A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Δ30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate.A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Δ30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys.Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Δ30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Δ30, designated rDEN2Δ30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Δ30 in SCID-HuH-7 mice.The rDEN2Δ30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for The rDEN2Δ30 and rDEN2Δ30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine. The increased prevalence of disease caused by the mosquito-borne dengue (DEN) viruses  has intensified the effort to generate a vaccine that would both confer protection and be economically feasible for use in countries with limited resources for healthcare . Risk faImmunity to the DEN viruses is primarily mediated by neutralizing antibodies directed against the envelope (E) glycoprotein, and most vaccine strategies aim to induce antibody against this major protective antigen. Live attenuated tetravalent vaccines appear to be the best vaccine candidates since they are economical to manufacture and they induce long-term immunity with the live attenuated yellow fever virus vaccine serving as a successful model flavivirus vaccine . SeveralWe have previously described attenuated and immunogenic monovalent vaccine candidates for DEN-1, DEN-2, DEN-3, and DEN-4 that were generated by two distinct recombinant methodologies. Using the first methodology, nucleotides 10478–10507 were deleted from the 3' UTR (Δ30) of a wild type cDNA clone for DEN-4 to generate a vaccine candidate, rDEN4Δ30, which is safe, attenuated, and immunogenic in rhesus monkeys and humans . IncorpoWe have found it prudent to pursue several strategies to develop a live attenuated virus vaccine for each dengue serotype recognizing that it has been a challenge to achieve a satisfactory balance between attenuation and immunogenicity ,18-20. TVero cells (African green monkey kidney) were propagated in OptiPro SFM  supplemented with 4 mM L-glutamine (Invitrogen). HuH-7 cells (human hepatoma) were maintained in D-MEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1 mM L-glutamine and 0.05 mg/ml gentamicin (Invitrogen). C6/36 cells  were maintained at 32°C in Minimal Essential Medium (MEM) containing Earle's salts and 25 mM HEPES buffer (Invitrogen) and supplemented with 10% FBS, 2 mM L-glutamine, and 0.1 mM non-essential amino acids (Invitrogen).Aedes albopictus mosquitoes, and subsequent passage in C6/36 cells before determination of genomic sequence.A dengue virus type 2 isolate, Tonga/74, was provided by Dr. Duane Gubler . The virus was isolated during a 1974 dengue outbreak in the South Pacific island of Tonga . The virViral RNA was isolated from DEN-2 Tonga/74 wild type virus using the QIAamp Viral RNA mini kit . Reverse transcription was performed using random hexamer primers and the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Overlapping PCR fragments of approximately 2000 base pairs were generated using DEN-2 specific primers and Advantage cDNA polymerase . Both strands of the resulting PCR fragments were sequenced directly on a 3100 Genetic Analyzer  using DEN-2 specific primers in BigDye terminator cycle sequencing reactions (Applied Biosystems) and the results were assembled into a consensus sequence. To determine the nucleotide sequence of the genomic 5' and 3' regions, the 5' cap nucleoside of the viral genome was removed with tobacco acid pyrophosphatase , followed by circularization of the genome using RNA ligase (Epicentre Technologies). An RT-PCR fragment spanning the ligation junction was generated and sequenced using DEN-2 primers. For the DEN-2 Tonga/74 consensus sequence, GenBank accession number AY744147 was assigned.SpeI restriction site at genomic nucleotide 2353; Fragment R was modified to contain a SpeI restriction site also at genomic nucleotide 2353, and, to stabilize the eventual full-length clone, two additional mutations at nucleotides 2362 – 2364 and 2397 were created to ensure that translation stop codons were present in all reading frames other than that used to synthesize the virus polyprotein; Fragment A was modified at nucleotide 3582 to ablate a naturally occurring SpeI restriction site and at nucleotide 4497 to ablate a naturally occurring KpnI restriction site; Fragment C was modified at nucleotide 9374 to ablate a naturally occurring KpnI restriction site; and Fragment Y, representing the 3' end of the genome was abutted to a KpnI restriction site. All mutations introduced into the cloned cDNA fragments were translationally-silent, thereby preserving the wild-type polyprotein sequence. Each fragment was added incrementally between the AscI and KpnI restriction sites of DEN-4 cDNA clone p4 (GenBank accession number: AY648301) to generate a full-length DEN-2 cDNA clone (p2) with the same vector background successfully used to generate rDEN-4 and rDEN4Δ30 virus [cDNA fragments of DEN-2 Tonga/74 were generated by reverse-transcription of the genome as indicated in Figure 30 virus . cDNA clUsing site-directed mutagenesis, an attenuating amino acid change characterized in the NS3 gene of DEN-4  was introduced into the p2Δ30 cDNA clone . A mutagAcc65I (isoschizomer of KpnI which cleaves leaving only a single 3' nucleotide) and were transcribed in vitro using the AmpliCap SP6 Message Maker kit . Purified transcripts were then transfected into Vero or C6/36 cells. Viruses recovered in C6/36 cells were passaged 3 times in Vero cells, and all viruses were biologically cloned by terminal dilution in Vero cells. The genomes of recombinant viruses used to infect rhesus monkeys were completely sequenced as described above to identify adventitious mutations that had accumulated during transfection and biological cloning.cDNA clones were linearized with Prkdcscid)  were injected intraperitoneally with 107 HuH-7 cells suspended in 0.2 ml phosphate-buffered saline. Tumors were detected in the peritoneum, and mice were infected by direct inoculation of the tumor with 104 PFU of virus in 0.05 ml Opti-MEM (Invitrogen). On day 7 post-infection, serum was obtained from cardiac blood and stored at -70°C. Virus titer in serum samples was determined by plaque assay in Vero cells.Four to six week-old SCID mice (Tac:Icr:Ha(ICR)-5 PFU virus diluted in L-15 medium (Invitrogen) or with a mock inoculum. Serum was collected on days 0–6, 8, 10, 12 and 28 after inoculation and stored at -70°C. Virus titer was determined for each serum sample by plaque assay in Vero cells and serum neutralizing antibody titer was determined for serum from days 0 and 28 by plaque reduction neutralization test. On day 28, monkeys were challenged with 105 PFU of DEN-2 Tonga/74, and serum was collected on days 29–34, 36, and 56. Virus titer was determined for serum from days 28–34 and 36 and serum neutralizing antibody titer was determined for serum from day 56.The DEN-2 viruses were evaluated in rhesus macaques using established methods . DEN virAedes aegypti and Toxorynchites amboinensis mosquitoes was evaluated as previously described [A. aegypti were fed blood meals containing serial 10-fold dilutions of virus. After 21 days, viral antigen was detected in head and midgut preparations by immunoflourescence assay using DEN-2-specific hyperimmune mouse ascitic fluid and fluorescein isothyocyanate conjugated goat anti-mouse IgG , and the mosquito infectious dose-50% (MID50) was determined. T. amboinensis were inoculated intrathoracically with a 0.2 ul dose containing serial ten-fold dilutions of virus and incubated for 14 days. Head preparations were made and antigen visualized as described above.Replication in escribed . Briefly10PFU/ml in Vero cells.A full-length cDNA clone, p2, was constructed that matched the genomic consensus sequence of the American genotype DEN-2 isolate, Tonga/74, with the exception of translationally-silent modifications made to facilitate cloning Figure . The pre10PFU/ml serum in SCID-HuH-7 mice, and an att phenotype in SCID-HuH-7 mice has been shown to be a predictor of reduced replication in rhesus monkeys [10PFU/ml (Table P < 0.05), and was similar to that observed for the well-characterized rDEN4Δ30 virus [As an initial evaluation of replication of the DEN-2 Tonga/74 virus and the rDEN-2 viruses, replication in SCID mice transplanted with HuH-7 human hepatoma cells (SCID-HuH-7 mice) was tested. Wild-type viruses from each