Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

Unsupervised segmentation of biomedical hyperspectral image data: tackling high dimensionality with convolutional autoencoders.

Information about the structure and composition of biopsy specimens can assist in disease monitoring and diagnosis. In principle, this can be acquired from Raman and infrared (IR) hyperspectral images (HSIs) that encode information about how a sample's constituent molecules are arranged in space. Each tissue section/component is defined by a unique combination of spatial and spectral features, but given the high dimensionality of HSI datasets, extracting and utilising them to segment images is non-trivial. Here, we show how networks based on deep convolutional autoencoders (CAEs) can perform this task in an end-to-end fashion by first detecting and compressing relevant features from patches of the HSI into low-dimensional latent vectors, and then performing a clustering step that groups patches containing similar spatio-spectral features together. We showcase the advantages of using this end-to-end spatio-spectral segmentation approach compared to i) the same spatio-spectral technique not trained in an end-to-end manner, and ii) a method that only utilises spectral features (spectral k-means) using simulated HSIs of porcine tissue as test examples. Secondly, we describe the potential advantages/limitations of using three different CAE architectures: a generic 2D CAE, a generic 3D CAE, and a 2D convolutional encoder-decoder architecture inspired by the recently proposed UwU-net that is specialised for extracting features from HSI data. We assess their performance on IR HSIs of real colon samples. We find that all architectures are capable of producing segmentations that show good correspondence with HE stained adjacent tissue slices used as approximate ground truths, indicating the robustness of the CAE-driven spatio-spectral clustering approach for segmenting biomedical HSI data. Additionally, we stress the need for more accurate ground truth information to enable a precise comparison of the advantages offered by each architecture.

In situ chemically specific mapping of agrochemical seed coatings using stimulated Raman scattering microscopy.

Providing sufficient, healthy food for the increasing global population is putting a great deal of pressure on the agrochemical industry to maximize crop yields without sustaining environmental damage. The growth and yield of every plant with sexual reproduction, depends on germination and emergence of sown seeds, which is affected greatly by seed disease. This can be most effectively controlled by treating seeds with pesticides before they are sown. An effective seed coating treatment requires a high surface coverage and adhesion of active ingredients onto the seed surface and the addition of adhesive agents in coating formulations plays a key role in achieving this. Although adhesive agents are known to enhance seed germination, little is understood about how they affect surface distribution of actives and how formulations can be manipulated to rationally engineer seed coating preparations with optimized coverage and efficacy. We show, for the first time, that stimulated Raman scattering microscopy can be used to map the seed surface with microscopic spatial resolution and with chemical specificity to identify formulation components distributed on the seed surface. This represents a major advance in our capability to rationally engineer seed coating formulations with enhanced efficacy.

Notch sensitivity jeopardizes titanium locking plate fatigue strength.

INTRODUCTION: Notch sensitivity may compromise titanium-alloy plate fatigue strength. However, no studies providing head-to-head comparisons of stainless-steel or titanium-alloy locking plates exist. MATERIALS AND METHODS: Custom-designed identically structured locking plates were made from stainless steel (F138 and F1314) or titanium alloy. Three screw-hole designs were compared: threaded screw-holes with angle edges (type I); threaded screw-holes with chamfered edges (type II); and non-threaded screw-holes with chamfered edges (type III). The plates' bending stiffness, bending strength, and fatigue life, were investigated. The stress concentration at the screw threads was assessed using finite element analyses (FEA). RESULTS: The titanium plates had higher bending strength than the F1314 and F138 plates (2.95:1.56:1) in static loading tests. For all metals, the type-III plate fatigue life was highest, followed by type-II and type-I. The type-III titanium plates had longer fatigue lives than their F138 counterparts, but the type-I and type-II titanium plates had significantly shorter fatigue lives. All F1314 plate types had longer fatigue lives than the type-III titanium plates. The FEA showed minimal stress difference (0.4%) between types II and III, but the stress for types II and III was lower (11.9% and 12.4%) than that for type I. CONCLUSIONS: The screw threads did not cause stress concentration in the locking plates in FEA, but may have jeopardized the fatigue strength, especially in the notch-sensitive titanium plates. Improvement to the locking plate design is necessary.

