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In this study, we investigated the intricate interplay between Trichoderma and the tomato genome, focusing on the transcriptional and metabolic changes triggered during the late colonization event. Microarray probe set (GSE76332) was utilized to analyze the gene expression profiles changes of the un-inoculated control (tomato) and Trichoderma-tomato interactions for identification of the differentially expressed significant genes. Based on principal component analysis and R-based correlation, we observed a positive correlation between the two cross-comaparable groups, corroborating the existence of transcriptional responses in the host triggered by Trichoderma priming. The statistically significant genes based on different p-value cut-off scores [(p adj -values or q-value); p adj -value < 0.05], [(p cal -values); p cal -value < 0.05; p cal  < 0.01; p cal  < 0.001)] were cross compared. Through cross-comparison, we identified 156 common genes that were consistently significant across all probability thresholds, and showing a strong positive corelation between p-value and q-value in the selected probe sets. We reported TD2, CPT1, pectin synthase, EXT-3 (extensin-3), Lox C, and pyruvate kinase (PK), which exhibited upregulated expression, and Glb1 and nitrate reductase (nii), which demonstrated downregulated expression during Trichoderma-tomato interaction. In addition, microbial priming with Trichoderma resulted into differential expression of transcription factors related to systemic defense and flowering including MYB13, MYB78, ERF2, ERF3, ERF5, ERF-1B, NAC, MADS box, ZF3, ZAT10, A20/AN1, polyol sugar transporter like zinc finger proteins, and a novel plant defensin protein. The potential bottleneck and hub genes involved in this dynamic response were also identified. The protein-protein interaction (PPI) network analysis based on 25 topmost DEG S (p cal -value < 0.05) and the Weighted Correlation Gene Network Analysis (WGCNA) of the 1786 significant DEGs (p cal- value < 0.05) we reported the hits associated with carbohydrate metabolism, secondary metabolite biosynthesis, and the nitrogen metabolism. We conclude that the Trichoderma-induced microbial priming re-programmed the host genome for transcriptional response during the late colonization event and were characterized by metabolic shifting and biochemical changes specific to plant growth and development. The work also highlights the relevance of statistical parameters in understanding the gene regulatory dynamics and complex regulatory networks based on differential expression, co-expression, and protein interaction networks orchestrating the host responses to beneficial microbial interactions., (© 2023. The Author(s).)

The study aimed to evaluate the efficacy of octyl-2-cyanoacrylate as well as sutures as a wound closure material. Whether octyl-2-cyanoacrylate can be used as an alternative to suture in the closure of facial wounds. Out of total of 19 wounds, 11 wounds were closed with sutures and tissue adhesive both, out of rest 8 wounds, 4 wounds were closed using sutures alone and in 4 wounds, closure was done with tissue adhesive alone. Therefore, a total of 15 facial wounds were divided into two groups: Group-I Cases in whom 3-0 Black silk suture was used for closure of the wound and Group-II Cases in whom Dermabond (Octyl-2-Cyanoacrylate) was used for closure of the wound. Results showed that Octyl-2-cyanoacrylate offered the benefit of decreased procedure time with less pain, no need for its removal, and better cosmetic outcome compared to sutures., Competing Interests: There are no conflicts of interest., (Copyright: © 2023 Journal of Pharmacy and Bioallied Sciences.)

Background: Trichoderma asperellum and T. harzianum were assessed in this study as a potential biological control against Colletotrichum truncatum. C. truncatum is a hemibiotrophic fungus that causes anthracnose disease in chilli thereby affecting plant growth and fruit yield. Scanning electron microscope (SEM) technique showed the beneficial interaction between chilli root-Trichoderma spp. inducing the plant growth promotion, mechanical barrier, and defense network under C. truncatum challenged conditions., Methods: Seeds bio-primed with T. asperellum, T. harzianum, and T. asperellum + T. harzianum promoted the plant growth parameters and strengthening of physical barrier via lignification on the wall of vascular tissues. Seed primed with bioagents were used for exploring the molecular mechanism of defense response in pepper against anthracnose to assess the temporal expression of six defense genes in the Surajmukhi variety of Capsicum annuum. QRT-PCR demonstrated induction of defense responsive genes in chilli pepper bioprimed with Trichoderma spp. such as plant defensin 1.2 (CaPDF1.2), superoxide dismutase (SOD), ascorbate peroxidase (APx), guaiacol peroxidase (GPx), pathogenesis related proteins PR-2 and PR-5., Results: The results showed that bioprimed seeds were assessed for T. asperellum, T. harzianum, and T. asperellum + T. harzianum-chilli root colonization interaction under in vivo conditions. The results of the scanning electron microscope revealed that T. asperellum, T. harzianum and T. asperellum + T. harzianum interact with chilli roots directly via the development of plant-Trichoderma interaction system. Seeds bio-primed with bioagents promoted the plant growth parameters, fresh and dry weight of shoot and root, plant height, leaf area index, number of leaves, stem diameter and strengthening of physical barrier via lignification on the wall of vascular tissues and expression of six defense related genes in pepper against anthracnose., Conclusions: Application of T. asperellum and T. harzianum and in combination of treatments enhanced the plant growth. Further, as seeds bioprimed with T. asperellum, T. harzianum and in combination with treatment of T. asperellum + T. harzianum induced the strengthening of the cell wall by lignification and expression of six defense related genes CaPDF1.2, SOD, APx, GPx, PR-2 and PR-5 in pepper against C. truncatum. Our study contributed for better disease management through biopriming with T. asperellum, T. harzianum and T. asperellum + T. harzianum. The biopriming possess enormous potential to promote plant growth, modulate the physical barrier, and induced the defense related genes in chilli pepper against anthracnose., (© 2023. The Author(s).)

White mold and stem rot is a common disease of Phaseolus vulgaris caused by Sclerotinia sclerotiorum. Biological control is a promising alternative for the control of this disease. In the present study, two Trichoderma spp., T. erinaceum and T. viride, and the consortium of both were evaluated as biocontrol agents against sclerotinia stem rot disease. The results revealed that T. erinaceum (NAIMCC-F-02171) and T. viride (NAIMCC-F-02500) when applied alone, significantly suppressed the infection rate of S. sclerotiorum and increased the rate of survival of plants by 74.5%. On the contrary, the combination of both the Trichoderma spp. was found to be more effective in reducing stem rot by 57.2% and increasing the survival of plants by 87.5% when compared to the individual Trichoderma applications. Further, the exogenous supplementation of Trichoderma activated antioxidative machineries, such as peroxidase, polyphenol oxidase, superoxide dismutase, catalase, and ascorbic acid in the plant. Besides, hydrogen peroxide and superoxide-free radical accumulation were also found to be reduced when T. erinaceum and T. viride were used either individually or in combination under the pathogen-challenged condition. Additionally, the photopigments in the bioprimed plants were markedly increased. Moreover, the combined inoculation of the two isolates yielded the highest records of growth parameters (root weight, shoot length, and leaf weight) compared with individual inoculation. Therefore, based on the above results, it was concluded that the combination of T. erinaceum and T. viride can be effectively used as an alternative to control white mold and stem rot caused by S. sclerotiorum., (© 2021 Wiley-VCH GmbH.)

