Effect on growth performance, carcass traits, and myostatin gene expression in Aseel chicken fed varied levels of dietary protein in isocaloric energy diets.

A study was conducted to assess the effect of feeding different crude protein (CP) levels with isocaloric metabolizable energy (ME) diets on growth performance, carcass traits, and myostatin (MSTN) gene expression of Aseel chicken during 0 to 16 weeks of age. A total of two hundred and ten day-old Aseel chickens were randomly allotted to seven dietary treatment groups. Each group had thirty chicks distributed into three replicates of ten chicks in each. Experimental diets were formulated to have varying levels of CP, viz. 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, and 21.5%, with isocaloric energy of 2800 kcal ME/kg diets of mash feed fed to birds in a completely randomized design. Different CP levels had a significant effect (P < 0.05) on the body weight gain (BWG) of Aseel chicken. At the end of 16 weeks of age, the group fed 21% CP gained 223.53 g more than the lowest CP (18.5%)-fed group. The different CP levels did not significantly (P > 0.05) influenced the feed intake of all treatment groups, but numerically highest feed intake was observed in the lowest CP (18.5%)-fed group. However, significant differences in feed efficiency (FE) appeared from the 13th week only with the 21.0% CP-fed group showing the best FE until the 16th week (3.86 to 4.06). The maximum dressing % (70.61) was observed by the 21% CP-fed group. The CP 21% diet down-regulated the MSTN gene expression in breast muscle tissue to 0.07 folds when compared to the diet of CP 20%. The best economical coordinates for maximum performance for Aseel chicken appeared to be CP of 21% and ME of 2800 kcal/kg to achieve the best FE of 3.86 at the earliest age of 13 weeks. In conclusion, 21% CP in an isocaloric diet of 2800 kcal ME/kg, in Aseel chickens, would be optimum to improve the growth performance at maximum in terms of BWG and FE up to 16 weeks of age.

Computational and experimental studies of Metallo organic framework on human epidermal cell line and anticancer potential.

The pentadentate ligand and the precursors were combined to form complexes by green approach. The ligand formation was confirmed by UV-Vis, FT-IR, 1H-NMR, and LC-MS. The optimised stable structure was obtained by molecular simulation studies and the complexes were interpreted by conductivity measurements, UV-Vis, FT-IR, magnetic susceptibility, VSM, and ESR spectral studies. The redox nature of the complexes was investigated by cyclic voltammetry. The cyclic voltammogram shows complexes exhibited single electron transfer from Cu+2/Cu+1. Complexes and penta-dentate ligand were screened for in vitro cytotoxicity by MTT assay method on A431 skin cancer cell line. The ligand structural stability and biological activity were confirmed by theoretical computational studies. The magnetic behaviour showed antiferromagnetic properties at low temperature. The complexes were used as high bar magnets. Similarly, the redox behaviour showed that the complexes could be used in electroplating techniques and sensors. Clinical application revealed that the complexes had effective cytotoxicity. From the data obtained, the complexes were in the form [MLR], where L was the penta-dentate ligand and R = [C6H5COO] & R = [C6H4COO (OH)].

Intragastric balloon as an adjunct to lifestyle programme in severely obese adolescents: impact on biomedical outcomes and skeletal health.

Intragastric Balloons are a temporary, reversible and safer option compared to bariatric surgery to promote significant weight loss, leading to improved metabolic outcomes. However, due to subsequent weight regain, alternative procedures are now preferred in adults. In adolescents, more amenable to lifestyle change, balloons may be an alternative to less reversible procedures. Our aim was to assess the tolerability and efficacy of the intragastric balloon in severely obese adolescents and the impact of associated weight loss on biomedical outcomes (glucose metabolism, blood pressure, lipid profiles) and bone density. A 2-year cohort study of 12 adolescents (BMI >3.5 s.d., Tanner stage >4) following 6 months intragastric balloon placement was carried out. Subjects underwent anthropometry, oral glucose tolerance test, and DEXA scans at 0, 6 and 24 months. The results showed clinically relevant improvements in blood pressure, insulin: glucose metabolism, liver function and sleep apnoea at 6 months. Changes were not sustained at 2 years though some parameters (Diastolic BP, HBA1c, insulin AUC) demonstrated longer-term improvement despite weight regain. Despite weight loss, bone mass accrual showed age appropriate increases. In conclusion, the intragastric balloon was safe, well tolerated and effective in supporting short-term weight loss and clinically relevant improvement in obesity-related complications, which resolved in some individuals. Benefits were not sustained in the majority at 2 years.