DEN serotype have been shown to replicate to approximately 6.0 log monkeys ,14,15,17Aedes aegypti fed on an infectious bloodmeal  and for Toxorynchites amboinensis inoculated intrathoracically  were lower than DEN-2 and rDEN-2, the differences were not as dramatic as had been observed for rDEN1Δ30 and rDEN4Δ30 when compared to their parent viruses [The replication (viremia), immunogenicity, and protective efficacy of the DEN-2 viruses in monkeys were studied. Monkeys inoculated with the DEN-2 Tonga/74 wild-type isolate were viremic for an average of 4.5 days with a mean peak titer of 2.1 log viruses ,14. The att mutation that has been characterized in another DEN serotype was imported into a homologous region in DEN-2. One such mutation, the 4995 mutation in DEN-4 NS3 at amino acid 158 (Ser→Leu), was previously incorporated into the DEN-4 vaccine candidate, rDEN4Δ30, and found to further attenuate the virus for SCID-HuH-7 mice and rhesus monkeys [10PFU/ml . The virus titer of rDEN2Δ30-4995 virus in SCID-HuH-7 mice was over 60-fold reduced from that of the rDEN-2 parent virus.Replication in the SCID-HuH-7 mouse model was used as an initial assessment of the rDEN2Δ30-4995 virus phenotype. Table Development of a live-attenuated tetravalent dengue vaccine has been complicated by two major factors. First, monovalent vaccine candidates that exhibit a satisfactory balance between attenuation and immunogenicity have been difficult to identify ,18-20. S10PFU/ml [10PFU/ml. However, the 10-fold reduction of replication of rDEN4Δ30 and rDEN2Δ30 in SCID-HuH-7 mice, compared to that of their respective wild type parents, was similar. To date, rDEN4Δ30 is the only Δ30 vaccine candidate that has been tested in humans, and it was found to be both safe and immunogenic [It was hoped that each of the four components of a tetravalent vaccine, consisting of DEN-1, -2, -3, and -4 wild type viruses, each with the common 30 nucleotide deletion mutation in the 3' UTR, would exhibit a similar level of attenuation in animal models -15. Unfo10PFU/ml ,14, the unogenic . If the To construct a further attenuated derivative of rDEN2Δ30, the 4995 mutation present in the NS3 gene of DEN-4 at amino acid 158 (Ser→Leu) was introduced into the homologous region of the NS3 protein of rDEN2Δ30 . AlthougEpidemiologic and molecular pathogenesis studies of DEN-2 strains support the concept that the DEN-2 Tonga/74 virus, from which the vaccine candidates were derived, may naturally have a lower level of virulence than other DEN-2 viruses. If the DEN-2 Tonga/74 parent virus is naturally attenuated to some degree, only a small incremental increase in attenuation might be required to satisfactorily attenuate it for humans. Gubler et al. investigated the 1974 outbreak of DEN-2 infection in the Pacific island of Tonga . In compSince the Tonga/74 outbreak, additional evidence has emerged that supports the suggestion that there are at least two circulating lineages of DEN-2 viruses that differ in virulence -29. The Aedes mosquitoes in comparison to Asian DEN-2 viruses [Aedes mosquitoes in our laboratory [Aedes aegypti mosquitoes fed an infectious blood meal. Since rDEN-2 and rDEN2Δ30 viruses were infectious by intrathoracic inoculation of Toxorynchites mosquitoes, the lack of infectivity for A. aegypti was likely caused solely by the inability of the DEN-2 Tonga/74 viruses to establish a midgut infection. Decreased infectivity for Aedes mosquitoes could serve to help limit transmission of the vaccine virus.The American genotype DEN-2 viruses exhibit decreased infectivity for  viruses ,25. Consboratory  whereas boratory . Howeverboratory ,27. At t10PFU/ml in Vero cells that would permit economical manufacture. Second, the viruses are derived from the DEN-2 Tonga/74 strain, a member of the American genotype, which has been associated with decreased virulence. Third, rDEN2Δ30 was attenuated for replication in SCID-HuH-7 mice and slightly attenuated for rhesus monkeys while inducing a protective neutralizing antibody response. Fourth, rDEN2Δ30-4995 was more attenuated in SCID-HuH-7 mice than rDEN2Δ30. Fifth, the DEN-2 Tonga/74 strain, like other members of the American genotype, is poorly infectious for Aedes aegypti mosquitoes which would help to limit uncontrolled transmission of the vaccine virus.The live-attenuated DEN-2 virus candidates described here, rDEN2Δ30 and rDEN2Δ30-4995, have several properties desired in a live attenuated virus vaccine for humans. First, both viruses reached a titer over 6.0 logThe authors declare that they have no competing interests.J.B. recovered viruses, conducted animal studies, and drafted the manuscript. C.H. and S.W. constructed the DEN-2 cDNA clone and C.H. performed sequencing. K.H. performed mosquito studies. B.M. and S.W. supervised the study and participated in planning and design. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


While the nucleus of a cell may be its command headquarters, mitochondria are equally vital—they are the power plants of the cell, and without them all cellular activity would quickly and irrevocably come to a halt. Testifying to their origins as once free-living bacteria, mitochondria have their own DNA, comprising 37 genes in humans on a single circular chromosome. Whether they invaded their ancestral hosts as parasites or were captured as subcellular collaborators, they have long since left their independent ways behind. Their meager complement of genes is far fewer than is needed to produce these complex organelles; it is clear from analyzing the nuclear genome that most of the mitochondria's presumed ancestral genes have been taken into the cell's nucleus, where they are under the strict control of their host.The transplanted mitochondrial genes have been faithfully doing their job under new management since they were first appropriated, probably hundreds of millions of years ago. However, not all of their DNA descendants have continued to make themselves so useful. For, in addition to many of the mitochondria's original genes, the human genome houses over 200 mitochondrial genetic fragments, useless pieces of code whose only remaining function is to be replicated generation after generation.Detritus from other sources is even more common within the genome, and most of it seems to be harmless. But in this issue, Ricchetti and colleagues show that mitochondrial fragments may not be quite so benign. They have continued to invade the human genome, even into the present day, and a large proportion of them take up residence within nuclear genes, possibly disrupting them and causing human diseases.Scanning the entire human genome, Ricchetti and colleagues found a total of 211 nuclear sequences of mitochondrial origin (NUMTs). Of these, they selected 42, which appeared to be the most recent integrations, for detailed study. Only 14 of them were also found in DNA from our closest relatives, chimpanzees, indicating that the rest arose after the human–chimp split approximately 5 million years ago. While 35 of the 42 were found in all humans tested, the rest were not, suggesting a still more recent origin for these among human populations.The authors also made two surprising discoveries about the location of these human-specific NUMTs. They were not evenly distributed across the entire genome; instead, for reasons that are unclear, there were a disproportionate number of them on two chromosomes—the Y chromosome, present only in males, and number 18. Furthermore, NUMTs were not randomly scattered among all the DNA of the chromosomes. Rather, they were much less likely to be found in non-coding “junk” DNA and much more likely to have inserted themselves within highly active genes. This phenomenon is likely to be related to the mechanism by which a NUMT enters the chromosome—it relies on the machinery that repairs breaks in the DNA, and these breaks are more common in genes that are frequently transcribed. Such insertions can cause disease, as shown by the recent discovery of a hemophilia patient with a NUMT interrupting his clotting factor gene.Much remains to be learned about the functional and temporal dynamics of NUMT insertions, but their potential for harm suggests that many NUMTS, unlike much of the rest of the flotsam that litters our genome, may be selected against quickly. Combined with their differential distribution among human ethnic groups, this may make them valuable markers for tracking both long- and short-term trends in human evolution and migration.


