New insights into sex chromosome evolution in anole lizards (Reptilia, Dactyloidae).

Anoles are a clade of iguanian lizards that underwent an extensive radiation between 125 and 65 million years ago. Their karyotypes show wide variation in diploid number spanning from 26 (Anolis evermanni) to 44 (A. insolitus). This chromosomal variation involves their sex chromosomes, ranging from simple systems (XX/XY), with heterochromosomes represented by either micro- or macrochromosomes, to multiple systems (X1X1X2X2/X1X2Y). Here, for the first time, the homology relationships of sex chromosomes have been investigated in nine anole lizards at the whole chromosome level. Cross-species chromosome painting using sex chromosome paints from A. carolinensis, Ctenonotus pogus and Norops sagrei and gene mapping of X-linked genes demonstrated that the anole ancestral sex chromosome system constituted by microchromosomes is retained in all the species with the ancestral karyotype (2n = 36, 12 macro- and 24 microchromosomes). On the contrary, species with a derived karyotype, namely those belonging to genera Ctenonotus and Norops, show a series of rearrangements (fusions/fissions) involving autosomes/microchromosomes that led to the formation of their current sex chromosome systems. These results demonstrate that different autosomes were involved in translocations with sex chromosomes in closely related lineages of anole lizards and that several sequential microautosome/sex chromosome fusions lead to a remarkable increase in size of Norops sagrei sex chromosomes.

Tracking chromosome evolution in southern African gerbils using flow-sorted chromosome paints.

Desmodillus and Gerbilliscus (formerly Tatera) comprise a monophyletic group of gerbils (subfamily Gerbillinae) which last shared an ancestor approximately 8 million years ago; diploid chromosome number variation among the species ranges from 2n = 36 to 2n = 50. In an attempt to shed more light on chromosome evolution and speciation in these rodents, we compared the karyotypes of 7 species, representing 3 genera, based on homology data revealed by chromosome painting with probes derived from flow-sorted chromosomes of the hairy footed gerbil, Gerbillurus paeba (2n = 36). The fluorescent in situ hybridization data revealed remarkable genome conservation: these species share a high proportion of conserved chromosomes, and differences are due to 10 Robertsonian (Rb) rearrangements (3 autapomorphies, 3 synapomorphies and 4 hemiplasies/homoplasies). Our data suggest that chromosome evolution in Desmodillus occurred at a rate of ~1.25 rearrangements per million years (Myr), and that the rate among Gerbilliscus over a time period spanning 8 Myr is also ~1.25 rearrangements/Myr. The recently diverged Gerbillurus (G. tytonis and G. paeba) share an identical karyotype, while Gerbilliscus kempi, G. afra and G. leucogaster differ by 6 Rb rearrangements (a rate of ~1 rearrangement/Myr). Thus, our data suggests a very slow rate of chromosomal evolution in Southern African gerbils.

Management of chronic kidney disease in the elderly.

Both chronic kidney disease (CKD) and end-stage renal disease are strongly age related. Although the morbidity and mortality of CKD have significantly improved in recent years because of a greater understanding of its pathophysiology and evidence-based approach to management, the application of this evidence to the elderly CKD patients is often fraught with difficulty. This is because, besides age, the clinical and biological variables that are widely prevalent in the elderly, such as multiple co-morbidities, functional impairments and polypharmacy, and quality of life and functional outcome measures, which are pertinent to this age group, have generally not been incorporated into the available evidence. This paper reviews the current evidence with a view to providing a framework for diagnosing and managing CKD in the elderly. Special references are made to age-related physiological changes in the renal system, assessment of renal function, and management of metabolic complications and end-stage renal disease.

Molecular cytogenetic characterization of the genome organization of the 6-banded armadillo (Euphractus sexcinctus).