Sugar starvation- and GA-inducible calcium-dependent protein kinase 1 feedback regulates GA biosynthesis and activates a 14-3-3 protein to confer drought tolerance in rice seedlings.

Germination followed by seedling growth constitutes two essential steps in the initiation of a new life cycle in plants, and in cereals, completion of these steps is regulated by sugar starvation and the hormone gibberellin. A calcium-dependent protein kinase 1 gene (OsCDPK1) was identified by differential screening of a cDNA library derived from sucrose-starved rice suspension cells. The expression of OsCDPK1 was found to be specifically activated by sucrose starvation among several stress conditions tested as well as activated transiently during post-germination seedling growth. In gain- and loss-of-function studies performed with transgenic rice overexpressing a constitutively active or RNA interference gene knockdown construct, respectively, OsCDPK1 was found to negatively regulate the expression of enzymes essential for GA biosynthesis. In contrast, OsCDPK1 activated the expression of a 14-3-3 protein, GF14c. Overexpression of either constitutively active OsCDPK1 or GF14c enhanced drought tolerance in transgenic rice seedlings. Hence, our studies demonstrated that OsCDPK1 transduces the post-germination Ca(2+) signal derived from sugar starvation and GA, refines the endogenous GA concentration and prevents drought stress injury, all essential functions to seedling development at the beginning of the life cycle in rice.

Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo.

BACKGROUND: Redundant skin laxity is a major feature of aging. Recently, radiofrequency has been introduced for nonablative tissue tightening by volumetric heating of the deep dermis. Despite the wide range of application based on this therapy, the effect of this technique on tissue and the subsequent tissue remodeling have not been investigated in detail. OBJECTIVE: Our objective is to evaluate the potential of non-linear optics, including multiphoton autofluorescence and second harmonic generation (SHG) microscopy, as a non-invasive imaging modality for the real-time study of radiofrequency-tissue interaction. METHODS: Electro-optical synergy device (ELOS) was used as the radiofrequency source in this study. The back skin of nude mouse was irradiated with radiofrequency at different passes. We evaluated the effect on skin immediately and 1 month after treatment with multiphoton microscopy. RESULTS: Corresponding histology was performed for comparison. We found that SHG is negatively correlated to radiofrequency passes, which means that collagen structural disruption happens immediately after thermal damage. After 1 month of collagen remodeling, SHG signals increased above baseline, indicating that collagen regeneration has occurred. Our findings may explain mechanism of nonablative skin tightening and were supported by histological examinations. CONCLUSIONS: Our work showed that monitoring the dermal heating status of RF and following up the detailed process of tissue reaction can be imaged and quantified with multiphoton microscopy non-invasively in vivo.

Bioaerosols as contributors to poor air quality in Taichung City, Taiwan.

Bioaerosols are a type of suspended sediments that contribute to poor air quality in Taiwan. Bioaerosols include allergens such as: fungi, bacteria, actinomycetes, arthropods and protozoa, as well as microbial products such as mycotoxins, endotoxins and glucans. When allergens and microbial products are suspended in the air, local air quality will be influenced adversely. In addition, when the particle size is small enough to pass through the respiratory tract entering the human body, the health of the local population is also threatened. Therefore, the purpose of this study is to attempt to understand the concentration and types of bacteria and the bacteria numbers for various particle size ranges during a study period of June 2005 to February 2006 in Taichung City, Central Taiwan. The results indicate that the total average bacterial concentration by using NA medium incubated for 48 h were 8.0 x 10(2), 1.4 x 10(3), 2.4 x 10(3) and 1.3 x 10(3), 1.9 x 10(3), 3.5 x 10(3) cfu/m(3) for CMES, TRIPS and RFS sampling sites during the daytime and nighttime period of June 2005 to February 2006. Moreover, the total average bacterial concentration by using R(2)A medium incubated for 48 h were 8.5 x 10(2), 1.5 x 10(3), 2.2 x 10(3) and 1.2 x 10(3), 1.7 x 10(3), 2.5 x 10(3) cfu/m(3) for CMES, TRIPS and RFS sampling sites the daytime and nighttime during the same sampling period. The total average bacteria concentration was the same in either NA or R(2)A medium for the same sampling times or sites. The total average bacterial concentration was higher in daytime period than that of nighttime period for CMES, TRIPS and RFS sampling sites. The high average bacterial concentration was found in the particle size range of 0.53-0.71 mm (average bioaerosol size was in the range of 2.1-4.7 microm) for each sampling site. Also, 20 kinds of bacteria exceeded levels for each sampling site and were classified as according to shape: rod, coccus and filamentous.

