First Exploration of Monopole-Driven Shell Evolution above the N=126 Shell Closure: New Millisecond Isomers in

Isomer spectroscopy of heavy neutron-rich nuclei beyond the N=126 closed shell has been performed for the first time at the Radioactive Isotope Beam Factory of the RIKEN Nishina Center. New millisecond isomers have been identified at low excitation energies, 985.3(19) keV in ^{213}Tl and 874(5) keV in ^{215}Tl. The measured half-lives of 1.34(5) ms in ^{213}Tl and 3.0(3) ms in ^{215}Tl suggest spins and parities 11/2^{-} with the single proton-hole configuration πh_{11/2} as leading component. They are populated via E1 transitions by the decay of higher-lying isomeric states with proposed spin and parity 17/2^{+}, interpreted as arising from a single πs_{1/2} proton hole coupled to the 8^{+} seniority isomer in the ^{A+1}Pb cores. The lowering of the 11/2^{-} states is ascribed to an increase of the πh_{11/2} proton effective single-particle energy as the second νg_{9/2} orbital is filled by neutrons, owing to a significant reduction of the proton-neutron monopole interaction between the πh_{11/2} and νg_{9/2} orbitals. The new ms isomers provide the first experimental observation of shell evolution in the almost unexplored N>126 nuclear region below doubly magic ^{208}Pb.

Depression-like behavior is dependent on age in male SAMP8 mice.

Aging is associated with an increased risk of depression in humans. To elucidate the underlying mechanisms of depression and its dependence on aging, here we study signs of depression in male SAMP8 mice. For this purpose, we used the forced swimming test (FST). The total floating time in the FST was greater in SAMP8 than in SAMR1 mice at 9 months of age; however, this difference was not observed in 12-month-old mice, when both strains are considered elderly. Of the two strains, only the SAMP8 animals responded to imipramine treatment. We also applied the dexamethasone suppression test (DST) and studied changes in the dopamine and serotonin (5-HT) uptake systems, the 5-HT2a/2c receptor density in the cortex, and levels of TPH2. The DST showed a significant difference between SAMR1 and SAMP8 mice at old age. SAMP8 exhibits an increase in 5-HT transporter density, with slight changes in 5-HT2a/2c receptor density. In conclusion, SAMP8 mice presented depression-like behavior that is dependent on senescence process, because it differs from SAMR1, senescence resistant strain.

Lack of Jun-N-terminal kinase 3 (JNK3) does not protect against neurodegeneration induced by 3-nitropropionic acid.

AIMS: 3-Nitropropionic acid (3-NP) is a toxin that replicates most of the clinical and pathophysiological symptoms of Huntington's disease, inducing neurodegeneration in the striatum due to the inhibition of mitochondrial succinate dehydrogenase. Different pathways have been implicated in the cell death induced by 3-NP in rodents. One of them is the Jun-N-terminal kinase (JNK) pathway, which may play a role in the neurodegenerative process in different diseases. Moreover, the lack of one isoform of JNK (JNK3) has been associated with neuroprotection in different experimental models of neurodegeneration. Therefore, in the present study the role of JNK3 in the experimental Huntington's model induced by 3-NP administration was evaluated. METHODS: 3-NP was intraperitoneally administered once a day for 3 days to wild-type and Jnk3-null mice. Coronal brain sections were used to determine cell death and astrogliosis in striatum. Western blots were performed to determine the involvement of different pathways in both wild-type and Jnk3-null mice. RESULTS: Although JNK activation was observed following 3-NP administration, the results indicate that the lack of JNK3 does not confer neuroprotection against 3-NP toxicity. Thus, other pathways must be involved in the neurodegeneration induced in this model. One of the possible pathways towards 3-NP-induced apoptosis could involve the calpains, as their activity was increased in wild-type and Jnk3-null mice. CONCLUSION: Although JNK3 is a key protein involved in cell death in different neurodegenerative diseases, the present study demonstrates that the lack of JNK3 does not confer neuroprotection against 3-NP-induced neuronal death.

AAV-mediated expression of secreted and transmembrane αKlotho isoforms rescues relevant aging hallmarks in senescent SAMP8 mice.

