Role and relevance of fish cell lines in advanced in vitro research.

INTRODUCTION: Cell line derived from fish has been established as a promising tool for studying many key issues of aquaculture covering fish growth, disease, reproduction, genetics, and biotechnology. In addition, fish cell lines are very useful in vitro models for toxicological, pathological, and immunological studies. The easier maintenance of fish cell lines in flexible temperature regimes and hypoxic conditions make them preferable in vitro tools over mammalian cell lines. Great excitement has been observed in establishing and characterizing new fish cell lines representing diverse fish species and tissue types. The well-characterized and authenticated cell lines are of utmost essential as these represent cellular functions very similar to in vivo state of an organism otherwise it would affect the reproducibility of scientific research. CONCLUSION: The fish cell lines have exhibited encouraging results in several key aspects of in vitro research in aquaculture including virology, nutrition and metabolism, production of vaccines, and transgenic fish production. The review paper reports the cell lines developed from fish, their characterization, and biobanking along with their potential applications and challenges in in vitro research.

Development of Escherichia coli-based gene expression profiling of sewage sludge leachates.

AIMS: The impact of municipal waste on pathogenic micro-organisms released into the environment is a public health concern. This study aims to evaluate the effects of sewage sludge and antibiotic contaminants on stress response, virulence and antibiotic resistance in a pathogenic Escherichia coli. METHODS AND RESULTS: The effects of sewage sludge leachates on uropathogenic E. coli CFT073 were determined by monitoring the expression of 45 genes associated with antibiotic/metal resistance, stress response and virulence using RT-qPCR. The E. coli gene expression was validated using subinhibitory concentrations of tetracycline and ciprofloxacin. E. coli exposed to sewage sludge or sewage sludge+fly ash leachates altered the expression of five antibiotic and metal resistance, three stress response and two virulence-associated genes. When antibiotics were combined with sludge or sludge+fly ash the antibiotic-associated gene expression was altered. CONCLUSIONS: E. coli treated with two sludge leachates had distinct gene expression patterns that were altered when the sludge leachates were combined with tetracycline, although to a lesser extent with ciprofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: The E. coli multigene expression analysis is a potential new tool for assessing the effects of pollutants on pathogenic microbes in environmental waters for improved risk assessment.

Notch and Cdk5 in Zebrafish Mindbomb Mutant: Co-regulation or Coincidence?

Notch signalling is critical for the development of the nervous system. In the zebrafish mindbomb mutants, disruption of E3 ubiquitin ligase activity inhibits Notch signalling. In these mutant embryos, precocious development of primary neurons leading to depletion of neural progenitor cells results in a neurogenic phenotype characterized by defects in neural patterning and brain development. Cyclin-dependent kinase 5 (Cdk5), a predominant neuronal kinase, is involved in a variety of essential functions of the nervous system. Most recently, mammalian studies on Notch and Cdk5 regulating each other's function have been emerging. The status of Cdk5 in the mindbomb mutant embryos with excessive primary neurons is not known. In situ hybridization of the zebrafish mindbomb mutant embryos uncovered a robust upregulation in Cdk5 expression but with a reduced Cdk5 activity. The implications of these findings in both the mammalian system and zebrafish are discussed in this mini-review to provide a glimpse into the relationship between Notch and Cdk5 that may explain certain neurodevelopmental defects associated with either mutations in ubiquitin ligase or altered expression of Cdk5.

Evaluation of dental implant insertion torque using a manual ratchet.

