An Indocyanine Green-Based Nanoprobe for In Vivo Detection of Cellular Senescence.

There is an urgent need to improve conventional cancer-treatments by preventing detrimental side effects, cancer recurrence and metastases. Recent studies have shown that presence of senescent cells in tissues treated with chemo- or radiotherapy can be used to predict the effectiveness of cancer treatment. However, although the accumulation of senescent cells is one of the hallmarks of cancer, surprisingly little progress has been made in development of strategies for their detection in vivo. To address a lack of detection tools, we developed a biocompatible, injectable organic nanoprobe (NanoJagg), which is selectively taken up by senescent cells and accumulates in the lysosomes. The NanoJagg probe is obtained by self-assembly of indocyanine green (ICG) dimers using a scalable manufacturing process and characterized by a unique spectral signature suitable for both photoacoustic tomography (PAT) and fluorescence imaging. In vitro, ex vivo and in vivo studies all indicate that NanoJaggs are a clinically translatable probe for detection of senescence and their PAT signal makes them suitable for longitudinal monitoring of the senescence burden in solid tumors after chemotherapy or radiotherapy.

Chemical characterization and source attribution of organic pollutants in industrial wastewaters from a Chinese chemical industrial park.

Accelerated urbanization and industrialization have led to an alarming increase in the generation of wastewater with complex chemical contents. Industrial wastewaters are often a primary source of water contamination. The chemical characterization of different industrial wastewater types is an essential task to interpret the chemical fingerprints of wastewater to identify pollution sources and develop efficient water treatment strategies. In this study, we conduct a non-target chemical analysis for the source characterization of different industrial wastewater samples collected from a chemical industrial park (CIP) located in southeast China. The chemical screening identified volatile and semi-volatile organic compounds that included dibutyl phthalate at a maximum concentration of 13.4 µg/L and phthalic anhydride at 35.9 µg/L. Persistent, mobile, and toxic (PMT) substances among the detected organic compounds were identified and prioritized as high-concern contaminants given their impact on drinking water resources. Moreover, a source analysis of the wastewater collected from the wastewater outlet station indicated that the dye production industry contributed the largest quantities of toxic contaminates (62.6%), and this result was consistent with the ordinary least squares and heatmap results. Thus, our study utilized a combined approach of a non-target chemical analysis, a pollution source identification method, and a PMT assessment of different industrial wastewater samples collected from the CIP. The results of the chemical fingerprints of different industrial wastewater types as well as the results of the PMT assessment benefit risk-based wastewater management and source reduction strategies.

Indicators of glucose dysregulation and the relationship with iron overload in Chinese children with beta thalassemia major.

INTRODUCTION: Patients with beta thalassemia major (TM) have a higher risk of diabetes and an abnormal oral glucose tolerance test (OGTT), but there is no single agree monitoring parameter that reflects glycemic status. The possible mechanisms include iron overload and blood transfusion, but they require further investigation. PURPOSE: This study explored the role of glycated hemoglobin A1c (HbA1c), fructosamine, and glycated albumin (GA) in evaluating the glucose dysregulation and to determine the potential relationship between iron deposition and glucose metabolism disorder in beta TM. METHODS: A cross-sectional study was performed on 118 patients with beta TM and the control group consisted of 33 healthy children with no statistical differences in age, sex, and body mass index (BMI). Fast plasma glucose (FPG), fast insulin (FINS), insulin resistance index (HOMA-IRI), and insulin sensitivity index (HOMA-ISI) were compared between the patient and control groups. HbA1c, GA, fructosamine, and serum ferritin (SF) were measured in the patient group. OGTT, as well as heart and liver magnetic resonance imaging (MRI) T2*, was performed. For all statistical analyses, SPSS 21.0 was used and p < 0.05 was accepted as statistically significant. RESULTS: FPG, FINS, and HOMA-IRI were significantly increased while HOMA-ISI decreased in the beta TM patients when compared with those in the control group. In patients with beta TM, 17 (14.41%) of patients had been diagnosed with diabetes, while 48 (40.68%) had both impaired fasting glucose and impaired glucose tolerance. HbA1c, GA, and fructosamine were increased according to the degree of abnormal glucose metabolism. Statistically significant differences were found in age, SF, and cardiac T2* between the abnormal and normal OGTT groups. CONCLUSION: HbA1c may be used as a significant measure for monitoring glycemic levels in patients with beta TM. Furthermore, GA and fructosamine were alternative indicators of glucose status. Patients with heart iron deposition or an SF > 4000 µg/L were prone to abnormal glucose metabolism, so chelation therapy should be reinforced.

