Formulation,Characterization, and Clinical Evaluation of Microemulsion Containing Clotrimazole for Topical Delivery

The objective of the present study was to formulate and evaluate microemulsion systems for topical delivery of clotrimazole (CTM). The solubility of CTM in various oils was determined to select the oil phase of the microemulsion systems. Pseudoternary phase diagrams were constructed to identify the area of microemulsion existence. Five CTM microemulsion formulations (M1–M5) were prepared and evaluated for their thermodynamic stability, pH, refractive index, droplet size, viscosity, and in vitro release across cellulose membrane. Among the prepared microemulsion formulations, M3 (lemon oil/Tween 80/n-butanol/water) and M4 (isopropyl myristate/Tween 80/n-butanol/water) microemulsion systems were found to be promising according to their physical properties and CTM cumulative percentage release. Gel form of M3 and M4 were prepared using 1% Carbopol 940 as the hydrogel matrix. Both formulations were evaluated in the liquid and gel forms for drug retention in the skin in comparison to the marketed CTM topical cream and their stability examined after storage at 40°C for 6 months. Microemulsion formulations achieved significantly higher skin retention for CTM over the CTM cream. Stability studies showed that M4 preparations were more stable than M3. The in vitro anti-fungal activity of M4 against Candida albicans was higher than that of the conventional cream. Moreover, clinical evaluation proved the efficacy and tolerability of this preparation in the treatment of various topical fungal infections.     

Construction of banana bunchy top nanovirus-DNA-3 encoding the coat protein gene and its introducing into banana plants cv. Williams

Banana (Musa sp.) is considered as one of the most important fruit crops worldwide as well as in Egypt. The main goal of this study was to construct the open reading frame (ORF) of banana bunchy top nanovirus (BBTV)-DNA-3 that encodes the viral coat protein (cp) gene for banana transformation. The previously sequenced BBTV-G-DNA-3-ORF that cloned into plasmid pH1 was used as a template for PCR amplification using two specific primers containing Bam H1 site. A new plasmid called pRHA1 containing the amplified ORF under the control of maize polyubiquitin (ubi) promoter was created. The bar gene (herbicide-resistance gene as a selectable marker) cassette (bar gene, Cauliflower mosaic caulimovirus (CaMV) 35S promoter and nos terminator) was released from plasmid pAB6 using Hind III-digestion and subcloned into the Hind III-digested plasmid pRHA1 to create the plasmid pRHA2 via the microprojectile bombardment transformation system. The plasmid pRHA2 was successfully introduced in the applied banana cultivar. Leaf painting test was conducted to confirm the expression of the bar gene in the putative transformed banana lines. The presence and expression of BBTV-G-cp gene were also detected using some molecular (polymerase chain reaction and dot blot using a cold DNA probe) and serological (ELISA and western blot) techniques, respectively, in the obtained transgenic banana lines.     

Attention-deficit hyperactive disorder presenting with school truancy in an adolescent: a case report

Attention-deficit hyperactive disorder (ADHD) is a psychiatric illness commonly diagnosed during the early years of childhood. In many adolescents with undiagnosed ADHD, presentation may not be entirely similar to that in younger children. These adolescents pose significant challenges to parents and teachers coping with their disability. Often adolescents with behavioural problems are brought to medical attention as a last resort. This case describes an adolescent who presented to a primary care clinic with school truancy. He was initially treated for depression with oppositional defiant disorder and sibling rivalry. Only following a careful detailed history and further investigations was the diagnosis of ADHD made. He showed a positive improvement with the use of methylphenidate for his ADHD and escitalopram for his depression. The success of his management was further supported by the use of behavioural therapy and parenting interventions. There is a need to increase public awareness of ADHD, especially among parents and teachers so that early intervention can be instituted in these children.     

From autoinhibition to inhibition in trans: the Raf-1 regulatory domain inhibits Rok-α kinase activity

The activity of Raf-1 and Rok-α kinases is regulated by intramolecular binding of the regulatory region to the kinase domain. Autoinhibition is relieved upon binding to the small guanosine triphosphatases Ras and Rho. Downstream of Ras, Raf-1 promotes migration and tumorigenesis by antagonizing Rok-α, but the underlying mechanism is unknown. In this study, we show that Rok-α inhibition by Raf-1 relies on an intermolecular interaction between the Rok-α kinase domain and the cysteine-rich Raf-1 regulatory domain (Raf-1reg), which is similar to Rok-α''s own autoinhibitory region. Thus, Raf-1 mediates Rok-α inhibition in trans, which is a new concept in kinase regulation. This mechanism is physiologically relevant because Raf-1reg is sufficient to rescue all Rok-α–dependent defects of Raf-1–deficient cells. Downstream of Ras and Rho, the Raf-1–Rok-α interaction represents a novel paradigm of pathway cross talk that contributes to tumorigenesis and cell motility.     

