A vacuolar cysteine proteinase, designated SH-EP, is expressed in the cotyledon of germinated Vigna mungo seeds and is responsible for the degradation of storage proteins. SH-EP is a characteristic vacuolar proteinase possessing a COOH-terminal endoplasmic reticulum (ER) retention sequence, KDEL. In this work, immunocytochemical analysis of the cotyledon cells of germinated V. mungo seeds was performed using seven kinds of antibodies to identify the intracellular transport pathway of SH-EP from ER to protein storage vacuoles. A proform of SH-EP synthesized in ER accumulated at the edge or middle region of ER where the transport vesicle was formed. The vesicle containing a large amount of proSH-EP, termed KV, budded off from ER, bypassed the Golgi complex, and was sorted to protein storage vacuoles. This massive transport of SH-EP via KV was thought to mediate dynamic protein mobilization in the cotyledon cells of germinated seeds. We discuss the possibilities that the KDEL sequence of KDEL-tailed vacuolar cysteine proteinases function as an accumulation signal at ER, and that the mass transport of the proteinases by ER-derived KV-like vesicle is involved in the protein mobilization of plants.

Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For Pantoea stewartii subsp. stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner. Two genes, esaI and esaR, encode essential regulatory proteins for quorum sensing. EsaI is the AHL signal synthase, and EsaR is the cognate gene regulator. esaI, DeltaesaR, and DeltaesaI-esaR mutations were constructed to establish the regulatory role of EsaR. We report here that strains containing an esaR mutation produce high levels of EPS independently of cell density and in the absence of the AHL signal. Our data indicate that quorum-sensing regulation in P. s. subsp. stewartii, in contrast to most other described systems, uses EsaR to repress EPS synthesis at low cell density, and that derepression requires micromolar amounts of AHL. In addition, derepressed esaR strains, which synthesize EPS constitutively at low cell densities, were significantly less virulent than the wild-type parent. This finding suggests that quorum sensing in P. s. subsp. stewartii may be a mechanism to delay the expression of EPS during the early stages of infection so that it does not interfere with other mechanisms of pathogenesis.

Extracellular polymeric substances are the construction materials for microbial aggregates such as biofilms, flocs ("planktonic biofilms") and sludge. Their major components are not only polysaccharides but also proteins and in some cases lipids, with minor contents of nucleic acids and other biopolymers. In the EPS, biofilm organisms can establish stable arrangements and function multicellularly as synergistic microconsortia. The matrix facilitates the retention of exoenzymes, cellular debris and genetic material; it can be considered as a microbial recycling yard. Gradients can develop due to the physiological activity and the fact that diffusive mass transport prevails over convective transport in the matrix. Biofilm cells tolerate higher concentrations of many biocides. The EPS matrix sequesters nutrients from the water phase. In photosynthetic communities, EPS molecules can function as light transmitters and provide photons to organisms located deeper in a microbial mat. The EPS matrix is a dynamic system, constructed by the organisms and responding to environmental changes. It enables the cells to function in a manner similar to multicellular organisms.

Transmission of a protozoan parasite from a vertebrate to invertebrate host is accompanied by cellular differentiation. The signals from the environment that trigger the process are poorly understood. The model parasite Trypanosoma brucei proliferates in the mammalian bloodstream and in the tsetse fly. On ingestion by the tsetse, the trypanosome undergoes a rapid differentiation that is marked by replacement of the variant surface glycoprotein (VSG) coat with GPI-anchored EP and GPEET procyclins. Here we show that a cold shock of DeltaT>> 15 degrees C is sufficient to reversibly induce high-level expression of the insect stage-specific EP gene in the mammalian bloodstream stages of T. brucei. The 3'-UTR of the EP mRNA is necessary and sufficient for the increased expression. During cold shock, EP protein accumulates in the endosomal compartment in the proliferating, slender, bloodstream stage, whereas the EP is present on the plasma membrane in the quiescent, stumpy, bloodstream stage. Thus, there is a novel developmentally regulated cell surface access control mechanism for a GPI-anchored protein. In addition to inducing EP expression, cold shock results in the acquisition of sensitivity to micromolar concentrations of cis-aconitate and citrate by stumpy but not slender bloodstream forms. The cis-aconitate and citrate commit stumpy bloodstream cells to differentiation to the procyclic stage along with rapid initial proliferation. We propose a hierarchical model of three events that regulate differentiation after transmission to the tsetse: sensing the temperature change, surface access of a putative receptor, and sensing of a chemical cue.

