Microneedle Array Design Determines the Induction of Protective Memory CD8+ T Cell Responses Induced by a Recombinant Live Malaria Vaccine in Mice

BACKGROUND: Vaccine delivery into the skin has received renewed interest due to ease of access to the immune system and microvasculature, however the stratum corneum (SC), must be breached for successful vaccination. This has been achieved by removing the SC by abrasion or scarification or by delivering the vaccine intradermally (ID) with traditional needle-and-syringes or with long microneedle devices. Microneedle patch-based transdermal vaccine studies have predominantly focused on antibody induction by inactivated or subunit vaccines. Here, our principal aim is to determine if the design of a microneedle patch affects the CD8(+) T cell responses to a malaria antigen induced by a live vaccine. METHODOLOGY AND FINDINGS: Recombinant modified vaccinia virus Ankara (MVA) expressing a malaria antigen was percutaneously administered to mice using a range of silicon microneedle patches, termed ImmuPatch, that differed in microneedle height, density, patch area and total pore volume. We demonstrate that microneedle arrays that have small total pore volumes induce a significantly greater proportion of central memory T cells that vigorously expand to secondary immunization. Microneedle-mediated vaccine priming induced significantly greater T cell immunity post-boost and equivalent protection against malaria challenge compared to ID vaccination. Notably, unlike ID administration, ImmuPatch-mediated vaccination did not induce inflammatory responses at the site of immunization or in draining lymph nodes. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the design of microneedle patches significantly influences the magnitude and memory of vaccine-induced CD8(+) T cell responses and can be optimised for the induction of desired immune responses. Furthermore, ImmuPatch-mediated delivery may be of benefit to reducing unwanted vaccine reactogenicity. In addition to the advantages of low cost and lack of pain, the development of optimised microneedle array designs for the induction of T cell responses by live vaccines aids the development of solutions to current obstacles of immunization programmes

Expression Patterns of Protein Kinases Correlate with Gene Architecture and Evolutionary Rates

Protein kinase (PK) genes comprise the third largest superfamily that occupy ∼2% of the human genome. They encode regulatory enzymes that control a vast variety of cellular processes through phosphorylation of their protein substrates. Expression of PK genes is subject to complex transcriptional regulation which is not fully understood.Our comparative analysis demonstrates that genomic organization of regulatory PK genes differs from organization of other protein coding genes. PK genes occupy larger genomic loci, have longer introns, spacer regions, and encode larger proteins. The primary transcript length of PK genes, similar to other protein coding genes, inversely correlates with gene expression level and expression breadth, which is likely due to the necessity to reduce metabolic costs of transcription for abundant messages. On average, PK genes evolve slower than other protein coding genes. Breadth of PK expression negatively correlates with rate of non-synonymous substitutions in protein coding regions. This rate is lower for high expression and ubiquitous PKs, relative to low expression PKs, and correlates with divergence in untranslated regions. Conversely, rate of silent mutations is uniform in different PK groups, indicating that differing rates of non-synonymous substitutions reflect variations in selective pressure. Brain and testis employ a considerable number of tissue-specific PKs, indicating high complexity of phosphorylation-dependent regulatory network in these organs. There are considerable differences in genomic organization between PKs up-regulated in the testis and brain. PK genes up-regulated in the highly proliferative testicular tissue are fast evolving and small, with short introns and transcribed regions. In contrast, genes up-regulated in the minimally proliferative nervous tissue carry long introns, extended transcribed regions, and evolve slowly.PK genomic architecture, the size of gene functional domains and evolutionary rates correlate with the pattern of gene expression. Structure and evolutionary divergence of tissue-specific PK genes is related to the proliferative activity of the tissue where these genes are predominantly expressed. Our data provide evidence that physiological requirements for transcription intensity, ubiquitous expression, and tissue-specific regulation shape gene structure and affect rates of evolution

Notes for genera: basal clades of Fungi (including Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota)

Compared to the higher fungi (Dikarya), taxonomic and evolutionary studies on the basal clades of fungi are fewer in number. Thus, the generic boundaries and higher ranks in the basal clades of fungi are poorly known. Recent DNA based taxonomic studies have provided reliable and accurate information. It is therefore necessary to compile all available information since basal clades genera lack updated checklists or outlines. Recently, Tedersoo et al. (MycoKeys 13:1--20, 2016) accepted Aphelidiomycota and Rozellomycota in Fungal clade. Thus, we regard both these phyla as members in Kingdom Fungi. We accept 16 phyla in basal clades viz. Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. Thus, 611 genera in 153 families, 43 orders and 18 classes are provided with details of classification, synonyms, life modes, distribution, recent literature and genomic data. Moreover, Catenariaceae Couch is proposed to be conserved, Cladochytriales Mozl.-Standr. is emended and the family Nephridiophagaceae is introduced