Senescence represents a stage in life associated with elevated incidence of morbidity and increased risk of mortality due to the accumulation of molecular alterations and tissue dysfunction, promoting a decrease in the organism's protective systems. Thus, aging presents molecular and biological hallmarks, which include chronic inflammation, epigenetic alterations, neuronal dysfunction, and worsening of physical status. In this context, we explored the AAV9-mediated expression of the two main isoforms of the aging-protective factor Klotho (KL) as a strategy to prevent these general age-related features using the senescence-accelerated mouse prone 8 (SAMP8) model. Both secreted and transmembrane KL isoforms improved cognitive performance, physical state parameters, and different molecular variables associated with aging. Epigenetic landscape was recovered for the analyzed global markers DNA methylation (5-mC), hydroxymethylation (5-hmC), and restoration occurred in the acetylation levels of H3 and H4. Gene expression of pro- and anti-inflammatory mediators in central nervous system such as TNF-α and IL-10, respectively, had improved levels, which were comparable to the senescence-accelerated-mouse resistant 1 (SAMR1) healthy control. Additionally, this improvement in neuroinflammation was supported by changes in the histological markers Iba1, GFAP, and SA ß-gal. Furthermore, bone tissue structural variables, especially altered during senescence, recovered in SAMP8 mice to SAMR1 control values after treatment with both KL isoforms. This work presents evidence of the beneficial pleiotropic role of Klotho as an anti-aging therapy as well as new specific functions of the KL isoforms for the epigenetic regulation and aged bone structure alteration in an aging mouse model.

Decrease of calbindin-d28k, calretinin, and parvalbumin by taurine treatment does not induce a major susceptibility to kainic acid.

Taurine, 2-aminoethanesulfonic acid, is present at high concentrations in many invertebrate and vertebrate systems, and it has several biological functions. In addition, it has been related to a neuroprotective role against several diseases, such as epilepsy. It has been reported that taurine induces a decrease of calbindin-D28k, calretinin, and parvalbumin protein levels in the hippocampus 3 days after administration. In the present work we hypothesized that the decrease of these proteins could alter the action of kainic acid (KA) and make mice more susceptible to excitotoxicity. Therefore, we treated mice with taurine and after 3 days treated them with KA. The results showed that taurine pretreatment did not induce a major susceptibility to KA. Moreover, neurodegeneration was reduced in pretreated mice. However, astrogliosis was similar to that observed in mice treated only with KA. The immunohistochemistries for calbindin-D28k, calretinin, and parvalbumin showed that these proteins were reduced as a consequence of KA treatment and of taurine treatment. However, mice pretreated with taurine prior to KA administration presented the same reduction in these proteins as mice treated with only taurine or only KA.

Role of matrix metalloproteinase-9 (MMP-9) in striatal blood-brain barrier disruption in a 3-nitropropionic acid model of Huntington's disease.

AIMS: 3-Nitropropionic acid (3-NPA) is a natural toxin that, when administered to experimental animals, reproduces the brain lesions observed in Huntington's disease, which mainly consist of selective neurodegeneration of the striatum. The lesions also include severe alterations to the blood-brain barrier (BBB), which increase its permeability to several substances including blood components and exogenous fluorescent dyes, and the concomitant degradation of some of its constituents such as endothelial cells, tight junction proteins and the basement membrane. We studied here the role of matrix metalloproteinases (MMPs)-2 and -9, also called gelatinases A and B, in the degradation of the BBB in the striatal lesions induced by the systemic administration of 3-NPA to Sprague-Dawley rats. METHODS: 3-NPA was intraperitoneally administered at a dose of 20 mg/kg once a day for 3 days. MMPs were studied by means of immunohistochemistry and in situ zymography. RESULTS: In 3-NPA-treated rats, MMP-9 was present in most of the degraded blood vessels in the injured striatum, while it was absent in vessels from non-injured tissue. In the same animals, MMP-2 staining was barely detected close to degraded blood vessels. The combination of MMP-9 immunostaining, in situ zymography and inhibitory studies of MMP-9 confirmed that net gelatinolytic activity detected in the degraded striatal blood vessels could be attributed almost exclusively to the active form of MMP-9. CONCLUSION: Our results highlight the prominent role of MMP-9 in BBB disruption in the striatal injured areas of this experimental model of Huntington's disease.