A postpartum haemorrhage package with condom uterine balloon tamponade: a prospective multi-centre case series in Kenya, Sierra Leone, Senegal, and Nepal.

OBJECTIVE: To evaluate the effectiveness and safety of an ultra-low-cost uterine balloon tamponade package (ESM-UBT™) for facility-based management of uncontrolled postpartum haemorrhage (PPH) in Kenya, Sierra Leone, Senegal, and Nepal. DESIGN: Prospective multi-centre case series. SETTING: Facilities in resource-scarce areas of Kenya, Sierra Leone, Nepal, and Senegal. POPULATION: Women with uncontrolled postpartum haemorrhage in 307 facilities across the four countries. METHODS: A standardised ESM-UBT package was implemented in 307 facilities over 29 months (1 September 2012 to 1 February 2015). Data were collected via a multi-pronged approach including data card completion, chart reviews, and provider interviews. Beginning in August 2014, women who had previously undergone UBT placement were sought and queried regarding potential complications associated with UBT use. MAIN OUTCOME MEASURES: All-cause survival, survival from PPH, and post-UBT use complications (surgery, hospitalisation, antibiotics for pelvic infection) associated with UBT use. RESULTS: 201 UBTs were placed for uncontrolled vaginal haemorrhage refractory to all other interventions. In all, 38% (71/188) of women were either unconscious or confused at the time of UBT insertion. All-cause survival was 95% (190/201). However, 98% (160/163) of women survived uncontrolled PPH if delivery occurred at an ESM-UBT online facility. One (1/151) potential UBT-associated complication (postpartum endometritis) was identified and two improvised UBTs were placed in women with a ruptured uterus. CONCLUSIONS: These pilot data suggest that the ESM-UBT package is a clinically promising and safe method to arrest uncontrolled postpartum haemorrhage and save women's lives. The UBT was successfully placed by all levels of facility-based providers. Future studies are needed to further evaluate the effectiveness of ESM-UBT in low-resource settings. TWEETABLE ABSTRACT: Evidence for ESM-UBT as a clinically promising and safe method to arrest uncontrolled PPH and save women's lives.

Management of the pulp in primary teeth--an update.

Management of the pulpal tissue in primary teeth is a clinical challenge facing dental practitioners on a regular basis. This article reviews the most common treatments used at the present time in the management of the pulp in deciduous teeth. It gives an overview of treatment options and the indications and contra-indications for the different treatment modalities. The evidence behind the medicaments used, their actions and success rates are discussed. Practical guidelines for choosing to retain or extract deciduous teeth and management of the primary tooth pulp with different clinical presentations are discussed. Areas of future research are highlighted.

From DNA damage to chromosome aberrations: joining the break.

Despite many years of experimental studies on radiation-induced chromosomal aberrations, and the recent progress in elucidating the molecular mechanisms of the DNA damage response, the link between DNA double-strand break repair and its expression as microscopically visible chromosomal rearrangements remains, in many ways, obscure. Some long standing controversies have partially been resolved to the satisfaction of most investigators, including the linearity of the dose-response for DNA double-strand break induction, the necessity of pairwise interaction of radiogenic damaged sites in the formation of exchange aberrations, and the importance of proximity between lesions in misrejoining. However, the contribution of different molecular DNA repair mechanisms (e.g., alternative end-joining pathways) and their impact on the kinetics of aberration formation is still unclear, as is the definition of "complex" radiogenic damaged sites - in either the chemical or spatial sense - which ostensibly lead to chromosome rearrangements. These topics have been recently debated by molecular biologists and cytogeneticists, whose opinions are summarized in this paper.