Production of complex extracellular polysaccharides (EPSs) by the nitrogen-fixing soil bacterium Sinorhizobium meliloti is required for efficient invasion of root nodules on the host plant alfalfa. Any one of three S. meliloti polysaccharides, succinoglycan, EPS II, or K antigen, can mediate infection thread initiation and extension (root nodule invasion) on alfalfa. Of these three polysaccharides, the only symbiotically active polysaccharide produced by S. meliloti wild-type strain Rm1021 is succinoglycan. The expR101 mutation is required to turn on production of symbiotically active forms of EPS II in strain Rm1021. In this study, we have determined the nature of the expR101 mutation in S. meliloti. The expR101 mutation, a spontaneous dominant mutation, results from precise, reading frame-restoring excision of an insertion sequence from the coding region of expR, a gene whose predicted protein product is highly homologous to the Rhizobium leguminosarum bv. viciae RhiR protein and a number of other homologs of Vibrio fischeri LuxR that function as receptors for N-acylhomoserine lactones (AHLs) in quorum-sensing regulation of gene expression. S. meliloti ExpR activates transcription of genes involved in EPS II production in a density-dependent fashion, and it does so at much lower cell densities than many quorum-sensing systems. High-pressure liquid chromatographic fractionation of S. meliloti culture filtrate extracts revealed at least three peaks with AHL activity, one of which activated ExpR-dependent expression of the expE operon.

Cyanobacterial extracellular polymeric substances (EPS) are mainly composed of high-molecular-mass heteropolysaccharides, with variable composition and roles according to the microorganism and the environmental conditions. The number of constituents - both saccharidic and nonsaccharidic - and the complexity of structures give rise to speculations on how intricate their biosynthetic pathways could be, and how many genes may be involved in their production. However, little is known regarding the cyanobacterial EPS biosynthetic pathways and regulating factors. This review organizes available information on cyanobacterial EPS, including their composition, function and factors affecting their synthesis, and from the in silico analysis of available cyanobacterial genome sequences, proposes a putative mechanism for their biosynthesis.

Electroporation (EP) has been used in basic research for the past 25 years to aid in the transfer of DNA into cells in vitro. EP in vivo enhances transfer of DNA vaccines and therapeutic plasmids to the skin, muscle, tumors, and other tissues resulting in high levels of expression, often with serological and clinical benefits. The recent interest in nonviral gene transfer as treatment options for a vast array of conditions has resulted in the refinement and optimization of EP technology. Current research has revealed that EP can be successfully used in many species, including humans. Clinical trials are currently under way. Herein, the transition of EP from basic science to clinical trials will be discussed.

OBJECTIVE

The objectives of our study were to determine whether dynamic contrast-enhanced MRI performed at 3 T and analyzed using a pharmacokinetic model improves the diagnostic performance of MRI for the detection of prostate cancer compared with conventional T2-weighted imaging, and to determine which pharmacokinetic parameters are useful in diagnosing prostate cancer.