Xenarthra, as the probable earliest offshoot of the placental tree, represents a key taxon for understanding mammalian phylogeny. To gain further insight into the chromosomal evolution and genome organization of the xenarthrans, we have established the first genome-wide comparative chromosome map between human and the 6-banded armadillo (Euphractussexcinctus, 2n = 58), a basal species on the Xenarthra branch, by reciprocal cross-species chromosome painting. In total, 22 human autosomal paints revealed 41 homologous segments in the euchromatic genome of E. sexcinctus. Our results provide further support for the notion that the 2 human homologous segmental associations, i.e. HSA 2/8 and 7a/10p, could constitute the synapomorphies that unite the xenarthrans. Moreover, we propose that the putative ancestral Xenarthra karyotype closely resemble the 2n = 54 karyotype of the E. sexcinctus, consisting of the equivalents of HSA1p, 1q, 2a, 2b, 2c/8c, 3/21, 4a, 4b/8b, 5, 6a, 6b, 7a/10p, 7b/16p, 8a, 9, 10q, 11, 12a/22a, 12b/22b, 13, 14/15, 16q/19q, 17, 18, 19p, 20, and X. In addition, we have analysed the C-banding patterns of E. sexcinctus, and cloned, FISHmapped and sequenced 7 novel repetitive DNA segments, providing further information on the complexity of genome architecture of E. sexcinctus.

Development of quantitative-fluorescence polymerase chain reaction for the rapid prenatal diagnosis of common chromosomal aneuploidies in 1,000 samples in Singapore.

INTRODUCTION: We aimed to develop a rapid quantitative-fluorescence polymerase chain reaction (QF-PCR) to detect common foetal aneuploidies in the Singapore population within 48 hours of sample collection in order to alleviate parental anxiety. METHODS: DNA from 1,000 foetal samples (978 amniotic fluids, 14 chorion villi and eight foetal blood samples) was analysed using a QF-PCR of 19 microsatellite markers located on chromosomes 13, 18, 21, X and Y. A total of 523 samples were archived before the QF-PCR analysis (archived), while QF-PCR was performed and the results obtained within 48 hours of sample collection in the remaining 477 samples (live). The results were confirmed with their respective karyotypes. RESULTS: In total, 47 autosomal trisomies (T) were found: 30 among the archived (three T13, 12 T18, 15 T21) and 17 among the live (four T18, 13 T21) samples. The QF-PCR results were verified with their respective karyotypes. We achieved 100 percent sensitivity (lower 95 percent confidence interval [CI], 92.8 percent) and specificity (lower 95 percent CI, 99.5 percent), and the time taken from sample collection to the obtaining of results for the 477 live samples was less than 48 hours. CONCLUSION: Prenatal diagnostic results of common chromosomal abnormalities can be released within 48 hours of sample collection using QF-PCR. Parental anxiety is alleviated and clinical management is enhanced with this short waiting time.

Array painting reveals a high frequency of balanced translocations in breast cancer cell lines that break in cancer-relevant genes.

Chromosome translocations in the common epithelial cancers are abundant, yet little is known about them. They have been thought to be almost all unbalanced and therefore dismissed as mostly mediating tumour suppressor loss. We present a comprehensive analysis by array painting of the chromosome translocations of breast cancer cell lines HCC1806, HCC1187 and ZR-75-30. In array painting, chromosomes are isolated by flow cytometry, amplified and hybridized to DNA microarrays. A total of 200 breakpoints were identified and all were mapped to 1 Mb resolution on bacterial artificial chromosome (BAC) arrays, then 40 selected breakpoints, including all balanced breakpoints, were further mapped on tiling-path BAC arrays or to around 2 kb resolution using oligonucleotide arrays. Many more of the translocations were balanced at 1 Mb resolution than expected, either reciprocal (eight in total) or balanced for at least one participating chromosome (19 paired breakpoints). Second, many of the breakpoints were at genes that are plausible targets of oncogenic translocation, including balanced breaks at CTCF, EP300/p300 and FOXP4. Two gene fusions were demonstrated, TAX1BP1-AHCY and RIF1-PKD1L1. Our results support the idea that chromosome rearrangements may play an important role in common epithelial cancers such as breast cancer.

Refining quantitative fluorescent polymerase chain reaction for prenatal detection of X chromosomal anomalies in the major Southeast Asian populations.

INTRODUCTION: This study aimed to refine the current quantitative fluorescent polymerase chain reaction (QF-PCR) screen to detect X chromosome anomalies for prenatal diagnosis in the major Southeast-Asian populations. METHODS: 100 amniotic fluid samples from Chinese, Malay and Indian origins were subjected to QF-PCR using the X chromosome markers, HPRT, X22 and AMXY, along with the autosomal marker D21S1411. RESULTS: Out of the 100 samples tested by markers X22 and HPRT, eight samples were homozygous for both markers, of which seven were resolved by comparison with the autosomal marker D21S1411. CONCLUSION: 99 percent of samples could be tested for X chromosome copy numbers, increasing the stringency for detection of X chromosome anomalies by QF-PCR. All results were confirmed by cytogenetics.