What mothers know, and want to know, about the complications of general anaesthesia.

BACKGROUND: Informed consent should be sought when performing anaesthesia on pregnant patients. There is no standard for consent for general anaesthesia on the delivery suite. This study was designed to assess post-partum women's awareness of the complications of general anaesthesia and the level of risk at which they felt these risks should be discussed. METHODS: One hundred and fifty parturients from two London hospitals who had undergone uncomplicated vaginal deliveries were asked on the first post-partum day about their knowledge of the potential complications of general anaesthesia for obstetrics. They were also asked about the level of risk at which they would wish to be informed before consenting to a general anaesthetic procedure. RESULTS: The knowledge of the risks of general anaesthesia among the parturients was poor, with awareness, allergy, nausea and vomiting being known by over 50%. Knowledge of difficult intubation and its consequences, dental damage, malignant hyperpyrexia and suxamethonium apnoea was known by less than 30% of the respondents. The level of risk at which mothers felt they should be informed was variable, with 50% wishing to know all risks up to 1 : 1000, and 19% wishing to know risks of greater than 1 : 1,000,000. All known risks were wished by nearly 30% of those questioned. CONCLUSIONS: Anaesthetists must be flexible when providing information to mothers about general anaesthesia and should provide more information to mothers if they wish it.

Impact of biomarkers for endothelial dysfunction and procoagulant state on 10-year cardiovascular risk in Type 2 diabetes.

AIMS: To estimate the coronary heart disease and cardiovascular disease risk associated with novel biomarkers in Type 2 diabetes mellitus. METHODS: We measured baseline peripheral blood concentrations of soluble E-selectin, factor XIIa, thrombin-antithrombin III complex and plasminogen activator inhibitor-1 in 86 patients with Type 2 diabetes free of known coronary heart disease. We used Cox proportional hazard models to estimate multivariable-adjusted hazard ratios associated with biomarker levels for 10-year coronary heart disease risk (n = 33 events) or total cardiovascular disease risk (n = 45 events). RESULTS: At baseline, mean (sd) age was 62 years (7 years); 62 were men; and 43 had microalbuminuria. Soluble E-selectin demonstrated cross-sectional relationships with glucose and factor XIIa was related to plasminogen activator inhibitor-1 and triglycerides (all P < 0.05). Baseline log soluble E-selectin was significantly related to incident coronary heart disease and cardiovascular disease. Hazard ratios (95% CIs) associated with a 1-unit increase in log soluble E-selectin in age- and sex-adjusted models were: coronary heart disease : 4.6 (95% CI 1.9-11.3), P = 0.001; cardiovascular disease: 3.6 (95% CI 1.7-7.4, P = 0.001); and in multivariable-adjusted models were: coronary heart disease: 2.9 (95% CI 1.2-7.1, P = 0.02); cardiovascular disease: 2.3 (95% CI 1.1-4.8), P = 0.02. Factor XIIa was significantly related to incident cardiovascular disease. The hazard ratios associated with a 1-unit increase in factor XIIa in age- and sex-adjusted models was 1.5 (95% CI 1.1-1.9, P = 0.003) and in a multivariable-adjusted model was 1.3 (95% CI 1.0-1.6, P = 0.047). Plasminogen activator inhibitor-1 and thrombin-antithrombin III complex were not related to cardiovascular disease events. CONCLUSIONS: In our study, soluble E-selectin and factor XIIa were significantly related to 10-year incident macrovascular events in patients with Type 2 diabetes. These preliminary findings call for replication in larger studies.

Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells.