Structural and Optical Properties of Textured Silicon Substrates by Three-Step Chemical Etching.

We implemented the fabrication of hybrid structures, including pyramids, etching holes, and inverted pyramidal cavities on silicon substrates, by three-step chemical etching. To achieve this, we utilized anisotropic wet etching as the first-step etching to form pyramids of various sizes. Subsequently, metal-assisted chemical etching was performed to develop aligned etching holes on the pyramidal structure. Ultimately, anisotropic wet etching was used again as the third-step etching for the etchant to penetrate holes to form inverted pyramidal cavities. Optimizing the three-step etching treatments, large-scale textured structures with low reflectance could be obtained, and they show potential for applications in sensors, solar cells, photovoltaics, and surface-enhanced Raman scattering (SERS). Examples of using the textured silicon substrates for SERS applications were given.

DNA damage responses and p53 in the aging process.

The genome is constantly attacked by genotoxic insults. DNA damage has long been established as a cause of cancer development through its mutagenic consequences. Conversely, radiation therapy and chemotherapy induce DNA damage to drive cells into apoptosis or senescence as outcomes of the DNA damage response (DDR). More recently, DNA damage has been recognized as a causal factor for the aging process. The role of DNA damage in aging and age-related diseases is illustrated by numerous congenital progeroid syndromes that are caused by mutations in genome maintenance pathways. During the past 2 decades, understanding how DDR drives cancer development and contributes to the aging process has progressed rapidly. It turns out that the DDR factor p53 takes center stage during tumor development and also plays an important role in the aging process. Studies in metazoan models ranging from Caenorhabditis elegans to mammals have revealed cell-autonomous and systemic DDR mechanisms that orchestrate adaptive responses that augment maintenance of the aging organism amid gradually accumulating DNA damage.

Air-Stable Half-Sandwich Iridium Complexes as Aerobic Oxidation Catalysts for Imine Synthesis.

Several N,O-coordinate half-sandwich iridium complexes, 1-5, containing constrained bulky ß-enaminoketonato ligands were prepared and clearly characterized. Single-crystal X-ray diffraction characterization of these complexes indicates that the iridium center adopts a distorted octahedral geometry. Complexes 1-5 showed good catalytic efficiency in the oxidative homocoupling of primary amines, dehydrogenation of secondary amines, and the oxidative cross-coupling of amines and alcohols, which furnished various types of imines in good yields and high selectivities using O2 as an oxidant under mild conditions. No distinctive substituent effects of the iridium catalysts were observed in these reactions. The diverse catalytic activity, broad substrate scope, mild reaction conditions, and high yields of the products made this catalytic system attractive in industrial processes.

A long lasting sunscreen controversy of 4-aminobenzoic acid and 4-dimethylaminobenzaldehyde derivatives resolved by ultrafast spectroscopy combined with density functional theoretical study.