METHODS

This prospective study included 50 consecutive patients with biopsy-proven prostate cancer who underwent imaging of the prostate on a 3-T scanner with a combination of a sensitivity-encoding (SENSE) cardiac coil and an endorectal coil. Scans were obtained at least 5 weeks after biopsy. T2-weighted turbo spin-echo images were obtained in three planes, and dynamic contrast-enhanced images were acquired during a single-dose bolus injection of gadopentetate dimeglumine (0.1 mmol/kg). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were estimated for T2-weighted and dynamic contrast-enhanced MRI. The following pharmacokinetic modeling parameters were determined and compared for cancer, inflammation, and healthy peripheral zone: K(trans) (forward volume transfer constant), k(ep) (reverse reflux rate constant between extracellular space and plasma), v(e) (the fractional volume of extracellular space per unit volume of tissue), and the area under the gadolinium concentration curve (AUGC) in the first 90 seconds after injection.

RESULTS

Pathologically confirmed cancers in the peripheral zone of the prostate were characterized by their low signal intensity on T2-weighted scans and by their early enhancement, early washout, or both on dynamic contrast-enhanced MR images. The overall sensitivity, specificity, PPV, and NPV of T2-weighted imaging were 94%, 37%, 50%, and 89%, respectively. The sensitivity, specificity, PPV, and NPV of dynamic contrast-enhanced MRI were 73%, 88%, 75%, and 75%, respectively. K(trans), k(ep), and AUGC were significantly higher (p < 0.001) in cancer than in normal peripheral zone. The ve parameter was not significantly associated with prostate cancer.

CONCLUSIONS

MRI of the prostate performed at 3 T using an endorectal coil produces high-quality T2-weighted images; however, specificity for prostate cancer is improved by also performing dynamic contrast-enhanced MRI and using pharmacokinetic parameters, particularly K(trans) and k(ep), for analysis. These results are comparable to published results at 1.5 T.

In Parkinson's disease (PD) and dementia with Lewy bodies (DLB) alpha-synuclein (alphaS) pathology is seen that displays a predictable topographic distribution. There are two staging/categorization systems, i.e. Braak's and McKeith's, currently in use for the assessment of alphaS pathology. The aim of these diagnostic strategies in pathology is, in addition to assess the stage/severity of pathology, to assess the probabilities of the related clinical symptomatology i.e. dementia and extrapyramidal symptoms (EPS). Herein, we assessed the applicability of these two staging/categorization systems and the frequency of dementia and EPS in a cohort of 226 alphaS-positive-subjects. These subject were selected from a large autopsy sample (n = 1,720), irrespective of the clinical presentation, based on the detection of alphaS-immunoreactivity (IR) in one of the most vulnerable nuclei; in the dorsal motor nucleus of vagus, substantia nigra and basal forebrain. The frequency of alphaS-IR lesions in this large cohort was 14% (248 out of 1,720). If applicable, each of the 226 subjects with all required material available was assigned a neuropathological stage/category of PD/DLB and finally the neuropathological data was analyzed in relation to dementia and EPS. 83% of subjects showed a distribution pattern of alphaS-IR that was compatible with the current staging/categorization systems. Around 55% of subjects with widespread alphaS pathology (Braak's PD stages 5-6) lacked clinical signs of dementia or EPS. Similarly, in respect to those subjects that fulfilled the McKeith criteria for diffuse neocortical category and displaying only mild concomitant Alzheimer's disease-related pathology, only 48% were demented and 54% displayed EPS. It is noteworthy that some subjects (17%) deviated from the suggested caudo-rostral propagation suggesting alternative routes of progression, perhaps due to concomitant diseases and genetic predisposition. In conclusion, our results do indeed confirm that current staging/categorization systems can readily be applied to most of the subjects with alphaS pathology. However, finding that around half of the subjects with abundant alphaS pathology remain neurologically intact is intriguing and raises the question whether we do assess the actual disease process.

OBJECTIVE

While all second-generation antipsychotics (SGAs) are promoted for having a low risk of extrapyramidal side effects (EPS), clinical observations suggest differences between the various agents. Nevertheless, this question has never been examined in a systematic review and meta-analysis of head-to-head comparisons.