Micronuclei (MN) and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMNcyt) assay. The molecular mechanisms leading to these events have been investigated over the past two decades using molecular probes and genetically engineered cells. In this brief review, we summarise the wealth of knowledge currently available that best explains the formation of these important nuclear anomalies that are commonly seen in cancer and are indicative of genome damage events that could increase the risk of developmental and degenerative diseases. MN can originate during anaphase from lagging acentric chromosome or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Malsegregation of whole chromosomes at anaphase may also lead to MN formation as a result of hypomethylation of repeat sequences in centromeric and pericentromeric DNA, defects in kinetochore proteins or assembly, dysfunctional spindle and defective anaphase checkpoint genes. NPB originate from dicentric chromosomes, which may occur due to misrepair of DNA breaks, telomere end fusions, and could also be observed when defective separation of sister chromatids at anaphase occurs due to failure of decatenation. NBUD represent the process of elimination of amplified DNA, DNA repair complexes and possibly excess chromosomes from aneuploid cells.

Effect of ethnicity on the hypnotic and cardiovascular characteristics of propofol induction.

We compared the propofol dose causing loss of verbal response and suppression of bispectral index to 50, between 50 white and 50 black patients, aged 18-65 years. Propofol was administered at 40 mg.kg⁻¹.h⁻¹ and reduced to 8 mg.kg⁻¹.h⁻¹ when bispectral index fell to 50. We recorded heart rate and mean arterial pressure for 15 min in total and calculated, for this period, maximal percentage change from baseline for each. A statistician, blinded to patient ethnicity, found mean (SD) propofol dose for loss of verbal response in white and black patients to be 1.41 (0.37) mg.kg⁻¹ and 1.16 (0.25) mg.kg⁻¹, respectively (p < 0.001). Corresponding figures for maximal percentage change in heart rate were 14.1 (12.6) % and 7.5 (14.0) % (p = 0.015). Other differences were non-significant. The dose of propofol required for loss of verbal response, but not for suppression of bispectral index to 50, is lower in black than in white patients.

Relationship between DNA repair and formation of sister chromatid exchanges and chromatid aberrations under the influence of poly(ADP-ribose) polymerase inhibition by 3-aminobenzamide.

The mechanisms of formation of sister chromatid exchanges (SCEs) and chromosome aberrations following inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide were studied in Chinese hamster ovary cell lines deficient in different repair pathways. The results confirm earlier findings that (a) the 'spontaneous' SCEs are formed due to the incorporated BrdU in the DNA, (b) 'spontaneous' and induced SCEs originate from different mechanisms, and (c) SCEs and chromatid exchanges are formed by different pathways.

DNA repair mechanisms involved in the removal of DBPDE-induced lesions leading to chromosomal alterations in CHO cells.

Polycyclic aromatic hydrocarbons (PAH) such as dibenzo[a,l]pyrene (DBP) are wide-spread environmental pollutants most probably mutagenic and carcinogenic to humans. Detailed data on the cytogenetic effects of anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) in mammalian cells are not available in the literature. The aim of this study is to elucidate the mechanisms involved in the induction of chromosomal aberrations and sister chromatid exchanges (SCEs) by DBPDE in mammalian cells. In order to achieve this a parental (AA8) and different DNA repair-deficient Chinese hamster ovary cell lines such as UV4, UV5, UV61 (nucleotide excision repair, NER), EM9 (base excision repair, BER), irs1SF (homologous recombination repair, HRR) and V3-3 (non-homologous end joining, NHEJ) were used. The most sensitive cell lines for DBPDE-induced chromosome aberrations were EM9 and irs1SF, while EM9 and V3-3 cell lines were the most sensitive in terms of SCEs induction. It can be suggested that the BER pathway plays an important role in the repair of lesions induced by DBPDE, affecting both chromosomal aberrations and SCEs induction. Moreover, the HRR pathway seems to play a role in cellular resistance to DBPDE mainly in terms of chromosomal aberration induction while the NHEJ pathway takes part affecting only the induction of SCEs.

Mauriac syndrome--a modern reality.

