Cell Wall Penetration Research Articles

Hardness and indentation modulus of resin-treated wood

This study assesses the potential of instrumented macrohardness testing for the fast and convenient characterisation of resin-treated wood. The effect of WPG (weight per cent gain; percentage mass of resin based on dry wood; high, low) and curing conditions (dry, wet) on hardness and indentation modulus of Scots pine (Pinus sylvestris L.) sapwood was investigated for two thermosetting resins, phenol formaldehyde and methylated melamine formaldehyde. Thickness of the treated specimens broadly confirmed the findings of earlier studies that dry curing conditions increase degree of cell wall penetration. Dry curing resulted in equal or lower hardness, but it mostly increased indentation modulus. Hardness improvement seems to depend on the overall reduction of the void volume fraction in the wood, while indentation modulus depends on cell wall modification. Therefore, indentation modulus is expected to show a much better correlation with dimensional stability and durability of resin-treated wood than hardness.

New Quinoxalinyl Chalcone Derivatives: Search for Potent Antimicrobial Agents

Owing to the prevalence of antimicrobial resistance and its arising threat rendered by the ineffectiveness of existing antibiotics, the present work was aimed to generate new 5/6 substituted quinoxalinyl chalcones derivatives. A total of 16 compounds were synthesized and characterized by IR, 1H NMR, 13C NMR and mass spectral studies. The antimicrobial activity against resistant pathogens was by micro-dilution assay method. The result revealed that compounds bearing 2,4-dichloro benzylidine and 4-fluoro benzylidine moiety demonstrated promising antibacterial and antifungal activities against the selected pathogenic bacteria and fungi. It was observed that chalcones of 3,4- dihydroquinoxalin-2(1H)-one exhibited better activity (MIC: 2.57 AŽÂ¼M) as compared to the chalcones of quinoxalin-2,3(1H,4H)-dione. The ClogP values of the synthesized compounds were in good agreement with antifungal activity indicated the significance of lipid solubility better cell wall penetration. Overall quinoxalinyl chalcones exhibited broad spectrum antimicrobial activity.Owing to the prevalence of antimicrobial resistance and its arising threat rendered by the ineffectiveness of existing antibiotics, the present work was aimed to generate new 5/6 substituted quinoxalinyl chalcones derivatives. A total of 16 compounds were synthesized and characterized by IR, 1H NMR, 13C NMR and mass spectral studies. The antimicrobial activity against resistant pathogens was by micro-dilution assay method. The result revealed that compounds bearing 2,4-dichloro benzylidine and 4-fluoro benzylidine moiety demonstrated promising antibacterial and antifungal activities against the selected pathogenic bacteria and fungi. It was observed that chalcones of 3,4- dihydroquinoxalin-2(1H)-one exhibited better activity (MIC: 2.57 AŽÂ¼M) as compared to the chalcones of quinoxalin-2,3(1H,4H)-dione. The ClogP values of the synthesized compounds were in good agreement with antifungal activity indicated the significance of lipid solubility better cell wall penetration. Overall quinoxalinyl chalcones exhibited broad spectrum antimicrobial activity.

Cell wall composition and penetration resistance against the fungal pathogen Colletotrichum higginsianum are affected by impaired starch turnover in Arabidopsis mutants.

Penetration resistance represents the first level of plant defense against phytopathogenic fungi. Here, we report that the starch-deficient Arabidopsis thaliana phosphoglucomutase (pgm) mutant has impaired penetration resistance against the hemibiotrophic fungus Colletotrichum higginsianum. We could not determine any changes in leaf cutin and epicuticular wax composition or indolic glucosinolate levels, but detected complex alterations in the cell wall monosaccharide composition of pgm. Notably, other mutants deficient in starch biosynthesis (adg1) or mobilization (sex1) had similarly affected cell wall composition and penetration resistance. Glycome profiling analysis showed that both overall cell wall polysaccharide extractability and relative extractability of specific pectin and xylan epitopes were affected in pgm, suggesting extensive structural changes in pgm cell walls. Screening of mutants with alterations in content or modification of specific cell wall monosaccharides indicated an important function of pectic polymers for penetration resistance and hyphal growth of C. higginsianum during the biotrophic interaction phase. While mutants with affected pectic rhamnogalacturonan-I (mur8) were hypersusceptible, penetration frequency and morphology of fungal hyphae were impaired on pmr5 pmr6 mutants with increased pectin levels. Our results reveal a strong impact of starch metabolism on cell wall composition and suggest a link between carbohydrate availability, cell wall pectin and penetration resistance.