4-Aminobenzoic acid (PABA) is one of the earliest patented and most commonly used sunscreen components. There is however a long-lasting controversy on its photo-protective efficacy owing to the lack of information on its protolytic equilibrium and photo-dynamics after absorption of ultraviolet radiation in physiologically relevant aqueous solution. The excitation dynamics in water also remains largely unknown for analogs of PABA such as 4-dimethylaminoacetophenone (DMAAP) and 4-dimethylaminobenzaldehyde (DMABA) which are recognized as prototypes for photo-induced twisted intramolecular charge transfer (TICT). Herein we report a combined application of femtosecond broadband time-resolved fluorescence and transient absorption coupled with density functional theoretical study for PABA, DMAAP, and DMABA under several solvent conditions with representative properties in terms of the pH, polarity and hydrogen bonding capacity. The results we gained demonstrate that, in a neutral aqueous solution, PABA taking the deprotonated anion form in the ground state undergoes rapid protonation after excitation, producing excited state species in the neutral form that may shift effectively by intersystem crossing (ISC) to the long-lasting triplet state capable of damaging nucleic acids. This provides evidence at the molecular level for the detrimental effect of PABA if used as a sunscreen ingredient. In contrast, our investigation on DMAAP and DMABA unveils an unusual solvent controlled deactivation dynamics rendered by the participation of the carbonyl oxygen associated nOπ* state featuring energy and structure strongly responsive to solvent properties. In particular, these molecules in water exhibit solute-solvent hydrogen bonding at the sites of the carbonyl oxygen and the amino nitrogen which is, respectively, weakened and strengthened after the excitation, leading to state reversal and formation of a nOπ* state with a peculiar non-planar structure. This quenches strongly the excitation, eliminates the TICT, suppresses the ISC and opens up the otherwise inaccessible internal conversion (IC) to account for ∼80% of the entire deactivation. The IC, observed to proceed at a rate of ∼2.5 ps, allows the effective recovery of the ground state, providing substantial protection against ultraviolet irradiation. Moreover, the revelation of highly solvent sensitive fluorescence emission from DMABA and DMAAP implies the potential application of these molecules as the functional element in the design of sensory materials for probing the polarity and hydrogen bonding character of the surrounding environment.

Immunological microenvironment alterations in follicles of women with proven severe endometriosis undergoing in vitro fertilization.

The purpose of this study was to test the hypothesis that different cytokine profiles may exist in the follicular fluid of endometriosis (EM) patients undergoing in vitro fertilization (IVF), as these differences may provide insights into the pathogenesis of the disease. This was a cross-sectional study conducted at the reproductive center of a medical university hospital. The study included 49 patients receiving IVF. 20 infertile women with proven EM and 29 women without diagnosed EM (control group) were evaluated. Follicular fluid (FF) and serum were collected at the time of follicle aspiration and the concentrations of 38 cytokines were determined by multiplexed immunoassay. The results indicated that the levels of IL-4, IL-13, IL-3 and IL-1α were significantly increased in the FF of women with EM, while levels of IFN-γ, IL-17A, MDC and MIP-1α were decreased compared with in the control subjects. In conclusions, the immune microenvironment of the FF in patients with EM is altered. This may contribute to the pathologic mechanism responsible for the poor outcome of IVF in patients with EM.

DNA damage in germ cells induces an innate immune response that triggers systemic stress resistance.

DNA damage responses have been well characterized with regard to their cell-autonomous checkpoint functions leading to cell cycle arrest, senescence and apoptosis. In contrast, systemic responses to tissue-specific genome instability remain poorly understood. In adult Caenorhabditis elegans worms germ cells undergo mitotic and meiotic cell divisions, whereas somatic tissues are entirely post-mitotic. Consequently, DNA damage checkpoints function specifically in the germ line, whereas somatic tissues in adult C. elegans are highly radio-resistant. Some DNA repair systems such as global-genome nucleotide excision repair (GG-NER) remove lesions specifically in germ cells. Here we investigated how genome instability in germ cells affects somatic tissues in C. elegans. We show that exogenous and endogenous DNA damage in germ cells evokes elevated resistance to heat and oxidative stress. The somatic stress resistance is mediated by the ERK MAP kinase MPK-1 in germ cells that triggers the induction of putative secreted peptides associated with innate immunity. The innate immune response leads to activation of the ubiquitin-proteasome system (UPS) in somatic tissues, which confers enhanced proteostasis and systemic stress resistance. We propose that elevated systemic stress resistance promotes endurance of somatic tissues to allow delay of progeny production when germ cells are genomically compromised.

The Protective Effects of the A/ZJU01/ PR8/2013 Split H7N9 Avian Influenza Vaccine Against Highly Pathogenic H7N9 in BALB/c Mice.