Most people threatened by AIDS, tuberculosis, and unsafe drinking water are poor and have little or no influence over the global politics of public health The rancour surrounding the Bangkok AIDS summit of July 2004 has exposed a series of fundamental disagreements surrounding the global politics of public health. These tensions range from access to cheaper lifesaving drugs to disputes over the role of poverty and gender equality in the promotion of sexual health.The world is now experiencing the most profound public health challenge of the last forty years: we have witnessed the appearance of new diseases such as Ebola, SARS, and in particular, AIDS, combined with the alarming resurgence of diseases previously thought to have been under control, such as malaria and tuberculosis ,2. The AYet the very idea of “public health” sits uncomfortably alongside the current emphasis of bio-medical science on the molecular realm of DNA coding and the development of lucrative Western markets for new pharmaceutical products such as sildenafil (Viagra). The needs of the majority—the global poor—scarcely feature within this tactical alliance between the biomedical sciences and corporate power. Since most people threatened by AIDS, tuberculosis, unsafe drinking water, and other health threats are poor, they have little or no influence over the global politics of public health.If we are to make sense of the current public health crisis, we need to explore interconnections between political, economic, and social developments that are ignored by the fragmentary emphasis of the biomedical sciences. The current impetus toward economic globalization is causing widespread social and economic disruption, ranging from wild currency fluctuations to the systematic collapse of viable agricultural systems An examination of the social impact of the global public health crisis shows that it is women and children who have been most badly affected. One of the least addressed dimensions to the externally imposed structural adjustment programmes on developing countries is the harmful impact on the sexual health of women caused by their increased economic dependence on men. Sexual health programmes based on abstinence, for example, ignore the difficulties women face in negotiating safer or non-penetrative forms of sex. It is married women in southern Africa and south Asia who make up the largest and most vulnerable group of women, since they are at risk of being infected by their husbands.A new generation of HIV-positive women activists, such as the Nigerian journalist and AIDS campaigner Rolake Odetoyinbo Nwagwu, are now engaged in a vital struggle to challenge the social attitudes and economic inequalities that have driven the devastating impact of AIDS on women and children in developing countries. The spread of AIDS in more traditional societies is closely linked with patriarchal power structures.. The sustenance of these structures is assured by the rise of poverty-fuelled ethnic and religious chauvinism, which undermines the prospects for developing more progressive approaches to social policy. And in regions where war or civil strife prevail, the vulnerability of women and children to sexual violence, economic exploitation, and disease is even greater so that we cannot consider public health questions separately from issues surrounding political stability and social justice.In order to understand better the political dynamics behind public health we need to recognize that the development of modern forms of governance has emerged in tandem with new approaches toward the administration of human populations. The rise of the 19th-century industrial city, for example, necessitated the development of much more sophisticated forms of urban governance in order to tackle the threat of epidemic disease and enable these new cities to function effectively as centres of economic activity. But these new spaces of public health control, which scholars such as Michel Foucault have so vividly described, had a shadowy “other”. This “other” was represented by the increasingly squalid conditions endured by the mass of the population in European colonies, exemplified by the devastating modern outbreaks of bubonic plague in cities such as Baghdad, Lagos, and Bombay The Italian political philosopher Giorgio Agamben has elaborated on this distinction between “inside” and “outside” under modern systems of governance to reveal the persistence of what he terms conditions of “bare life” within even the most sophisticated legal and political systems A striking manifestation of this systematic exclusion of the poor from medical care is provided by the Bush administration's efforts to stymie access to affordable antiretroviral drugs. A dramatic standoff in 2001 between the global pharmaceutical industry and public health activists in South Africa, following the import of cheap generic antiretroviral drugs manufactured in Brazil, led to the historic Doha Declaration on intellectual property rights and public health In order to widen access to affordable drugs, the current production of generic medicines will have to be massively expanded, but the Bush administration and its corporate allies in the pharmaceutical industry has worked assiduously to undermine the potential impact of the Doha agreement Of the $15 billion pledged by the Bush administration in the fight against AIDS most of this money will be focused on 15 selected countries. The countries were chosen by their willingness to abide by bilateral trade deals to prevent the production of cheaper generic drugs and their willingness to stress the centrality of abstinence as a strategy for AIDS prevention (a strategy that the religious Right has pushed for) ,17. It sThe US government has sought to highlight the inadequacies of health care infrastructure in developing countries as a further justification for the diversion of attention from the cost of drugs. But primary health care services have themselves been undermined by the structural adjustment and trade policies promoted by Western financial institutions and their corporate backers. The global politics of AIDS is therefore caught in a neo-liberal vicious spiral from which it is impossible to disentangle the needs for social and institutional reform in the worst affected regions from the challenge of widening access to available treatments.Public health pioneers of the past, such as the German bacteriologist Robert Koch, were not only scientists but also political advocates for social change. Improvements in health care were perceived as part of a nexus of reforms that ranged from better housing and nutrition to an extension of voting rights to ensure that the poor had adequate political representation.The contemporary politics of public health needs to be considered, however, in relation to wider discourses on security and human welfare that are quite different from those of the 19th century. The current Western preoccupation with the threat of international terrorism, for example, has distracted attention from the much more real threats to human well-being created by poverty and ill health. The vast resources—both financial and logistical—now being poured into “security” contrast starkly with the inadequate funding provided for the Global Fund for HIV/AIDS, Tuberculosis, and Malaria. The increasingly unilateral and self-interested stance of the US illustrates the tensions between attempts to build international forms of governance for health care and the continuing centrality of national interests or global institutions that explicitly represent the economic power of a relatively small group of nations The paradox is that widening global disparities in wealth and poverty play a role in fostering the social and political conditions in which religious fanaticism and hatred for the West can flourish. So any “war on terror” that fails to address the causes of poverty, despair, and insecurity in the lives of the world's poor will ultimately create a more dangerous world for everyone.


A recent paper suggests that genes can interact in networks to limit variation of phenotype. Similar principles might apply to the regulation of ion channels in nerve cells Individuals within a wild population show remarkably little morphological variation, given the amount of environmental variation they encounter during development and the amount of genetic variation within the population. This phenotypic constancy led to the proposal that individuals were somehow buffered, or canalized, against genetic and environmental variation . ClearlyDrosophila and Arabidopsis have suggested that Hsp90 (heat shock protein 90), a member of a family of proteins expressed at high temperatures (heat shock), may be an excellent candidate for bringing about canalization (Hsp83 (the gene encoding Hsp90) show increased levels of morphological abnormalities; (2) individuals treated with a pharmacological inhibitor of Hsp90 show severe morphological abnormalities; (3) the normal function of Hsp90 is to stabilise the tertiary structure of signal transduction molecules involved in developmental pathways; and (4) this function may be compromised by environmental factors, e.g., heat shock.Experiments in both lization . SeveralHsp90 may not, however, be uniquely placed to act as an evolutionary buffer producing canalization. Recent theoretical work has suggested that canalization may be an emergent property of complex gene networks and may not require specific mechanisms of protein stabilisation and environmental coupling such as those provided by Hsp90 . Siegal Thus, populations derived from ‘knockouts’ express phenotypic variation that was not expressed by the wild-type network, suggesting that any of the genes within the network may buffer genetic variation. This suggests that at least one aspect of generating evolutionary buffering is not unique to Hsp90. But can genes that, unlike Hsp90, are not conditional upon the environment act as evolutionary buffers? To test this, Sacchromyces cerevisiae, in which each gene may be ‘knocked out’ in turn and the expression of the remaining genes determined, to demonstrate that their simulations also had application to biological gene networks. Using these data, they showed that ‘knockouts’ show greater variability in gene expression than wild-type yeast, suggesting that buffering has been disrupted.The simulations described by + and K+ channels as well as numerous Cl−, Ca2+, and voltage-independent leak channels. The electrical properties of a single neuron are dependent, though not exclusively, upon the suite of ion channels expressed within that neuron. The properties of a neural network, which generates behaviour, are determined both by the intrinsic expression patterns of ion channels within neurons and the connectivity between neurons. The nervous system develops as an interaction between experience and genetically programmed events. One mechanism by which this interaction is achieved is ion channel compensation  may be compensated for by regulating the expression of other ion channels. ‘Knockout’ of one of these compensating ion channels may reveal the change in voltage-dependent Na+ channel properties, resulting in a shift in the output of the neuron. This hypothesis has several testable predictions. For example, ‘knocking out’ an ion channel should increase the variation in the activity of particular neurons among individuals in a population. This variation in neural activity may produce an effect on the behaviour of the whole organism.It is possible, therefore, that the networks of genes regulating ion channel expression may act as evolutionary buffers. The relationship between neural activity and the network of ion channel encoding genes may stabilise the neural activity in relation to both the genetic and environmental variation. The stabilisation of neural activity may have consequences for the generation of adaptive behaviour, which is constructed from neural activity. It is possible that ion channels could canalize the evolution of the nervous system by reducing behavioural variation and therefore removing the substrate on which natural selection may act. For example, changes in voltage-dependent NaDrosophila photoreceptors (Studying canalization in ion channel gene networks may have significant advantages over studying developmental gene networks because it is relatively straightforward to measure the amounts of ion channels expressed in single identified neurons, to alter the expression of individual ion channels, and to relate these alterations to behaviour. I am currently pursuing the impact of ion channel compensation in eceptors , 2003c.