Single-wall and multi-wall carbon nanotubes promote rice root growth by eliciting the similar molecular pathways and epigenetic regulation.

Organisms are constantly exposed to environmental stimuli and have evolved mechanisms of protection and adaptation. Various effects of nanoparticles (NPs) on crops have been described and some results confirm that NPs could enhance plant growth at the physiological and genetic levels. This study comparatively analysed the effect of carbon nanotubes (CNTs) on rice growth. The results showed that single-wall CNTs were located in the intercellular space while multi-wall CNTs penetrated cell walls in roots. CNTs could promote rice root growth through the regulation of expression of the root growth related genes and elevated global histone acetylation in rice root meristem zones. These responses were returned to normal levels after CNTs were removed from medium. CNTs caused the similar histone acetylation and methylation statuses across the local promoter region of the Cullin-RING ligases 1 (CRL1) gene and increased micrococcal nuclease accessibility of this region, which enhanced this gene expression. The authors results suggested that CNTs could cause plant responses at the cellular, genetic, and epigenetic levels and these responses were independent on interaction modes between root cells and CNTs.

The bacteriophage ϕ29 tail possesses a pore-forming loop for cell membrane penetration.

Most bacteriophages are tailed bacteriophages with an isometric or a prolate head attached to a long contractile, long non-contractile, or short non-contractile tail. The tail is a complex machine that plays a central role in host cell recognition and attachment, cell wall and membrane penetration, and viral genome ejection. The mechanisms involved in the penetration of the inner host cell membrane by bacteriophage tails are not well understood. Here we describe structural and functional studies of the bacteriophage ϕ29 tail knob protein gene product 9 (gp9). The 2.0 Å crystal structure of gp9 shows that six gp9 molecules form a hexameric tube structure with six flexible hydrophobic loops blocking one end of the tube before DNA ejection. Sequence and structural analyses suggest that the loops in the tube could be membrane active. Further biochemical assays and electron microscopy structural analyses show that the six hydrophobic loops in the tube exit upon DNA ejection and form a channel that spans the lipid bilayer of the membrane and allows the release of the bacteriophage genomic DNA, suggesting that cell membrane penetration involves a pore-forming mechanism similar to that of certain non-enveloped eukaryotic viruses. A search of other phage tail proteins identified similar hydrophobic loops, which indicates that a common mechanism might be used for membrane penetration by prokaryotic viruses. These findings suggest that although prokaryotic and eukaryotic viruses use apparently very different mechanisms for infection, they have evolved similar mechanisms for breaching the cell membrane.

Microstructural and Micromechanical Characterization of Modified Urea-Formaldehyde Resin Penetration into Wood

Characterization of the adhesive penetration behavior in wood is highly desired for optimizing the manufacturing processes and product properties. In this study, modified urea-formaldehyde (UF) adhesive was used to prepare glued laminated timber (Cryptomeria fortunei Hooibrenk). The depth of gross penetration was measured by fluorescence microscopy (FM), which showed the UF passed through 1.5 to 3.5 earlywood tracheids (with an average penetration depth of 88.95 ± 27.49 μm) or 0.5 to 4.0 latewood tracheids (with an average penetration depth of 36.39 ± 15.14 μm). In addition, the distribution of cell wall penetration was observed clearly by confocal laser scanning microscopy (CLSM). The adhesive was found to diffuse into the cell walls of surface tissues embedded in the UF. To verify the results from CLSM, the mechanical properties of cell walls with and without adhesive penetration were measured through nanoindentation (NI). The reduced elastic modulus of exposed cell walls (18.10 GPa) was roughly equal to that of fully filled cell walls (17.68 GPa) but significantly greater than that of reference ones (15.71 GPa). The hardness showed a similar variation trend for these three types of cell walls. Combining the three techniques, both the microstructure and micromechanics of the adhesive penetration behavior can be quantitatively identified in a complementary manner.