In the present study, Paenibacillus dendritiformis , Trichoderma harzianum, and Trichoderma asperellum were appraised as potential biocontrol agents that induce resistance in chilli ( Capsicum annuum ) against the devastating pathogen Colletotrichum truncatum, which causes anthracnose. Bright-field and scanning electron micrographs showed the hyphal degradation, lysis, and abnormal swelling in C. truncatum against P. dendritiformis in a dual plate assay. Under greenhouse conditions, chilli seeds pretreated with P. dendritiformis, T. asperellum, T. harzianum, and T. asperellum + T. harzianum by soil soak method inflicted an induced systemic resistance (ISR) in chilli against a C. truncatum -challenged condition. In chilli, the disease index percentage was significantly reduced in the T. asperellum + T. harzianum -treated seeds, followed by the T. harzianum- , T. asperellum-, and P. dendritiformis -treated seeds as compared to the untreated and challenged, respectively. Chilli seeds were primed with T. asperellum + T. harzianum (78.67%), which revealed maximum disease protection under the challenged condition, followed by T. harzianum (70%), T. asperellum (64%), and P. dendritiformis (56%) as compared to untreated and C. truncatum -challenged (6%) condition served as control. The seeds that were pretreated with biocontrol agents (BCAs) inflicted ISR against C. truncatum by enhancing the activity of defence-related enzymes (superoxide dismutase (SOD), peroxidase (POX), polyphenol oxidase (PPO), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and phenylalanine ammonia-lyase (PAL)), accumulating phenolic compounds, and increasing the relative chlorophyll content in chilli. Nitroblue tetrazolium (NBT) and 3,3'-Diaminobenzidine (DAB) stains were used to detect the accumulation of superoxide anion and hydrogen peroxide that appeared nearby the fungal infection sites. The accumulation of reactive oxygen species (O 2 - and H 2 O 2 ) in the pathogen-inoculated leaves was a maximum of 48 hpi, followed by P. dendritiformis, T. asperellum, T. harzianum, and T. asperellum + T. harzianum treated tissue upon C. truncatum -challenged condition as compared to the control. Overall, our results showed the potential of T. harzianum, T. asperellum, and P. dendritiformis as biocontrol agents that prevent infection by C. truncatum and inflict an induced systemic resistance in chilli by enhancing the biosynthesis of phenolic compounds, defence and antioxidative enzymes, and reducing the lesion development and reactive oxygen species accumulation. This is the first report of induced systemic resistance against anthracnose in chilli obtained by application of T. harzianum , T. asperellum and P. dendritiformis , through seed priming.

Aim: The aim of this study was to compare 2 mm conventional miniplates with three-dimensional (3D) strut plates in the treatment of mandibular fractures treated in the Department of Oral and Maxillofacial Surgery of a college in India, during 2012-2015., Materials and Methods: All 20 patients with mandible fracture requiring open reduction and internal fixation of the fracture were reported and reviewed in the Department of Oral and Maxillofacial Surgery of the same dental college in India, during 2012-2015. We classified the data according to the gender, age, etiology, fracture site, and method of treatment of the fractures. Fixation done in 10 patients with 2 mm conventional miniplates kept in Group I, while 10 patients with 3D strut miniplate system considered in Group II., Results: Assessment of patient was done under the following parameters with follow-up at regular intervals at 2 nd day postoperatively and at 1 st , 3 rd , and 6 th weeks postoperatively: pain, swelling, infection, interincisal mouth opening in millimeters, paresthesia/anesthesia, mobility between fracture fragments, overall occlusion, need for any supplemental fixation, fate of implant, and complications., Conclusions: 3D titanium miniplates showed similar results when compared to standard titanium miniplates with advantages such as reduction in operative time, ease of placement, and cost-effective over the conventional plate system., Competing Interests: There are no conflicts of interest., (Copyright: © 2020 Annals of Maxillofacial Surgery.)

The beneficial association and interaction of rhizocompetent microorganisms are widely used for plant biofertilization and amelioration of stress-induced damage in plants. To explore the regulatory mechanism involved in plant defense while associating with beneficial microbial species, and their interplay when co-inoculated with pathogens, we evaluated the response of tomato defense-related WRKY gene transcripts. The present study was carried out to examine the qRT-PCR-based relative quantification of differentially expressed defense-related genes in tomato ( Solanum lycopersicum L.; variety S-22) primed with Trichoderma erinaceum against the vascular wilt pathogen ( Fusarium oxysporum f. sp. lycopersici ). The tissue-specific and time-bound expression profile changes under the four different treatments "(unprimed, Fol challenged, T. erinaceum primed and Fol + T. erinaceum )" revealed that the highest upregulation was observed in the transcript profile of SlWRKY31 (root) and SlWRKY37 (leaf) in T. erinaceum bioprimed treated plants at 24 h with 16.51- and 14.07-fold increase, respectively. In contrast, SlWRKY4 showed downregulation with the highest repression in T. erinaceum bioprimed root (24 h) and leaf (48 h) tissue samples with 0.03 and 0.08 fold decrease, respectively. Qualitative expression of PR proteins (chitinases and glucanases) was found elicited in T. erinaceum primed plants. However, the antioxidative activity of tomato superoxide dismutase and catalase increased with the highest upregulation of SOD and SlGPX1 in Fol + T. erinaceum treatments. We observed that these expression changes were accompanied by 32.06% lesser H 2 O 2 production in T. erinaceum bioprimed samples. The aggravated defense response in all the treated conditions was also reflected by an increased lignified stem tissues. Overall, we conclude that T. erinaceum bio-priming modulated the defense transcriptome of tomato after the Fol challenged conditions, and were accompanied by enhanced accumulation of defense-related WRKY transcripts, increased antioxidative enzyme activities, and the reinforcements through a higher number of lignified cell layers.