Neck dissection has traditionally played an important role in the management of patients with regionally advanced head and neck squamous cell carcinoma (HNSCC) treated with radical radiotherapy alone. However, with the incorporation of chemotherapy in the therapeutic strategy for advanced HNSCC and resultant improvement in outcome the routine use of post chemo-radiotherapy neck dissection is being questioned.Published data for this review was identified by systematically searching MEDLINE, CANCERLIT & EMBASE databases from 1995 until date with restriction to the English language.There is lack of high quality evidence on the role of planned neck dissection in advanced HNSCC treated with chemo-radiotherapy. A systematic literature search could identify only one small randomized controlled trial (Level I evidence) addressing this issue, albeit with major limitations. Upfront neck dissection followed by chemo-radiotherapy resulted in better disease-specific survival as compared to chemoradiation only. Several single arm prospective and retrospective reports were also identified with significant heterogeneity and often-contradictory conclusions.Planned neck dissection after radical chemo-radiotherapy achieves a high level of regional control, but its ultimate benefit is limited to a small subset of patients only. Unless there are better non-invasive ways to identify residual viable disease, the role of such neck dissection shall remain debatable. A large randomized controlled trial addressing this issue is needed to clarify its role and provide evidence-based answers. The optimal management of the neck in loco-regionally advanced head & neck squamous cell carcinomas (HNSCC) following primary chemo-radiotherapy remains controversial ,2. TradiPublished data for this review was identified by systematically searching the MEDLINE, CANCERLIT & EMBASE databases from 1995 until date with restriction to the English language. "Head & Neck cancer" OR "HNSCC" was combined with "chemo-radiotherapy" OR "chemo-radiation" as Medical Subject Heading (MeSH) terms and each of the following phrase used as text words: "adjuvant neck dissection"; "planned neck dissection"; and "neck management". Relevant cross-references were also considered.There is only one small randomized control trial (American Society of Clinical Oncology  Level I In absence of high quality evidence, the best available evidence tempered with clinical judgment often guides decision-making. Two of the recently published reports ,12 somewArgiris  et al evBrizel  et al idApart from the afore-mentioned two reports, there are a few reasonably large studies (involving >50 patients: Table The potential benefit of planned neck dissection after a course of intensive chemo-radiotherapy in terms of improved regional control with or without an impact on survival needs to be weighed against the expected morbidity associated with the surgical procedure ,14,25. OPlanned neck dissection after radical chemo-radiotherapy achieves a high level of regional control, but its ultimate benefit is limited to a small subset of patients only. The morbidity of such dissection is small, but significant. Its impact on survival is yet to be completely realized. In the majority of patients it is either unnecessary because there is no residual disease in the neck or futile because of unsalvageable primary recurrence or distant metastases. Nevertheless, it is recommended that planned neck dissection be performed for patients with less than a complete response in the neck after combined modality therapy to optimize regional control, provided the primary is controlled and there is no evidence of distant metastases. It should also be performed as part of salvage surgery for locally persistent or residual disease at primary site. The criterion for planned neck dissection for patients with advanced nodal disease with a CCR in the neck following chemo-radiotherapy should incorporate not only the nodal staging but also the actual size of the involved lymph nodes. Unless there are better non-invasive ways to identify residual viable disease, which could include functional imaging like Positron Emission Tomography and biological assays like hypoxia markers, the role of such neck dissection shall remain debatable. A large randomized controlled trial across several institutions addressing these issues is needed to clarify the role of planned neck dissection in advanced HNSCC treated with primary chemo-radiotherapy and provide evidence-based answers.No source of funding involved in this reviewNone declared.Dr JP proposed the idea of systematic review on the issueDr TG did the literature search & prepared the manuscriptDr JP critically reviewed and revised the manuscript


There have been contradictory reports regarding the B cell helping function of CD57+ GC-Th cells in production of immunoglobulin (Ig). Furthermore, the cytokine and co-stimulation requirement for their helper activity remains largely unknown. To clarify and gain more insight into their function in helping B cells, we systematically investigated the capacity of human tonsil CD57+ GC-Th cells in inducing B cell Ig synthesis.The function of CD57+ GC-Th cells are highly efficient in helping B cell production of all four subsets of Ig  compared to other T-helper cells located in germinal centers or interfollicular areas. CD57+ GC-Th cells were particularly more efficient than other T cells in helping GC-B cells but not naïve B cells. CD57+ GC-Th cells induced the expression of activation-induced cytosine deaminase (AID) and class switch recombination in developing B cells. IgG1-3 and IgA1 were the major Ig isotypes induced by CD57+ GC-Th cells. CD40L, but not IL-4, IL-10 and IFN-γ, was critical in CD57+ GC-Th cell-driven B cell production of Ig. However, IL-10, when added exogenously, significantly enhanced the helper activity of CD57+ GC-Th cells, while TGF-β1 completely and IFN-γ partially suppressed the CD57+ GC-Th cell-driven Ig production.We demonstrated that CD57+CD4+ T cells in the germinal centers of human lymphoid tissues are the major T helper cell subset for GC-B cells in Ig synthesis. Their helper activity is consistent with their capacity to induce AID and class switch recombination, and can be regulated by CD40L, IL-4, IL-10 and TGF-β.CD57 CD57+ G of IL-2 ,18. CD57on in GC . Based uh2 cells . Using apression . The mic5+ cells ,23 and d5+ cells . Becauseientists ,25-27. T+ GC-Th cells in inducing B cell Ig synthesis in naïve vs. germinal center B (GC-B) cells in comparison with other T cell subsets in human tonsils. We show that CD57+ GC-Th cells are more efficient than other germinal center or interfollicular T cells in supporting B cell production of Ig. CD57+ GC-Th cells, when compared to other T cells, have better helper activity for GC-B cells than for naïve B cells. CD57+ GC-Th cells induced the expression of activation-induced cytosine deaminase (AID) and CSR in developing B cells. CD40L, but not other major cytokines, is critical for the helper activity of CD57+ GC-Th cells. IL-10 positively and