Discovery of 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazoles as Novel DNA Gyrase Inhibitors Targeting the ATP-Binding Site.

Bacterial DNA gyrase and topoisomerase IV are essential enzymes that control the topological state of DNA during replication and validated antibacterial drug targets. Starting from a library of marine alkaloid oroidin analogues, we identified low micromolar inhibitors of Escherichia coli DNA gyrase based on the 5,6,7,8-tetrahydroquinazoline and 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole scaffolds. Structure-based optimization of the initial hits resulted in low nanomolar E. coli DNA gyrase inhibitors, some of which exhibited micromolar inhibition of E. coli topoisomerase IV and of Staphylococcus aureus homologues. Some of the compounds possessed modest antibacterial activity against Gram positive bacterial strains, while their evaluation against wild-type, impA and ΔtolC E. coli strains suggests that they are efflux pump substrates and/or do not possess the physicochemical properties necessary for cell wall penetration. Our study provides a rationale for optimization of this class of compounds toward balanced dual DNA gyrase and topoisomerase IV inhibitors with antibacterial activity.

Modifying wood with bio-polyesters: analysis and performance

Oligomers of lactic and glycolic acid (OLA, OGA) and poly(butylene succinate/adipate) (OBS, OBA) were impregnated and heated into wood for dimensional stabilisation. These bio-polyesters variously provide bulking and lumen filling treatments. Solvent extraction of treated wood showed that ca. 50% of the polymer remains in wood with in situ polymerisation evidenced by GPC. OGA readily penetrates the wood cell wall, which occurred only on heating OLA, whereas OBS/OBA was lumen filling with any cell wall penetration only evident after water soaking. Stability of OLA/OGA treated wood was improved, but did not withstand extended leaching, whereas OBS/OBA treated wood presented higher stability once leached. Bending resistance was influenced by OLA/OGA treatments, but original MOE values were recovered after leaching. Overall, the results show the bio-polyester treatment can confer specific properties of the modified wood to suit selected end use applications.

Plant cell wall in pathogenesis, parasitism and symbiosis.

Plant cell wall in pathogenesis, parasitism and symbiosis.

Active invasion of bacteria into living fungal cells.

The rice seedling blight fungus Rhizopus microsporus and its endosymbiont Burkholderia rhizoxinica form an unusual, highly specific alliance to produce the highly potent antimitotic phytotoxin rhizoxin. Yet, it has remained a riddle how bacteria invade the fungal cells. Genome mining for potential symbiosis factors and functional analyses revealed that a type 2 secretion system (T2SS) of the bacterial endosymbiont is required for the formation of the endosymbiosis. Comparative proteome analyses show that the T2SS releases chitinolytic enzymes (chitinase, chitosanase) and chitin-binding proteins. The genes responsible for chitinolytic proteins and T2SS components are highly expressed during infection. Through targeted gene knock-outs, sporulation assays and microscopic investigations we found that chitinase is essential for bacteria to enter hyphae. Unprecedented snapshots of the traceless bacterial intrusion were obtained using cryo-electron microscopy. Beyond unveiling the pivotal role of chitinolytic enzymes in the active invasion of a fungus by bacteria, these findings grant unprecedented insight into the fungal cell wall penetration and symbiosis formation.

Fungi open new possibilities for anaerobic fermentation of organic residues

Large amounts of fibre-rich organic waste material from public green and private gardens have to be treated environmentally friendly; however, this fibre-rich biomass has low biogas yields. This study investigated the presence of fungi in full-scale biogas plants as well as in laboratory reactors and elucidated the importance of fungi for the biogas process. The dominating members of the eukaryotic community were identified by analyzing 18S rRNA gene and internal transcribed spacer 1 (ITS1) region fragments of clone libraries. These identifications were accompanied by diverse microscopic techniques such as fluorescence microscopy and conventional scanning electron microscopy. Cells of presumably fungal origin were characterized by intensive fluorescence and were about 1 order of magnitude larger than prokaryotic cells. Molecular techniques enabled to identify fungi from the subphyla Agaricomycotina, Mucoromycotina, Pezizomycotina, Pucciniomycotina and Saccharomycotina and from the class Neocallimastigomycetes. Members of these groups can be important for microbial degradation of complex compounds, due to the ability to penetrate cell walls, and thus open the cells for the influx of bacteria, further enhancing degradation. Optimal treatment of biowaste depends on the amount of lignocelluloses. Targeted application of fungi to the biogas process will open wider possibilities for anaerobic treatment of fibre-rich biomass and can result in better biomass utilization as a renewable energy resource. Due to higher temperature optima of fungal cellulolytic enzymes, the thermophilic process is suggested for anaerobic degradation of fibre-rich biomass.