We report two cases of adolescent females with poorly controlled diabetes mellitus who were found to have gross hepatomegaly on annual review. With the additional findings of short stature (in one case), delayed puberty and a Cushingoid habitus they were diagnosed with Mauriac syndrome. Within our diabetes service we have incorporated regular abdominal examinations for all children and young people with long standing, poorly controlled diabetes (HbA1c persistently >9.5%). A brief review of the literature is included.

NanoFerrite particle based radioimmunonanoparticles: binding affinity and in vivo pharmacokinetics.

Dextran and PEG-coated iron oxide nanoparticles (NP), when suitably modified to enable conjugation with molecular targeting agents, provide opportunities to target cancer cells. Monoclonal antibodies, scFv, and peptides conjugated to 20 nm NP have been reported to target cancer for imaging and alternating magnetic field (AMF) therapy. The physical characteristics of NPs can affect their in vivo performance. Surface morphology, surface charge density, and particle size are considered important factors that determine pharmacokinetics, toxicity, and biodistribution. New NanoFerrite (NF) particles having improved specific AMF absorption rates and diameters of 30 and 100 nm were studied to evaluate the variation in their in vitro and in vivo characteristics in comparison to the previously studied 20 nm superparamagnetic iron oxide (SPIO) NP. SPIO NP 20 nm and NF NP 30 and 100 nm were conjugated to (111)In-DOTA-ChL6, a radioimmunoconjugate. Radioimmunoconjugates were conjugated to NPs using 25 microg of RIC/mg of NP by carbodiimide chemistry. The radioimmunonanoparticles (RINP) were purified and characterized by PAGE, cellulose acetate electrophoresis (CAE), live cell binding assays, and pharmacokinetics in athymic mice bearing human breast cancer (HBT 3477) xenografts. RINP (2.2 mg) were injected iv and whole body; blood and tissue data were collected at 4, 24, and 48 h. The preparations used for animal study were >90% monomeric by PAGE and CAE. The immunoreactivity of the RINP was 40-60% compared to (111)In-ChL6. Specific activities of the doses were 20-25 microCi/2.2 mg and 6-11 microg of mAb/2.2 mg of NP. Mean tumor uptakes (% ID/g +/- SD) of each SPIO 20 nm, NF 30 nm, and 100 nm RINP at 48 h were 9.00 +/- 0.8 (20 nm), 3.0 +/- 0.3 (30 nm), and 4.5 +/- 0.8 (100 nm), respectively; the ranges of tissue uptakes were liver (16-32 +/- 1-8), kidney (7.0-15 +/- 1), spleen (8-17 +/- 3-8), lymph nodes 5-6 +/- 1-2), and lung (2.0-4 +/- 0.1-2). In conclusion, this study demonstrated that 100 nm NF NP could be conjugated to (111)In-mAb so that the resulting RINP had characteristics suitable for AMF therapy. Although 100 nm RINP targeted tumor less than 20 nm SPIO RINP, their heating capacity is typically 6 times greater, suggesting the 100 nm NF RINP could still deliver better therapy with AMF.

Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes.

We have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5' part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3' part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

Influence of green tea on enzymes of carbohydrate metabolism, antioxidant defense, and plasma membrane in rat tissues.

OBJECTIVE: Green tea, consumed worldwide since ancient times, is considered beneficial to human health. We hypothesized that green tea would enhance antioxidant defenses and specific metabolic activities of rat intestine, liver, and kidney to improve their functions. METHODS: The effect of green tea given to rats in the diet or drinking water for 25 d was determined on blood chemistry and on activities of enzymes of carbohydrate metabolism, brush border membrane, and antioxidant defense. RESULTS: Serum glucose, cholesterol, phosphate, and body weight decreased, whereas the activities of lactate and malate dehydrogenases and glucose-6- and fructose 1,6-bis-phosphatases increased in the intestine and kidney but slightly changed in the liver. Activity of glucose-6-phosphate dehydrogenase profoundly increased in the renal cortex but decreased in other tissues. Lipid peroxidation increased in the intestine and renal medulla and decreased in the renal cortex and liver; catalase increased in all tissues but the medulla. Superoxide dismutase activity decreased in the intestine but increased in renal tissues. Activities of brush border membrane enzymes in general increased in the intestine and kidney. CONCLUSION: Green tea consumption resulted in enhanced enzyme activities of carbohydrate metabolism and antioxidant defenses, which may lead to improved health.