Ultra-high resolution array painting facilitates breakpoint sequencing.

OBJECTIVE: To describe a considerably advanced method of array painting, which allows the rapid, ultra-high resolution mapping of translocation breakpoints such that rearrangement junction fragments can be amplified directly and sequenced. METHOD: Ultra-high resolution array painting involves the hybridisation of probes generated by the amplification of small numbers of flow-sorted derivative chromosomes to oligonucleotide arrays designed to tile breakpoint regions at extremely high resolution. RESULTS AND DISCUSSION: How ultra-high resolution array painting of four balanced translocation cases rapidly and efficiently maps breakpoints to a point where junction fragments can be amplified easily and sequenced is demonstrated. With this new development, breakpoints can be mapped using just two array experiments: the first using whole-genome array painting to tiling resolution large insert clone arrays, the second using ultra-high-resolution oligonucleotide arrays targeted to the breakpoint regions. In this way, breakpoints can be mapped and then sequenced in a few weeks.

Prenatal detection of unbalanced chromosomal rearrangements by array CGH.

BACKGROUND: Karyotype analysis has been the standard method for prenatal cytogenetic diagnosis since the 1970s. Although highly reliable, the major limitation remains the requirement for cell culture, resulting in a delay of as much as 14 days to obtaining test results. Fluorescent in situ hybridisation (FISH) and quantitative fluorescent PCR (QF-PCR) rapidly detect common chromosomal abnormalities but do not provide a genome wide screen for unexpected imbalances. Array comparative genomic hybridisation (CGH) has the potential to combine the speed of DNA analysis with a large capacity to scan for genomic abnormalities. We have developed a genomic microarray of approximately 600 large insert clones designed to detect aneuploidy, known microdeletion syndromes, and large unbalanced chromosomal rearrangements. METHODS: This array was tested alongside an array with an approximate resolution of 1 Mb in a blind study of 30 cultured prenatal and postnatal samples with microscopically confirmed unbalanced rearrangements. RESULTS: At 1 Mb resolution, 22/30 rearrangements were identified, whereas 29/30 aberrations were detected using the custom designed array, owing to the inclusion of specifically chosen clones to give increased resolution at genomic loci clinically implicated in known microdeletion syndromes. Both arrays failed to identify a triploid karyotype. Thirty normal control samples produced no false positive results. CONCLUSIONS: Analysis of 30 uncultured prenatal samples showed that array CGH is capable of detecting aneuploidy in DNA isolated from as little as 1 ml of uncultured amniotic fluid; 29/30 samples were correctly diagnosed, the exception being another case of triploidy. These studies demonstrate the potential for array CGH to replace conventional cytogenetics in the great majority of prenatal diagnosis cases.

The RASSF1A isoform of RASSF1 promotes microtubule stability and suppresses tumorigenesis.

The RASSF1A isoform of RASSF1 is frequently inactivated by epigenetic alterations in human cancers, but it remains unclear if and how it acts as a tumor suppressor. RASSF1A overexpression reduces in vitro colony formation and the tumorigenicity of cancer cell lines in vivo. Conversely, RASSF1A knockdown causes multiple mitotic defects that may promote genomic instability. Here, we have used a genetic approach to address the function of RASSF1A as a tumor suppressor in vivo by targeted deletion of Rassf1A in the mouse. Rassf1A null mice were viable and fertile and displayed no pathological abnormalities. Rassf1A null embryonic fibroblasts displayed an increased sensitivity to microtubule depolymerizing agents. No overtly altered cell cycle parameters or aberrations in centrosome number were detected in Rassf1A null fibroblasts. Rassf1A null fibroblasts did not show increased sensitivity to microtubule poisons or DNA-damaging agents and showed no evidence of gross genomic instability, suggesting that cellular responses to genotoxins were unaffected. Rassf1A null mice showed an increased incidence of spontaneous tumorigenesis and decreased survival rate compared with wild-type mice. Irradiated Rassf1A null mice also showed increased tumor susceptibility, particularly to tumors associated with the gastrointestinal tract, compared with wild-type mice. Thus, our results demonstrate that Rassf1A acts as a tumor suppressor gene.

Corrosion behaviour of high copper dental amalgams.