Regulation of GSK-3beta by calpain in the 3-nitropropionic acid model.

Glycogen synthase kinase-3beta (GSK-3beta) is a crucial component in the cascade of events that culminate in a range of neurodegenerative diseases. It is controlled by several pathways, including calpain-mediated cleavage. Calpain mediates in cell death induced by 3-nitropropionic acid (3-NP), but GSK-3beta regulation has not been demonstrated. Here we studied changes in total GSK-3beta protein levels and GSK-3beta phosphorylation at Ser-9 in this model. The 3-NP treatment induced GSK-3beta truncation. This regulation was dependent on calpain activation, since addition of calpeptin to the medium prevented this cleavage. While calpain inhibition prevented 3-NP-induced neuronal loss, inhibition of GSK-3beta by SB-415286 did not. Furthermore, inhibition of cdk5, a known target of calpain involved in 3-NP-induced cell death, also failed to rescue neurons in our model. Our results point to a new target of calpain and indicate possible cross-talk between calpain and GSK-3beta in the 3-NP toxicity pathway. On the basis of our findings, we propose that calpain may modulate 3-NP-induced neuronal loss.

Taurine treatment inhibits CaMKII activity and modulates the presence of calbindin D28k, calretinin, and parvalbumin in the brain.

Taurine, 2-aminoethanesulfonic acid, is present at high concentrations in many invertebrate and vertebrate systems and has several biological functions. In addition, it has been related to a neuroprotective role against several diseases such as epilepsy. In the present work, we treated mice with taurine and examined its effects on the expression of proteins in the hippocampus associated with calcium regulation. Taurine treatment alters the presence of calbindin-D28k, calretinin, and parvalbumin in the brain, mainly in the hippocampus. It also reduced CaMKII activity, indicating that taurine could alter calcium signaling pathways. However, the activity of calpain, a protease related to apoptosis induced by calcium signalling, did not change. The concentration of taurine in the hippocampus was also unaffected by the treatment. These results provide new insight into the role of taurine in calcium homeostasis.

Consensus statement of the Spanish Association Of Urology on the use of meshes in pelvic organ prolapse. / Consenso de la Asociación Española de Urología sobre el uso de mallas en la cirugía del prolapso de órganos pélvicos.

INTRODUCTION: Recently the Food and Drug Administration has banned the use of transvaginal meshes for the surgical treatment of pelvic organ prolapse (POP) in the United States. This has caused a worldwide impact on the management of pelvic floor pathology by different specialists. OBJECTIVE: To achieve a consensus on the use of meshes in the surgical treatment of POPs. ACQUISITION OF DATA/EVIDENCE: A Committee of experts of the Spanish Association of Urology (AEU) was organized to review the literature and analyze the safety and efficacy of the use of polypropylene meshes in POP surgery. RESULTS/EVIDENCE FROM THE LITERATURE: The evidence reflects that the use of meshes, compared to the use of native tissues, offers better efficacy at the expense of new complications and a higher rate of surgical reviews, these being minor in the hands of expert surgeons. CONCLUSIONS: POP surgery must be performed by experienced surgeons, properly trained and in referral centers. The patient should receive correct information about the different treatment options. Transvaginal meshes should only be indicated in complex cases and in recurrences after POP surgery. AEU PROPOSAL: Creation of a clinical guideline and a national registry for long-term evaluation. Preparation of an Informed Consent available to all professionals and patients, as well as a specific training plan to achieve better training in complex pelvic floor surgery.

Prevention of epilepsy by taurine treatments in mice experimental model.

An experimental model based on kainic acid (KA) injections replicates many phenomenological features of human temporal lobe epilepsy, the most common type of epilepsy in adults. Taurine, 2-aminoethanesulfonic acid, present in high concentrations in many invertebrate and vertebrate systems, is believed to serve several important biological functions. In addition, it is believed to have a neuroprotective role against several diseases. In the present study, an experimental mouse model based on taurine pretreatment prior to KA administration has been improved to study whether taurine has a neuroprotective effect against KA-induced behavior and cell damage. Under different treatments tested, taurine's most neuroprotective effects were observed with intraperitoneal taurine injection (150 mg/kg dosage) 12 hr before KA administration. Thus, a reduction in or total absence of seizures, together with a reduction in or even disappearance of cellular and molecular KA-derived effects, was detected in mice pretreated with taurine compared with those treated only with KA. Moreover, the use of tritiated taurine revealed taurine entry into the brain, suggesting possible changes in intracellular:extracellular taurine ratios and the triggering of pathways related to neuroprotective effects.