METHODS

We searched the register of the Cochrane schizophrenia group (last search May 2007), supplemented by MEDLINE (last search July 2009) for randomized, blinded studies comparing the following SGAs in the treatment of schizophrenia or related disorders: amisulpride, aripiprazole, clozapine, olanzapine, quetiapine, risperidone, sertindole, ziprasidone, and zotepine. At least 3 reviewers extracted the data independently. The primary outcome was "use of antiparkinson medication." The results were combined in a meta-analysis.

RESULTS

We included 54 studies with 116 arms. Risperidone was associated with more use of antiparkinson medication than clozapine, olanzapine, quetiapine, and ziprasidone. Ziprasidone showed more use of antiparkinson medication than olanzapine and quetiapine and zotepine more than clozapine. There was no significant difference between amisulpride and its comparators (olanzapine, risperidone, or ziprasidone). Quetiapine showed significantly less use of antiparkinson medication than the 3 other SGAs it was compared with (olanzapine, risperidone, and ziprasidone). Scale-derived data (Barnes Akathisia Scale and Simpson Angus Scale) were limited.

CONCLUSIONS

Our meta-analysis demonstrates that there are differences between the SGAs in their ability to induce EPS that clinicians consider warrant treatment with antimuscarinic drugs. Even though the differences were relatively small, they might be important for individual patients and should be taken into account in drug choice.

Male gametes are produced throughout reproductive life by a classic stem cell mechanism. However, little is known about the molecular mechanisms for lineage production that maintain male germ-line stem cell (GSC) populations, regulate mitotic amplification divisions, and ensure germ cell differentiation. Here we utilize the Drosophila system to identify genes that cause defects in the male GSC lineage when forcibly expressed. We conducted a gain-of-function screen using a collection of 2050 EP lines and found 55 EP lines that caused defects at early stages of spermatogenesis upon forced expression either in germ cells or in surrounding somatic support cells. Most strikingly, our analysis of forced expression indicated that repression of bag-of-marbles (bam) expression in male GSC is important for male GSC survival, while activity of the TGF beta signal transduction pathway may play a permissive role in maintenance of GSCs in Drosophila testes. In addition, forced activation of the TGF beta signal transduction pathway in germ cells inhibits the transition from the spermatogonial mitotic amplification program to spermatocyte differentiation.

Biofilms, accumulations of microorganisms at interfaces, have been described for every aqueous system supporting life. The structure of these microbial communities ranges from monolayers of scattered single cells to thick, mucous structures of macroscopic dimensions (microbial mats; algal-microbial associations; trickling filter biofilms). During recent years the structure of biofilms from many different environments has been documented and evaluated by use of a broad variety of microscopic, physico-chemical and molecular biological techniques, revealing a generally complex 3D structure. Parallel to these investigations more and more complex mathematical models and simulations were developed to explain the development, structures, and interactions of biofilms. The forces determining the spatial structure of biofilms, including microcolonies, extracellular polymeric substances (EPS), and channels, are still the subject of controversy. To achieve conclusive explanations for the structures observed in biofilms the cooperation of both fields of investigation, modelling and experimental research, is necessary. The expanding field of molecular techniques not only allows more and more detailed documentation of the spatial distribution of species, but also of functional activities of single cells in their biofilm environment. These new methods will certainly reveal new insights in the mechanisms involved in the developmental processes involved in the formation and behavior of biofilms.