BACKGROUND: Dental implant insertion torque is crucial for the success of the implant and the prosthesis. This in-vivo study was undertaken to determine the average insertion torque being applied to the dental implant while surgically placing it with a non-calibrated manual ratchet. METHODS: Three dental surgeons placed a total of 45 dental implants (Touareg, ADIN, Afula, Israel) in 42 selected patients. Each surgeon placed 15 implants. Standardised protocols were followed to prepare the site to place the dental implant. Each implant was placed using a manual non-calibrated implant ratchet first. Once the implant was nearly placed, a manual calibrated torque gauge ratchet was used to place the implant in its final position and at that instance, the maximum final torque applied was noted on the torque gauge scale. RESULTS: The mean dental implant insertion torque applied by three surgeons using a non-calibrated manual ratchet was estimated to be 63.26 Ncm with a standard error of 6.80 i.e. (63.26 + 6.8), which was significantly higher than the baseline of 35 Ncm (p < 0.0001). The mean dental implant torque applied by Surgeon 1, 2 and 3, respectively, was 65.93 Ncm, 62.60 Ncm and 62.13 Ncm and this difference amongst them was found to be statistically insignificant (p > 0.05) and each of them had reached more than the baseline level of 35 Ncm individually and significantly (p < 0.0001). CONCLUSION: Without the use of torque measuring devices, an average surgeon may achieve an average insertion torque of 63.26 + 6.8 Ncm.

Radial glial cell: critical functions and new perspective as a steroid synthetic cell.

The radial glial cell (RGC) is a glial cell type in the central nervous system of all vertebrates. Adult teleost fish have abundant RGCs in the brain in contrast to mammals. Adult fish RGCs have many important functions, including forming a structural scaffold to guide neuronal migration and serving as the progenitor cells in the brain to generate neurons. The role of the RGC in adult neurogenesis explains the high regenerative capacity of adult fish brain. There is increasing evidence from several species that some glial cells produce or metabolize steroids. It is now well-known that teleost RGCs express aromatase and produce estrogens from androgen precursors, which may be important for local neuroendocrine functions and regulation of neurogenesis. The question of whether RGCs are capable of de novo steroid synthesis from cholesterol remains unanswered. However, the expression of steroidogenic acute regulatory protein, and the key enzyme cytochrome P450 17alpha-hydroxylase in primary cultures of goldfish RGCs indicate the potential to produce 17α-hydroxy-pregnenolone and thus other steroid intermediates. The possibility of synthesizing additional non-estrogenic steroids may indicate new functions for the RGC.

Genetic basis of dental agenesis--molecular genetics patterning clinical dentistry.

Tooth agenesis is one of the most common congenital malformations in humans. Hypodontia can either occur as an isolated condition (non-syndromic hypodontia) or can be associated with a syndrome (syndromic hypodontia), highlighting the heterogeneity of the condition. Though much progress has been made to identify the developmental basis of tooth formation, knowledge of the etiological basis of inherited tooth loss is still lacking. To date, the mutation spectra of non-syndromic form of familial and sporadic tooth agenesis in humans have revealed defects in various such genes that encode transcription factors, MSX1 and PAX9 or genes that code for a protein involved in canonical Wnt signaling (AXIN2), and a transmembrane receptor of fibroblast growth factors (FGFR1). The aim of this paper is to review the current literature on the molecular mechanisms responsible for selective hypodontia in humans and to present a detailed overview of causative genes and syndromes associated with hypodontia.

Evaluation of shear bond strength of composite resin bonded to alloy treated with sandblasting and electrolytic etching.

Conservation of natural tooth structure precipitated the emergence of resin-retained fixed partial dentures. The weakest link in this modality is the bond between resin cement and alloy of the retainer. Various alloy surface treatment have been recommended to improve alloy-resin bond. This in vitro study was carried out to observe changes in the Nickel-Chromium alloy (Wiron 99, Bego) surface following sandblasting or electrolytic etching treatment by scanning electron microscope (SEM) and to evaluate the shear bond strength of a resin luting cement bonded to the surface treated alloy. 80 alloy blocks were cast and divided into four groups of 20 each. In groups-A & B, the test surfaces were treated by sandblasting with 50 and 250 µm sized aluminium oxide particles respectively. In groups-C & D, the test surfaces were first treated by sandblasting with 50 and 250 µm sized aluminium oxide particles respectively followed by electrolytic etching. Test surfaces were observed under SEM at 1,000× magnification. Two alloy blocks of each group were luted together by a resin luting cement (Rely X, 3M) and their shear bond strength was tested. The mean shear bond strength in MPa of groups-A to D were 6.44 (±0.74), 8.18 (±0.51), 14.45 (±0.59) and 17.43 (±1.20) respectively. Group-D showed bond strength that is more than clinically acceptable bond strength. It is recommended that before luting resin-retained fixed partial dentures, the fitting surface of the retainer should be electrolytically etched to achieve adequate micromechanical retention.