Aim/objective: The mandibular third molar is the most frequently impacted tooth with incidence varies from 9.5% to 68% in different populations. Hence, the aim was to study the prevalence and pattern of mandibular impacted third molar among Delhi-National Capital Region (NCR) population., Materials and Methods: The study was conducted with data collected from registered hospitals and dental clinics of Delhi NCR region. The study represents a retrospective analysis of panoramic radiographs and intraoral periapical radiograph of patients at these centers from June 2014 to June 2016., Results: Out of 960 patients with the third molar investigated, a total of 250 patients having impacted mandibular third molar (152 [60.8%] males and 98 [39.2%]) females between June 2014 and June 2016 were included in the study. The age ranged from 20 to 55 years, with a mean age of 27.6 years and the standard deviation was 5.8 years. The prevalence of impacted mandibular third molars for this study was 26.04%., Conclusion: This study demonstrated that males (60.8%) were more likely to present with impacted mandibular third molars than females (39.2%). The prevalence of third molar impactions was almost the same on both the left (45.8%) and right (54.2%) sides. This study also noted that mesioangular impactions (49.2%) were the most common type of impaction. The least common form of impactions was the transverse types (2%). The prevalence of impacted mandibular third molars for this study was 26.04%., Competing Interests: There are no conflicts of interest.

Vascular wilt of tomato caused by Fusarium oxysporum f.sp. lycopersici (FOL) is one of the most devastating diseases, that delimits the tomato production worldwide. Fungal short-chain dehydrogenases/reductases (SDRs) are NADP(H) dependent oxidoreductases, having shared motifs and common functional mechanism, have been demonstrated as biochemical targets for commercial fungicides. The 1,3,6,8 tetra hydroxynaphthalene reductase (T4HNR) protein, a member of SDRs family, catalyzes the naphthol reduction reaction in fungal melanin biosynthesis. We retrieved an orthologous member of T4HNR, (complexed with NADP(H) and pyroquilon from Magnaporthe grisea ) in the FOL (namely; FOXG&#95;04696) based on homology search, percent identity and sequence similarity (93% query cover; 49% identity). The hypothetical protein FOXG&#95;04696 (T4HNR like) had conserved T-G-X-X-X-G-X-G motif (cofactor binding site) at N-terminus, similar to M. grisea (1JA9) and Y-X-X-X-K motif, as a part of the active site, bearing homologies with two fungal keto reductases T4HNR ( M. grisea ) and 17-β-hydroxysteroid dehydrogenase from Curvularia lunata (teleomorph: Cochliobolus lunatus PDB ID: 3IS3). The catalytic tetrad of T4HNR was replaced with ASN 115 , SER 141 , TYR 154 , and LYS 158 in the FOXG&#95;04696. The structural alignment and superposition of FOXG&#95;04696 over the template proteins (3IS3 and 1JA9) revealed minimum RMSD deviations of the C alpha atomic coordinates, and therefore, had structural conservation. The best protein model (FOXG&#95;04696) was docked with 37 fungicides, to evaluate their binding affinities. The Glide XP and YASARA docked complexes showed discrepancies in results, for scoring and ranking the binding affinities of fungicides. The docked complexes were further refined and rescored from their docked poses through 50 ns long MD simulations, and binding free energies (ΔG bind ) calculations, using MM/GBSA analysis, revealed Oxathiapiprolin and Famoxadone as better fungicides among the selected one. However, Famoxadone had better interaction of the docked residues, with best protein ligand contacts, minimum RMSD (high accuracy of the docking pose) and RMSF (structural integrity and conformational flexibility of docking) at the specified docking site. The Famoxadone was found to be acceptable based on in silico toxicity and in vitro growth inhibition assessment. We conclude that the FOXG&#95;04696, could be employed as a novel candidate protein, for structure-based design, and screening of target fungicides against the FOL pathogen.

The WRKY transcription factors have indispensable role in plant growth, development and defense responses. The differential expression of WRKY genes following the stress conditions has been well demonstrated. We investigated the temporal and tissue-specific (root and leaf tissues) differential expression of plant defense-related WRKY genes, following the infection of Fusarium oxysporum f. sp. lycopersici (Fol) in tomato. The genome-wide computational analysis revealed that during the Fol infection in tomato, 16 different members of WRKY gene superfamily were found to be involved, of which only three WRKYs (SolyWRKY4, SolyWRKY33, and SolyWRKY37) were shown to have clear-cut differential gene expression. The quantitative real time PCR (qRT-PCR) studies revealed different gene expression profile changes in tomato root and leaf tissues. In root tissues, infected with Fol, an increased expression for SolyWRKY33 (2.76 fold) followed by SolyWRKY37 (1.93 fold) gene was found at 24 hrs which further increased at 48 hrs (5.0 fold). In contrast, the leaf tissues, the expression was more pronounced at an earlier stage of infection (24 hrs). However, in both cases, we found repression of SolyWRKY4 gene, which further decreased at an increased time interval. The biochemical defense programming against Fol pathogenesis was characterized by the highest accumulation of H2O2 (at 48 hrs) and enhanced lignification. The functional diversity across the characterized WRKYs was explored through motif scanning using MEME suite, and the WRKYs specific gene regulation was assessed through the DNA protein docking studies The functional WRKY domain modeled had β sheets like topology with coil and turns. The DNA-protein interaction results revealed the importance of core residues (Tyr, Arg, and Lys) in a feasible WRKY-W-box DNA interaction. The protein interaction network analysis revealed that the SolyWRKY33 could interact with other proteins, such as mitogen-activated protein kinase 5 (MAPK), sigma factor binding protein1 (SIB1) and with other WRKY members including WRKY70, WRKY1, and WRKY40, to respond various biotic and abiotic stresses. The STRING results were further validated through Predicted Tomato Interactome Resource (PTIR) database. The CELLO2GO web server revealed the functional gene ontology annotation and protein subcellular localization, which predicted that SolyWRKY33 is involved in amelioration of biological stress (39.3%) and other metabolic processes (39.3%). The protein (SolyWRKY33) most probably located inside the nucleus (91.3%) with having transcription factor binding activity. We conclude that the defense response following the Fol challenge was accompanied by differential expression of the SolyWRKY4(↓), SolyWRKY33(↑) and SolyWRKY37(↑) transcripts. The biochemical changes are occupied by elicitation of H2O2 generation and accumulation and enhanced lignified tissues.