Differentiation of normal and cancerous lung tissues by multiphoton imaging.

We utilize multiphoton microscopy for the label-free diagnosis of noncancerous, lung adenocarcinoma (LAC), and lung squamous cell carcinoma (SCC) tissues from humans. Our results show that the combination of second-harmonic generation (SHG) and multiphoton excited autofluorescence (MAF) signals may be used to acquire morphological and quantitative information in discriminating cancerous from noncancerous lung tissues. Specifically, noncancerous lung tissues are largely fibrotic in structure, while cancerous specimens are composed primarily of tumor masses. Quantitative ratiometric analysis using MAF to SHG index (MAFSI) shows that the average MAFSI for noncancerous and LAC lung tissue pairs are 0.55+/-0.23 and 0.87+/-0.15, respectively. In comparison, the MAFSIs for the noncancerous and SCC tissue pairs are 0.50+/-0.12 and 0.72+/-0.13, respectively. Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from noncancerous tissues.

The effects of DNA formulation and administration route on cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung cancer animal model.

BACKGROUND: Tyrosine kinase inhibitor gefitinib is effective against lung cancer cells carrying mutant epidermal growth factor receptor (EGFR); however, it is not effective against lung cancer carrying normal EGFR. The breaking of immune tolerance against self epidermal growth factor receptor with active immunization may be a useful approach for the treatment of EGFR-positive lung tumors. Xenogeneic EGFR gene was demonstrated to induce antigen-specific immune response against EGFR-expressing tumor with intramuscular administration. METHODS: In order to enhance the therapeutic effect of xenogeneic EGFR DNA vaccine, the efficacy of altering routes of administration and formulation of plasmid DNA was evaluated on the mouse lung tumor (LL2) naturally overexpressing endogenous EGFR in C57B6 mice. Three different combination forms were studied, including (1) intramuscular administration of non-coating DNA vaccine, (2) gene gun administration of DNA vaccine coated on gold particles, and (3) gene gun administration of non-coating DNA vaccine. LL2-tumor bearing C57B6 mice were immunized four times at weekly intervals with EGFR DNA vaccine. RESULTS: The results indicated that gene gun administration of non-coating xenogenic EGFR DNA vaccine generated the strongest cytotoxicity T lymphocyte activity and best antitumor effects. CD8(+) T cells were essential for anti-tumor immunity as indicated by depletion of lymphocytes in vivo. CONCLUSION: Thus, our data demonstrate that administration of non-coating xenogenic EGFR DNA vaccine by gene gun may be the preferred method for treating EGFR-positive lung tumor in the future.

Isolation of the acrylamide denitrifying bacteria from a wastewater treatment system manufactured with polyacrylonitrile fiber.

Acrylamide has carcinogenicity and toxicity, so its discharge to natural water and soil systems might have an adverse impact on water quality, endangering public health and welfare. The investigation attempts to isolate acrylamide denitrifying bacteria from a wastewater treatment system manufactured with polyacrylonitrile (PAN) fiber. The goal is to elucidate the effectiveness of isolated pure strain and PAN mixed strains in treating acrylamide from synthetic wastewater. The results reveal that Ralstonia eutropha TDM-3 was isolated from the wastewater treatment system manufactured with PAN fiber. The PAN mixed strains and R. eutropha TDM-3 can consume up to 1446 mg/L acrylamide to denitrify from synthetic wastewater. Complete acrylamide removal depended on the supply of sufficient electron acceptors (nitrate). Strain R. eutropha TDM-3, Azoarcus sp. pF6, Azoarcus sp. T, and Herbaspirillum sp. G8A1 are related closely, according to the phylogenetic analyses of 16S rDNA sequences.