Microwave, infrared-microwave double resonance, and theoretical studies of C2H4···H2S complex.

In this manuscript, rotational spectra of four new isotopologues of the S-H···π bonded C2H4···H2S complex, i.e., C2D4···H2S, C2D4···D2S, C2D4···HDS, and (13)CCH4···H2S have been reported and analyzed. All isotopologues except C2D4···HDS show a four line pattern whereas a doubling of the transition frequencies was observed for C2D4···HDS. These results together with our previous report on the title complex [M. Goswami, P. K. Mandal, D. J. Ramdass, and E. Arunan, Chem. Phys. Lett. 393(1-3), 22-27 (2004)] confirm that both subunits (C2H4 and H2S) are involved in large amplitude motions leading to a splitting of each rotational transition to a quartet. Further, the results also confirm that the motions which are responsible for the observed splittings involve both monomers. Molecular symmetry group analysis, considering the interchange of equivalent H atoms in H2S and C2H4 could explain the observed four line pattern and their intensities in the microwave spectrum. In addition, hydride stretching fundamentals of the complex were measured using coherence-converted population transfer Fourier Transform Microwave-infrared (IR-MW double resonance) experiments in the S-H and C-H stretch regions. Changes in the tunneling splittings upon vibrational excitation are consistent with the isotopic dependence of pure rotational transitions. A complexation shift of 2.7-6.5 cm(-1) has been observed in the two fundamental S-H stretching modes of the H2S monomer in the complex. Vibrational pre-dissociation in the bound S-H stretch has been detected whereas the instrument-limited line-shapes in other S-H and C-H stretches indicate slower pre-dissociation rate. Some local perturbations in the vibrational spectra have been observed. Two combination bands have been observed corresponding to both the S-H stretching fundamentals and what appears to be the intermolecular stretching mode at 55 cm(-1). The tunneling splitting involved in the rotation of C2H4 unit has been deduced to be 1.5 GHz from the IR-MW results. In addition, ab initio barrier heights derived for different motions of the monomers support the experimental results and provide further insight into the motions causing the splitting.

Genetic characterization of two hill stream fish species Barilius bendelisis (Ham.1807) and Barilius barna (Ham.1822) using RAPD markers.

Genetic structure of four wild populations of two hill stream fishes Barilius bendelisis (Ham.1807) and B. barna (Ham. 1822) from Uttarakhand, India, was studied using RAPD markers. Eight selective primers provided distinct and consistent RAPD profiles in both the species, producing a total of 47 and 35 scorable bands in B. bendelisis and B. barna respectively. The bands in the range 666-4,830 bp were scored for consistent results. The RAPD profiles generated by all the eight primers revealed varying degrees of polymorphism (25.00-50.00 %). The average genetic diversity (h) was estimated as 0.1661 and 0.1606 among the four populations of B. bendelisis and B. barna respectively.

Crossed beam reaction of phenyl and D5-phenyl radicals with propene and deuterated counterparts--competing atomic hydrogen and methyl loss pathways.