This study evaluated the corrosion behaviour of two high copper dental amalgam alloys [Dispersalloy (Dentsply-Caulk) and Tytin (Kerr)] in different electrolytes. Amalgam specimens were prepared, coupled to a copper wire, cemented into glass tubes and polished to a 600-grit finish. A corrosion cell was prepared using a carbon counter-electrode, a standard calomel electrode as the reference and amalgam as the working electrode. The alloys were tested in the following mediums at 37 degrees C: (i) artificial saliva based on Fusayama's solution (FS), (ii) artificial saliva with citric acid adjusted to pH 4.0 (FC) and (iii) 1% sodium chloride solution (SC). Corrosion potentials (E(corr)) and corrosion rates (I(corr)) were determined using potentiostatic and impedance spectroscopy methods. Data was subjected to anova/Scheffe's post hoc test at 0.05 significance level. For both alloys, the corrosion potential in FS was significantly greater than in SC. Corrosion potential of Tytin in FS and SC was also significantly greater than in FC. The corrosion rate of Dispersalloy in FC was significantly greater than in FS and SC. For Tytin, corrosion rate in SC was significantly greater than in FS and FC. Although no significant difference in corrosion potential/rate was observed between the alloys when tested in FS, significant differences were observed when electrochemical testing was carried out in FC and SC. The corrosion behaviour of high copper amalgam alloys are both material and environment dependent. Certain food substances may increase the corrosion of high copper amalgams.

Applications of combined DNA microarray and chromosome sorting technologies.

The sequencing of the human genome has led to the availability of an extensive mapped clone resource that is ideal for the construction of DNA microarrays. These genomic clone microarrays have largely been used for comparative genomic hybridisation studies of tumours to enable accurate measurement of copy number changes (array-CGH) at increased resolution. We have utilised these microarrays as the target for chromosome painting and reverse chromosome painting to provide a similar improvement in analysis resolution for these studies in a process we have termed array painting. In array painting, chromosomes are flow sorted, fluorescently labelled and hybridised to the microarray. The complete composition and the breakpoints of aberrant chromosomes can be analysed at high resolution in this way with a considerable reduction in time, effort and cytogenetic expertise required for conventional analysis using fluorescence in situ hybridisation. In a similar way, the resolution of cross-species chromosome painting can be improved and we present preliminary observations of the organisation of homologous DNA blocks between the white cheeked gibbon chromosome 14 and human chromosomes 2 and 17.

The Alvarado score and acute appendicitis.

INTRODUCTION: This is a retrospective study to assess the accuracy of the Alvarado score in predicting appendicitis for patients with right iliac fossa pain. MATERIALS AND METHODS: This is a retrospective study of consecutive patients with suspected appendicitis. The Alvarado score was computed from the admission notes and correlated with the final outcome of the patient. Patients discharged without surgery were reviewed in the outpatient's clinic to ascertain that they did not need surgery. RESULTS: There were 148 patients in the study and 63 had appendectomies with intention to treat appendicitis. The normal appendicectomy rate was 21%. The number of patients with score 1-4, 5-6, 7-8 and 9-10 were 46, 40, 44 and 18, respectively, while the number of appendicitis in each group were 0, 2 (5%), 30 (68%) and 18 (100%), respectively. The positive and negative predictive values of Alvarado's scores of 7 or more were 77% and 97.6%, respectively. CONCLUSION: Alvarado score is a useful tool in the diagnosis of appendicitis, especially at both ends of the scale.

Helicobacter pylori in familial clusters based on antibody profile.

Studies have shown a high prevalence of Helicobacter pylori infection in close communities and that intrafamilial spread during early childhood may be a route of transmission. A total of 72 household members from 21 families were enrolled in this study. Sera from individuals showed 50/72 (69.4%) seropositive for IgG against H. pylori by ELISA. Western blots showed diversity in the protein profiles with molecular masses ranging from approximately 8 to 130 kDa. Cohen's kappa statistical analysis of the blot patterns showed that nine families demonstrated similar profiles (100%), while 4 other families showed varying similarities (17-50%). The results support the hypothesis of intrafamilial transmission of H. pylori. Furthermore, serological studies can be used as an effective approach to determine the familial status in relation to H. pylori infection.

Macrophages/microglia as 'sensors' of injury in the pineal gland of rats following a non-penetrative blast.