Bloom DNA Helicase Facilitates Homologous Recombination between Diverged Homologous Sequences

Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. Here, we demonstrate the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM^−/− cells. In addition, BLM^−/− cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, we measured the frequency of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM^−/− cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.The RecQ helicases, a subfamily of DNA helicases, carry out the unwinding of duplex DNA in the 3′ to 5′ direction. Homologs of RecQ have been identified in a wide range of organisms, from budding yeast to humans (reviewed in Ref. 1). There are five human RecQ family proteins: Blm, Wrn, RecQ1, RecQ4, and RecQ5. The BLM, WRN, and RECQ4 genes are mutated in Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively (1–3). A hallmark of Bloom syndrome cells is the drastic increase in the level of sister chromatid exchange (SCE),⁴ which results from homologous recombination (HR) associated with cross-over of the DNA damage caused during DNA replication (4, 5). It is therefore believed that Blm acts as an anti-recombination factor and inhibits aberrant recombination. This idea is supported by the observation that Sgs1, the yeast ortholog of Blm, facilitates the resolution of aberrant joint molecules during meiotic HR (6, 7) and following replication blockage (8).HR plays a critical role in the maintenance of genome stability by repairing DNA double-strand breaks (DSBs) and releasing replication blockages at damaged template strands (9, 10). The current model for HR-mediated DSB repair is that DSBs are processed to produce a 3′ single-stranded overhang, along which Rad51 is polymerized (11, 12). The resulting Rad51-DNA filament undergoes homology search and strand invasion into intact homologous duplex DNA, leading to the formation of the D-loop structure. DNA synthesis from the invading strand followed by dissociation from the homologous duplex DNA and subsequent re-annealing of the newly synthesized strand with the other end of the DSB completes the repair. This type of HR, referred to as synthesis-dependent strand anneal (SDSA), results in sequence transfer from the intact template sequence (donor) to the damaged DNA (recipient), and accounts for the majority of mitotic HR (11, 13). Extensive strand exchange of the D-loop, on the other hand, leads to the generation of Holliday junction (HJ) intermediates. SDSA does not cause cross-overs, whereas HR involving the Holliday junction often causes cross-overs, such as SCE and meiotic HR. An increase in the level of SCE in Bloom syndrome cells therefore supports the idea that Blm suppresses the formation of HJ as well as recombinogenic DNA lesions. This idea is supported by the biochemical evidence of the Blm-dependent resolution of Holliday junctions (14). On the other hand, in Drosophila, DmBlm is known to facilitate the repair of DSB by promoting SDSA (15, 16). However, the role of Blm in SDSA in the other higher eukaryotic cells has not been defined.BLM^−/− cells established from the chicken DT40 B lymphocyte line exhibit a marked increase in the frequency of both SCE and targeted integration (17–19), as do human Bloom syndrome cells (20, 21). In this study, using the chicken DT40 cells, we investigated the role of Blm in SDSA induced by defined DNA damage. To this end, we evaluated this type of SDSA using two phenotypic assays designed to analyze Ig gene conversion and DSB-induced gene targeting. Ig gene conversion diversifies the Ig variable (V) gene through HR during in vitro passage. This reaction is initiated by activation-induced cytidine deaminase-mediated uracil formation at the functional rearranged V-region (22–24). Uracil is converted to an abasic site, probably leading to a single-strand gap (25). This lesion in the functional rearranged VJ_λ stimulates the nonreciprocal sequence transfer of a single nucleotide to several hundred nucleotides, from an array of “pseudo-V_λ” regions (donor), located upstream from the functional rearranged VJ_λ, to the rearranged V region (recipient) (26–28) (see Fig. 1A). Because donor and recipient segments have an ∼10% sequence divergence, sequential Ig gene conversion events are able to substantially diversify Ig V segments. Ig gene conversion is raised only by SDSA without the formation of a Holliday junction. Hence, phenotypic analysis of Ig gene conversion provides a unique opportunity to selectively examine the role of Blm in activation-induced cytidine deaminase-induced SDSA. Moreover, nucleotide sequence analysis of Ig gene conversion products can evaluate the accuracy of HR. Like Ig gene conversion, DSB-induced gene targeting is mediated only by SDSA. The induction of DSBs by a rare-cutting endonuclease, I-SceI, at the endogenous locus, increases the frequency of gene targeting by 3 orders of magnitudes, and the frequency of gene targeting can be evaluated by measuring the reconstitution of a marker gene (29) (see Fig. 1B).Open in a separate windowFIGURE 1.Schematic diagram of assay systems used in this study. A, principle of the Ig gene conversion assay. The predominantly sIgM-negative DT40 clone contains a frameshift in its rearranged V-J_λ segments, which can be repaired by pseudogene-templated conversion events. The rate of Ig gene conversion can be measured in subclones by flow cytometric analysis of sIgM staining. B, phenotypic assays of Ig gene conversion and DSB-induced gene targeting. Pseudo-V genes and the targeting fragment act as donors for the rearranged V_λ segment and S2neo, respectively.We here show that the loss of Blm drastically reduces the rate of Ig gene conversion without compromising its accuracy or affecting the length of the gene conversion tracts, indicating that Blm plays a role in the promotion of SDSA. This is an unexpected result, because Blm is in fact believed to suppress general HR reactions, particularly recombination between diverged homologous sequences. To understand the function of Blm in SDSA, we analyzed the effect of heterologous sequences near a DSB site on HR-dependent DSB repair. The data demonstrate that Blm can promote SDSA when there is sequence divergence between the damaged recipient DNA and the homologous donor sequence. Thus, Blm has both positive and negative effects on HR, depending upon the type of DNA damage and the step of the HR reaction.     

