SSR individual identification system construction and population genetics analysis for Chamaecyparis formosensis.

Chamaecyparis formosensis is an endemic species of Taiwan, threatened from intensive use and illegal felling. An individual identification system for C. formosensis is required to provide scientific evidence for court use and deter illegal felling. In this study, 36 polymorphic simple sequence repeat markers were developed. By applying up to 28 non-linked of the developed markers, it is calculated that the cumulative random probability of identity (CPI) is as low as 1.652 × 10-12, and the identifiable population size is up to 60 million, which is greater than the known C. formosensis population size in Taiwan. Biogeographical analysis data show that C. formosensis from four geographic areas belong to the same genetic population, which can be further divided into three clusters: SY (Eastern Taiwan), HV and GW (Northwestern Taiwan), and MM (Southwestern Taiwan). The developed system was applied to assess the provenance of samples with 88.44% accuracy rate and therefore can serve as a prescreening tool to reduce the range required for comparison. The system developed in this study is a potential crime-fighting tool against illegal felling.

Development and technical application of SSR-based individual identification system for Chamaecyparis taiwanensis against illegal logging convictions.

Chamaecyparis taiwanensis is an endemic plant suffering illegal logging in Taiwan for its high economic value. Lack of direct evidence to correlate stump and timber remains a hurdle for law enforcement. In this report, 23 polymorphic Genomic Simple Sequence Repeat (gSSR) and 12 Expressed Sequence Tag (EST)-SSR markers were developed and their transferability was assessed. The individual identification system built from selected non-linkage 30 SSR markers has a combined probability of identity as 5.596 × 10-12 equivalents to identifying an individual in a population of up to 18 million C. taiwanensis with 99.99% confidence level. We also applied the system in an actual criminal case by selecting 19 of these markers to correlate illegally felled timbers and victim trees. Our data demonstrate that molecular signals from three timbers hit with three victim trees with confidence level more than 99.99%. This is the first example of successfully applying SSR in C. taiwanensis as a court evidence for law enforcement. The identification system adapted advanced molecular technology and exhibits its great potential for natural resource management on C. taiwanensis.

Microbial Functional Responses to Cholesterol Catabolism in Denitrifying Sludge.

The 2,3-seco pathway, the pathway for anaerobic cholesterol degradation, has been established in the denitrifying betaproteobacterium Sterolibacterium denitrificans. However, knowledge of how microorganisms respond to cholesterol at the community level is elusive. Here, we applied mesocosm incubation and 16S rRNA sequencing to reveal that, in denitrifying sludge communities, three betaproteobacterial operational taxonomic units (OTUs) with low (94% to 95%) 16S rRNA sequence similarity to Stl. denitrificans are cholesterol degraders and members of the rare biosphere. Metatranscriptomic and metabolite analyses show that these degraders adopt the 2,3-seco pathway to sequentially catalyze the side chain and sterane of cholesterol and that two molybdoenzymes-steroid C25 dehydrogenase and 1-testosterone dehydrogenase/hydratase-are crucial for these bioprocesses, respectively. The metatranscriptome further suggests that these betaproteobacterial degraders display chemotaxis and motility toward cholesterol and that FadL-like transporters may be the key components for substrate uptake. Also, these betaproteobacteria are capable of transporting micronutrients and synthesizing cofactors essential for cellular metabolism and cholesterol degradation; however, the required cobalamin is possibly provided by cobalamin-de novo-synthesizing gamma-, delta-, and betaproteobacteria via the salvage pathway. Overall, our results indicate that the ability to degrade cholesterol in sludge communities is reserved for certain rare biosphere members and that C25 dehydrogenase can serve as a biomarker for sterol degradation in anoxic environments. IMPORTANCE Steroids are ubiquitous and abundant natural compounds that display recalcitrance. Biodegradation via sludge communities in wastewater treatment plants is the primary removal process for steroids. To date, compared to studies for aerobic steroid degradation, the knowledge of anaerobic degradation of steroids has been based on only a few model organisms. Due to the increase of anthropogenic impacts, steroid inputs may affect microbial diversity and functioning in ecosystems. Here, we first investigated microbial functional responses to cholesterol, the most abundant steroid in sludge, at the community level. Our metagenomic and metatranscriptomic analyses revealed that the capacities for cholesterol approach, uptake, and degradation are unique traits of certain low-abundance betaproteobacteria, indicating the importance of the rare biosphere in bioremediation. Apparent expression of genes involved in cofactor de novo synthesis and salvage pathways suggests that these micronutrients play important roles for cholesterol degradation in sludge communities.

Isolation and characterization of SSR and EST-SSR loci in Chamaecyparis formosensis (Cupressaceae).

