dependent accumulation. Analysis by reverse-phase HPLC displayed a decline in ergosterol production in a dose-dependent manner when exposed to increasing concentrations of T. violacea extract. Quantification of the sterol intermediates of the ergosterol pathway indicated a definite accumulation of 2,3-oxidosqualene. The results prove that the plant extract affected ergosterol synthesis by inhibiting oxidosqualene cyclase. This prevented the formation of downstream intermediates of the ergosterol pathway ultimately resulting in inhibition of ergosterol production.

Nanotechnology and the future of condoms in the prevention of sexually transmitted infections.

Objective: The human immunodeficiency virus (HIV) is among the utmost destructive viruses humankind has ever faced in almost four decades. It carries with it profound socioeconomic and public health implications. Unfortunately, there is, currently, no effective cure for HIV infections. This review discusses the various types of condoms, microbicides, and the potential use of nanoparticle-coated condoms as a means of diminishing the risk of HIV transmission and sexually transmitted infections (STIs) during sexual intercourse. Methods: We identified 153 articles from 1989 to 2015 indexed in various journal platforms, reports, and magazines. Using the PRISMA guidelines as proxy in performing the research review process, only 53 articles were selected. Ideally, articles that failed to describe the nature and types of condoms, condom failures, nanoparticle-coated condoms, microbicides, and HIV prevention were excluded. Results and Discussion: In general, it has been shown that antiretroviral therapy (ART) currently available can only limit transmission and acquisition of HIV strains. Apart from ART treatment, the use of condoms has been identified globally as a cost-effective intervention for reducing the spread of HIV and other STIs. However, while condoms are supposed to be effective, reliable, and easy to use, research has shown that they are attributable to 20% failures including breakages. Nevertheless, other studies have shown that coating condoms with nanoparticles is an important and effective method for reducing condom breakage and HIV/STI transmission during sexual intercourse. Conclusions: A review of literature cited in this paper has shown that nanotechnology-based condom systems have the potential to prevent the spread of HIV and STIs. Furthermore, the antimicrobial nature of some nanoparticles could provide a safe and efficient way to disrupt and/or inactivate different STIs - including viral, bacterial, and fungal diseases.

Objectif: Le virus d'immunodéficience acquise (VIH) est l'un des virus les plus destructeurs auquel l'humanité toute entière a eu à faire face durant ces quatre décennies. Loin d'être un sujet de santé publique à prendre à la légère, il traine dans son sillage des profondes conséquences socio-économiques. Malheureusement, il n'ya pas de traitement effectif à ce jour contre les infections liées au VIH. Cette revue fait donc un bref étalage des différents types de condoms, de microbicides et de l'utilisation des condoms enrobés de nanoparticules comme méthodes de prévention de la transmission du VIH et des maladies sexuellement transmissibles (MST) pendant les rapports sexuels. Méthode: Nous avons identifié153 articles datant de 1989 à 2015 et indexés dans plusieurs plateformes de revues scientifiques et de rapports. Utilisant les indications de PRISMA comme proxy dans le processus de recherche, seulement 53 articles ont été sélectionnés. Ceux qui n'ont pas traité de la nature et des types de condoms, de l'échec de l'utilisation des condoms, des condoms enrobés de nanoparticules, des microbicides et de la prévention du VIH ont été exclus. Résultat et Discussion: Dans l'ensemble, il a été démontré que la thérapie antirétrovirale (TAR) disponible présentement limite uniquement la transmission des souches de VIH. En plus des traitements par TAR, l'utilisation des condoms a été reconnue de façon globale comme méthode appropriée et efficace de réduction de la dissémination du VIH et des MST. Cependant, nonobstant le fait que les condoms soient considérés comme moyens effectifs, fiables, efficaces et faciles à utiliser, il a été démontré par les chercheurs que 20% des cas d'échecs sont attribués à la déchirure des condoms. Néanmoins, aucune étude n'a démontré que les condoms enrobés de nanoparticules offrent une meilleure option face aux déchirures et à la réduction de la transmission du VIH et des MST lors des rapports sexuels. Conclusion: La revue de littérature citée dans cet article a démontré que les condoms enrobés de nanoparticules ont le potentiel de prévenir la dissémination du VIH et des MST. Dans le même ordre d'idée, la nature antimicrobienne de certaines nanoparticules pourrait contribuer efficacement et sûrement à éliminer les différentes MST y compris celles d'origine virale, bactérienne ou fongique. Mots clés: Thérapie antirétrovirale, condom, syndrome d'immunodéficience acquise /infection sexuellement transmissible, microbicide, nano-composites antimicrobiens, prévention.

Validation of hisH4 and cox5 reference genes for RT-qPCR analysis of gene expression in Aspergillus flavus under aflatoxin conducive and non-conducive conditions.