Development of submergence-tolerant rice cultivars: the Sub1 locus and beyond

Background and Aims

Submergence is a recurring problem in the rice-producing rainfed lowlands of south and south-east Asia. Developing rice cultivars with tolerance of submergence and with agronomic and quality traits acceptable to farmers is a feasible approach to address this problem. The objectives of this study were to (a) develop mega varieties with Sub1 introgression that are submergence tolerant, (b) assess the performance of Sub1 in different genetic backgrounds, (c) determine the roles of the Sub1A and Sub1C genes in conferring tolerance, and (d) assess the level of tolerance in F₁ hybrids heterozygous for the Sub1A-1-tolerant allele.

Methods

Tolerant varieties were developed by marker-assisted backcrossing through two or three backcrosses, and their performance was evaluated to determine the effect of Sub1 in different genetic backgrounds. The roles of Sub1A and Sub1C in conferring the tolerant phenotype were further investigated using recombinants identified within the Sub1 gene cluster based on survival and gene expression data.

Key Results

All mega varieties with Sub1 introgression had a significantly higher survival rate than the original parents. An intolerant Sub1C allele combined with the tolerant Sub1A-1 allele did not significantly reduce the level of tolerance, and the Sub1C-1 expression appeared to be independent of the Sub1A allele; however, even when Sub1C-1 expression is completely turned off in the presence of Sub1A-2, plants remained intolerant. Survival rates and Sub1A expression were significantly lower in heterozygotes compared with the homozygous tolerant parent.

Conclusions

Sub1 provided a substantial enhancement in the level of tolerance of all the sensitive mega varieties. Sub1A is confirmed as the primary contributor to tolerance, while Sub1C alleles do not seem important. Lack of dominance of Sub1 suggests that the Sub1A-1 allele should be carried by both parents for developing tolerant rice hybrids.Key words: Oryza sativa, Sub1, marker-assisted backcrossing, mega varieties, submergence tolerance, recombinant, hybrid, abiotic stress     

Untangling the taxonomic ambiguities of the spotted seerfish Scomberomorus guttatus with the description of a new species from India

Scomberomorus guttatus has been subjected to a series of synonym assignations over the years. Its taxonomy has been mired with ambiguities due to the greater-than-average morphological variations observed in samples from different regions. An integrated taxonomic revision with molecular support indicated that the species that was thought to be a single entity, in reality, is a complex of three distinct species. They are morphologically distinct with respect to the body depth and elongation, cephalic morphometry, and meristic characteristics. Otolith morphometry and phylogenetic evidence further established the taxonomic divergence within the spotted seerfish complex. The phylogenetic characteristic as indicated by the mitochondrial Cytochrome c Oxidase subunit I (COI) sequence unveiled that S. guttatus had a high intraspecific divergence of 11.1% from its two identical congeners and a divergence of 2.34% between the congeners, indicating scope for categorizing them as separate species. Based on the morphological and molecular evidence, S. guttatus is redescribed; the senior synonym Scomberomorus leopardus is resurrected as a valid species; and a new species Scomberomorus avirostrus n. sp. is described with keys for species distinction.     

Exploring Indian Rosewood as a promising biogenic tool for the synthesis of metal nanoparticles with tailor-made morphologies

Stem bark extracts of Indian Rosewood, a traditionally used Indian medicinal plant, were used as highly efficient multifunctional green chemicals/biogenic agents in the rapid synthesis of stable, monometallic Ag and Au nanoparticles and their corresponding bimetallic alloy nanoparticles with interesting shapes and morphological characteristics. We determined that the high efficiency of these extracts is due to the presence of complex multifunctional molecules, such as polyphenolics and hydroxyflavonoids, which are involved in the reduction of Au^III and Ag^I ions to zerovalent metallic nanoparticles and the stabilization of their corresponding nanoparticles.