Out of the various mycotoxigenic food and feed contaminant, the fungal species belonging to Penicillium genera, particularly Penicillium roqueforti is of great economic importance, and well known for its crucial role in the manufacturing of Roquefort and Gorgonzola cheese. The mycotoxicosis effect of this mold is due to secretion of several metabolites, of which PR toxin is of considerable importance, with regard to food quality and safety challenges issues. The food products and silages enriched with PR toxin could lead into damage to vital internal organs, gastrointestinal perturbations, carcinogenicity, immunotoxicity, necrosis, and enzyme inhibition. Moreover, it also has the significant mutagenic potential to disrupt/alter the crucial processes like DNA replication, transcription, and translation at the molecular level. The high genetic diversities in between the various strains of P. roqueforti persuaded their nominations with Protected Geographical Indication (PGI), accordingly to the cheese type, they have been employed. Recently, the biosynthetic mechanism and toxicogenetic studies unraveled the role of ari1 and prx gene clusters that cross-talk with the synthesis of other metabolites or involve other cross-regulatory pathways to negatively regulate/inhibit the other biosynthetic route targeted for production of a strain-specific metabolites. Interestingly, the chemical conversion that imparts toxic properties to PR toxin is the substitution/oxidation of functional hydroxyl group (-OH) to aldehyde group (-CHO). The rapid conversion of PR toxin to the other derivatives such as PR imine, PR amide, and PR acid, based on conditions available reflects their unstability and degradative aspects. Since the PR toxin-induced toxicity could not be eliminated safely, the assessment of dose-response and other pharmacological aspects for its safe consumption is indispensable. The present review describes the natural occurrences, diversity, biosynthesis, genetics, toxicological aspects, control and prevention strategies, and other management aspects of PR toxin with paying special attention on economic impacts with intended legislations for avoiding PR toxin contamination with respect to food security and other biosafety purposes.

Plant defense against their pathogens can be induced by a complex network of different inducers. The present study investigates the synergistic effect of Trichoderma harzianum, exogenous salicylic acid (SA) and methyl jasmonate (MeJA) over the response and regulation of the antioxidant defense mechanisms and lipid peroxidation in tomato plants against Fusarium wilt disease. In the present work, tomato plants were infected by Fusarium oxysporum f. sp. lycopersici 3 days after inoculated with T. harzianum and/or sprayed daily for 3 days with chemical inducers (SA and MeJA). Plants were analysed at 0, 24, 48, 72 and 96 h after inoculation with Fusarium oxysporum f. sp. lycopersici. Infection of tomato plants by pathogen led to strong reduction in the dry weight of roots and shoots with the enhanced concentration of H 2 O 2 and varying degree of lipid peroxidation. Concurrently, exogenous SA, when applied with pathogen greatly enhanced H 2 O 2 content as well as activities of antioxidant enzymes except catalase (CAT) and ascorbate peroxidase (APx). The pathogen challenged plants pretreated with T. harzianum and MeJA together exhibited less lipid peroxidation and as well as the elevated level of ascorbic acid and enhanced activities of antioxidant enzymes. All applied treatments protected tomato seedlings against Fusarium wilt disease but the percentage of protection was found higher in plants pretreated with the combination of T. harzianum and chemical inducers.

Alternaria is an important fungus to study due to their different life style from saprophytes to endophytes and a very successful fungal pathogen that causes diseases to a number of economically important crops. Alternaria species have been well-characterized for the production of different host-specific toxins (HSTs) and non-host specific toxins (nHSTs) which depend upon their physiological and morphological stages. The pathogenicity of Alternaria species depends on host susceptibility or resistance as well as quantitative production of HSTs and nHSTs. These toxins are chemically low molecular weight secondary metabolites (SMs). The effects of toxins are mainly on different parts of cells like mitochondria, chloroplast, plasma membrane, Golgi complex, nucleus, etc. Alternaria species produce several nHSTs such as brefeldin A, tenuazonic acid, tentoxin, and zinniol. HSTs that act in very low concentrations affect only certain plant varieties or genotype and play a role in determining the host range of specificity of plant pathogens. The commonly known HSTs are AAL-, AK-, AM-, AF-, ACR-, and ACT-toxins which are named by their host specificity and these toxins are classified into different family groups. The HSTs are differentiated on the basis of bio-statistical and other molecular analyses. All these toxins have different mode of action, biochemical reactions and signaling mechanisms to cause diseases. Different species of Alternaria produced toxins which reveal its biochemical and genetic effects on itself as well as on its host cells tissues. The genes responsible for the production of HSTs are found on the conditionally dispensable chromosomes (CDCs) which have been well characterized. Different bio-statistical methods like basic local alignment search tool (BLAST) data analysis used for the annotation of gene prediction, pathogenicity-related genes may provide surprising knowledge in present and future.

Fungal glucose oxidase (GOD) is widely employed in the different sectors of food industries for use in baking products, dry egg powder, beverages, and gluconic acid production. GOD also has several other novel applications in chemical, pharmaceutical, textile, and other biotechnological industries. The electrochemical suitability of GOD catalyzed reactions has enabled its successful use in bioelectronic devices, particularly biofuel cells, and biosensors. Other crucial aspects of GOD such as improved feeding efficiency in response to GOD supplemental diet, roles in antimicrobial activities, and enhancing pathogen defense response, thereby providing induced resistance in plants have also been reported. Moreover, the medical science, another emerging branch where GOD was recently reported to induce several apoptosis characteristics as well as cellular senescence by downregulating Klotho gene expression. These widespread applications of GOD have led to increased demand for more extensive research to improve its production, characterization, and enhanced stability to enable long term usages. Currently, GOD is mainly produced and purified from Aspergillus niger and Penicillium species, but the yield is relatively low and the purification process is troublesome. It is practical to build an excellent GOD-producing strain. Therefore, the present review describes innovative methods of enhancing fungal GOD production by using genetic and non-genetic approaches in-depth along with purification techniques. The review also highlights current research progress in the cost effective production of GOD, including key advances, potential applications and limitations. Therefore, there is an extensive need to commercialize these processes by developing and optimizing novel strategies for cost effective GOD production.