TGF-β1 negatively regulate the helper activity of CD57+ GC-Th cells.To clarify and gain more insight into their function in helping B cells, we systematically investigated the capacity of human tonsil CD57+ CD4+ T cells are located in germinal centers surrounded by IgD+ naïve B cells  surrounding GC. Although some are found in IFA, CD69+ CD4+ T cells were also preferentially found in GC , CD4+CD57-CD69+ cells (mainly in GC and a minor proportion in IFA), and CD4+CD57-CD69- cells (mainly in IFA).We examined the distribution of T helper cell subsets in human tonsils based upon the expression of CD4, CD57 and CD69. As reported previously ,19,28,29C Figure . In cont+ T cell population was fractionated into CD57+ GC-Th cells , total CD57- T cells, CD57-CD69+ T cells and CD57-CD69- T cells  in the composition of Ig that they induced.The relative composition of IgM, IgG, IgA and IgE produced in response to CD57r Figure . Class-s+ GC-Th cells have the capacity to induce AID in B cells. Naïve B cells were co-cultured with CD57+ GC-Th cells, and AID expression was examined  were compared, AID expression preceded the expression of IgG3 transcript by 1–2 days in culture , whereas CD4+CD57-CD69- interfollicular T cells did not express CD40L at significant levels. There was no significant difference in the expression of surface CD40L between CD57+ and CD57- CD69+ cells.Cytokines and CD40L regulate B cell maturation and Ig production. We examined whether CD40L, IL-4, IL-10 and IFN-γ play any roles in the CD57E Figure . In this+ GC-Th cells  employed in our study appear to favor the detection of the suppressive function of TGF-β. Taken together, these results imply that Th1, Th2 and regulatory T cells, if present in germinal centers, could positively or negatively control the function of CD57+ GC-Th cells in regulation of humoral immune responses. Indeed, there are regulatory T cells in GCs that express surface TGF-β and can effectively suppress the function of CD57+ GC-Th cells [CD40L appears to be essential for the helper activity of CD57tivation . Anotherynthesis . We did Th cells .+ GC-Th cells in supporting CSR and Ig synthesis in B cells, and revealed the factors that regulate their activity, thereby substantiating the so-far inconclusive function of CD57+ GC-Th cells. The fact that these T cells have preferential and efficient helper activity for GC-B cells and are specifically localized in GCs in large numbers suggests that CD57+ GC-Th cells are probably the major T helper subset responsible for supporting B cell differentiation for Ig production in germinal centers.Our results demonstrated the capacity of CD57+ T cells (purity >97%) were isolated by depleting non-CD4+ T cells using a magnetic bead depletion method . After staining of the isolated CD4+ T cells with appropriate antibodies, CD57+ GC-Th cells (purity >95%) were isolated by a positive magnetic selection method (Miltenyi Biotec). CD4+CD57-CD69+ and CD4+CD57-CD69- T cell subsets (purity >95%) were further isolated from the CD57- T cell fraction by magnetically selecting CD69+ T cells (Miltenyi Biotec). Total B cells were isolated by rosetting with 2-amino-ethylisothiouronium bromide (AET)-treated sheep red blood cells followed by CD4+ T cell depletion (CD19+ cells > 99.5%). Naïve B cells (CD19+IgD+ cells >99%) were isolated from the total B cell fraction by depleting CD38+ T cells followed by positive magnetic selection of IgD+ B cells. CD19+CD38+IgD+/- GC-B cells (purity >95%) were isolated from the tonsil CD19+ B cells as described before [Mononuclear cells were prepared by density gradient centrifuge on histopaque 1077  from human tonsil pathological specimens obtained from young patients (3–10 yr) undergoing tonsillectomy to relieve obstruction of respiratory passages and improve drainage of the middle ear at Sagamore Surgical Center . The use of human pathological specimens in this study was approved by the institutional review board at Purdue University. CD45 T and 105 B cells were co-cultured, unless indicated otherwise, in each well of 48-well plates in the presence of Staphylococcal enterotoxin B . Cells were incubated in 5% CO2 incubators at 37°C for 3–8 days. Recombinant IL-4, IL-10, and TGF-β1 were purchased from R&D systems . Recombinant IFN-γ was obtained from BD Pharmingen . Purified CD154-blocking antibody (24–31) was obtained from Ancell Corporation . IL-4-blocking antibody (MP4-25D2) was purchased from BD Pharmingen. Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems. All antibodies and reagents added to culture were azide-free. Cytokines were added at saturating concentrations: IL-4 (40 ng/ml), IL-10 (40 ng/ml), IFN-γ (200 ng/ml) and TGF-β1 (10 ng/ml). Neutralizing antibodies were added at following concentrations: anti-CD40L (20 μg/ml), anti-IL-4 (5 μg/ml), anti-IL-10 (5 μg/ml), anti-IFN-γ (2.5 μg/ml) and isotype antibody (5 μg/ml).All cell cultures were performed in RPMI1640 medium supplemented with 10% FBS, gentamycin, streptomycin, and penicillin. To cross-link the B cell receptors, isolated B cells were incubated for 2 h at 4°C with Sepharose-conjugated rabbit Ab to human Ig μ chain and human Ig (H + L) chain , and then washed with cold PBS. 10T cells were stained with anti-human CD57 , anti-human CD69 , anti-human CD4 , and anti-human CD3 . B cells were stained with anti-CD19 , anti-human IgD , anti-human CD38 , and anti-human CD3 . Stained cells were analyzed using a 4-color FACSCalibur™ (BD Biosciences).Frozen sections of tonsils were acetone-fixed and stained using antibodies to CD57 , CD69 , IgD  and/or CD4 . Stained sections were analyzed using a confocal microscopy system (Bio-Rad MRC 1024UV and Nikon Diaphot 300 microscope) at Purdue Cytometry Lab.Culture supernatants were assayed by ELISA as previously described . The senTotal RNA was extracted from cultured cells with Trizol reagent , and was reverse-transcribed into cDNAs with SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's protocol. The primer pairs used in this study were designed by Cerutti et al. : IgM, FRTotal RNA was extracted from freshly isolated or cultured cells using Trizol reagent , and was reverse-transcribed into cDNAs with SuperScript™ II Reverse Transcriptase. RT-PCR amplification of AID was performed using the two primers: AID-forward (5'-GAT GAA CCG GAG GAA GTT TC-3') and AID-reverse (5'-TCA GCC TTG CGG TCC TCA CAG-3'), which generated a specific 351 bp PCR product after 30 cycles of PCR reaction . β-actin was also amplified as a control.Student's paired t-test was used. P values smaller than 0.05 were considered significant.GC, germinal center; GC-Th cells, germinal center T helper cells; GC-B cells, germinal center B cells; AID, activation-induced cytosine deaminase; CSR, class switch recombination; SEB, staphylococcal enterotoxin B.CHK conceived, coordinated the study, analyzed the results and wrote the text. JRK, HWL and SGK participated in experiments, data analysis, making figures and proofreading the manuscript. PH provided specimens and helped perform the study. All authors read and approved the manuscript.