We conducted the crossed molecular beams reactions of the phenyl and D5-phenyl radicals with propylene together with its partially deuterated reactants at collision energies of ~45 kJ mol(-1) under single collision conditions. The scattering dynamics were found to be indirect and were mainly dictated by an addition of the phenyl radical to the sterically accessible CH(2) unit of the propylene reactant. The resulting doublet radical isomerized to multiple C(9)H(11) intermediates, which were found to be long-lived, decomposing in competing methyl group loss and atomic hydrogen loss pathways with the methyl group loss leading to styrene (C(6)H(5)C(2)H(3)) and the atomic hydrogen loss forming C(9)H(10) isomers cis/trans 1-phenylpropene (CH(3)CHCHC(6)H(5)) and 3-phenylpropene (C(6)H(5)CH(2)C(2)H(3)). Fractions of the methyl versus hydrogen loss channels of 68 ± 16% : 32 ± 10% were derived experimentally, which agrees nicely with RRKM theory. As the collision energy rises to 200 kJmol(-1), the contribution of the methyl loss channel decreases sharply to typically 25%; the decreased importance of the methyl group loss channel was also demonstrated in previous crossed beam experiments conducted at elevated collision energies of 130-193 kJ mol(-1). The presented work highlights the interesting differences of the branching ratios with rising collision energies in the reaction dynamics of phenyl radicals with unsaturated hydrocarbons related to combustion processes. The facility of forming styrene, a common molecule found in combustion against the elusiveness of forming the cyclic indane molecule demonstrates the need to continue to explore the potential surfaces through the combinative single collision experiment and electronic structure calculations.

Development and characterization of a cell line TTCF from endangered mahseer Tor tor (Ham.).

Tor tor is an important game and food fish of India with a distribution throughout Asia from the trans-Himalayan region to the Mekong River basin to Malaysia, Pakistan, Bangladesh and Indonesia. A new cell line named TTCF was developed from the caudal fin of T. tor for the first time. The cell line was optimally maintained at 28°C in Leibovitz-15 (L-15) medium supplemented with 20% fetal bovine serum (FBS). The propagation of TTCF cells showed a high plating efficiency of 63.00%. The cytogenetic analysis revealed a diploid count of 100 chromosomes at passage 15, 30, 45 and 60 passages. The viability of the TTCF cell line was found to be 72% after 6 months of cryopreservation in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 578- and 655-bp sequences of 16S rRNA and cytochrome oxidase subunit I (COI) genes of mitochondrial DNA (mtDNA) respectively. TTCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids. Further, immunocytochemistry studies confirm its fibroblastic morphology of cells. Genotoxicity assessment of H2O2 in TTCF cell line revealed the utility of TTCF cell line as in vitro model for aquatic toxicological studies.

Development of an ES-like cell culture system (RESC) from rohu, Labeo rohita (Ham.).

An embryonic stem (ES)-like cell culture system RESC from a commercially important freshwater carp, Labeo rohita, was developed using blastula stage embryos. The cells were cultured in Leibovitz-15 (L-15) medium in gelatin-coated cell culture flask supplemented with 15 % fetal bovine serum along with 10 ng ml(-1) basic fibroblast growth factor at 28 °C under feeder-free conditions. The ES-like cells were characterized by their unique morphology, alkaline phosphatase activity, embryoid body formation tendency, expression of transcription factor Oct4, and consistent chromosome count. The RESC cells when treated with retinoic acid differentiated into cells of different lineages. The RESC developed from mid-blastula embryos of L. rohita would be a useful tool for cellular differentiation and gene expression studies.

Evaluation of the satisfaction level of patients visiting dental emergency services.

Context: Inadequate data is available of patient satisfaction in dental emergency departments in India. Aim: This study was undertaken with the aim to evaluate the satisfaction level of patients visiting emergency services of a dental institute in an Indian city. Settings and Design: A questionnaire-based cross-sectional exploratory study was designed over a period of 2 months. Subjects and Methods: A total of 51 subjects visiting the dental emergency services after routine working hours participated in this questionnaire-based study and submitted their responses. Statistical Analysis Used: Pearson's Chi-square test. Results: A statistically significant correlation (P < 0.05) was observed between effectiveness of the treatment given in terms of relief from complaints with the experience at reception, rating the hospital in terms of overall waiting time for any service with ambience (P = 0.031), between effectiveness of the treatment given in terms of control/relief from complaints (P = 0.00), 'rating patient's experience with "on-duty doctor" (response time, behavior, appearance, attitude etc.), rating the hospital in terms of overall waiting time for any service (P = 0.010), experience' with nursing staff (responsive, courteous, polite) and rating the hospital in terms of overall waiting time for any service. Conclusions: Emergency care where patients were satisfied included reception, greeting while entering the department, ambience of the hospital and the Emergency Department, and good experience with the on-duty doctor, nursing staff, and security. Waiting time for treatment at emergency care was less. Recommending this hospital to others was statistically significant with the experience of the patient with the staff.