PREMISE OF THE STUDY: Simple sequence repeat (SSR) and expressed sequence tag (EST)-SSR markers were developed as tools for marker-assisted selection of Chamaecyparis formosensis and for the molecular differentiation of cypress species. METHODS AND RESULTS: Based on the SSR-enriched genomic libraries and transcriptome data of C. formosensis, 300 primer pairs were selected for initial confirmation, of which 19 polymorphic SSR and eight polymorphic EST-SSR loci were chosen after testing in 92 individuals. The number of alleles observed for these 27 loci ranged from one to 17. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.000 to 0.903, respectively. Most markers also amplified in C. obtusa var. formosana. CONCLUSIONS: The developed SSR and EST-SSR sequences are the first reported markers specific to C. formosensis. These markers will be useful for individual identification of C. formosensis and to distinguish cypress species such as C. obtusa var. formosana.

Cross-Linked Cholecyst-Derived Extracellular Matrix for Abdominal Wall Repair.

Abdominal wall repair frequently utilizes either nondegradable or biodegradable meshes, which are found to stimulate undesirable biological tissue responses or which possess suboptimal degradation rate. In this study, a biologic mesh prototype made from carbodiimide cross-linked cholecyst-derived extracellular matrix (EDCxCEM) was compared with small intestinal submucosa (Surgisis®), cross-linked bovine pericardium (Peri-Guard®), and polypropylene (Prolene®) meshes in an in vivo rabbit model. The macroscopic appearance and stereological parameters of the meshes were evaluated. Tailoring the degradation of the EDCxCEM mesh prevents untimely degradation, while allowing cellular infiltration and mesh remodeling to take place in a slower but predictable manner. The results suggest that the cross-linked biodegradable cholecyst-derived biologic mesh results in no seroma formation, low adhesion, and moderate stretching of the mesh. In contrast to Surgisis, Peri-Guard, and Prolene meshes, the EDCxCEM mesh showed a statistically significant increase in the volume fraction (Vv) of collagen (from 34% to 52.1%) in the central fibrous tissue region at both day 28 and 56. The statistically high length density (Lv), of blood vessels for the EDCxCEM mesh at 28 days was reflected also by the higher cellular activity (high Vv of fibroblast and moderate Vv of nuclei) indicating remodeling of this region in the vicinity of a slowly degrading EDCxCEM mesh. The lack of mesh area stretching/shrinkage in the EDCxCEM mesh showed that the remodeled tissue was adequate to prevent hernia formation. The stereo-histological assays suggest that the EDCxCEM delayed degradation profile supports host wound healing processes including collagen formation, cellular infiltration, and angiogenesis. The use of cross-linked CEM for abdominal wall repair is promising.

A clinically relevant in vivo model for the assessment of scaffold efficacy in abdominal wall reconstruction.

An animal model that allows for assessment of the degree of stretching or contraction of the implant area and the in vivo degradation properties of biological meshes is required to evaluate their performance in vivo. Adult New Zealand rabbits underwent full thickness subtotal unilateral rectus abdominis muscle excision and were reconstructed with the non-biodegradable Peri-Guard®, Prolene® or biodegradable Surgisis® meshes. Following 8 weeks of recovery, the anterior abdominal wall tissue samples were collected for measurement of the implant dimensions. The Peri-Guard and Prolene meshes showed a slight and obvious shrinkage, respectively, whereas the Surgisis mesh showed stretching, resulting in hernia formation. Surgisis meshes showed in vivo biodegradation and increased collagen formation. This surgical rabbit model for abdominal wall defects is advantageous for evaluating the in vivo behaviour of surgical meshes. Implant area stretching and shrinkage were detected corresponding to mesh properties, and histological analysis and stereological methods supported these findings.

Anticataractogenesis Mechanisms of Curcumin and a Comparison of Its Degradation Products: An in Vitro Study.

Curcumin (Cur) exhibits anticataractogenesis activity. This study aimed to compare the activities of Cur with those of its degradation products in a series of in vitro lens protein turbidity assays. The results show that Cur (200 µM) ameliorates selenite-induced crystallin aggregation, and the mean OD value was 0.10 ± 0.02 (p < 0.05), which was significantly different from controls (0.15 ± 0.01) after incubating for 3 days. However, Cur did not significantly inhibit calcium-induced proteolysis after incubating for 3 days. Such results were supported by isothermal titration calorimetry observation that Cur binds with selenite but not with calcium. Presence of Cur and the degradation products examined (ferulic acid, cinnamic acid, vanillin, and vanillic acid) indicates significantly protective activities on lens γ-crystallins after UVC exposure for 3 h. Among the compounds examined, only ferulic acid exhibited a significant inhibitory effect against UVB-induced turbidity with a mean OD of 0.32 ± 0.01 (p < 0.05), which was significantly different from controls (0.49 ± 0.02). The previously reported anticataract effects of Cur may stem not only from Cur but also from its degradation products through various cataractogenesis mechanisms in vitro.