In the present study, we have evaluated the comparative biochemical defense response generated against Alternaria alternata and its purified toxins viz. alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA). The necrotic lesions developed due to treatment with toxins were almost similar as those produced by the pathogen, indicating the crucial role of these toxins in plant pathogenesis. An oxidative burst reaction characterized by the rapid and transient production of a large amount of reactive oxygen species (ROS) occurs following the pathogen infection/toxin exposure. The maximum concentration of hydrogen peroxide (H 2 O 2 ) produced was reported in the pathogen infected samples (22.2-fold) at 24 h post inoculation followed by TeA (18.2-fold), AOH (15.9-fold), and AME (14.1-fold) in treated tissues. 3,3'- Diaminobenzidine staining predicted the possible sites of H 2 O 2 accumulation while the extent of cell death was measured by Evans blue dye. The extent of lipid peroxidation and malondialdehyde (MDA) content was higher (15.8-fold) at 48 h in the sample of inoculated leaves of the pathogen when compared to control. The cellular damages were observed as increased MDA content and reduced chlorophyll. The activities of antioxidative defense enzymes increased in both the pathogen infected as well as toxin treated samples. Superoxide dismutase (SOD) activity was 5.9-fold higher at 24 h post inoculation in leaves followed by TeA (5.0-fold), AOH (4.1-fold) and AME (2.3-fold) treated leaves than control. Catalase (CAT) activity was found to be increased upto 48 h post inoculation and maximum in the pathogen challenged samples followed by other toxins. The native PAGE results showed the variations in the intensities of isozyme (SOD and CAT) bands in the pathogen infected and toxin treated samples. Ascorbate peroxidase (APx) and glutathione reductase (GR) activities followed the similar trend to scavenge the excess H 2 O 2 . The reduction in CAT activities after 48 h post inoculation demonstrate that the biochemical defense programming shown by the host against the pathogen is not well efficient resulting in the compatible host-pathogen interaction. The elicitor (toxins) induced biochemical changes depends on the potential toxic effects (extent of ROS accumulation, amount of H 2 O 2 produced). Thus, a fine tuning occurs for the defense related antioxidative enzymes against detoxification of key ROS molecules and effectively regulated in tomato plant against the pathogen infected/toxin treated oxidative stress. The study well demonstrates the acute pathological effects of A. alternata in tomato over its phytotoxic metabolites.

Aims and Objectives: Transverse preputial onlay island flap urethroplasty (TPOIF) was described initially for distal hypospadias, but has seen extended application for proximal hypospadias. We describe a set of modifications in the technique and results in a large series of proximal hypospadias., Materials and Methods: All children who underwent TPOIF repair for proximal hypospadias (proximal penile, penoscrotal and scrotal) from June 2006 to June 2013 by a single surgeon were prospectively followed till June, 2014. A standard technique and postoperative protocol were followed. Salient points to be emphasized in the technique: (1) dissection of the dartos pedicle till penopubic junction to prevent penile torsion, (2) incorporation of the spongiosum in the urethroplasty, (3) midline urethral plate incision in glans (hinging the plate), (4) Dartos blanket cover on whole urethroplasty., Results: Out of 136 children with proximal hypospadias, 92 children who underwent TPOIF formed the study group. Out of 92 children, 48 (52 %) children required a tunica albuginea plication for chordee correction. In total, 16 (17 %) patients developed 24 complications and 11 children (12 %) required second surgeries: fistula closure in 7 (with meatoplasty in 5), glansplasty for glans dehiscence in 2 and excision of diverticulum in 2. Two children required a third surgery. Only 5 children had a noticeable penile torsion (less than 30 degree), and 7 had a patulous meatus., Conclusions: Transverse preputial onlay island flap urethroplasty can deliver reliable cosmetic and functional outcomes in proximal hypospadias.

There are multiple causes of lung abscess, but the differential rarely includes pyelonephritis as a primary cause leading to lung abscess resulting from the development of a nephrobronchial fistula. The patient had no urinary symptoms or abdominal pain and the etiology of lung abscess was only incidentally discovered after chest CT revealed extension of pleural fluid below the diaphragm.

Undescended testis (UDT) is seen in 3% to 5% of all newborn boys. Complications such as infertility and malignant transformation have been well documented in UDT. However, torsion of a UDT can also occur and the diagnosis is often missed or delayed, leading to loss of testis. This event may occur even before the currently recommended age for surgery, which is at 6-9 months. We present a case series of six children with torsion of undescended testes and their subsequent diagnosis and management. The risk of torsion of UDT is understated. Paediatricians should be educated about this complication and torsion should be included in the differential diagnosis when a boy with an empty scrotum presents with acute abdomen or red and tender swelling in the groin, as early detection and intervention can help salvage the testes.

Pharmaceutical powder engineering often involves forming interfaces between the drug and a suitable polymer. The structure at the interface plays a critical role in the properties and performance of the composite. However, interface structures have not been well understood due to a lack of suitable characterization tool. In this work, we have used ellipsometry and neutron reflectometry to characterize the structure of such interfaces in detail. Ellipsometry provided a quick estimate of the number of layers and their thicknesses, whereas neutron reflectometry provided richer structural information such as density, thickness, roughness, and intermixing of different layers. The combined information allowed us to develop an accurate model about the layered structure and provided information about intermixing of different layer components. Systematic use of these characterization techniques on several model systems suggests that the nature of the polymer had a small effect on the interfacial structure, while the solvent used in polymer coating had a large effect. These results provide useful information on the efforts of engineering particle properties through the control of the interfacial chemistry.

A study of the interaction of four endoglucanases with amorphous cellulose films by neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) is reported. The endoglucanases include a mesophilic fungal endoglucanase (Cel45A from H. insolens), a processive endoglucanase from a marine bacterium (Cel5H from S. degradans ), and two from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima ). The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. The endoglucanases displayed highly diverse behavior. Cel45A and Cel5H, which possess carbohydrate-binding modules (CBMs), penetrated and digested within the bulk of the films to a far greater extent than Cel9A and Cel5A, which lack CBMs. While both Cel45A and Cel5H were active within the bulk of the films, striking differences were observed. With Cel45A, substantial film expansion and interfacial broadening were observed, whereas for Cel5H the film thickness decreased with little interfacial broadening. These results are consistent with Cel45A digesting within the interior of cellulose chains as a classic endoglucanase, and Cel5H digesting predominantly at chain ends consistent with its designation as a processive endoglucanase.

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.

The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that anionic lipid membranes can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40s presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40s strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases.