Evidence of calpain/cdk5 pathway inhibition by lithium in 3-nitropropionic acid toxicity in vivo and in vitro.

Lithium reduced striatal neurodegeneration induced in rats by 3-nitropropionic acid inhibiting calpain activation. Lithium prevented an increase in cdk5 activity, as shown by the levels of the co-activator p35. Myocite enhancer factor 2 (MEF2), a downstream substrate for cdk5 with pro-survival activity, showed increased phosphorylation. In primary cultures of neurons treated with 3-NP, lithium also reduced protease activity mediated by calpain, cdk5 activation and cellular death. These observations indicate that lithium has a neuroprotective effect. Lithium treatment also reduced the intracellular increase in calcium induced by 3-NP. The finding that lithium mediates the modulation of the calpain/cdk5 pathway further supports its use in the treatment of neurodegenerative diseases.

Resveratrol Modulates and Reverses the Age-Related Effect on Adenosine-Mediated Signalling in SAMP8 Mice.

Resveratrol (RSV) is a natural compound present in berries, grapes and red wine that has shown some neuroprotective properties, but the mechanism by which RSV exhibits its protective role is not very well understood yet. Little is known about the effect of RSV on adenosinergic system, a system regulated in an age-dependent manner in SAMP8 mice, widely considered as an Alzheimer's model. Therefore, the aim of the present work was to assess whether RSV intake was able to modulate the adenosine-mediated signalling in SAMP8 mice. Data showed herein clearly demonstrate the ability of RSV to modulate adenosine receptor gene expression as well as transduction pathway mediated by receptors expressed on plasma membrane. Interestingly, this polyphenol was able to reverse the age-related loss of adenosine A1 receptors and its corresponding signalling pathway. Moreover, adenosine A2A receptors were not modulated by aging or RSV, but A2A-mediated signalling was completely desensitized after RSV treatment compared to untreated mice. Enzymes involved on adenosine metabolism, such as 5'-nucleotidase and adenosine deaminase, were found to be reduced after RSV treatment, but adenosine levels remained unchanged. Nevertheless, an age-related decrease on 5'-nucleotidase activity and adenosine and related metabolite levels was observed. In conclusion, our data show that RSV modulates adenosine-mediated signalling, strongly suggesting that the role of RSV via adenosine receptor signalling and its modulation of neurotransmission in neurodegenerative diseases should be considered as new therapeutic target for RSV neuroprotective effect.

Apoptotic mechanisms involved in neurodegenerative diseases: experimental and therapeutic approaches.

Alzheimer's disease (AD) and Parkinson's disease (PD) are two of the most significant neurodegenerative disorders in the developed world. However, although these diseases were described almost a century ago, the molecular mechanisms that lead to the neuronal cell death associated with these diseases are not yet clear, and vigorous research efforts have failed to identify effective treatment options. In the present review, we evaluate the potential mechanisms underlying apoptosis and neuronal death in neurodegenerative disorders. A role for mitochondria in the release of proapoptotic proteins, such as cytochrome c and apoptosis-inducing factor (AIF) etc., is discussed along with key processes involving oxidative stress and activation of glutamate receptors. We also deliberate the implication of DNA damage, primarily p53 induction and reentry in the cell cycle. Finally, we postulate that multitargeting therapies comprising antioxidants, cell cycle inhibitors and modulating agents of COX-2 or c-JUN kinase pathways could be suitable strategies to prevent or delay the process of neuronal cell death in neurodegenerative disorders. Thus, the aim of this review is to discuss the pathways involved in the pathogenesis of neurodegenerative diseases such as AD, PD and Huntington's disease (HD). Furthermore, current and future pharmacotherapeutics will be considered.

Resveratrol modulates response against acute inflammatory stimuli in aged mouse brain.