Bioaerosols study in central Taiwan during summer season.

Suspended particles, of which bioaerosols are one type, constitute one of the main reasons to cause severe air quality in Taiwan. Bioaerosols include allergens such as fungi, bacteria, actinomycetes, arthropods and protozoa, as well as microbial products such as mycotoxins, endotoxins and glucans. When allergens and microbial products are suspended in the air, local air quality will be influenced severely. In addition, when the particle size is small enough to pass through the respiratory tract entering the human body, the health of the local population is also threatened. Therefore, the purpose of this study attempted to understand the concentration and types of bacteria during summer period at four sampling sites in Taichung city, central Taiwan. The results indicated that total average bacterial concentration by using R2A medium incubated for 48 h were 7.3 x 10(2) and 1.2 x 10(3) cfu/m3 for Chung-Ming elementary sampling site during daytime and night-time period of summer season. In addition, total average bacterial concentration by using R2A medium incubated for 48 h were 2.2 x 10(3) and 2.5 x 10(3) cfu/m3 for Taichung refuse incineration plant sampling site during daytime and night-time period of summer season. As for Rice Field sampling site during daytime and night-time period of summer season, the results also reflected that the total average bacterial concentration by using R2A medium incubated for 48 h were 3.4 x 10(3) and 3.5 x 10(3) cfu/m3. Finally, total average bacterial concentration by using R2A medium incubated for 48 h were 1.6 x 10(3) and 1.9 x 10(3) cfu/m3 for Central Taiwan Science Park sampling site during daytime and night-time period of summer season. Moreover, the average bacterial concentration increased as the incubated time in a growth medium increased for particle sizes of 0.65-1.1, 1.1-2.1, 2.1-3.3, 3.3-4.7 and 4.7-7.0 microm. The total average bacterial concentration has no significant difference for day and night sampling period at any sampling site for the expression of bacterial concentration in term of order. The high average bacterial concentration was found in the particle size of 0.53-0.71 mm (average bioaerosol size was in the range of 2.1-4.7 microm) for each sampling site. Besides, there were exceeded 20 kinds of bacteria for each sampling site and the bacterial shape were rod, coccus and filamentous.

Isolation of the epsilon-caprolactam denitrifying bacteria from a wastewater treatment system manufactured with acrylonitrile-butadiene-styrene resin.

epsilon-Caprolactam has high COD and toxicity, so its discharge to natural water and soil systems may lead to an adverse environmental effect on water quality, endangering public health and welfare. This investigation attempts to isolate epsilon-caprolactam denitrifying bacteria from a wastewater treatment system manufactured with acrylonitrile-butadiene-styrene (ABS) resin. The goal is to elucidate the effectiveness of isolated pure strain and ABS mixed strains in treating epsilon-caprolactam from synthetic wastewater. The results reveal that Paracoccus versutus MDC-3 was isolated from the wastewater treatment system manufactured with ABS resin. The ABS mixed strains and P. versutus MDC-3 can consume up to 1539mg/l epsilon-caprolactam to denitrify from synthetic wastewater. Complete epsilon-caprolactam removal depended on the supply of sufficient electron acceptors (nitrate). Strain P. versutus MDC-3, Hyphomicrobium sp. HM, Methylosinus pucelana and Magnetospirillum sp. CC-26 are related closely, according to the phylogenetic analyses of 16S rDNA sequences.

Isolation and physiological characterization of the pentachlorophenol degrading bacterium Sphingomonas chlorophenolica.