Nanoscale glucan polymer network causes pathogen resistance

Successful defence of plants against colonisation by fungal pathogens depends on the ability to prevent initial penetration of the plant cell wall. Here we report that the pathogen-induced (1,3)-β-glucan cell wall polymer callose, which is deposited at sites of attempted penetration, directly interacts with the most prominent cell wall polymer, the (1,4)-β-glucan cellulose, to form a three-dimensional network at sites of attempted fungal penetration. Localisation microscopy, a super-resolution microscopy technique based on the precise localisation of single fluorescent molecules, facilitated discrimination between single polymer fibrils in this network. Overexpression of the pathogen-induced callose synthase PMR4 in the model plant Arabidopsis thaliana not only enlarged focal callose deposition and polymer network formation but also resulted in the exposition of a callose layer on the surface of the pre-existing cellulosic cell wall facing the invading pathogen. The importance of this previously unknown polymeric defence network is to prevent cell wall hydrolysis and penetration by the fungus. We anticipate our study to promote nanoscale analysis of plant-microbe interactions with a special focus on polymer rearrangements in and at the cell wall. Moreover, the general applicability of localisation microscopy in visualising polymers beyond plant research will help elucidate their biological function in complex networks.

3D-QSAR and Cell Wall Permeability of Antitubercular Nitroimidazoles against Mycobacterium tuberculosis

Inhibitory activities of monocyclic nitroimidazoles against Mycobacterium tuberculosis (Mtb) deazaflavin-dependent nitroreductase (DDN) were modeled by using docking, pharmacophore alignment and comparative molecular similarity indices analysis (CoMSIA) methods. A statistically significant model obtained from CoMSIA was established based on a training set using pharmacophore-based molecular alignment. The leave-one out cross-validation correlation coefficients q2 (CoMSIA) were 0.681. The CoMSIA model had a good correlation (/CoMSIA = 0.611) between the predicted and experimental activities against excluded test sets. The generated model suggests that electrostatic, hydrophobic and hydrogen bonding interactions all play important roles for interaction between ligands and receptors. The predicted cell wall permeability (logPapp) for substrates with high inhibitory activity against Mtb were investigated. The distribution coefficient (logD) range was 2.41 < logD < 2.89 for the Mtb cell wall membrane permeability. The larger the polar surface area is, the better the permeability is. A larger radius of gyration (rgry) and a small fraction of rotatable bonds (frtob) of these molecules leads to higher cell wall penetration ability. The information obtained from the in silico tools might be useful in the design of more potent compounds that are active against Mtb.

Finite Element Analysis of Single Cell Wall Cutting by Piezoelectric-Actuated Vibrating Rigid Nanoneedle

We proposed a piezoelectric-actuated rigid nanoneedle for single cell wall cutting. A fabricated Tungsten (W) nanoneedle was assembled with a commercial piezoelectric actuator laterally and perpendicularly. The body of the nanoneedle is cylindrical while the shape of the tip is conical. Calibration of the assembled device was conducted using both experimental and numerical approaches. Experimentally, we have obtained a 4.5 μm displacement of the nanoneedle from an input potential of 100 V toward the Y axis. Results also showed that, at lateral configuration, nanoneedle tip is more stable than at perpendicular configuration. For the perpendicular configuration, we observed a fractional displacement of 1.2 pm, but in the lateral configuration we did not experience any fractional displacement. As a result, for a precise cell wall cutting we choose the lateral configuration of the nanoneedle tip. W303 yeast cell model was used as a sample for single cell wall cutting. Yeast cell wall is more likely to behave as a Mooney-Rivlin hyperelastic material. Two different frequencies of vibrating nanoneedle were used for cell wall cutting operation; 1 and 10 Hz. For a constant penetration depth of 1.2 μm, penetration velocity was 7 and 24 μm/s, respectively. Investigated results showed that the faster nanoneedle causes less damage to the cell wall during cutting operation. Additionally, further finite element (FE) analysis was performed to observe the effect of tip edge angle for cell wall cutting. Three different edge angles, 10°, 15°, and 20° were used to perform cell wall cutting. Single cell wall penetration force increases with the increasing of tip edge angle and the diameters of the nanoneedle.