With upcoming age, the capability to fight against harmful stimuli decreases and the organism becomes more susceptible to infections and diseases. Here, the objective was to demonstrate the effect of dietary resveratrol in aged mice in potentiating brain defenses against LipoPolySaccharide (LPS). Acute LPS injection induced a strong proinflammatory effect in 24-months-old C57/BL6 mice hippocampi, increasing InterLeukin (Il)-6, Tumor Necrosis Factor-alpha (Tnf-α), Il-1ß, and C-X-C motif chemokine (Cxcl10) gene expression levels. Resveratrol induced higher expression in those cytokines regarding to LPS. Oxidative Stress (OS) markers showed not significant changes after LPS or resveratrol, although for resveratrol treated groups a slight increment in most of the parameters studies was observed, reaching signification for NF-kB protein levels and iNOS expression. However, Endoplasmic Reticulum (ER) stress markers demonstrated significant changes in resveratrol-treated mice after LPS treatment, specifically in eIF2α, BIP, and ATF4. Moreover, as described, resveratrol is able to inhibit the mechanistic Target of Rapamycin (mTOR) pathway and this effect could be linked to (eIF2α) phosphorylation and the increase in the expression of the previously mentioned proinflammatory genes as a response to LPS treatment in aged animals. In conclusion, resveratrol treatment induced a different cellular response in aged animals when they encountered acute inflammatory stimuli.

Glycogen synthase kinase-3 is involved in the regulation of the cell cycle in cerebellar granule cells.

Recent studies have demonstrated that neuronal reentry in the cell cycle and specifically the expression of the transcription factor E2F-1, constitutes a pathway that may be involved in neuronal apoptosis after serum and potassium withdrawal. Other enzymes such as glycogen synthase kinase-3beta (GSK-3beta) are also involved in this apoptotic stimulus, and thus in the process of neuronal cell death. Primary cerebellar granule cells (CGNs) were used in this study to determine whether pharmacological inhibition of GSK-3beta is involved in neuronal modulation of the cell cycle, and specifically in the regulation of E2F-1 and retinoblastoma protein (Rb). CGNs showed a dramatic increase in GSK-3beta activity after 2h of serum and potassium deprivation. Immunoblot and activity assays revealed that lithium and SB415286 inhibit fully the activation of GSK-3beta and attenuate the expression of cyclin D, cyclin E, pRb phosphorylation and the transcription factor E2F-1. These data were confirmed using AR-014418, a selective GSK-3beta inhibitor that prevents the expression of cell-cycle proteins. Our data indicate that GSK-3beta inhibition regulates, in part, the cell cycle in CGNs by inhibiting Rb phosphorylation and thus inhibiting E2F-1 activity. However, the selective inhibition of GSK-3beta with AR-A014418 had not effect on cell viability or apoptosis mediated by S/K withdrawal. Furthermore, our results suggest that selective GSK-3beta inhibition is not sufficient to protect against apoptosis in this S/K withdrawal model, indicating that Li(+) and SB415286 neuroprotective effects are mediated by the inhibition of additional targets to GSK3beta. Therefore, there is a connection between cell cycle and GSK-3beta activation and that these, along with other mechanisms, are involved in the molecular paths leading to the apoptotic process of rat CGNs triggered by S/K withdrawal.

Comparative analysis of the effects of resveratrol in two apoptotic models: inhibition of complex I and potassium deprivation in cerebellar neurons.

The mechanism involved in neuronal apoptosis is largely unknown. Studies performed on neuronal cell cultures provide information about the pathways which orchestrate the process of neuronal loss and potential drugs for the treatment of neurological disorders. In the present study we select resveratrol, a natural antioxidant, as a potential drug for the treatment of neurodegenerative diseases. We evaluate the neuroprotective effects of resveratrol in two apoptotic models in rat cerebellar granule neurons (CGNs): the inhibition of mitochondrial complex I using 1-methyl-4-phenylpyridinium (MPP(+)) (an in vitro model of Parkinson's disease) and serum potassium withdrawal. We study the role of the mammalian silent information regulator 2 (SIRT1) in the process of neuroprotection mediated by resveratrol. Because recent studies have demonstrated that SIRT1 is involved in cell survival and has antiaging properties, we also measured changes in the expression of this protein after the addition of these two apoptotic stimuli. MPP(+)--induced loss of cell viability and apoptosis in CGNs was prevented by the addition of RESV (1 microM to 100 microM). However, the neuroprotective effects were not mediated by the activation of SIRT1, since sirtinol-an inhibitor of this enzyme--did not attenuate them. Furthermore MPP(+) decreases the protein expression of SIRT1. RESV did not prevent serum potassium withdrawal-induced apoptosis although it did completely attenuate oxidative stress production by these apoptotic stimuli. Furthermore, serum potassium withdrawal increases the expression of SIRT1. Our results indicate that the antiapoptotic effects of RESV in MPP(+) are independent of the stimulation of SIRT1 and depend on its antioxidant properties. Furthermore, because SIRT1 is involved in neuronal survival depending on the apoptotic stimuli, changes in the expression of SIRT1 could be involved in the regulation of the apoptotic route.