Mutation Research: the origin.

This brief commentary reflects on the founding of the journal Mutation Research. It includes a photograph of the participants in the scientific conference held in 1962 at the University of Leiden, The Netherlands, at which Professor F.H. Sobels proposed the establishment of the Journal.

Impact of structurally modifying hyaluronic acid on CD44 interaction.

CD44 is a widely-distributed type I transmembrane glycoprotein that binds hyaluronic acid (HA) in most cell types, including primary tumor cells and cancer-initiating cells and has roles in cell migration, cell-cell, and cell-matrix adhesion. HA-derived conjugates and nanoparticles that target the CD44 receptor on cells have been reported for targeted delivery of therapeutics and imaging agents. Altering crucial interactions of HA with CD44 active sites holds significant importance in modulating targeting ability of hyaluronic acid to other cancer types that do not express the CD44 receptor or minimizing the interaction with CD44+ cells that are not target cells. The approach adopted here was deacetylation of the N-acetyl group and selective sulfation on the C6-OH on the HA polymer, which form critical interactions with the CD44 active site. Major interactions identified by molecular modeling were confirmed to be hydrogen bonding of the C6-OH with Tyr109 and hydrophobic interaction of the N-acetyl group with Tyr46, 83 and Ile 92. Modified HA was synthesized and characterized and its interactions were assessed by in vitro and molecular modeling approaches. In vitro techniques included flow cytometry and fluorescence polarization, while in silico approaches included docking and binding calculations by a MM-PBSA approach. These studies indicated that while both deacetylation and sulfation of HA individually decrease CD44 interaction, both chemical modifications are required to minimize interaction with CD44+ cells. The results of this study represent the first step to effective retargeting of HA-derived NPs for imaging and drug delivery.

Microelectrode array recordings of cardiac action potentials as a high throughput method to evaluate pesticide toxicity.

The threat of environmental pollution, biological warfare agent dissemination and new diseases in recent decades has increased research into cell-based biosensors. The creation of this class of sensors could specifically aid the detection of toxic chemicals and their effects in the environment, such as pyrethroid pesticides. Pyrethroids are synthetic pesticides that have been used increasingly over the last decade to replace other pesticides like DDT. In this study we used a high-throughput method to detect pyrethroids by using multielectrode extracellular recordings from cardiac cells. The data from this cell-electrode hybrid system was compared to published results obtained with patch-clamp electrophysiology and also used as an alternative method to further understand pyrethroid effects. Our biosensor consisted of a confluent monolayer of cardiac myocytes cultured on microelectrode arrays (MEA) composed of 60 substrate-integrated electrodes. Spontaneous activity of these beating cells produced extracellular field potentials in the range of 100 microV to nearly 1200 microV with a beating frequency of 0.5-4 Hz. All of the tested pyrethroids; alpha-Cypermethrin, Tetramethrin and Tefluthrin, produced similar changes in the electrophysiological properties of the cardiac myocytes, namely reduced beating frequency and amplitude. The sensitivity of our toxin detection method was comparable to earlier patch-clamp studies, which indicates that, in specific applications, high-throughput extracellular methods can replace single-cell studies. Moreover, the similar effect of all three pyrethroids on the measured parameters suggests, that not only detection of the toxins but, their classification might also be possible with this method. Overall our results support the idea that whole cell biosensors might be viable alternatives when compared to current toxin detection methods.

Chromosomal locations of satellite DNA in the MSWBS ascites tumor cell line.

In situ hybridization of mouse satellite complementary RNA to the chromosomes of the mouse ascites tumor line MSWBS revealed that the distribution of satellite DNA sequences paralleled the location of constitutive heterochromatin as defined by the C-banding technique.