Aim: Phimosis is a common paediatric urological disorder and often necessitates circumcision. We prospectively evaluated local steroid therapy (LST) as the first choice therapy for such children., Methods: Two hundred and sixty symptomatic boys up to 15 years of age (mean 34 months) with phimosis were started on betamethasone dipropionate (0.05%) application on gently stretched prepuce twice a day. Follow-up visits were arranged at the end of weeks 1, 2 and 4 and 6 months. Grade of phimosis was objectively graded., Results: Ninety one percent of the boys showed a successful outcome at the end of 4 weeks; 72% responded in first week, further 16% responded in week 2, and only 2.6% achieved alleviation of phimosis on further application of LST beyond 2 weeks. Fourty two (17.8%) boys had a recurrence of phimosis on a long-term follow-up (mean - 25.4 months, range 6-48 months); thus, the long-term success rate was 77%, while 60 (23%) boys underwent surgery., Conclusion: Local steroid therapy is safe and successful in alleviating symptomatic tight foreskin in a large majority of children. The response can be seen as early as 1 week; most of the children respond by week 2 and continuing therapy further may not be very effective., (© 2011 The Author(s)/Acta Paediatrica © 2011 Foundation Acta Paediatrica.)

Silicon photonic microring resonators have established their potential for label-free and low-cost biosensing applications. However, the long-term performance of this optical sensing platform requires robust surface modification and biofunctionalization. Herein, we demonstrate a conjugation strategy based on an organophosphonate surface coating and vinyl sulfone linker to biofunctionalize silicon resonators for biomolecular sensing. To validate this method, a series of glycans, including carbohydrates and glycoconjugates, were immobilized on divinyl sulfone (DVS)/organophosphonate-modified microrings and used to characterize carbohydrate-protein and norovirus particle interactions. This biofunctional platform was able to orthogonally detect multiple specific carbohydrate-protein interactions simultaneously. Additionally, the platform was capable of reproducible binding after multiple regenerations by high-salt, high-pH, or low-pH solutions and after 1 month storage in ambient conditions. This remarkable stability and durability of the organophosphonate immobilization strategy will facilitate the application of silicon microring resonators in various sensing conditions, prolong their lifetime, and minimize the cost for storage and delivery; these characteristics are requisite for developing biosensors for point-of-care and distributed diagnostics and other biomedical applications. In addition, the platform demonstrated its ability to characterize carbohydrate-mediated host-virus interactions, providing a facile method for discovering new antiviral agents to prevent infectious disease.

Temperature-sensitive hydrogel polymers are utilized as responsive layers in various applications. Although the polymer's native characteristics have been studied extensively, details concerning its properties during interaction with biorelated structures are lacking. This work investigates the interaction between a thermoresponsive polymer cushion and different lipid membrane capping layers probed by neutron reflectometry. N-isopropylacrylamide copolymerized with methacroylbenzophenone first supported a lipid bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and subsequently 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The polymer-membrane systems were investigated above and below the polymer transition temperature (37 and 25°C). Although the same cushion supported each lipid membrane, the polymer hydration profile and thickness were markedly different for DPPE and DPPC systems. Because DPPE and DPPC have different bending rigidities, these results establish that the polymer-membrane interaction is critically mediated by the mechanics of the membrane, providing better insight into cell-hydrogel interactions., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Improving the efficiency of enzymatic hydrolysis of cellulose is one of the key technological hurdles to reduce the cost of producing ethanol and other transportation fuels from lignocellulosic material. A better understanding of how soluble enzymes interact with insoluble cellulose will aid in the design of more efficient enzyme systems. We report a study involving neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) of the interaction of a fungal enzyme extract ( T. viride ) and an endoglucanse from A. niger with amorphous cellulose films. The use of amorphous cellulose is motivated by that the fact that several biomass pretreatments currently under investigation disrupt the native crystalline structure of cellulose and increase the amorphous content. NR reveals the profile of water through the film at nanometer resolution and is highly sensitive to interfacial roughness, whereas QCM-D provides changes in mass and film stiffness. NR can be performed using either H(2)O- or D(2)O-based aqueous reservoirs. NR measurement of swelling of a cellulose film in D(2)O and in H(2)O revealed that D/H exchange on the cellulose chains must be taken into account when a D(2)O-based reservoir is used. The results also show that cellulose films swell slightly more in D(2)O than in H(2)O. Regarding enzymatic digestion, at 20 °C in H(2)O buffer the T. viride cocktail rapidly digested the entire film, initially roughening the surface, followed by penetration and activity throughout the bulk of the film. In contrast, over the same time period, the endoglucanase was active mainly at the surface of the film and did not increase the surface roughness.

Polymer-supported single lipid bilayers are models to study configurations of cell membranes. We used off-specular neutron scattering to quantify in-plane height-height correlations of interfacial fluctuations of such a lipid bilayer. As temperature decreased from 37 °C to 25 °C, the polymer swells and the polymer-supported lipid membrane deviates from its initially nearly planar structure. A correlation length characteristic of capillary waves changes from 30 μm at 37 °C to 11 μm at 25 °C, while the membrane bending rigidity remains roughly constant in this temperature range., (© 2011 American Physical Society)

Imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been used to study protein bound to a photolithographically-patterned, commercial poly(ethylene glycol) (PEG)-based polymer film. The effect of different ion sources on the fragmentation pattern from this sample was analyzed with respect to the surface sensitivity of characteristic protein fragments and contrast in the ion images. The method demonstrates that, under similar fluence (below the static limit), Bi(3) (+) provides better surface sensitivity for low mass fragments and the best image contrast as compared to Bi(1) (+) and C(60) (+) cluster sources. Principal component analysis (PCA) was utilized to process depth profiles for this sample and shows that a primary ion fluence of approximately 20 × 10(12) ions/cm(2) is required to etch through the adsorbed protein layer.

The nonperturbative nature of neutron reflectometry (NR) coupled with its isotopic sensitivity has made it an ideal candidate for the study of model biological membranes at the solid-liquid interface. In this article, methods are presented for the creation and characterization of supported model membranes which can mimic many of the critical attributes of cell membranes. It is demonstrated that NR can characterize the structure, composition and organization of model membranes deposited on solid, nanoporous and polymer supports. Additionally, in situ NR measurements of the interactions between model membranes and external stimuli are presented. Finally, an investigation of the adherence region of live mouse fibroblast cells is described.

Organophosphonic acid self-assembled monolayers (SAMs) on oxide surfaces have recently seen increased use in electrical and biological sensor applications. The reliability and reproducibility of these sensors require good molecular organization in these SAMs. In this regard, packing, order, and alignment in the SAMs is important, as it influences the electron transport measurements. In this study, we examine the order of hydroxyl- and methyl-terminated phosphonate films deposited onto silicon oxide surfaces by the tethering by aggregation and growth method using complementary, state-of-art surface characterization tools. Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy and in situ sum frequency generation (SFG) spectroscopy are used to study the order of the phosphonate SAMs in vacuum and under aqueous conditions, respectively. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry results show that these samples form chemically intact monolayer phosphonate films. NEXAFS and SFG spectroscopy showed that molecular order exists in the octadecylphosphonic acid and 11-hydroxyundecylphosphonic acid SAMs. The chain tilt angles in these SAMs were approximately 37° and 45°, respectively.

