High-throughput DNA sequencing of environmentally insulted latent fingerprints after visualization with nanoscale columnar-thin-film technique.

The goals of this study were to (a) ascertain human identity capabilities of DNA obtained from latent fingerprints that have been first environmentally insulted and then developed by the deposition of a columnar thin film (CTF), and (b) to determine if the CTF process and material are detrimental to single nucleotide polymorphism (SNP) analysis. Fingerprints were deposited on five different types of substrates and aged for one day, 7 days or 30 days while being environmentally insulted under one of the four conditions: 16.6 °C and 60% relative humidity (RH) (Condition A), 24.5 °C and 60% RH (Condition B), 35 °C and 67% RH (Condition C) and a cold condition (Condition D). Then CTF technique was then on 59% of these fingerprints. DNA samples from 805 fingerprints were extracted, quantified, subjected to manual library preparation using the Precision ID Identity Panel, and underwent high-throughput sequencing. The Ion S5™ platform was employed to sequence 124 SNP amplicons. SNPs were successfully sequenced from 802/805 samples. Total read depth was consistent across environmental conditions, and majority of samples had 100% profile completeness and 100% concordance. Anecdotally, libraries that were amplified with a higher cycle number had more 'Major Allele Frequency' flags compared to samples amplified with 23 cycle numbers, possibly due to stochastic effects. Neither the substrates nor the CTF process and materials inhibit downstream DNA analysis. DNA of low quality and quantity from the chosen samples can be sequenced using the Precision ID Identity Panel on the Ion S5™ platform which performed well, however, a different approach may be needed if spurious alleles are suspected.

Reducing biomass recalcitrance by heterologous expression of a bacterial peroxidase in tobacco (Nicotiana benthamiana).

Commercial scale production of biofuels from lignocellulosic feed stocks has been hampered by the resistance of plant cell walls to enzymatic conversion, primarily owing to lignin. This study investigated whether DypB, the lignin-degrading peroxidase from Rodococcus jostii, depolymerizes lignin and reduces recalcitrance in transgenic tobacco (Nicotiana benthamiana). The protein was targeted to the cytosol or the ER using ER-targeting and retention signal peptides. For each construct, five independent transgenic lines were characterized phenotypically and genotypically. Our findings reveal that expression of DypB in the cytosol and ER does not affect plant development. ER-targeting increased protein accumulation, and extracts from transgenic leaves showed higher activity on classic peroxidase substrates than the control. Intriguingly, in situ DypB activation and subsequent saccharification released nearly 200% more fermentable sugars from transgenic lines than controls, which were not explained by variation in initial structural and non-structural carbohydrates and lignin content. Pyrolysis-GC-MS analysis showed more reduction in the level of lignin associated pyrolysates in the transgenic lines than the control primarily when the enzyme is activated prior to pyrolysis, consistent with increased lignin degradation and improved saccharification. The findings reveal for the first time that accumulation and in situ activation of a peroxidase improves biomass digestibility.

Within gut physicochemical variation does not correspond to distinct resident fungal and bacterial communities in the tree-killing xylophage, Anoplophora glabripennis.

Insect guts harbor diverse microbial assemblages that can be influenced by multiple factors, including gut physiology and interactions by the host with its environment. The Asian longhorned beetle (A. glabripennis; Cerambycidae: Lamiinae) is an invasive tree-killing insect that harbors a diverse consortium of fungal and bacterial gut associates that provision nutrients and facilitate lignocellulose digestion. The physicochemical conditions of the A. glabripennis gut and how these conditions may influence the microbial composition across gut regions are unknown. In this study, we used microsensors to measure in situ oxygen concentrations, pH, and redox potential along the length of the A. glabripennis larval gut from two North American populations. We then analyzed and compared bacterial and fungal gut communities of A. glabripennis within individual hosts along the length of the gut using 16S and ITS1 amplicon sequencing. The A. glabripennis midgut lumen was relatively anoxic (<0.01kPa) with a pH gradient from 5.5 to 9, moving anterior to posterior. Redox potential was higher in the anterior midgut relative to posterior regions. No differences in physicochemistry were measured between the two populations of the beetle, but the two populations harbored different communities of bacteria and fungi. However, microbial composition of the A. glabripennis gut microbiota did not differ among gut regions despite physicochemical differences. Unlike other insect systems that have distinct gut compartmentalization and corresponding microbial assemblages, the A. glabripennis gut lacks dramatic morphological modifications, which may explain why discrete microbial community structures were not found along the digestive system.