Many chlorophenols tend to persist in the environment, and they may become public health hazards. Among chlorophenols, pentachlorophenol (PCP) is a priority pollutant that has been used widely as a general biocide in commercial wood treatment. Owing to the rapid industrial growth, serious soil and water pollutions by chlorophenols has been reported in Taiwan. In this study, 10 indigenous PCP-degrading bacterial strains were isolated from a PCP-degrading mixed culture, and the potential of both the pure and mixed cultures for PCP degradation compared. Moreover, the physiological characteristics and optimum growth conditions of the PCP-degrading bacteria were investigated. One of the isolated bacterial strains with good potential for PCP degradation was characterized and identified as Sphingomonas chlorophenolica by 16S rDNA gene analysis. The result of the optimum growth temperatures revealed that this organism was a mesophile. The optimum pH for PCP removal by S. chlorophenolica was between 6.9 and 7.6. Increase in concentration of PCP has a negative effect on the biodegradation potential of S. chlorophenolica and PCP concentration above 600 mg l(-1) was inhibitory to its growth. The results of this study indicate that this S. chlorophenolica strain has a better potential for PCP degradation compared to the enriched mixed culture. The physiological characterization of the isolates also indicates the possible application of this strain for bioremediation of sites contaminated with PCP.

Degradation of chlorophenols using pentachlorophenol-degrading bacteria Sphingomonas chlorophenolica in a batch reactor.

Chlorophenols are common environmental contaminants that have been used as the major component in wide-spectrum biocides in industry and agriculture. Many chlorophenols tend to persist in the environment and may become public health hazards. This research studied the ability of the pentachlorophenol (PCP)-degrading bacterium Sphingomonas chlorophenolica to degrade and dechlorinate other chlorophenols. In addition, the characteristics of S. chlorophenolica were also investigated. When S. chlorophenolica cells were preincubated with PCP, the lag phase PCP degradation periods became shorter and the PCP concentrations that could be removed became higher. S. chlorophenolica was able to completely degrade 2,3,6-trichlorophenol (2,3,6-TCP), 2,4,6-trichlorophenol (2,4,6-TCP), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP), and PCP within 38.1, 15.1, 11.8, and 11.8 h, and to release concentrations of 50.1, 60.9, 63.7, and 58.5 mg/L chloride at the same period of time. In the presence of supplementary carbon sources, the PCP removal efficiency increased with the presence of glucose or pyruvate. However, the removal efficiency of 75 mg/L 2,4-dichlorophenol did not increase with supplemental carbon sources.

Removal of nitriles from synthetic wastewater by acrylonitrile utilizing bacteria.

This study describes the ability of two bacteria strains, isolated from an ABS resin manufacturing wastewater treatment system, to remove high acrylonitrile concentrations. Straight chain aliphatic nitrile compound (propionitrile, allyl cyanide); branch chain aliphatic nitrile compound (isobutyronitrilc) and aromatic nitrile compound (benzonitrile) removal by Comamonas testosteroni and Acidovorax sp. was also investigated. The results are: Comamonas testosteroni and Acidovorax sp. can remove acrylonitrile completely up to 1904.8 mg/L within 66.6 h and 1538.5 mg/L within 143.3 h, respectively. Comamonas testosteroni degraded acrylonitrile faster than Acidovorax sp., and accumulated much more acrylic acid in the medium than Acidovorax sp. The addition of acrylamide to the medium was removed by Comamonas testosteroni and Acidovorax sp. Both strains utilized acrylic acid, a metabolic by-product of acrylonitrile, as a carbon source and ammonium nitrogen, a metabolic by-product of acrylonitrile, as a nitrogen source for growth. Comamonas testosteroni and Acidovorax sp. removed propionitrile completely up to 1352.8 mg/L and 975.7 mg/L within 166.9 h, respectively. With isobutyronitrile as a substrate, more than 97.8% could be removed within 169.8 h by Comamonas testosteroni up to 1352.8 mg/L, and by Acidovorax sp. up to 975.7 mg/L. These two strains could remove allyl cyanide completely up to 775 mg/L within 49.1 h and benzonitrile completely up to 442.7 mg/L within 31.1 h. Because Comamonas testosteroni and Acidovorax sp. have good degrading ability for removing nitrile compounds they are expected to play an important role in the treatment of nitrile compounds.