Inhibition of cyclin-dependent kinases is neuroprotective in 1-methyl-4-phenylpyridinium-induced apoptosis in neurons.

The biochemical pathways involved in neuronal cell death in Parkinson's disease are not completely characterized. Mitochondrial dysfunction, specifically alteration of the mitochondrial complex I, is the primary target of the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) induced apoptosis in neurons. In the present study, we examine the role of caspase-dependent and -independent routes in MPP+-induced apoptosis in rat cerebellar granule neurons (CGNs). We show a distinct increase in the expression of the cell cycle proteins cyclin D, cyclin E, cdk2, cdk4 and the transcription factor E2F-1 following a MPP+ treatment of CGNs. Flavopiridol (FLAV), a broad inhibitor of cyclin-dependent kinases (CDKs), attenuated the neurotoxic effects of MPP+ and significantly attenuates apoptosis mediated by MPP+ 200 microM. Likewise, the antioxidant vitamin E (vit E) increases neuronal cell viability and attenuates apoptosis induced by MPP+. Moreover, the expression levels of cyclin D and E2F-1 induced by this parkinsonian neurotoxin were also attenuated by vit E. Since, the broad-spectrum caspase inhibitor zVAD-fmk did not attenuate MPP+-induced apoptosis in CGNs, our data provide a caspase-independent mechanism mediated by neuronal reentry in the cell cycle and increased expression of the pro-apoptotic transcription factor E2F-1. Our results also suggest a potential role of oxidative stress in neuronal reentry in the cell cycle mediated by MPP+. Finally, our data further support the therapeutic potential of flavopiridol, for the treatment of Parkinson's disease.

Kainate induces AKT, ERK and cdk5/GSK3beta pathway deregulation, phosphorylates tau protein in mouse hippocampus.

Acute treatment with kainate 30 mg/kg (KA) produced behavioral alterations and reactive gliosis. However, it did not produce major death of mouse hippocampal neurons, indicating that concentrations were not cytotoxic. KA caused rapid and temporal Erk phosphorylation (at 6h) and Akt dephosphorylation (1-3 days). Concomitantly, the activation of GSK3beta was increased 1-3 days after KA. After 7 days, a reduction in GSK3beta activation was observed. Caspase-3 activity increased, but to a lesser extent than calpain activation (measured by fluorimetry and calpain-cleaved alpha-spectrin). As calpain is involved in cdk5 activation, and cdk5 is related to GSK3beta, the cdk5/p25 pathway was examined. Results showed that the p25/p35 ratio in KA-injected mice for 3 days was 73.6% higher than control levels. However, no changes in cdk5 expression were detected. Both Western blot and immunohistochemistry against p-Tau(Thr(231)) indicated an increase at this phosphorylated site of tau protein. Indeed an increase in p-Tau(Ser(199)) and p-Tau(Ser(396)) was observed by Western blot. Our results demonstrate that tau hyperphosphorylation, induced by KA, is due to an increase in GSK3beta/cdk5 activity in combination with an inactivation of Akt. This indicates that the calpain/cdk5 pathway for tau phosphorylation has a potential role in delayed apoptotic death evoked by excitotoxicity. Moreover, the subsequent activation of caspase and calpain proteases leads to dephosphorylation of tau, thus increasing microtubular destructuration. Taken together, our results provide new insights in the activation of several kinase-pathways implicated in cytoskeletal alterations that are a common feature of neurodegenerative diseases.

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.