Biodegradation of native feather keratin by Bacillus subtilis recombinant strains

A mixed culture containing two recombinant Bacillus subtilis strains; was used to hydrolyze 1% chicken feather; both were previously transformed with late-expressed and early expressed alkaline protease (aprE) carrying plasmids pS1 and p5.2, respectively. Proteolytic and keratinolytic activities of the mixed culture increased in parallel with those of the culture of B. subtilis DB100 (p5.2), and both were higher than that of B. subtilis (pS1) cultures. On the other hand, data indicated that degradation of feather by the recombinant strains B. subtilis DB100 (p5.2), was greatly enhanced when using a previously optimized medium. High levels of free amino groups as well as soluble proteins were also obtained. The concentration of amino acids was considerably increased during the fermentation process. It was found that, the amino acids Phe, Gly and Tyr were the major amino acids liberated in the cultures initiated by both strains. Results render these recombinant strains suitable for application in feather biodegradation large scale processes.     

Genotypic detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis strains by DNA sequencing: a randomized trial

Background

Tuberculosis is a growing international health concern. It is the biggest killer among the infectious diseases in the world today. Early detection of drug resistance allows starting of an appropriate treatment. Resistance to drugs is due to particular genomic mutations in specific genes of Mycobacterium tuberculosis(MTB). The aim of this study was to identify the presence of Isoniazid (INH) and Rifampicin(RIF) drug resistance in new and previously treated tuberculosis (TB) cases using DNA sequencing.

Methods

This study was carried out on 153 tuberculous patients with positive Bactec 460 culture for acid fast bacilli.

Results

Of the 153 patients, 105 (68.6%) were new cases and 48 (31.4%) were previously treated cases. Drug susceptibility testing on Bactec revealed 50 resistant cases for one or more of the first line antituberculous. Genotypic analysis was done only for rifampicin resistant specimens (23 cases) and INH resistant specimens (26 cases) to detect mutations responsible for drug resistance by PCR amplification of rpoB gene for rifampicin resistant cases and KatG gene for isoniazid resistant cases. Finally, DNA sequencing was done for detection of mutation within rpoB and KatG genes. Genotypic analysis of RIF resistant cases revealed that 20/23 cases (86.9%) of RIF resistance were having rpoB gene mutation versus 3 cases (13.1%) having no mutation with a high statistical significant difference between them (P < 0.001). Direct sequencing of Kat G gene revealed point mutation in 24/26 (92.3%) and the remaining 2/26 (7.7%) had wild type KatG i.e. no evidence of mutation with a high statistical significant difference between them (P < 0.001).

Conclusion

We can conclude that rifampicin resistance could be used as a useful surrogate marker for estimation of multidrug resistance. In addition, Genotypic method was superior to that of the traditional phenotypic method which is time-consuming taking several weeks or longer.     

AglH,a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius,is capable of complementing the eukaryal Alg7

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D₁₀₀), IV (F₂₂₀) and V (F₂₆₄) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

     

Aqueous extract of tobacco induces mitochondrial potential dependent cell death and epithelial-mesenchymal transition in gingival epithelial cells

Smokeless tobacco habits are detrimental to oral health. A correlation between tobacco use and local epithelial tissue damage exists. Yet, the underlying cellular mechanism is not precisely characterized. This study assessed the dose-dependent action of Smokeless tobacco extract on gingival epithelial cells. Gingival tissue was taken from 5 healthy donors. Gingival epithelial cells were isolated by an enzymatic method and cultured up to passage 2. The cultured cells were treated with smokeless tobacco extract at 10%, 25%, 50%, and 75% volume concentration. After 48 h of incubation, MTT assay, Annexin V/PI assay, and DiIC1(5) assay were used to evaluate viability, apoptosis, and mitochondrial potential of the cells. RT-qPCR was used to determine the expression of BAX, BCL2, ECAD, NCAD, and TWIST. The Smokeless tobacco extract reduced cell viability by disrupting the mitochondrial potential and inducing apoptosis. Further, the Smokeless tobacco extract induced a dose-dependent epithelial-mesenchymal-transition in gingival epithelial cells. Apoptotic cellular death caused by tobacco extract on the gingival epithelial system was dependant on the mitochondrial potential of the cell. The results demonstrate that smokeless tobacco causes detrimental metabolic alterations of the periodontium.Featured applicationThis study elucidates the mechanism by which Smokeless tobacco products cause cellular damage to the gingival epithelium. The use of Smokeless tobacco products can lead to major cellular and surface changes in the gingiva and its appearance. The consequences of these changes are not limited to oral cancer but also increases a person’s risk for dental and periodontal disease.     

The protozoan inositol phosphorylceramide synthase: a novel drug target that defines a new class of sphingolipid synthase

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilize phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalyzed by IPC synthase. Because of the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome data bases of these species. By utilizing bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species, strongly indicating that IPC synthase activity is reconstituted. Furthermore, the gene product can be specifically inhibited by an anti-fungal-targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme that defines a new class of protozoan sphingolipid synthase. The identification and characterization of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side affects.     