BACKGROUND/AIMS: Since the first case of novel H7N9 infection was reported, China has experienced five epidemics of H7N9. During the fifth wave, a highly pathogenic H7N9 strain emerged. In order to assess whether the H7N9 vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) was effective in protecting against highly pathogenic H7N9, we conducted this study. METHODS: Groups of mice were immunized twice by intraperitoneal injection with 500 µl of either split vaccine alone or MF59-adjuvanted vaccine. Serum was collected 2 weeks after the second vaccine booster. The hemagglutinin inhibition test was conducted on vaccine seed and highly pathogenic H7N9 to evaluate the neutralization of highly pathogenic H7N9. We also immunized mice and challenged them with highly pathogenic H7N9. Mice were observed for illness, weight loss, and death at 1 week and 2 weeks post-infection. Then, the mice were sacrificed and lungs were removed. Antibody responses were assessed and pathological changes in the lung tissue were evaluated. RESULTS: The ability of serum to neutralize highly pathogenic H7N9 was reduced. In mice, highly pathogenic H7N9 was more virulent than A/Zhejiang/DTID-ZJU01/2013(H7N9). After challenge with highly pathogenic H7N9, all mice died while mice challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) all recovered. The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine was able to protect against infection with highly pathogenic H7N9 in mice, with or without MF59. Moreover, H7N9 vaccine adjuvanted with MF59 produced high antibody levels, which lead to better protection. CONCLUSIONS: The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) is effective in protecting against highly pathogenic H7N9. H7N9 vaccine adjuvanted with MF59 offers better protection against infection with highly pathogenic H7N9. In order to make the H7N9 vaccine applicable to humans, further clinical trials are required to evaluate MF59 adjuvanted vaccine. Meanwhile, the vaccine strain should be updated based on the highly pathogenic H7N9 gene sequence.

Process optimization for the rapid production of adenoviral vectors for clinical trials in a disposable bioreactor system.

Recombinant adenoviral (Ad) vectors are highly efficient gene transfer vectors widely used in vaccine development and immunotherapy. To promote the industrial application of Ad vectors, studies focusing on reducing the cost of manufacturing, shortening the preclinical research period, and improving the quality of products are needed. Here, we describe a highly efficient and economical process for producing Ad vector in a novel, single-use bioreactor system suitable for clinical trials. A mini-bioreactor was used for parameter optimization and development of medium replacement protocols for Ad5-GFP production before scale-up. HEK293 cell culture and virus infection were monitored in a disposable AmProtein Current Perfusion Bioreactor and Bioflo310 bioreactor using optimized parameters and medium replacement protocols. The total cell number increased from 2.0 × 109 to 3.2 × 1010 after 6 days of culture. The total number of viral particles obtained in a single batch was 1.2 × 1015. These results demonstrate the efficiency and suitability of this system for Ad vector production for research and GMP applications.

Nanoporous TiO2/SnO2/Poly(3,4-ethylene-dioxythiophene): Polystyrenesulfonate Composites as Efficient Counter Electrode for Dye-Sensitized Solar Cells.

A nanoporous composite film combined of conducting inorganic template (TiO2/SnO2) and conducting polymer catalyst (poly(3,4-ethylenedioxythiophene):polystyrenesulfonate, PEDOT: PSS) was developed as an alternative counter electrode for dye-sensitized solar cell (DSSC) through low-temperature process. The TiO2/SnO2 template was first fabricated by coating a homogeneous TiO2 nanoparticles blended paste containing a SnCl4 aqueous solution on the conductive substrate, followed by annealing at 150 °C. The counter electrode was then completed by spin-coating the PEDOT: PSS aqueous solution into the template and drying at 80 °C. The obtained TiO2/SnO2/ PEDOT: PSS (TSP) composite film exhibits more excellent catalytic activity for the tri-iodide reduction than the pristine PEDOT: PSS film, resulting in the significant improvements in the fill factor and efficiency of the cells. The values of the fill factor and efficiency respectively increase from 0.564 and 4.79% to 0.699 and 6.54%. Noted that the photovoltaic performances of the TSP based DSSC is very similar to those of the Pt based one. The fill factor and efficiency of the later are 0.696 and 6.48%, respectively. The outstanding properties of the TSP composite film used as the counter electrode can be ascribed to its prominent synergistic effects. In the TSP composite film, the conducting TiO2 is applied as the main skeleton material with the in-situ formed SnO2 as a binder to construct a nanoporous structure for the PEDOT: PSS coating and also to provide numerous high-speed conductive paths for the electron transportation from the substrate to the PEDOT: PSS coating, and the PEDOT: PSS adhered on the TiO2/SnO2 skeleton mainly acts as the catalyst to enlarge its surface area allowing for more active sites for the tri-iodide reduction.