Specific complex interactions between soil bacteria belonging to Rhizobium, Sinorhizobium, Mesorhizobium, Phylorhizobium, Bradyrhizobium and Azorhizobium commonly known as rhizobia, and their host leguminous plants result in development of root nodules. Nodules are new organs that consist mainly of plant cells infected with bacteroids that provide the host plant with fixed nitrogen. Proper nodule development requires the synthesis and perception of signal molecules such as lipochitooligosaccharides, called Nod factors that are important for induction of nodule development. Bacterial surface polysaccharides are also crucial for establishment of successful symbiosis with legumes. Sugar polymers of rhizobia are composed of a number of different polysaccharides, such as lipopolysaccharides (LPS), capsular polysaccharides (CPS or K-antigens), neutral beta-1, 2-glucans and acidic extracellular polysaccharides (EPS). Despite extensive research, the molecular function of the surface polysaccharides in symbiosis remains unclear. This review focuses on exopolysaccharides that are especially important for the invasion that leads to formation of indetermined (with persistent meristem) type of nodules on legumes such as clover, vetch, peas or alfalfa. The significance of EPS synthesis in symbiotic interactions of Rhizobium leguminosarum with clover is especially noticed. Accumulating data suggest that exopolysaccharides may be involved in invasion and nodule development, bacterial release from infection threads, bacteroid development, suppression of plant defense response and protection against plant antimicrobial compounds. Rhizobial exopolysaccharides are species-specific heteropolysaccharide polymers composed of common sugars that are substituted with non-carbohydrate residues. Synthesis of repeating units of exopolysaccharide, their modification, polymerization and export to the cell surface is controlled by clusters of genes, named exo/exs, exp or pss that are localized on rhizobial megaplasmids or chromosome. The function of these genes was identified by isolation and characterization of several mutants disabled in exopolysaccharide synthesis. The effect of exopolysaccharide deficiency on nodule development has been extensively studied. Production of exopolysaccharides is influenced by a complex network of environmental factors such as phosphate, nitrogen or sulphur. There is a strong suggestion that production of a variety of symbiotically active polysaccharides may allow rhizobial strains to adapt to changing environmental conditions and interact efficiently with legumes.

Adaptive regulatory T cells (Tr1) are induced in the periphery upon encountering cognate antigens. In cancer, their frequency is increased; however, Tr1-mediated suppression mechanisms are not yet defined. Here, we evaluate the simultaneous involvement of ectonucleotidases (CD39/CD73) and cyclooxygenase 2 (COX-2) in Tr1-mediated suppression. Human Tr1 cells were generated from peripheral blood mononuclear cell-derived, sorted CD4(+)CD25(-) T cells and incubated with autologous immature dendritic cells, irradiated COX-2(+) or COX-2(-) tumor cells, and IL-2, IL-10, and IL-15 (each at 10-15 IU/ml) for 10 days as described (Bergmann, C., Strauss, L., Zeidler, R., Lang, S., and Whiteside, T. L. (2007) Cancer Immunol. Immunother. 56, 1429-1442). Tr1 were phenotyped by multicolor flow cytometry, and suppression of proliferating responder cells was assessed in carboxyfluorescein diacetate succinimidyl ester-based assays. ATP hydrolysis was measured using a luciferase detection assay, and levels of adenosine or prostaglandin E(2) (PGE(2)) in cell supernatants were analyzed by mass spectrometry or ELISA, respectively. Intracellular cAMP levels were measured by enzyme immunoassay. The COX-2(+) tumor induced a greater number of Tr1 than COX-2(-) tumor (p < 0.05). Tr1 induced by COX-2(+) tumor were more suppressive, hydrolyzed more exogenous ATP (p < 0.05), and produced higher levels of adenosine and PGE(2) (p < 0.05) than Tr1 induced by COX-2(-) tumor. Inhibitors of ectonucleotidase activity, A(2A) and EP(2) receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE(4) inhibitor, increased the intracellular cAMP level in responder cells and their susceptibility to Tr1-mediated suppression. Tr1 present in tumors or the peripheral blood of head and neck squamous cell carcinoma patients co-expressed COX-2, CD39, and CD73. A concomitant inhibition of PGE(2) and adenosine via the common intracellular cAMP pathway might be a novel approach for improving results of immune therapies for cancer.