A crossed molecular beams and ab initio study on the formation of C6H3 radicals. an interface between resonantly stabilized and aromatic radicals.

The crossed molecular beams reaction of dicarbon molecules, C(2)(X(1)Σ(g)(+)/a(3)Π(u)) with vinylacetylene was studied under single collision conditions at a collision energy of 31.0 kJ mol(-1) and combined with electronic structure calculations on the singlet and triplet C(6)H(4) potential energy surfaces. The investigations indicate that both reactions on the triplet and singlet surfaces are dictated by a barrierless addition of the dicarbon unit to the vinylacetylene molecule and hence indirect scattering dynamics via long-lived C(6)H(4) complexes. On the singlet surface, ethynylbutatriene and vinyldiacetylene were found to decompose via atomic hydrogen loss involving loose exit transition states to form exclusively the resonantly stabilized 1-hexene-3,4-diynyl-2 radical (C(6)H(3); H(2)CCCCCCH; C(2v)). On the triplet surface, ethynylbutatriene emitted a hydrogen atom through a tight exit transition state located about 20 kJ mol(-1) above the separated stabilized 1-hexene-3,4-diynyl-2 radical plus atomic hydrogen product; to a minor amount (<5%) theory predicts that the aromatic 1,2,3-tridehydrobenzene molecule is formed. Compared to previous crossed beams and theoretical investigations on the formation of aromatic C(6)H(x) (x = 6, 5, 4) molecules benzene, phenyl, and o-benzyne, the decreasing energy difference from benzene via phenyl and o-benzyne between the aromatic and acyclic reaction products, i.e., 253, 218, and 58 kJ mol(-1), is narrowed down to only ∼7 kJ mol(-1) for the C(6)H(3) system (aromatic 1,2,3-tridehydrobenzene versus the resonantly stabilized free radical 1-hexene-3,4-diynyl-2). Therefore, the C(6)H(3) system can be seen as a "transition" stage among the C(6)H(x) (x = 6-1) systems, in which the energy gap between the aromatic isomer (x = 6, 5, 4) is reduced compared to the acyclic isomer as the carbon-to-hydrogen ratio increases and the acyclic isomer becomes more stable (x = 1, 2).

Development and characterization of a continuous cell line PSCF from Puntius sophore.

A continuous Puntius sophore caudal-fin (PSCF) cell line of the pool barb Puntius sophore, an important freshwater food and ornamental fish of Asia and South East Asia, was developed from the caudal fin for the first time. The cell line was optimally maintained at 28° C in Leibovitz-15 (L-15) medium supplemented with 10% foetal bovine serum (FBS). The cytogenetic analysis revealed a diploid count of 50 chromosomes at 25th and 50th passage and 52 chromosomes at passage 70, 85 and 100. The viability of the PSCF cell lines was 75% after 6 months of storage in liquid nitrogen (-196° C). The most striking feature of the PSCF cells was its high increased growth ratio as evident from the population doubling time of 25 h at passage 100. The origin of the cell lines was confirmed by the amplification of 581 and 655 bp fragments of 16 S rRNA and cytochrome oxidase subunit I (COI) of mitochondrial DNA (mtDNA) genes, respectively. The PSCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids and the expression of GFP gene in the cells indicated the potential utility of the cells in gene expression studies.

Genetic characterization of Metapenaeus affinis (H. M. Edwards, 1837) using RAPD markers.