The influence of curing conditions on the chemical distribution in wood modified with thermosetting resins

Scots pine (Pinus sylvestris L.) sapwood was impregnated with aqueous solutions of phenol formaldehyde and methylated melamine formaldehyde resins and subsequently cured in an oven. One set of specimens was cured in plastic bags to avoid drying (wet curing) while another set of samples was heated and water was allowed to freely evaporate (dry curing). Macroscopic resin distribution was investigated using X-ray densitometry and infrared spectroscopy (FTIR-ATR). During dry curing, the resins migrated to the wood surface resulting in a gradient. Wet curing resulted in even distribution of the resins because it was immobilized due to condensation and precipitation in the wood. Neither densitometry nor FTIR-ATR was found to be generally applicable for investigating resin distribution in modified wood. Wet curing resulted in low cell wall bulking as compared to dry curing, probably because resin precipitated before drying. Storing impregnated wood prior to curing under non-drying conditions (“diffusion phase”) also reduced cell wall penetration and bulking.

Direct and rapid quantum dots labelling of Escherichia coli cells

Here, we supplied a direct and rapid quantum dots labelling method of bacterial cells in food, water and environmental contaminations. Outer layers of Escherichia coli cells prevent cells from direct interactions with molecules and objects such as quantum dots. Permeabilization treatment of E. coli cells may facilitate macromolecules penetrate cell walls and improve internal bacterial quantum dots (QDs) labelling. In this work, we investigated direct internal QDs labelling of E. coli cells permeabilized using three methods including chloroform-SDS treatment, lysozyme-EDTA treatment and osmotic shock treatment. Effects of permeabilization were analysed by scanning electronic microscopy and measuring activity of alkaline phosphatase (PhoA) released from periplasm. Internal bacterial QDs labelling was monitored by flow cytometry and fluorescent microscopy. After chloroform-SDS or lysozyme-EDTA treatment, cells could be directly labelled with QDs. No interaction was observed between osmotic shock treated cells and QDs. The mechanism of cell permeabilization explaining different labelling efficiency has been established. The QDs labelling approach presented in this work provides a simple, rapid and sensitive detection method for multiple purpose bacterial analysis in combination with biological techniques.

Histopathological assessment of wheat seedling tissues infected by Fusarium pseudograminearum

Histopathological assessment of infection by the crown rot pathogen Fusarium pseudograminearum in wheat seedling tissues was performed using fluorescence microscopy. The coleoptiles and leaf sheaths of four host cultivars of differing susceptibility were examined. Leaf sheaths were most frequently penetrated via stomata, indicated by initial lesions forming at the guard cells. Internally, cell wall penetration was facilitated by penetration structures which appeared as hyphal swellings or septate foot‐shaped appressoria. Colonization of leaf sheaths resulted in the re‐emergence of hyphae from stomata on both surfaces of the sheath. These hyphae are hypothesized to have two major roles; first as exploratory hyphae for colonization of new tissues, and secondly as sites of profuse conidial production. The formation of conidia on the leaf sheath surface was only recorded on the most susceptible bread wheat genotype. No other major differences in host–pathogen interactions were observed among these cultivars. Almost all cell types in the leaf sheath tissues were extensively colonized, except for the vascular bundles and silica cells. This investigation provides the first comprehensive assessment of F. pseudograminearum infection structures and growth patterns during the infection of wheat seedlings.