To investigate the role of retinoid X receptors (RXRs) in epidermal homeostasis, we generated RXRalphabeta(ep-/-) somatic mutants in which both RXRalpha and RXRbeta are selectively ablated in epidermal keratinocytes of adult mice. These mice develop a chronic dermatitis mimicking that observed in atopic dermatitis (AD) patients. In addition, they exhibit immunological abnormalities including elevated serum levels of IgE and IgG, associated with blood and tissue eosinophilia, indicating that keratinocyte-selective ablation of RXRs also generates a systemic syndrome similar to that found in AD patients. Furthermore, the profile of increased expression of cytokines and chemokines in skin of keratinocyte-selective RXRalphabeta-ablated mutants was typical of a T helper 2-type inflammation, known to be crucially involved in human AD pathogenesis. Finally, we demonstrate that thymic stromal lymphopoietin, whose expression is rapidly and strongly induced in RXRalphabeta-ablated keratinocytes, plays a key role in initiating the skin and systemic AD-like pathologies.

Cerebral concussion is both the most common and most puzzling type of traumatic brain injury (TBI). It is normally produced by acceleration (or deceleration) of the head and is characterized by a sudden brief impairment of consciousness, paralysis of reflex activity and loss of memory. It has long been acknowledged that one of the most worthwhile techniques for studying the acute pathophysiology of concussion is by the recording of neurophysiological activity such as the electroencephalogram (EEG) and sensory evoked potentials (EPs) from experimental animals. In the first parts of this review, the majority of such studies conducted during the past half century are critically reviewed. When potential methodological flaws and limitations such as anesthetic protocols, infliction of multiple blows and delay in onset of recordings were taken into account, two general principles could be adduced. First, the immediate post-concussive EEG was excitatory or epileptiform in nature. Second, the cortical EP waveform was totally lost during this period. In the second parts of this review, five theories of concussion which have been prominent during the past century are summarized and supportive evidence assessed. These are the vascular, reticular, centripetal, pontine cholinergic and convulsive hypotheses. It is concluded that only the convulsive theory is readily compatible with the neurophysiological data and can provide a totally viable explanation for concussion. The chief tenet of the convulsive theory is that since the symptoms of concussion bear a strong resemblance to those of a generalized epileptic seizure, then it is a reasonable assumption that similar pathobiological processes underlie them both. Further, it is demonstrated that EPs and EEGs recorded acutely following concussive trauma are indeed the same or similar to those obtained following the induction of a state of generalized seizure activity (GSA). According to the present incarnation of the convulsive theory, the energy imparted to the brain by the sudden mechanical loading of the head may generate turbulent rotatory and other movements of the cerebral hemispheres and so increase the chances of a tissue-deforming collision or impact between the cortex and the boney walls of the skull. In this conception, loss of consciousness is not orchestrated by disruption or interference with the function of the brainstem reticular activating system. Rather, it is due to functional deafferentation of the cortex as a consequence of diffuse mechanically-induced depolarization and synchronized discharge of cortical neurons. A convulsive theory can also explain traumatic amnesia, autonomic disturbances and the miscellaneous collection of symptoms of the post-concussion syndrome more adequately than any of its rivals. In addition, the symptoms of minor concussion (a.k.a. being stunned, dinged, or dazed) are often strikingly similar to minor epilepsy such as petit mal. The relevance of the convulsive theory to a number of associated problems is also discussed. These include the relationship between concussion and more serious types of closed head injury, the utility of animal models of severe brain trauma, the etiology of the cognitive deficits which may linger long after a concussive injury, the use of concussive (captive bolt) techniques to stun farm animals prior to slaughter and the question of why some animals (such as the woodpecker) can tolerate massive accelerative forces without being knocked out.