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Not AvailableBasidiocarps (fruiting bodies) of wood rotting fungi were inoculated into mycological agar slants and incubated for 20 days at 30°C along with the available isolates. Microscopic observations revealed few characteristic features belonging to basidiomycetes. Twenty of these novel isolates were screened for laccase production after growing them on Potato Dextrose Agar, Malt Extract and Mycological Agar for seven days at 30°C. Three different indicator compounds viz., 0.1% Syringaldehyde; 0.1% Guaiacol and 0.1% (w/v) ABTS were added to the solid media in order to detect ligninolytic enzymes. Eight strains produced detectable amounts of laccase in culture supernatant using Malt extract broth and Kirk's Basal medium, as detected by activity on ABTS. Evaluation of the selected eight cultures, using seven different buffers (Sodium acetate buffer pH - 4.5, pH - 5.0 and pH - 5.2; Glycine –HCl buffer; Succinate buffer; Sodium tartrate buffer and Sodium dihydrogen phosphate-citric acid buffer) failed to show significant variation in the activity (P>0.05). Though sonication of fungal biomass yielded higher enzyme activity, it was not significant (P>0.05). Based on the intensity of the color formation and Quantitative ABTS enzyme assay of the 8 positive isolates, four isolates, NI-04, NI-07, NI-09 and NI-03 showed 1843, 1383, 1530 and 386 units of laccase activity with the corresponding intensity of the color developments. Based on the results of the complete screening, NI-09, NI-04 and NI-07 proved to be the most promising white rot basidiomycetes strains. Results obtained were very encouraging. The enzyme produced can be purified and used for heterologous vector mediated transformation, and thus available for industrial and biological bioprocess useNot Availabl

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Not AvailableThe rumen microbial utilization of energy-rich cell walls of crop residues is hindered by the presence of lignin, which limitsits overall digestion process and significantly influences animal performance. In the frame of the development of a bioprocess using the competences of white rot fungi to enhance digestibility of crop residues by delignification, a new strain of Schizophyllum commune NI-07 was isolated. The sole lignolytic activity detected in submerged culture was laccase which increased 3-fold after immobilization of fungus on polyurethane foam cubes. The enzyme was purified 42-fold by employing ammonium sulphate precipitation and size exclusion chromatography on Sephadex G-50 to a specific activity of 15930 U/mg of protein and had a molecular mass of 75 kDa. The laccase obtained from submerged culture medium after cul- tivation of immobilized S. commune NI-07, exhibited considerably higher pH and thermostability and affinity for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) compared to those obtained without immobilization of the fungus. Highly significant (P < 0.05) improvement in the in vitro dry matter digestibility was obtained in 5 common crop residues treated with fungal laccases. We prove the potential of laccase obtained from S. commune NI-07 in enhancement of digestibility of crop residues by way of delignification

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Not AvailableLaccase enzymes plays a vital role in innumerable biotechnological applications and hence their large scale production has stimulated considerable research. In the present study, degenerate universal primer pairs were employed to isolate laccase gene from a wild isolate of laccase producing white rot fungi Schizophyllum commune. Primer pairs for this fungal isolate were also designed using laccase specific consensus sequences for fungi. The PCR product of 1000 bp 2 amplicon was visualized on agarose gel using the degenerate primer pair Cu1F/Cu3R. Matching of the sequenced gel purified DNA sequence resembled most of the putative phosphatases involved in cell cycle with 100% identity to S. commune. Here we report the false negative results obtained upon use of laccase specific degenerate primers as well as other primers specific for the genus. These failed to contribute towards isolation of laccase gene even after optimization of PCR conditions in terms of reaction volume, annealing temperature, number of cycles, touchdown PCR and gradient PCR. These findings constitute a practical guide for researchers addressing amplification of transcripts of this biotechnologically important enzyme from the not so well characterized genus of Schizophyllum.Not Availabl

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Not AvailableThe present study was conducted to evaluate the effect of exogenous lignolytic enzymes harvested from immobilized Coriolus versicolor and Ganoderma lucidium on body weight, in vivo digestibility, rumen fermentation and rumen enzymes in sheep. Four groups of sheep (6-each) were fed 350 g concentrate mixture to meet the energy and protein requirement as per ICAR standards. The control sheep received ad lib. ragi straw treated with production media devoid of enzyme (G1). Test group 1 (G2) received ad lib. ragi straw treated with C. versicolor enzyme media (Enz.1) in a 1:2.5 (w/v) ratio, group 2 (G3) received ad lib. straw treated with G. lucidium enzyme media (Enz.2) in a 1:2.5 (w/v) ratio. Group 3 (G4) received ad lib. amount of straw treated with a combination of Enz.1 and Enz.2 in an equal volume in a 1:2.5 (w/v) ratio. After 40 d feeding an ADG (gd-1) of 112.5 and 107.5 was recorded in G2 and G3 as compared to 97.5 of control. A 5% increase in dry matter digestibility (DMD) of 77 .36±4.28 and 77.04±5.69% was obtained in G2 and G3. Treatment of straw with a combination of enzymes (G4) failed to show increase in either ADG or DMD. Rumen fermentation pattern did show any significant difference. The fiber degrading enzymes elicited enhancement of activity in sheep fed the supplemental enzymes. Applying lignin degrading enzymes as feed supplements for enhancing digestibility of crop residues holds promise in the immediate future.Not Availabl