New York Times crossword puzzle, you probably have a gift for insight. The puzzles always have an underlying hint to solving them, but on Saturdays that clue is insanely obtuse. If you had all day, you could try a zillion different combinations and eventually figure it out. But with insight, you'd experience the usual clueless confusion, until—voilà—the fog clears and you get the clue, which suddenly seems obvious. The sudden flash of insight that precedes such “Aha!” moments is characteristic of many types of cognitive processes besides problem-solving, including memory retrieval, language comprehension, and various forms of creativity. Although different problem-solving strategies share many common attributes, insight-derived solutions appear to be unique in several ways. In this issue, researchers from Northwestern and Drexel Universities report on studies revealing a unique neural signature of such insight solutions.If you're one of those insufferable people who can finish the Saturday Eureka!, which Archimedes reportedly shouted upon realizing that water displacement could be used to compute density. Illustrating the strong emotional response elicited by such a sudden insight, Archimedes is said to have run home from the baths in euphoric glee—without his clothes.Mark Jung-Beeman, John Kounios, Ed Bowden, and their colleagues recount the storied origin of the term Among other characteristics that typically distinguish insight from “noninsight” solutions, people feel stuck before insight strikes; they can't explain how they solved the problem and might say they were not even thinking about it; the solution appears suddenly and is immediately seen as correct. But are the neural processes involved in arriving at a solution through insight actually distinct from those related to more mundane problem-solving?Recent findings suggest that people think about solutions, at an unconscious level, prior to solving insight problems, and that the right cerebral hemisphere (RH) appears to be preferentially involved. Jung-Beeman et al. predicted that a particular region of the RH, called the anterior superior temporal gyrus (aSTG), is likely involved in insight because it seems critical for tasks that require recognizing broad associative semantic relationships—exactly the type of process that could facilitate reinterpretation of problems and lead to insight.pine, crab, sauce) and asked to think of one word that would form a compound word or phrase for each of the words (can you figure it out?). Neural activity was mapped with fMRI while the participants were given 124 similar word problems—which can be solved quickly with or without insight, and evoke a distinct Aha! moment about half the time they're solved. Subjects pressed a button to indicate whether they had solved the problem using insight, which they had been told leads to an Aha! experience characterized by suddenness and obviousness.To test this hypothesis, Jung-Beeman et al. mapped both the location and electrical signature of neural activity using functional magnetic resonance imaging (fMRI) and the electroencephalogram (EEG). In the first experiment, thirteen people were given three words , the authors conclude that the increase does not simply reflect the emotional jolt associated with insight.In a second experiment, 19 new participants engaged in the same type of problem-solving tasks as the first group while their brain waves were measured with an EEG. The researchers then analyzed the EEG recordings to look for differences between insight and noninsight solutions in brain wave activity. The researchers found that 0.3 seconds before the subjects indicated solutions achieved through insight, there was a burst of neural activity of one particular type: high-frequency (gamma band) activity that is often thought to reflect complex cognitive processing. This activity was also mapped to the aSTG of the RH, providing compelling convergence across experiments and methods.at least one additional component. Further, the fact that the effect occurred in RH aSTG suggests what that process may be: integration of distantly related information. Distinct neural processes, the authors conclude, underlie the sudden flash of insight that allows people to “see connections that previously eluded them.”Problem-solving involves a complex cortical network to encode, retrieve, and evaluate information, but these results show that solving verbal problems with insight requires


Mechanisms that affect recovery from fetal and neonatal hypoxic-ischemic (H-I) brain injury have not been fully elucidated. The incidence of intrapartum asphyxia is approximately 2.5%, but the occurrence of adverse clinical outcome is much lower. One of the factors which may account for this relatively good outcome is the process of neurogenesis, which has been described in adult animals. We used a neonatal mouse model to assess new cells in the hippocampus after H-I injury.Neonatal mice underwent permanent unilateral carotid ligation on the seventh postnatal day followed by exposure to 8% hypoxia for 75 minutes. The presence of new cells was determined by bromodeoxyuridine (BrdU) incorporation into cells with sacrifice of the animals at intervals. Brain sections were stained for BrdU in combination with neuronal, glial, endothelial and microglial stains.We found a significant increase in BrdU-positive cells in the neonatal mouse hippocampus in the injured area compared to the non-injured area, most prominent in the dentate gyrus (DG) . Among the cells which showed differentiation, those which were stained as either microglial or endothelial cells showed a peak increase at three days after the injury in the DG, injured versus non-injured side . As in the adult animal, neurogenesis was significantly increased in the DG with injury , and this increase was subsequent to the appearance of the other dividing cells. Numbers of new oligodendrocytes were significantly higher in the DG on the non-injured side , suggesting that oligodendrocyte synthesis was reduced in the injured hippocampus.These findings demonstrate that the neonatal animal responds to brain injury with neurogenesis, much like the adult animal. In addition, H-I insult leads to more neurogenesis than hypoxia alone. This process may play a role in the recovery of the neonatal animal from H-I insult, and if so, enhancement of the process may improve recovery. The incidence of intrapartum asphyxia was recently determined to be 2.5% in a large population of singleton pregnancies . Even soNew neuronal growth has been demonstrated in several studies of adult H-I models, including in humans -10, but Figure The DG on the hypoxic-ischemic side of the brain exhibited significantly higher numbers of BrdU-positive cells at P10, P17, and P42 than either CA1 or CA3. BrdU/RCA-positive cells were significantly higher at P10 in the DG. The morphology and location of the BrdU/RCA-positive cells suggested that they were endothelial or microglial cells. BrdU/NeuN-positive cells were higher at P17 and P42 in the DG , followed rapidly by the appearance of BrdU/RCA-positive cells, again mainly in the DG, and then by BrdU/NeuN-positive cells (putative neurons) developing only in the DG (at 42 days of age). There were minimal numbers of BrdU/GFAP-positive cells observed at all points.Ischemia has been shown to increase neurogenesis in several adult animal models of brain ischemia. Following global cerebral ischemia in adult gerbils, neurogenesis has been shown to increase in the hippocampal SGZ, while newly born neurons were demonstrated in the CA1 pyramidal cell layer . In anotThe data strongly suggested that the development of oligodendrocytes (BrdU/CNPase-positive cells) was impaired in the DG on the hypoxic-ischemic side of the brain, with significantly fewer of these cells observed in the hypoxic-ischemic side as compared to the hypoxia alone side at 10 days. This finding is in contradiction to findings reported by Zaidi et al , who demWhite matter damage in neonates is thought to be in part due to vulnerability of the immature oligodendrocyte to H-I damage . In seveWe found a significant, early increase in BrdU-positive cells expressing RCA, an endothelial cell marker and microglial marker, on the side of the brain affected by hypoxic-ischemic injury. The mechanisms of angiogenesis in the brain after H-I injury are not well understood, and there is also little known about angiogenesis in neonatal animal models. Vascular endothelial growth factor (VEGF) is endothelial cell specific and has been implicated in hypoxia-mediated angiogenesis . VEGF haThe present study did not demonstrate an increase in BrdU-positive cells co-labeling for an astrocyte marker, GFAP, after H-I injury. Astrocytes have been demonstrated to express apoptotic enzymes within hours after H-I injury in neonatal rats . In neonAs evidenced by the variability reported in the astrocytic response to H-I brain injury, the response of this cell in this environment needs to be better characterized. Because astrocytes help to maintain the extracellular milieu needed for neuronal formation and function, it is possible that their early loss after H-I injury may contribute to subsequent neuronal degeneration. Future studies in our neonatal mouse model could be geared towards better understanding the early response of astrocytes. Postponing evaluation until three days post-injury in this model likely resulted in underestimation of the astrocytes' role in this injury repair process. Chronic hypoxia has been demonstrated to induce astrocytes into more immature phenotypes, which do not express normal levels of GFAP . The abiAlthough the BrdU-positive