The pineal gland of adult rats was examined immunohistochemically and electron microscopically following exposure of the animals to a single blast equivalent to 110 kg TNT explosive. The most dramatic feature in rats killed at 7, 14 and 21 days after the blast was the upsurge of a large number of macrophages/microglia intensely immunostained with OX-42, OX-18, OX-6 and ED1 antibodies. These antibodies recognise the complement type three (CR3) receptors, major histocompatibility complex class I and class II (MHC I and MHC II) antigens and monocyte/macrophage antigens. Cell counts in OX-42 immunostained sections showed a two-fold increase at these intervals but returned to normal values at 28 days. The immunolabelled cells appeared extremely hypertrophic after the blast when compared with those in normal rats. In the latter and in rats killed at 28 days after the blast, immunoreactive cells were sparsely distributed. Ultrastructural study confirmed a wider occurrence of perivascular macrophages/microglia after the blast and the cells were laden with massive amounts of phagosomes resembling degenerating pinealocyte processes. It is concluded that the seemingly quiescent macrophages/microglia present normally in pineal gland were activated by the external blast force. The induced changes including the increase in cell numbers and endocytosis, however, were reversible in longer surviving animals.

Ultrastructural changes of macroglial cells in the rat brain following an exposure to a non-penetrative blast.

The present study examined the ultrastructural changes in astrocytes and oligodendrocytes in rats following an exposure to a non-penetrative blast. At 1 and 7 days after the blast, the astrocytes in the cerebral and cerebellar cortex were hypertrophied; their end-feet associated with the blood vessels were also swollen, bearing sparsely distributed organelles. The above changes were not observed in experimental rats when the survival interval was prolonged. It is concluded from this study that the blast could have disrupted the integrity of the blood-brain barrier resulting in possible abnormal entry of serum-derived substances thereby leading to astrocytic hypertrophy. The reversible nature of the changes is evidenced by the seemingly normal appearance of astrocytes in rats killed at 14, 21 and 28 days after the blast. Oligodendrocytes remained unaffected at various time intervals after the blast.

Studies of the choroid plexus and its associated epiplexus cells in the lateral ventricles of rats following an exposure to a single non-penetrative blast.

The choroid plexus in rats exhibited ultrastructural changes following a non-penetrative blast. The immunophenotypic features of epiplexus cells associated with the choroid plexus epithelium were also altered. In rats killed at 1 and 7 days after the blast, the intercellular spaces between the epithelial cells were greatly widened, coupled with the massive eruption and possible extrusion of the apical cytoplasm into the ventricular lumen. Associated with these changes was the passage of some monocytes/lymphocytes across the epithelium. The incidence of such a migratory phenomenon was more frequent in rats killed 7 days after the blast. In rats killed 14 days after the blast, the ultrastructural changes of the epithelial cells became less pronounced. At 21 and 28 days after the blast, the ultrastructure of the choroid plexus was comparable to that of normal specimens. The immunoreactivity of epiplexus cells in terms of their cell number and staining intensity with the monoclonal antibodies OX-42, OX-18, OX-6 and ED1 was noticeably augmented at 7 and 14 days after the blast; this, however subsided at 21 and 28 days. It is concluded that the choroid plexus is extremely sensitive to a blast wave as manifested by its structural alterations and the vigorous expression of CR3 receptors and MHC antigens by the epiplexus cells. It is suggested that a possible immune response might have been triggered in the cerebrospinal fluid ventricular system following the blast.

The response of neurons and microglia to blast injury in the rat brain.

Rats subjected to a single non-penetrative blast were examined for possible neuronal damage and glial reaction by immunohistochemistry and electron microscopy. The most dramatic feature in rats killed between 1 and 14 days after the blast was the widespread response of microglial cells in various parts of the brain in which the cells were hypertrophied and their surface antigens, like complement type three receptors (CR3), were upregulated. The blast wave also induced the vigorous expression of major histocompatibility complex (MHC) class I and II (Ia) antigen. In rats killed 21 days after the blast, the elevated immunoreactivity of microglia had subsided and at 28 days both the microglial external morphology and immunoreactivity were comparable to those of normal animals. In rats killed 4-7 days after the blast, the neurons in the cerebral and cerebellar cortex appeared normal except for the occurrence of some 'darkened' dendrites. The incidence of 'darkened' dendrites was most common in rats killed at day 14 but they were absent at 21 and 28 days. Microglial cells were closely associated with some of the 'darkened' dendrites. Results in this study show that a non-penetrative blast in rats provokes a widespread microglial activation suggesting increased endocytosis and immunological responses. However, it remains uncertain whether such a drastic response was a direct activation of the cells by the blast wave or elicited indirectly by some chemical factors released from the damaged brain tissues.