Influence of the viscosity of UF resins on the radial and tangential penetration into poplar wood and on the shear strength of adhesive joints

Abstract This paper is the final part of a series reporting on the penetration (P) and bonding behaviour of urea-formaldehyde (UF) resins of different viscosities based on different degrees of condensation (DCs). The various resins were applied onto radial and tangential wood surfaces, and solid wood assemblies were prepared by hot pressing. Pt and Pr were observed by evaluation of photomicrographs of the bond lines. The mean and maximum depth of penetration was inversely proportional to the DC. The average depth of Pt was found to be slightly higher than Pr because of the abundance of pits on the radial walls. However, the maximum P was higher in radial direction, due to the elliptic shape of vessels in poplar as well as due to rays improving the resin flow. The lap shear strength (σm) of the same bonded assemblies was determined. Despite the fact that the wood failure always was very high, the σm increased with the better penetration of the resins with low viscosity. The higher strength values measured has been interpreted as a fortification effect of the interphase by bulk resin penetration, as cell wall penetration cannot be expected due to the large size of such resin molecules.

The use of GFP-transformed isolates to study infection of banana with Fusarium oxysporum f. sp. cubense race 4

Fusarium oxysporum f. sp. cubense (Foc) is the causal pathogen of Fusarium wilt of banana. To understand infection of banana roots by Foc race 4, we developed a green fluorescent protein (GFP)-tagged transformant and studied pathogenesis using fluorescence microscopy and confocal laser scanning microscopy. The transformation was efficient, and GFP expression was stable for at least six subcultures with fluorescence clearly visible in both hyphae and spores. The transformed Foc isolate also retained its pathogenicity and growth pattern, which was similar to that of the wild type. The study showed that: (i) Foc race 4 was capable of invading the epidermal cells of banana roots directly; (ii) potential invasion sites include epidermal cells of root caps and elongation zone, and natural wounds in the lateral root base; (iii) in banana roots, fungal hyphae were able to penetrate cell walls directly to grow inside and outside cells; and (iv) fungal spores were produced in the root system and rhizome. To better understand the interaction between Foc race 4 and bananas, nine banana cultivars were inoculated with the GFP-transformed pathogen. Root exudates from these cultivars were collected and their effect on conidia of the GFP-tagged Foc race 4 was determined. Our results showed that roots of the Foc race 4-susceptible banana plants were well colonized with the pathogen, but not those of the Foc race 4-resistant cultivars. Root exudates from highly resistant cultivars inhibited the germination and growth of the Fusarium wilt pathogen; those of moderately resistant cultivars reduced spore germination and hyphal growth, whereas the susceptible cultivars did not affect fungal germination and growth. The results of this work demonstrated that GFP-tagged Foc race 4 isolates are an effective tool to study plant–fungus interactions that could potentially be used for evaluating resistance in banana to Foc race 4 by means of root colonization studies. Banana root exudates could potentially also be used to identify cultivars in the Chinese Banana Germplasm Collection with resistance to the Fusarium wilt pathogen.

Role of selected dark septate endophyte species and other hyphomycetes as saprobes on moss gametophytes

Dark septate endophytes (DSEs) live asymptomatically in the roots of vascular plants, are common in arctic and alpine areas, and are thought to play a quasimycorrhizal role. It is not known, however, whether they precede or arrive with their hosts. Previously reported enzymatic abilities of Phialocephala fortinii suggest that DSEs can live on organic debris in the soil, but there is little direct or experimental evidence for this. Phialocephala fortinii , Leptodontidium orchidicola , Cadophora melinii , Cadophora luteo-olivacea , and Lecythophora sp. were inoculated onto autoclaved Hylocomium splendens gametophytes and incubated for 3 months to determine if they degrade this organic material based on observations made using light and scanning electron microscopy. All fungi were able to colonize the bryophyte tissue to some extent. Lecythophora sp. and L. orchidicola penetrated cells by forming bore holes. Cadophora luteo-olivacea and P. fortinii were also observed inside cells, but bore holes through bryophyte cell walls were not observed. Cadophora melinii sporulated and grew abundantly on the surface of gametophytes but did not appear to penetrate cell walls. Phialocephala fortinii and L. orchidicola formed sclerotia in the gametophytes similar to those formed in roots. These results suggest that DSE fungi can persist and produce propagules, i.e., sclerotia and conidia, in the absence of host roots. These observations support the hypothesis that DSE fungi are able to precede their hosts during primary succession events.