The interaction of beta-cyclodextrin (beta-CD) with mixed bilayers composed of sphingomylein and cholesterol (Chol) above and below the accepted stable complexation ratio (67:33) was investigated. Membranes with the same (symmetric) and different (asymmetric) compositions in their inner and outer leaflets were deposited at surface pressures of 20, 30, and 40 mN/m at the solid-liquid interface. Using neutron reflectometry, membranes of various global molar ratios (defined as the sum of the molar ratios of the inner and outer leaflets), were characterized before and after beta-CD was added to the subphase. The structure of bilayers with global molar ratios at or above the stable complexation ratio was unchanged by beta-CD, indicating that beta-CD is unable to remove sphingomyelin or complexed Chol. However, beta-CD removed all uncomplexed Chol from bilayers composed of global molar ratios below the stable complexation ratio. The removal of Chol by beta-CD was independent of the initial structure of the membranes as deposited, suggesting that asymmetric membranes homogenize by the exchange of molecules between leaflets. The interaction of beta-CD with the aforementioned membranes was independent of the deposition surface pressure except for a symmetric 50:50 membrane deposited at 40 mN/m. The scattering from 50:50 bilayers with higher packing densities (deposited at 40 mN/m) was unaffected by beta-CD, suggesting that the removal of Chol can depend on both the composition and packing density of the membrane., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Covalent modification of surfaces with carbohydrates (glycans) is a prerequisite for a variety of glycomics-based biomedical applications, including functional biomaterials, glycoarrays, and glycan-based biosensors. The chemistry of glycan immobilization plays an essential role in the bioavailability and function of the surface bound carbohydrate moiety. However, the scarcity of analytical methods to characterize carbohydrate-modified surfaces complicates efforts to optimize glycan surface chemistries for specific applications. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive technique suited for probing molecular composition at the biomaterial interface. Expanding ToF-SIMS analysis to interrogate carbohydrate-modified materials would increase our understanding of glycan surface chemistries and advance novel tools in the nascent field of glycomics. In this study, a printed glycan microarray surface was fabricated and subsequently characterized by ToF-SIMS imaging analysis. A multivariate technique based on principal component analysis (PCA) was used to analyze the ToF-SIMS dataset and reconstruct ToF-SIMS images of functionalized surfaces. These images reveal chemical species related to the immobilized glycan, underlying glycan-reactive chemistries, gold substrates, and outside contaminants. Printed glycoarray elements (spots) were also interrogated to resolve the spatial distribution and spot homogeneity of immobilized glycan. The bioavailability of the surface-bound glycan was validated using a specific carbohydrate-binding protein (lectin) as characterized by Surface Plasmon Resonance Imaging (SPRi). Our results demonstrate that ToF-SIMS is capable of characterizing chemical features of carbohydrate-modified surfaces and, when complemented with SPRi, can play an enabling role in optimizing glycan microarray fabrication and performance.

This study assesses the capability of high-resolution surface analytical tools to distinguish immobilized antibody orientations on patterned surfaces designed for antibody affinity capture. High-fidelity, side-by-side copatterning of protein A (antibody Fc domain affinity reagent) and fluorescein (antibody Fab domain hapten) was achieved photolithographically on commercial amine-reactive hydrogel polymer surfaces. This was verified from fluorescence imaging using fluorescently labeled protein A and intrinsic fluorescence from fluorescein. Subsequently, dye-labeled murine antifluorescein antibody (4-4-20) and antibody Fab and Fc fragments were immobilized from solution onto respective protein A- and fluorescein- copatterned or control surfaces using antibody-ligand affinity interactions. Fluorescence assays support specific immobilization to fluorescein hapten- and protein A-patterned regions through antigen-antibody recognition and natural protein A-Fc domain interactions, respectively. Affinity-based antibody immobilization on the two different copatterned surfaces generated side-by-side full antibody "heads-up" and "tails-up" oriented surface patterns. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis, sensitive to chemical information from the top 2 to 3 nm of the surface, provided ion-specific images of these antibody patterned regions, imaging and distinguishing characteristic ions from amino acids enriched in Fab domains for antibodies oriented in "heads-up" regions, and ions from amino acids enriched in Fc domains for antibodies oriented in "tails-up" regions. Principal component analysis (PCA) improved the distinct TOF-SIMS amino acid compositional and ion-specific surface mapping sensitivity for each "heads-up" versus "tails-up" patterned region. Characteristic Fab and Fc fragment immobilized patterns served as controls. This provides first demonstration of pattern-specific, antibody orientation-dependent surface maps based on antibody domain- and structure-specific compositional differences by TOF-SIMS analysis. Since antibody immobilization and orientation are critical to many technologies, orientation characterization using TOF-SIMS could be very useful and convenient for immobilization quality control and understanding methods for improving the performance of antibody-based surface capture assays.

Photolithographically prepared surface patterns of two affinity ligands (biotin and chloroalkane) specific for two proteins (streptavidin and HaloTag®, respectively) are used to spontaneously form high-fidelity surface patterns of the two proteins from their mixed solution. High affinity protein-surface self-selection onto patterned ligands on surfaces exhibiting low non-specific adsorption rapidly yields the patterned protein surfaces. Fluorescence images after protein immobilization show high specificity of the target proteins to their respective surface patterned ligands. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging further supports the chemical specificity of streptavidin and HaloTag® for their surface patterned ligands from mixed protein solutions. However, ToF-SIMS did detect some non-specific adsorption of bovine serum albumin, a masking protein present in excess in the adsorbing solutions, on the patterned surfaces. Protein amino acid composition, surface coverage, density and orientation are important parameters that determine the relative ToF-SIMS fragmentation pattern yields. ToF-SIMS amino acid-derived ion fragment yields summed to produce surface images can reliably determine which patterned surface regions contain bound proteins, but do not readily discriminate between different co-planar protein regions. Principal component analysis (PCA) of these ToF-SIMS data, however, improves discrimination of ions specific to each protein, facilitating surface pattern discrimination and contrast.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a surface analysis technique well-suited to detect and identify trace surface species. With the latest analyzers, ion sources and data analysis methods, imaging ToF-SIMS provides detailed 2-D and 3-D surface reactivity maps. Coupling multivariate analysis methods such as principal component analysis (PCA) with ToF-SIMS provides a powerful method for differentiating spatial regions with different chemistries. ToF-SIMS and PCA are used in this study to image and analyze a two-component photolithograph-patterned surface chemistry currently published and commercialized for bioassays, bio-chips and cell-based biosensors. A widely used reactive surface coupling chemistry, N-hydroxysuccinimide (NHS), and 2-methoxyethylamine (MeO) were co-patterned into adjacent regions on a commercial microarray polymer coating using standard photolithography methods involving deposition, patterning and removal of a routinely used photoresist material. After routine processing, ToF-SIMS and PCA of the patterned surface revealed significant residual photoresist material remaining at the interface of the NHS/MeO patterns, as well as lower concentrations of residual photoresist material remaining within the MeO-containing regions, providing spatial mapping and residue analysis not evident from other characterization techniques. As detection of surface photoresist residue remains an inherent challenge in photolithographic processing of a wide array of materials, the use of ToF-SIMS coupled with PCA is shown to be a high-resolution characterization tool with the high sensitivity and specificity required for surface quality control measurements following photolithography and pattern development relevant to many current processes.