Visualizing the Attack of RNase Enzymes on Dendriplexes and Naked RNA Using Atomic Force Microscopy

Cationic polymers such as poly(amidoamine), PAMAM, dendrimers have been used to electrostatically complex siRNA molecules forming dendriplexes for enhancing the cytoplasmic delivery of the encapsulated cargo. However, excess PAMAM dendrimers is typically used to protect the loaded siRNA against enzymatic attack, which results in systemic toxicity that hinders the in vivo use of these particles. In this paper, we evaluate the ability of G4 (flexible) and G5 (rigid) dendrimers to complex model siRNA molecules at low +/− ratio of 2/1 upon incubation for 20 minutes and 24 hours. We examine the ability of the formed G4 and G5 dendriplexes to shield the loaded siRNA molecules and protect them from degradation by RNase V1 enzymes using atomic force microscopy (AFM). Results show that G4 and G5 dendrimers form similar hexagonal complexes upon incubation with siRNA molecules for 20 minutes with average full width of 43±19.3 nm and 62±8.3 at half the maximum height, respectively. AFM images show that these G4 and G5 dendriplexes were attacked by RNase V1 enzyme leading to degradation of the exposed RNA molecules that increased with the increase in incubation time. In comparison, incubating G4 and G5 dendrimers with siRNA for 24 hours led to the formation of large particles with average full width of 263±60 nm and 48.3±2.5 nm at half the maximum height, respectively. Both G4 and G5 dendriplexes had a dense central core that proved to shield the loaded RNA molecules from enzymatic attack for up to 60 minutes. These results show the feasibility of formulating G4 and G5 dendriplexes at a low N/P (+/−) ratio that can resist degradation by RNase enzymes, which reduces the risk of inducing non-specific toxicity when used in vivo.     

Mandibular symphysis dimensions in different sagittal and vertical skeletal relationships

BackgroundThe aim of this cross-sectional study was to compare the dimensions of mandibular symphysis (MS) between gender and the different sagittal and vertical skeletal relationships.Material and MethodsPre-treatment records of orthodontic patients were divided according to gender, sagittal (Class I, II and III) and vertical (decreased, average and increased mandibular plane [MP] angle) skeletal relationships. Measurements of MS parameters were performed on lateral cephalograms using IMAGEJ software. Comparisons between MS parameters and gender and the different skeletal relationships was performed using multifactorial and one-way ANOVA, and independent sample t-tests.ResultsA total of 104 records (25 males and 79 females) fulfilled the inclusion criteria. Males had significantly greater MS surface area, dentoalveolar length, skeletal symphysis length, total symphysis length, vertical symphysis dimension and symphysis convexity (p < 0.05). Skeletal Class II patients had significantly greater dentoalveolar and skeletal symphysis lengths while Class III had greater chin length, vertical symphysis dimension and symphysis convexity (p < 0.05). Patients with decreased vertical dimension had greater skeletal symphysis length (p = 0.026) and those with an average vertical relationship had greater chin length (p < 0.001).ConclusionsThe morphology of the mandibular symphysis is affected by gender, sagittal and vertical skeletal patterns. Males had increased mandibular symphysis surface area and linear dimensions. Class II patients had greater dentoalveolar length. Chin length was greater in patients with an average MP angle.     

PtxtPME1 and homogalacturonans influence xylem hydraulic properties in poplar

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under‐ or over‐express a xylem‐abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under‐ and over‐expressing aspen lines developed xylem with lower‐specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.     

Circadian phase modulates the enhancing effect of the Egyptian Moringa peregrina extract on learning and memory in mice

One of the most important issues nowadays is memory disorders. The exact circadian modulation of learning and memory is still under investigation. The present study was carried out to evaluate the probable enhancing and neuroprotective effects of the Egyptian Moringa peregrina; administered at two different Zeitgeber times (ZT), on learning and memory in mice. M. peregrina dried leaves were aqueously extracted and intranasally (IN) administered at four doses (viz. 12.5, 25, 50, and 100 mg/kg). Twenty animal groups (n = 8/group) were treated with M. peregrina at ZT 3:00 vs. 15:00 representing the rest (day) vs. active (night) phases, respectively. Additionally, sub-chronic effect of daily IN administration of the 25 mg/kg dose for two weeks was examined at ZT 3:00 vs. 15:00. Memory performance was assessed after 1, 2 weeks of treatment and a 3rd recovery week. Memory test was performed at ZT 17:00 by measuring the percentage of time spent in novel arm of Y-maze. Dose-response curve revealed that both 25 and 50 mg/kg doses administered at ZT 3:00 significantly improved memory efficiency. Rest phase administration significantly enhanced memory functions after 1, 2 weeks as well as after the recovery week. Moreover, IN pretreatment with M. peregrina (25 mg/kg) revealed a protective effect against scopolamine-induced amnesia. In conclusion, these findings clearly indicated that M. peregrina possesses valuable enhancing and protective effects on learning and memory processes in mice which is circadian-phase dependent.