cells in our study were co-labeled with stains identifying four separate types of cells, there were many BrdU-positive cells that were not identified by these four stains. The co-labeling studies we performed accounted for only about 20% of all the BrdU-positive cells. The BrdU labeling protocol we used consisted of BrdU injections over the course of the experiment, rather than a one time injection at the time of H-I injury, which resulted in the staining of more cells than would be found following a single injection. In this manner, we were able to evaluate how each cell type varied its number and location at different intervals after the injury. This provided information about the cellular response to hypoxic-ischemic brain injury in both the acute and long-term settings. The role and identity of the non-co-labeled cells remains unclear. It is probable that these were immature cell types, not yet expressing the markers for which we evaluated.The validity of cells staining for BrdU representing new or dividing cells versus apoptotic or necrotic cells is also an issue to be considered. As a result, TUNEL staining was performed on several hippocampal sections. At P10, three days after H-I injury, TUNEL staining does reveal apoptotic and necrotic nuclei. However, by P42, this finding is virtually non-existent, suggesting that any BrdU-positive cells are present due to cell proliferation or growth. . The pups were then placed with the dams for two hours prior to placement in an 8% oxygen chamber partially immersed in a water bath at 37° for 75 minutes. This procedure was described previously in the rat by Levine  and Riceet al , and thel [et al . Within Paraffin was removed from slides using xylene, followed by rehydration in an alcohol dilution series. Slides were soaked in 0.1% Triton-X 100 in PBS for ten minutes to increase permeability of fixed tissue, followed by rinsing in 1X PBS. Antigen retrieval was performed using a microwave method. Slides were incubated for twenty minutes after slow boiling for ten minutes and then rinsed in PBS and blocked using 2% normal calf serum for 20 minutes.Sheep anti-BrdU antibody (Biodesign #M20105S), diluted 1:200 in PBS, was selected for the visualization of new cells. Co-labeling runs used mouse anti-NeuN (Chemicon #MAB377) 1:2000 for the visualization of neurons, rabbit anti-GFAP (DAKO # Z0334), diluted 1:300 in PBS for the visualization of astrocytes, biotinylated RCA (Vector #B-1085) diluted 1:300, for the visualization of endothelial cells and microglia cells, and CNPase (Sigma #C-5922), diluted 1:200, for the visualization of oligodendrocytes. All primary antibodies except NeuN were allowed to incubate at room temperature for one hour.Elite ABC Kit #PK-6102), diluted 1:200 in 1.5% normal horse serum, was applied to each section and incubated for thirty minutes at room temperature. Slides were washed in a PBS series before applying Vector Elite ABC reagent and incubation for thirty minutes at room temperature. Sections were washed again in PBS and TSA amplification reagent (NEN #SAT700) with biotinylated tyramide 1:200 in amplification buffer was applied. Sections were incubated for eight minutes at room temperature.Primary NeuN antibody was allowed to incubate for thirty minutes at room temperature, and washed three times in a 1X PBS solution. Biotinylated anti-mouse antibody , diluted 1:400, was used to visualize BrdU in all cases. FITC anti-rabbit (Jackson # 711-095-152), diluted 1:100, was used to visualize GFAP. Strepavidin FITC (Jackson #016-010-084), diluted 1:100, was used to visualize biotinylated RCA and NeuN. FITC anti-mouse (Jackson #715-095-151), diluted 1:100, was used to visualize CNPase.To reduce background, slides were rinsed in 4 changes of PBS for 5 minutes each. Secondary antibodies were applied and incubated for 1 hour. Slides were rinsed in PBS, and immersed in Hoechst Stain for 8 minutes. Another rinse series in PBS was applied before coverslipping, using Vectashield Mounting medium. (Vector #H-1000)Cells were counted in 4–6 animals at each time interval. For each animal, the cells were counted from the injury area in the anterior hippocampus   in CA1, To confirm dual labeling, confocal imaging was performed in selected cells with a Zeiss 510 laser scanning confocal microscope with the use of Zeiss software. The objective used was Zeiss63x C-Apochromat 1.2 NA. To excite the FITC fluorochrome (green), a 488-nm laser line generated by an argon laser was used, and for the Cy3 fluorochrome (red), a 543-nm laser line from a HeNe laser was used. Filter sets used were a bandpass 500- to 600- filter ("green" channel) and a long-pass 585- to 650-nm filter ("red" channel). We identified cells that were labeled for BrdU and the neuronal marker NeuN. These cells were then examined by confocal microscopy, and 1-μm step Z series were obtained. , according to manufacturer recommendations. Therefore, after rehydration, the tissue was treated with Proteinase K (20 μg/mL) and incubated for 15 minutes. Endogenous peroxidases were quenched with hydrogen peroxide. Sections were then treated with equilibration buffer and TdT enzyme treatment. Next, the sections were stained with anti-dioxigenin peroxidase conjugate for 30 minutes, and the slides were developed with stable DAB.Nested, repeated measures analysis of variance was used to examine differences in number of cells between times of sacrifice , side of brain , region of the hippocampus  and antibody type . A second analysis was performed using only BrdU-positive cells. Post hoc analyses were performed using the adjusted least square means and applying a Bonferroni correction to the overall alpha level. Statistical significance was assessed using an alpha level of 0.05. All analyses were performed using SAS 8.2.Ricinus communis agglutinin I –s RCA, neuronal nuclear marker – NeuNhypoxic-ischemic – H-I, bromodeoxyuridine – BrdU, subventricular zone – SVZ, subgranular zone – SGZ, dentate gyrus – DG, glial fibrillary acidic protein – GFAP, 2'3'-cyclic nucleotide 3'-phosphodiesterase -CNPase,


Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed.Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied.Using real-time RT-PCR to study the expression of 10 poplar (genus Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments. For many years the vast majority of gene expression studies have employed non-quantitative or semi-quantitative RNA gel blots and RT-PCR analysis. Real-time PCR technology has removed many of the difficulties associated with quantitative gene expression studies , and reaReal-time qRT-PCR offers a robust means for precisely quantifying changes in gene expression over a wide dynamic range. It is also applicable to experiments where RNA amounts are limiting, such as for micro-dissected tissues. However, selection of an appropriate normalization method is crucial for reliable quantitative gene expression results ,6. The pNormalization to total RNA content poses a number of problems. It is difficult to quantify small amounts of RNA, and variation in RT and PCR reaction efficiencies are not accounted for by this method. Similarly, normalization to an external RNA standard is problematic due to RNA instability. The most commonly used method is relative quantitation, whereby gene expression level is normalized to that of an internal reference gene. While this avoids the problems and limitations of absolute quantitation, selection of a proper internal control–gene expressed at a nearly constant level in all tissue samples being investigated–is required. Failure to use an appropriate control gene may result in biased gene expression profiles, as well as low precision. The consequences may be that only gross changes in expression level are declared statistically significant, or that patterns of expression are erroneously characterized.18S rRNA is frequently used as an internal control, it is far from ideal. It requires the use of total RNA and random primers for the RT reaction, and is expressed at very high levels; some means for attenuating 18S expression might be needed when weakly expressed genes are studied. In addition, there can be imbalances in rRNA and mRNA fractions between different samples, and 18S is not always expressed at a constant level in all conditions [18S expression levels appear to be affected to a lesser extent by partial RNA degradation than are mRNA expression levels [Until recently, internal controls (often referred to as housekeeping or maintenance genes), were selected based on stability of expression in qualitative studies , via low-sensitivity assays such as densitometry of hybridized blots, or via semi-quantitative RT-PCR. None of these will be adequate for identifying reliable internal controls for real-time qRT-PCR. For example, expression profiling via real-time qRT-PCR of 10 commonly used human internal control genes revealed different degrees and patterns of expression among 13 tissue types, and no single gene was a suitable universal control for all tissue types . AlthougStudies in mammalian and microbial systems, where real-time qRT-PCR has been most extensively applied to date, have begun to include evaluations of various housekeeping genes for normalization -11. VandPopulus trichocarpa × P. deltoides, cottonwood hybrid) tissues collected at different developmental stages, and at different times of the year. The goal of our studies was to detect changes associated with seasonal development and tree aging for several regulatory genes. We therefore undertook a study to compare the stability of several potential control genes. We found that the genes tested exhibited very different degrees of