The Comparative Studies of Binding Activity of Curcumin and Didemethylated Curcumin with Selenite: Hydrogen Bonding vs Acid-Base Interactions.

In this report, the in vitro relative capabilities of curcumin (CCM) and didemethylated curcumin (DCCM) in preventing the selenite-induced crystallin aggregation were investigated by turbidity tests and isothermal titration calorimetry (ITC). DCCM showed better activity than CCM. The conformers of CCM/SeO3(2-) and DCCM/SeO3(2-) complexes were optimized by molecular orbital calculations. Results reveal that the selenite anion surrounded by CCM through the H-bonding between CCM and selenite, which is also observed via IR and NMR studied. For DCCM, the primary driving force is the formation of an acid-base adduct with selenite showing that the phenolic OH group of DCCM was responsible for forming major conformer of DCCM. The formation mechanisms of selenite complexes with CCM or DCCM explain why DCCM has greater activity than CCM in extenuating the toxicity of selenite as to prevent selenite-induced lens protein aggregation.

RGD surface functionalization of the hydrophilic acrylic intraocular lens material to control posterior capsular opacification.

Posterior Capsular Opacification (PCO) is the capsule fibrosis developed on implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LECs) undergoing Epithelial Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including the patient's age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs have shown that the former has more severe PCO. On the other hand, we have previously demonstrated that the adhesion of LECs is favored on hydrophobic compared to hydrophilic materials. By combining these two facts and contemporary knowledge in PCO development via the EMT pathway, we propose a biomimetically inspired strategy to promote LEC adhesion without de-differentiation to reduce the risk of PCO development. By surface grafting of a cell adhesion molecule (RGD peptide) onto the conventional hydrophilic acrylic IOL material, the surface-functionalized IOL can be used to reconstitute a capsule-LEC-IOL sandwich structure, which has been considered to prevent PCO formation in literature. Our results show that the innovative biomaterial improves LEC adhesion, while also exhibiting similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties compared to the starting material. In addition, compared to the hydrophobic IOL material, our bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression, which is the crucial pathway to induce PCO. The in vitro assays suggest that this biomaterial has the potential to reduce the risk factor of PCO development.

Biointerface multiparametric study of intraocular lens acrylic materials.

PURPOSE: To compare hydrophilic and hydrophobic acrylic materials designed for intraocular lenses in a multiparametric investigation in a liquid environment to highlight their properties in terms of adhesion forces, lens epithelial cell (LEC) adhesion, and tissue response as indicators of the risk for posterior capsule opacification (PCO) development. SETTING: University of Liège, Liège, Belgium. DESIGN: Experimental study. METHODS: The hydrophobicity and surface adhesion force were assessed using contact-angle and atomic force microscopy measurements. The bioadhesiveness of the disks and the tissue response were determined by in vitro experiments using bovine serum albumin and porcine LECs and by in vivo rabbit subcutaneous implantation, respectively. RESULTS: Increasing surface hydrophobicity led to a greater surface-adhesion force and greater LEC adhesion. After 1 month, the rabbit subcutaneous implants showed a similar thin layer of fibrous capsule surrounding the disks without extensive inflammation. A layer of rounded cells in contact with disks was detected on the hydrophobic samples only. CONCLUSIONS: Hydrophobic acrylic disks that have been associated with a reduced risk for PCO in clinical studies showed increased tackiness. FINANCIAL DISCLOSURES: Proprietary or commercial disclosures are listed after the references.

Balanced detection for spectral domain optical coherence tomography.

The use and advantages of applying balanced-detection (BD) operation method to high speed spectral domain optical coherence tomography (SDOCT) are presented in this study, which we believe is the first such demonstration. Compared to conventional SDOCT, BD-SDOCT provides two unique advantages. First, the method can suppress background noise and autocorrelation artifacts in biological tissues. Second, it is a power-efficient method which is particularly helpful for high speed SDOCT to eliminate random intensity noise and to achieve shot noise limited detection. This performance allows in vivo three-dimensional tissue visualization with high imaging quality and high speed.

Balanced detection spectral domain optical coherence tomography with a multiline single camera for signal-to-noise ratio enhancement.