We investigated hydroxyalkylphosphonate monolayers as a novel platform for the biofunctionalization of silicon-based field effect sensor devices. This included a detailed study of the thin film properties of organophosphonate films on Si substrates using several surface analysis techniques, including AFM, ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), X-ray reflectivity, and current-voltage characteristics in electrolyte solution. Our results indicate the formation of a dense monolayer on the native silicon oxide that has excellent passivation properties. The monolayer was biofunctionalized with 12 mer peptide nucleic acid (PNA) receptor molecules in a two-step procedure using the heterobifunctional linker, 3-maleimidopropionic-acid-N-hydroxysuccinimidester. Successful surface modification with the probe PNA was verified by XPS and contact angle measurements, and hybridization with DNA was determined by fluorescence measurements. Finally, the PNA functionalization protocol was translated to 2 microm long, 100 nm wide Si nanowire field effect devices, which were successfully used for label-free DNA/PNA hybridization detection.

High-fidelity surface functional group (e.g., N-hydroxysuccinimide (NHS) reactive ester) patterning is readily and reliably achieved on commercial poly(ethylene glycol) (PEG)-based polymer films already known to exhibit high performance non-fouling properties in full serum and in cell culture conditions. NHS coupling chemistry co-patterned with methoxy-capped PEG using photolithographic methods is directly spatially imaged using imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal components statistical analysis. Patterned NHS surface reactive zones are clearly resolved at high sensitivity despite the complexity of the polymer matrix chemistry. ToF-SIMS imaging also reveals the presence of photo-resist residue remaining from typical photolithography processing methods. High cross-correlation between various ion-derived ToF-SIMS images is observed, providing sensitive chemical corroboration of pattern chemistry and biological reactivity in complex milieu. Surface-specific protein coupling is observed first by site-selective reaction of streptavidin with NHS patterns, followed by identical patterns of biotinylated Alexa-labeled albumin coupling. This suggests that streptavidin immobilized on the patterns remains bioactive. Fluorescently labeled full serum is shown to react selectively with NHS-reactive regions, with minimal signal from methoxy-capped regions. Insufficient serum is adsorbed under any conditions to these surfaces to support cell attachment in serum-containing media. This reflects the high intrinsic non-adsorptive nature of this chemistry. Fibroblasts attach and proliferate in serum culture only when a cell adhesion peptide (RGD) is first grafted to NHS regions on the PEG-based surfaces. Longer-term serum-based cell culture retains high cell-pattern fidelity that correlates with chemical imaging of both the NHS and RGD patterns and also lack of cell adhesion to methoxy-capped regions. Cell staining shows orientation of adherent cells within the narrow patterned areas. Cell patterns are consistently retained beyond 15 days in serum media.

The attachment of a bifunctional iodo-organo-phosphinate compound to gold (Au) surfaces via chemisorption of the iodine atom is described and used to chelate a redox-active metal cluster via the phosphinate group. XPS, AFM, and electrochemical measurements show that (4-iodo-phenyl)phenyl phosphinic acid (IPPA) forms a tightly bound self-assembled monolayer (SAM) on Au surfaces. The surface coverage of an IPPA monolayer on Au was quantified by an electrochemical method and found to be 0.40 +/- 0.03 nmol/cm2, roughly corresponding to 0.4 monolayers. We show that the Au/IPPA SAM, but not the underivatized Au, adsorbs Mn4O4(Ph2PO2)6 from solution by a phosphinate exchange reaction to yield Au/IPPA/Mn4O4(Ph2PO2)5 SAM. The resulting SAM is firmly bound and not removed by sonication, as confirmed by manganese XPS (Mn 2p1/2) and by AFM. Electrochemistry confirms that Mn4O4(Ph2PO2)6 is anchored on the Au/IPPA surface and that redox chemistry can be mediated between the electrode and the surface-attached complex. Mn4O4(Ph2PO2)6 contains the reactive Mn4O46+ cubane core, a redox-active bioinspired catalyst.

Phosphonate-steel interactions have been industrially significant for decades, but details of the phosphonate-steel interface have not yet been characterized. Self-assembled monolayers of phosphonic acids were formed on stainless steel 316L by room-temperature solution deposition. The acids are covalently bound to the surface as phosphonates in a bidentate manner, as determined by diffuse reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Complete coverage of the surface is confirmed by contact angle measurement and atomic force microscopic imaging. This method of monolayer formation contrasts the requirement for heating and long reaction times found to be necessary to form phosphonate monolayers on other metal oxide substrates, such as titanium and silicon.

Differential charging is often regarded as a problem in X-ray photoelectron spectroscopic studies, especially for insulating or partially conducting samples. Application of a positive bias can reduce the effect of differential charging by attracting stray electrons from the system, thereby compensating for the electron loss. On the other hand, differential charging effect can be enhanced by the application of a negative bias to the sample during spectrum acquisition. The successful use of the differential charging technique to distinguish between multi- and monolayer organophosphonate films on oxide-covered silicon has been reported. A detailed description of this technique is now presented which shows how differential charging can be used as an important tool for the characterization of self-assembled films deposited on various surfaces. The dependence of this technique on the conductivity of the substrate has been investigated by studying the spectral behavior of the deposited films of phosphonic acid on conducting, semiconducting, and insulating samples (stainless steel, silicon, and glass). Application of either positive or negative dc electrical bias affects the carbon core-level (C1s) line shape and intensity, which is dependent on the atom's physical location above the surface.