Genetic structure of four populations of Metapenaeus affinis from Maharashtra, Orissa, Kerala and Tamil Nadu in India was studied using RAPD markers. Five selective primers provided distinct and consistent RAPD profiles in all the four populations. The bands in the range 225-1,900 bp were scored for consistent results. The RAPD profiles generated by all the five primers revealed varying degrees of polymorphism, ranging from 25.00% (primer E-03) to 65.00% (primer E-06). Nei's (Nei M, Natl Acad Sci Proc USA 70:3321-3323, 1973) genetic diversity (h) among the four populations varied from 0.2565 ± 0.2146 (Orissa population) to 0.3576 ± 0.1897 (Maharashtra population).

Development and characterization of two new cell lines from common carp, Cyprinus carpio (Linn).

Two new cell lines (CCF and CCH) were established from fin and heart tissues of common carp, Cyprinus carpio. The cells were optimally maintained in Leibovitz-15 medium supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml of basic fibroblastic growth factor (bFGF). The effects of temperature, concentration of FBS and bFGF on the growth of CCF and CCH cells were examined. The temperature ranged from 24 to 32°C for good growth of the cells. The growth rate of cells was higher in medium containing 10% FBS and the addition of bFGF to the medium significantly increased the growth rate. The CCF cells were found to be epithelial, while the CCH cells were fibroblastic in nature. The cytogenetic analysis of the cell lines revealed a diploid number of 100 chromosomes in C. carpio. The viability of CCF and CCH cell lines were 70 and 72%, respectively, after six months of storage in liquid nitrogen (-196° C). Molecular characterization of the cell lines using 16S rRNA and Cytochrome Oxidase Subunit I (COI) revealed the origin of the cell lines. These new cell lines will be useful for isolation of fish viruses and other in vitro biotechnological studies.

Frequency Dependent Charge Transport and Spin State Switching Characteristics of Fe(phen)2(NCS)2 in Polymer.

We report on the bistability in spin states of spin crossover (SCO) compound Fe(phen)2(NCS)2 in polymer (polypyrrole) by frequency (1-100 kHz) and temperature dependent (305-457 K) electrical conductivity measurements. The structure and growth of SCO compounds in conducting polymer are obtained by scanning electron microscopy, X-ray diffraction and optical absorption measurements. The thermal dependence of ac conductivity σ(ω) shows the clear formation of a hysteresis loop in its cooling and heating cycle due to the difference in conductivity in high spin and low spin state. The size, shape and width of the hysteresis loops are found to be critically dependent on the applied frequency and/or the ratio between SCO and polymer. The ac conductivity is found to exhibit a dispersive behavior following Jonscher's law: σ(ω) ∝ ωn below a critical frequency ωc, above which it is found to monotonically decrease with increasing frequency. The thermal dependence of the exponent n and ωc is also explored. The charge transport phenomena are explained in the framework of hopping of charge carriers. The data reveals that addition of polymer can play an important role to tune the conductivity of SCO compounds and its spin state dependence characteristics which may be quite helpful for fabricating future spin-based devices. Temperature dependent magnetic susceptibility measurement also confirms the spin transition behavior of the SCO/ppy composite samples. These SCO/ppy composite samples can be taken as the reliable nanomaterials fabricated with the concept of future spin based nanoarchitectonics.

Molecular identification and phylogenetic relationships of seven Indian Sciaenids (Pisces: Perciformes, Sciaenidae) based on 16S rRNA and cytochrome c oxidase subunit I mitochondrial genes.

The partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) mitochondrial genes were analyzed for species identification and phylogenetic relationships among the commercially important Indian sciaenids (Otolithes cuvieri, Otolithes ruber, Johnius dussumieri, Johnius elongatus, Johnieops vogleri, Otolithoides biauritus and Protonibea diacanthus). Sequence analysis of both genes revealed that the seven species fell into three distinct groups, which were genetically distant from each other and exhibited identical phylogenetic resolution. Partial sequences of both the genes provided sufficient phylogenetic information to distinguish the seven sciaenids indicating the usefulness of mtDNA-based approach in species identification.