In this study, the use and advantages of balanced detection (BD) in spectral domain optical coherence tomography (SD-OCT) are demonstrated. A-scans are calculated as a combination of two phase-opposed interferometric spectra acquired simultaneously by using a multiline single camera spectrometer. Not only does this system suppress artifacts due to autocorrelation, but also the signal of interest is increased by a factor of 2 as experimentally verified. Our BD-based SD-OCT gives a signal-to-noise ratio improvement of 8-14 dB for the peak within 1 mm compared to standard SD-OCT using a single detection scheme. This method is validated by experimental measurement of a glass plate.

Anti-UVC irradiation and metal chelation properties of 6-benzoyl-5,7-dihydroxy-4-phenyl-chromen-2-one: an implications for anti-cataract agent.

Coumarin derivative 1, 5,7-dihydroxy-6-(3-methyl-1-butyryl)-4-phenyl-chromen- 2-one, has been reported to possess radical scavenging activity and DNA protection. We have synthesized a series of coumarins with structural modifications at positions C4, C5, C6 and C7 and evaluated them for their anti-UVC properties. Coumarin 7, 6-benzoyl-5,6-dihydroxy-4-phenyl-chromen-2-one, was found to have the most potent activity in protecting porcine γ-crystallin against UVC insults. Results of fluorescence assays indicated that compound 7 was capable of decreasing the loss of intensity while lens crystallins and DNA PUC19 were irradiated with UVC. Presence of compound 7 decreased hydroxyl radical levels determined by probe 1b and the free iron concentrations determined by Ferrozine reagent. The chelation assay showed that compound 7 was chelated to metal via 6-CO and 5-OH on the benzopyrone ring. The observed protective effects of compound 7 towards crystallins from insults of UVC and free radicals may be due to its iron-chelating activity and its peak absorption at 254 nm.

Role of pirenoxine in the effects of catalin on in vitro ultraviolet-induced lens protein turbidity and selenite-induced cataractogenesis in vivo.

PURPOSE: In this study, we investigated the biochemical pharmacology of pirenoxine (PRX) and catalin under in vitro selenite/calcium- and ultraviolet (UV)-induced lens protein turbidity challenges. The systemic effects of catalin were determined using a selenite-induced cataractogenesis rat model. METHODS: In vitro cataractogenesis assay systems (including UVB/C photo-oxidation of lens crystallins, calpain-induced proteolysis, and selenite/calcium-induced turbidity of lens crystallin solutions) were used to screen the activity of PRX and catalin eye drop solutions. Turbidity was identified as the optical density measured using spectroscopy at 405 nm. We also determined the in vivo effects of catalin on cataract severity in a selenite-induced cataract rat model. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied to analyze the integrity of crystallin samples. RESULTS: PRX at 1,000 µM significantly delayed UVC-induced turbidity formation compared to controls after 4 h of UVC exposure (p<0.05), but not in groups incubated with PRX concentrations of <1,000 µM. Results were further confirmed by SDS-PAGE. The absolute γ-crystallin turbidity induced by 4 h of UVC exposure was ameliorated in the presence of catalin equivalent to 1~100 µM PRX in a concentration-dependent manner. Samples with catalin-formulated vehicle only (CataV) and those containing PRX equivalent to 100 µM had a similar protective effect after 4 h of UVC exposure compared to the controls (p<0.05). PRX at 0.03, 0.1, and 0.3 µM significantly delayed 10 mM selenite- and calcium-induced turbidity formation compared to controls on days 0~4 (p<0.05). Catalin (equivalent to 32, 80, and 100 µM PRX) had an initial protective effect against selenite-induced lens protein turbidity on day 1 (p<0.05). Subcutaneous pretreatment with catalin (5 mg/kg) also statistically decreased the mean cataract scores in selenite-induced cataract rats on post-induction day 3 compared to the controls (1.3±0.2 versus 2.4±0.4; p<0.05). However, catalin (equivalent to up to 100 µM PRX) did not inhibit calpain-induced proteolysis activated by calcium, and neither did 100 µM PRX. CONCLUSIONS: PRX at micromolar levels ameliorated selenite- and calcium-induced lens protein turbidity but required millimolar levels to protect against UVC irradiation. The observed inhibition of UVC-induced turbidity of lens crystallins by catalin at micromolar concentrations may have been a result of the catalin-formulated vehicle. Transient protection by catalin against selenite-induced turbidity of crystallin solutions in vitro was supported by the ameliorated cataract scores in the early stage of cataractogenesis in vivo by subcutaneously administered catalin. PRX could not inhibit calpain-induced proteolysis activated by calcium or catalin itself, and may be detrimental to crystallins under UVB exposure. Further studies on formulation modifications of catalin and recommended doses of PRX to optimize clinical efficacy by cataract type are warranted.