variation in expression among tissue samples, and that a statistical and graphical method helped us to select the genes best suited for the developmental studies we were conducting. This approach, which is very similar to a classical method used by plant breeders to assess the relative stability in yield of different varieties [In this study, we used real-time qRT-PCR to examine the expression of 10 housekeeping genes in a diversity of poplar , translational initiation factor, elongation factor, and rRNA. Searches of the literature revealed that members of all classes have been used as internal controls for studies of plant gene expression using RNA gel blots or RT-PCR assays. Poplar genes belonging to these gene families were identified via searches of the EST database Table . The expT). CT is the cycle at which a significant increase in amount of PCR product (measured by increase in fluorescence) occurs, generally the middle of the exponential phase of amplification.Within a single experiment, aliquots of the same cDNA synthesis reaction were used for real-time PCR amplification of each of the 10 genes and all gene primer and cDNA combinations were amplified in triplicate in a single PCR run. The entire experiment was then repeated a second time and results combined for statistical analysis. Quantitation via real-time PCR is based on cycle threshold  for each gene are given in Table 18S as an internal control for expression studies using these and other tissue samples and had noticed that 18S CT values sometimes varied considerably (data not shown). This may have been largely due to the high abundance of 18S transcripts. Use of 18S as an internal control for studies of genes expressed at relatively low levels required additional dilution of the cDNA templates for 18S amplification relative to the gene being studied. In the present study, the amount of cDNA was the same for all PCR reactions, but 18S primer concentrations were 50 nM, while all other gene primer concentrations were 600 nM. As expected, 18S was the most abundant (lowest CT) housekeeping transcript; TUA was the least abundant.Mean CT values to examine variation among tissues and RT-PCR experiments. Examination of the distribution of the residual values from ANOVA indicated that assumptions concerning homogeneity of variance and normality of data were adequately met (data not shown). The ANOVA F-test of differences among tissues indicated that five of the genes showed significant variation in expression among the tissue samples. The degree of residual variation, as reflected in the mean square error (MSE), residuals, or coefficient of variation (CV), varied widely. Four genes had CVs below 5%, and two had CVs at or above 25% , whereas TUA is expressed at a much lower level (mean CT = 28.9)  Table . For theol genes . A potenOur primers were designed based on a limited EST set that likely did not include all family members, and ESTs vary in sequence quality. Thus, primers could have amplified more than one family member or primer mismatches due to EST sequence errors could have lowered PCR efficiency. Although gel and real-time PCR dissociation curve analyses did not indicate that multiple genes were amplified with our primer sets, these analyses might not detect multiple amplicons from different family members that are the same size and have the same PCR efficiency. As discussed above this is not necessarily a detriment–amplification of multiple family members might result in a more stable internal control than single gene amplification. In addition, the upcoming release of a large poplar unigene set and annotated genome sequence  will impUsing ANOVA and linear regression analysis, we demonstrated that levels of expression stability among a number of potential control genes can vary widely, and that it is not difficult, costly or labor-intensive to test a number of genes. Moreover, such validation tests might have the additional benefit of revealing technical problems, such as excessive variability in RT and PCR efficiency due to RNA quality or inconsistent pipetting.For some experiments, choice of an internal control is straightforward. For example, a number of housekeeping genes should be satisfactory controls for comparisons of transgene expression level in the same tissue type from different transgenic lines grown under identical conditions. However, this is not the case for studies that compare gene expression among different tissue or cell types, at different developmental stages, or under different environmental conditions, as were represented in our study of trees in field environments over a period of seven months. For such studies, internal controls should be carefully tested and validated. Statistical confirmation of internal controls for qRT-PCR should enable previously indiscernible small changes in expression level to be reliability detected.P. trichocarpa × P. deltoidies) over a seven-month period in 2001  with modifications. Tissues (0.2 g) were ground to a fine powder with mortar and pestle in liquid N2. The powder was added to a tube containing 1 ml of RNeasy RLT buffer and 0.01 g soluble polyvinylpyrrolidone , and homogenized using a polytron for approximately 30 sec. Four volumes of 5 M Potassium acetate, pH 6.5 was added to the homogenate, the mixture was incubated on ice for 15 min, and the precipitate removed by a 15 min centrifugation  at 4°C. Supernatant was transferred to two 1.5 ml microcentrifuge tubes and 0.5 volume of 100% EtOH was added. Samples were transferred to RNeasy mini columns and the remaining steps were as directed by the manufacturer's instructions for plant RNA isolation (steps 6–11). RNA was quantified using spectrophotometric OD260 measurements and quality was assesed by OD260/ OD 280 ratios and by electrophoresis on 1% formaldehyde agarose gels followed by ethidium bromide staining. RNAs were stored at -80°C.Tissues were collected from five or six year-old ramets  of a single female poplar hybrid clone  according to the manufacturer's protocol, and two-step real-time RT-PCR performed. cDNA was synthesized from 5 μg of RNA using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen) with random hexamer primers according to the manufacturer's instructions, except that the initial 65°C denaturation step was omitted. The cDNAs were diluted 1:5 with nuclease-free water. Aliquots of the same cDNA sample were used with all primer sets for real-time PCR, and amplification reactions with all primer sets were performed in the same PCR run. Reactions were done in a 25 μl volume containing 600 nM of each primer, 6.5 μl of cDNA sample (≈320 ng of input RNA) and 1X SYBR Green PCR master mix (Applied Biosystems). For 18S amplification, primer concentration was 50 nm. Real-time PCR was performed on the ABI Prism 7700 Sequence Detection System (Applied Biosystems) in a 96-well reaction plate using the parameters recommended by the manufacturer . Each PCR reaction was performed in triplicate and no-template controls were included. Specificity of the amplifications was verified at the end of the PCR run using ABI Prism Dissociation Curve Analysis Software. The entire experiment, including both the RT and real-time PCR steps, was repeated, giving a total of two experimental replications.T values) from the ABI PRISM 7700 Sequence Detection System were analyzed in Microsoft Excel. Single factor ANOVA and regression analysis using the least squares method were performed using the Excel Analysis ToolPak. Assumptions concerning homogeneity of variance and normality were evaluated from inspection of residuals  from the ANOVA  Generate data from multiple analyses of gene expression via quantitative RT PCR that can be assumed to be statistically independent (or nearly so), including from multiple independent samples that bracket the experimental conditions of interest.2) Conduct ANOVA to examine the extent of variation among samples, and  test their significance using appropriate F-ratios. Examine plot of residuals vs. mean expression level, or use a statistical test, to check normality of data.3) Genes showing high variance among tissues in ANOVA, especially if accompanied by large mean square errors (or coefficients of variation), are to be avoided as controls.4) Calculate mean expression level over all genes studied for each sample type as an index of both experimental and biological conditions that promote high levels of measured expression. Use of a large number of genes and tissue samples  are desirable where estimates of stability are to be compared between studies.5) Regress mean expression level for each gene in each sample type over the mean for the sample type. The estimated slope and mean square residual (deviation from regression prediction) provide estimates of the degree to which the gene is sensitive to general expression-promoting conditions (slope) and whose expression continues to be difficult to predict .6) Assuming that both constancy over sample types (low slope) and high predictability (low coefficient of variation) are desirable, a stability index can be created as their product , and the gene with the lowest value chosen.7) Alternatively, visually inspect regression and residual plots to select genes that would be most suitable as controls for specific sets of experimental conditions.AMB developed the molecular methods, participated in design of the study, and drafted the manuscript. IAY designed primers, carried out the real time RT-PCR experiments, performed the statistical analysis and drafted the manuscript. SHS conceived the analysis, and participated in its design. All authors read, helped to edit, and approved the final manuscript.