Relative dose intensity and therapy efficacy in different breast cancer molecular subtypes: a retrospective study of early stage breast cancer patients treated with neoadjuvant chemotherapy.

To investigate the relationship between chemotherapy dose intensity and therapy efficacy of different molecular subtypes. Clinical and pathological features of the patients with breast cancer were retreived from the hospital records. 315 patients were analyzed (251 showed clinical response, 38 acquired pCR). Patients with positive ER status, negative PR status, higher Ki67 level and higher RTDI had better therapy response. 13.5 and 84.5 % were identified the benchmark of Ki67 and RTDI, respectively. As the result of interior-subgroup comparison, luminal subgroups acquired better response rate when RTDI ≥ 84.5 %. In patients of luminal breast cancer, tumor size change arose from increasing of dose intensity and finally showed reached a plateau after RTDI ≥ 95 % (r (2) = 0.303, p < 0.001). As the result of intersubgroup comparison, TNBC patients were more likely to acquired better clinical and pathology response when RDTI < 84.5 %. Ki67 change arose sharply from increasing of dose intensity when RDTI < 84.5 % (r (2) = 0.656, p < 0.001), whereas the regression curve showed a terminal plateau in patients of RDTI ≥ 84.5 % (r (2) = 0.427, p < 0.001). Given lower RTDI, luminal patients are less likely to achieve response, and TNBC patients are associated with higher response rate. Dissimilar of therapy efficacy between luminal subtype and TNBC becomes inconspicuous as RTDI rises. Chemosensitivity may associate with dose intensity, especially in luminal subtypes, and tailored therapeutic strategies should be considered.

Acoustic detection of biosonar activity of deep diving odontocetes at Josephine Seamount High Seas Marine Protected Area.

The temporal occurrence of deep diving cetaceans in the Josephine Seamount High Seas Marine Protected Area (JSHSMPA), south-west Portugal, was monitored using a passive acoustic recorder. The recorder was deployed on 13 May 2010 at a depth of 814 m during the North Atlantic Treaty Organization Centre for Maritime Research and Experimentation cruise "Sirena10" and recovered on 6 June 2010. The recorder was programmed to record 40 s of data every 2 min. Acoustic data analysis, for the detection and classification of echolocation clicks, was performed using automatic detector/classification systems: M3R (Marine Mammal Monitoring on Navy Ranges), a custom matlab program, and an operator-supervised custom matlab program to assess the classification performance of the detector/classification systems. M3R CS-SVM algorithm contains templates to detect beaked whales, sperm whales, blackfish (pilot and false killer whales), and Risso's dolphins. The detections of each group of odontocetes was monitored as a function of time. Blackfish and Risso's dolphins were detected every day, while beaked whales and sperm whales were detected almost every day. The hourly distribution of detections reveals that blackfish and Risso's dolphins were more active at night, while beaked whales and sperm whales were more active during daylight hours.

Discrimination of frequency-modulated Baleen whale downsweep calls with overlapping frequencies.

Automatic classification of fin, sei, and blue whale frequency modulated downsweeps has been a challenging task for bioacousticians. These calls overlap in frequency range and have similar time durations. The traditional spectrogram methodology, the Short Time Fourier Transform, tends to be ineffective because of the large temporal ambiguities needed to achieve the necessary frequency resolution to study the fine time-frequency (TF) structures. Spectrograms generated with the Pseudo Wigner-Ville Distribution (PWVD) provide much higher simultaneous TF resolution compared with the traditional method. The PWVD allows bioacousticians to study the fine TF structures of the sound, such as the instantaneous frequency, instantaneous bandwidth, contour slope, etc. These features set the foundation of identifying sounds that are usually considered difficult to discriminate using the traditional method. Wigner-Ville distribution of the baleen whale downsweeps showed distinguishable characteristics; for example, the TF contour of fin and sei whales exhibited concave and convex shapes, which have never been reported in the literature. A Support Vector Machine classifier was trained and tested based on the parameters extracted from the PWVD.

Temporal signal processing of dolphin biosonar echoes from salmon prey.