AcsA-AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769.

The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.

Isolation and characterization of two cellulose morphology mutants of Gluconacetobacter hansenii ATCC23769 producing cellulose with lower crystallinity.

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of ß-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.

Identification and characterization of non-cellulose-producing mutants of Gluconacetobacter hansenii generated by Tn5 transposon mutagenesis.

The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel(-)) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript "Ax" indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel(-) mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel(-) mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding ß-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis.

Pankiller effect of prolonged exposure to menadione on glioma cells: potentiation by vitamin C.

Menadione (Vitamin K3) has anti-tumoral effects against a wide range of cancer cells. Its potential toxicity to normal cells and narrow therapeutic range limit its use as single agent but in combination with radiation or other anti-neoplastic agents can be of therapeutic use. In this paper, we first evaluated the early (within 3 h) effect of menadione on ongoing DNA replication. In normal rat cerebral cortex mini-units menadione showed an age dependent anti-proliferative effect. In tissue mini-units prepared from newborn rats, menadione inhibited ongoing DNA replication with an IC (50) of approximately 10 µM but 50 µM had no effect on mini-units from prepared adult rat tissue. The effect of short (72 h) and prolonged exposure (1-2 weeks) to menadione alone in the DBTRG.05MG human glioma cells line and in combination with vitamin C was studied. After short period of exposure data show that menadione alone or in combination with vitamin C provided similar concentration-response curves (and IC(50) values). Prolonged exposure to these drugs was evaluated by their ability to kill 100% of glioma cells and prevent regrowth when cells are re-incubated in drug-free media. In this long-term assay, menadione:vitamin C at a ratio 1:100 showed higher anti-proliferative activity when compared to each drug alone and allowed to reduce each drug concentration between 2.5 to 5-fold. Similar anti-proliferative effect was demonstrated in 8 patient derived glioblastoma cell cultures. Our data should be able to encourage further advanced studies on animal models to evaluate the potential use of this combination therapy for glioma treatment.

Knocking out salicylate biosynthesis genes in Mycobacterium smegmatis induces hypersensitivity to p-aminosalicylate (PAS).

Because of the emergence of strains of Mycobacterium tuberculosis resistant to first-line antituberculosis agents, one of the second-line drugs, p-aminosalicylate (PAS), has regained importance in the treatment of tuberculosis. The mode of action of PAS, however, remains controversial as to whether it inhibits mycobactin or folate biosynthesis. To unravel this, we have studied the effect of PAS on wild-type Mycobacterium smegmatis and its mutants (gene knockouts of the salicylate pathway -trpE2, entC and entD). The wild type had no sensitivity to PAS (MIC>400 µg mL(-1) ), whereas the mutants were hypersensitive, with 1 µg mL(-1) inhibiting growth. The sulphonamides, trimethoprim and dapsone, had little effect on the growth of either the mutants or the wild type. In addition, PAS at 0.5 µg mL(-1) increased the accumulation of salicylate with the wild type and mutants. These results support our hypothesis that PAS targets the conversion of salicylate to mycobactin, thus preventing iron acquisition from the host.

Roles of trpE2, entC and entD in salicylic acid biosynthesis in Mycobacterium smegmatis.

Mycobacterium smegmatis acquires extracellular iron using exochelin, mycobactin and carboxymycobactin. The latter two siderophores are synthesized from salicylic acid, which, in turn, is derived from chorismic acid in the shikimic acid pathway. To understand the conversion mechanism of chorismic acid to salicylic acid in M. smegmatis, knockout mutants of the putative key genes, trpE2, entC and entD, were created by targeted mutagenesis. By enzymatic assays with the cell-free extracts of the various knockout mutants, we have shown that TrpE2 converts chorismic acid into isochorismic acid and is thus an isochorismate synthase. The gene products of both entC and entD are involved in the conversion of isochorismic acid into salicylic acid, and hence correspond to salicylate synthase.