Aspergillus flavus is an environmental pathogen that produces highly carcinogenic aflatoxins. Biosynthesis of aflatoxins is affected by external factors such as pH, temperature, carbon source and nitrogen source. Real-Time PCR (RT-qPCR) is a powerful technique used to detect minute changes in gene expression of a target gene in comparison to one or more reference genes. Several candidate genes were analysed to determine their suitability for use as reference genes for analysing gene expression in A. flavus via RT-qPCR under various aflatoxin conducive and non-conducive conditions. BestKeeper analysis indicated that histone H4 (hisH4) and cytochrome C oxidase subunit V (cox5) were suitable reference genes for analysis of gene expression in A. flavus via RT-qPCR. This was further confirmed by REST2009 analysis of hisH4 and cox5 stability. Furthermore, REST2009 was used to predict which gene or gene combination would be the best reference gene/s for RT-qPCR expression analysis under each treatment condition tested in this study.

Aqueous extracts of Tulbaghia violacea inhibit germination of Aspergillus flavus and Aspergillus parasiticus conidia.

Aspergillus flavus and Aspergillus parasiticus are important plant pathogens and causal agents of pre- and postharvest rots of corn, peanuts, and tree nuts. These fungal pathogens cause significant crop losses and produce aflatoxins, which contaminate many food products and contribute to liver cancer worldwide. Aqueous preparations of Tulbaghia violacea (wild garlic) were antifungal and at 10 mg/ml resulted in sustained growth inhibition of greater than 50% for both A. flavus and A. parasiticus. Light microscopy revealed that the plant extract inhibited conidial germination in a dose-dependent manner. When exposed to T. violacea extract concentrations of 10 mg/ml and above, A. parasiticus conidia began germinating earlier and germination was completed before that of A. flavus, indicating that A. parasiticus conidia were more resistant to the antifungal effects of T. violacea than were A. flavus conidia. At a subinhibitory extract dose of 15 mg/ml, hyphae of both fungal species exhibited increased granulation and vesicle formation, possibly due to increased reactivity between hyphal cellular components and T. violacea extract. These hyphal changes were not seen when hyphae were formed in the absence of the extract. Transmission electron microscopy revealed thickening of conidial cell walls in both fungal species when grown in the presence of the plant extract. Cell walls of A. flavus also became considerably thicker than those of A. parasiticus, indicating differential response to the extract. Aqueous preparations of T. violacea can be used as antifungal treatments for the control of A. flavus and A. parasiticus. Because the extract exhibited a more pronounced effect on A. flavus than on A. parasiticus, higher doses may be needed for control of A. parasiticus infections.

Purification and partial characterization of ostrich skeletal muscle cathepsin D and its activity during meat maturation.

Cathepsin D was purified from ostrich (Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45°C, and was strongly inhibited by pepstatin-A (K(i)=3.07×10(-9)M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112±8.57% at day 30 (mean value from 5 ostriches).

Molecular profiling demonstrates limited diversity amongst geographically separate strains of Ustilago scitaminea.

Intraspecies diversity within Ustilago scitaminea isolates from South Africa, Reunion Island, Hawaii and Guadeloupe was assessed by RAPDs, bE mating-type gene detection, rDNA sequence analysis, microscopy and germination and morphological studies. Except for sequence data, the other analyses yielded no differences in the isolates that could be used in a phylogenetic separation. Mycelial DNA of the SA isolate shared 100% sequence identity with that of mycelial DNA cultured from in vitro produced teliospores of the parent cultivar. Overall the ITS1 and ITS2 regions were found to have 96.1% and 96.9% sequence identity with a total of 17 and 21 base changes, respectively, amongst the isolates. The Reunion Island isolate was shown to be most distantly related by 3.6% to the other isolates, indicating a single clonal lineage. The lack of germination in teliospores from Guadeloupe may be attributed to changes in temperature and humidity during transportation.

Internal Transcribed Spacer Regions 1 and 2 and Random Amplified Polymorphic DNA Analysis of Didymella bryoniae and Related Phoma Species Isolated from Cucurbits.

ABSTRACT Didymella bryoniae (anamorph Phoma cucurbitacearum) is the causal agent of gummy stem blight, although other Phoma species are often isolated from cucurbit plants exhibiting symptoms of the disease. The molecular and phylogenetic relationships between D. bryoniae and these Phoma species are unknown. Isolates of D. bryoniae and Phoma obtained from cucurbits grown at various geographical locations in the United States were subjected to random amplified polymorphic DNA (RAPD) analysis and internal transcribed spacer (ITS) sequence analysis (ITS-1 and ITS-2) to determine the molecular and phylogenetic relationships within and between these fungi. Using RAPD fingerprinting, 59 isolates were placed into four phylogenetic groups, designated RAPD group (RG) I, RG II, RG III, and RG IV. D. bryoniae isolates clustered in either RG I (33 isolates), RG II (12 isolates), or RG IV (one isolate), whereas all 13 Phoma isolates clustered to RG III. There was greater than 99% sequence identity in the ITS-1 and ITS-2 regions between isolates in RG I and RG II, whereas isolates in RG III, P. medicaginis ATCC 64481, and P. exigua ATCC 14728 clustered separately. On muskmelon seedlings, a subset of RG I isolates were highly virulent (mean disease severity was 71%), RG II and RG IV isolates were slightly virulent (mean disease severity was 4%), and RG III isolates were nonpathogenic (disease severity was 0% for all isolates). The ITS sequences indicate that RG I and RG II are both D. bryoniae, but RAPD fingerprints and pathogenicity indicate that they represent two different molecular and virulence subgroups.