Killer whales project short broadband biosonar clicks. The broadband nature of the clicks provides good temporal resolution of echo highlights and allows for the discriminations of salmon prey. The echoes contain many highlights as the signals reflect off different surfaces and parts of the fish body and swim bladder. The temporal characteristics of echoes from salmon are highly aspect dependent and six temporal parameters were used in a support vector machine to discriminate between species. Results suggest that killer whales can classify salmon based on their echoes and provide some insight as to which features might enable the classification.

[Analysis of chromosomal abnormalities and a report of eight new karyotypes among children in genetic counseling].

OBJECTIVE: To study the relationship between abnormal karyotypes and clinical phenotypes among children in genetic counseling in Guangxi Zhuang Autonomous Region, China. METHODS: We studied 601 children who visited Guangxi Zhuang Autonomous Region Women and Children Care Hospital for genetic counseling between January 2009 and July 2012. Blood samples were cultured routinely for karyotype analysis with G banding as well as clinical analysis. RESULTS: Out of 601 patients, 329 (54.7%) had chromosomal abnormalities, and 8 new abnormal human karyotypes were found. Among 329 children with abnormal karyotypes, 317 (96.4%) had an abnormal number of chromosomes, and 12 (3.6%) had abnormal chromosomal structure. Abnormal karyotypes were clinically manifested by Down's syndrome (74.5%), growth retardation (10.9%), and mental retardation (3.0%). CONCLUSIONS: Eight rare abnormal karyotypes were found in the study, providing new resources for the genetic studies and etiological analysis of growth retardation, mental retardation, gonadal dysgenesis, and multiple congenital anomalies in children.

Efficacy and safety of leuprorelin 3-month depot (11.25 mg) for idiopathic central precocious puberty treatment of Chinese girls: a single-center retrospective study.

OBJECTIVES: This study aimed to evaluate the efficacy and safety of 3-month leuprorelin acetate (3-month LA, 11.25 mg) for the treatment of idiopathic central precocious puberty (ICPP) in Chinese girls. METHODS: We conducted a single-center retrospective study in China on 28 girls with ICPP who received at least one year of 3-month LA treatment. Data from anthropometry, biochemistry, bone age (BA), and pelvic ultrasonography were assessed before and every 6 months during medication. RESULTS: At CPP diagnosis, the mean chronological age (CA) was 7.8±0.8 years, with bone age advancement (BA-CA) of 1.5±0.8 years. After treatment initiation, growth velocity decreased significantly from 8.5±1.6 cm/year to 5.8±1.1 cm/year at month 12 (p<0.001). GnRH-stimulated peak LH ≤3IU/L, the primary efficacy criterion, was observed in 27 out of 28 (96.4 %) children at month 3. Basal estradiol <20 pg/mL was achieved by all 28 girls (100 %) at month 6 and remained stable at month 12. Basal follicle-stimulating hormone (FSH) decreased from 4.1±3.5 to 1.7±0.9 (p<0.001), and basal LH was also significantly reduced from 3.3±6.5 to 0.7±0.8 (p=0.035) at month 12. The mean predicted adult height (PAH) at treatment initiation was 152.7±5.8 cm, it increased significantly to 157.5±5.5 cm (p=0.007) after one-year treatment. Pubertal development was slowed in most patients, and in some cases, it was even reversed. Only one patient (3.6 %) reported local intolerance. CONCLUSIONS: Three-month leuprorelin acetate is a safe and effective treatment for suppressing the pituitary-gonadal axis and restoring impaired adult height in Chinese girls.

Automated extraction and classification of time-frequency contours in humpback vocalizations.

A time-frequency contour extraction and classification algorithm was created to analyze humpback whale vocalizations. The algorithm automatically extracted contours of whale vocalization units by searching for gray-level discontinuities in the spectrogram images. The unit-to-unit similarity was quantified by cross-correlating the contour lines. A library of distinctive humpback units was then generated by applying an unsupervised, cluster-based learning algorithm. The purpose of this study was to provide a fast and automated feature selection tool to describe the vocal signatures of animal groups. This approach could benefit a variety of applications such as species description, identification, and evolution of song structures. The algorithm was tested on humpback whale song data recorded at various locations in Hawaii from 2002 to 2003. Results presented in this paper showed low probability of false alarm (0%-4%) under noisy environments with small boat vessels and snapping shrimp. The classification algorithm was tested on a controlled set of 30 units forming six unit types, and all the units were correctly classified. In a case study on humpback data collected in the Auau Chanel, Hawaii, in 2002, the algorithm extracted 951 units, which were classified into 12 distinctive types.

[The effect of OPTC-shRNA on the bovine retinal pigment epithelial cells and hyalocytes co-culture collagen gel contraction system].

OBJECTIVE: To investigate the OPTC-shRNA inhibiting effect on the opticin expression by the bovine hyalocytes and retina pigment epithelial (RPE) cells co-culture collagen gel contraction system. METHODS: Experimental study. The OPTC-shRNA expression vector was designed and transfected into bovine RPE cells cultured in vitro. The relative expression and the inhibition rate of the opticin protein were measured by Western blot on days 3, 5 and 7. An in vitro cells co-culture bovine type I collagen gel contraction assay was constructed consisting of the hyalocytes and RPE cells. Six groups were established in this experiments:OPTC-shRNA plasmid transfected RPE cells and hyalocytes (group A), empty plasmid transfected RPE cells and hyalocytes (group B), non-transfected RPE cells and hyalocytes (group C), non-transfected RPE cells (group D), only hyalocytes (group E), and no cells (group F). The collagen gel contractile activities of these groups were compared by One-way ANOVA, SNK-q tests and regression analysis;and the influence of the hyalocytes density variance on the collagen gel contraction in groups A, B and C were also analyzed. RESULTS: The OPTC-shRNA expression vector with significant inhibition effect was constructed and transfected into bovine RPE cells successfully. The results of Western blot analysis showed that the inhibitory rates on the opticin expression on days 3, 5 and 7 were (83.91 ± 2.88), (84.71 ± 4.27) and (82.85 ± 2.72)%, respectively. Furthermore, the differences among days 3, 5 and 7 were insignificant (F = 1.15, P > 0.05). On day 3, the gel contraction rates for the sub-groups with various hyalocytes densities (2×10(7), 1×10(8) and 5×10(8)/L) in groups A, B and C were: group A: (23.52 ± 2.08), (56.00 ± 1.02), (61.62 ± 1.73)%; group B: (16.56 ± 2.01), (36.41 ± 1.33), (49.56 ± 1.75)%; group C: (15.75 ± 1.37), (37.45 ± 1.14), (48.45 ± 1.97)%. The gel contraction rates for groups D and E were (12.18 ± 0.95)% and (10.95 ± 0.93)%, respectively; no gel contraction was observed in group F. Pairwise comparisons of the gel contraction rates were performed by SNK-q test among groups A, B and C for various hyalocyte densities. In the 2×10(7)/L cell density group, the differences between groups A and B or C were significant (q = 11.38, 2.72, respectively, P both < 0.05), the differences between B and C were insignificant (q = 1.34, P > 0.05). In the 1×10(8)/L cell density group, the differences between groups A and B or C were significant (q = 8.83, 46.22, respectively, P both < 0.05), the differences between B and C were insignificant (q = 1.34, P > 0.05). In the 5×10(8)/L cell density group, the differences between groups A and B or C were significant (q = 48.83, 46.22, respectively, P both < 0.05), the differences between groups B and C were insignificant (q = 1.74, P > 0.05). Pairwise comparisons of the sub-groups with different hyalocyte densities in groups A, B and C (comparisons of 2×10(7)/L and 1×10(8)/L, 2×10(7)/L and 5×10(8)/L, 2×10(7)/L and 2×10(7)/L, respectively), the differences were all significant (group A:q = -55.97, -65.66, -9.69, respectively; group B: q = -34.53, -57.41, -22.88, respectively; group C: q = -41.94, -63.19, -21.25, P all < 0.05). Furthermore, the regression analysis was performed between the hyalocyte density and the collage gel contraction rates in each group. The results showed that there was a positive correlation between the gel contraction rates of the co-culture collagen gel contraction system and its hyalocyte density (groups A, B, C: r = 0.919, 0.981, 0.937, respectively, P all < 0.05). Pairwise comparison of groups D and E, D and F, E and F by SNK-q test revealed q = 54.87, 49.33, 5.54, respectively, P all < 0.05. CONCLUSION: Opticin is capable of regulating the contraction of bovine hyalocytes and RPE cells co-culture collagen gel.