The clinicopathologic characteristics of 69 cases of eccrine porocarcinoma (EP) have been studied. Seven cases of purely in situ disease are included. Forty patients were female, 29 male with ages ranging from 29 to 91 years (mean 73 years). The lower extremity represented the single most common site (44%). Other common sites were the trunk (15 cases, 24%) and head (11 cases, 18%). The histologic diagnosis of EP was predicated on the basis of an irregular tumor at least partly formed of characteristic poromatous basaloid epithelial cells displaying ductal differentiation, and significant cytologic atypia. Forty-seven tumors (68%) contained mature well-formed eccrine ducts having an eosinophilic luminal cuticle, with the remaining tumors containing small ill-formed ducts and/or intracytoplasmic lumina. All ducts were discernible via light microscopy and in 49 cases were highlighted with DPAS stain and/or CEA/EMA immunocytochemistry. A variant with a broad pushing tumor margin and marked nuclear pleomorphism showed some resemblance to proliferative bowenoid dysplasia. In 11 cases (18%) the tumors appeared to arise in continuity with a benign preexistent poroma. A variety of histologic patterns were displayed including clear, squamous, and spindle cell differentiation, mucus cell metaplasia, and colonization by melanocytes. Lymphovascular invasion was present in 9 cases (15%). Three cases showed pagetoid extension of malignant cells (epidermotropism) and appeared to be multifocal. Follow-up was available in 54 patients (78%) with 9 (17%) experiencing local recurrence, 10 developing lymph node metastases (19%), and 6 (11%) experiencing distant metastases or death. Mitoses, the presence of lymphovascular invasion, and tumor depth >7 mm were associated with a poorer prognosis. Dividing tumors into those with a "pushing" or "infiltrating" advancing margin was also predictive of outcome with the latter having an increased risk of local recurrence. This report, the largest series of EP to date, suggests that the incidence of aggressive behavior is less than popularly believed. Furthermore, EP can display a wide variety of histologic patterns that may lead to diagnostic error in the unwary. The large number of cases in this series enables a reliable evaluation of prognostic parameters. A more aggressive clinical course may be indicated by more than 14 mitoses per high power field (hazard ratio [HR] for death 17.0, 95% confidence interval [CI] 2.71-107), lymphovascular invasion by tumor (HR 4.41, CI 1.13-17.2), and depth >7 mm (HR 5.49, CI 1.0-30.3). Thus, mitoses, lymphovascular invasion, and tumor depth should be evaluated in these tumors. We also suggest that tumors presenting an "infiltrative" advancing margin are particularly prone to local recurrence and require wide excision with close attention to the surgical margins by the reporting pathologist.

Obesity is an important risk factor for esophageal adenocarcinoma (EAC), and elevated serum leptin is characteristic of obesity. We hypothesized that leptin may have biological effects in promoting esophageal adenocarcinoma and examined the effects of leptin on the OE33 Barrett's-derived EAC line. Proliferation was assessed by dimethylthiazoldiphenyltetra-zoliumbromide and 5-bromo-2'-deoxyuridine incorporation assays and apoptosis by ELISA of intracellular nucleosomes. Intracellular signaling was examined using specific pharmacological inhibitors and direct detection of phosphorylated active kinases. Expression of the long and short leptin receptors by OE33 cells was confirmed by RT-PCR, Western blotting and immunocytochemistry. Leptin stimulated OE33 cell proliferation in a dose-dependent manner and inhibited apoptosis. These effects were dependent on cyclooxygenase (COX)-2 and replicated by adding prostaglandin E2 (PGE2). The effects of PGE2 and leptin were abolished by the EP-4 antagonist AH23848. ERK, p38 MAPK, phosphatidylinositol 3'-kinase/Akt, and Janus tyrosine kinase (JAK)-2 were activated upstream of COX-2 induction, whereas the epidermal growth factor receptor and c-Jun NH2-terminal kinase (JNK) were downstream of COX-2. The activation of ERK and Akt but not p38 MAPK was JAK2 dependent. PGE2 stimulated phosphorylation of JNK in an EGF receptor-dependent manner, and activation of the epidermal growth factor receptor required protein kinase C, src, and matrix metalloproteinase activities. We conclude that leptin stimulates cell proliferation and inhibits apoptosis in OAC cells via ERK, p38 MAPK, phosphatidylinositol 3'-kinase/Akt, and JAK2-dependent activation of COX-2 and PGE2 production. Subsequent PGE2-mediated transactivation of the epidermal growth factor receptor and JNK activation are essential to the leptin effects. These effects may contribute to the greatly increased risk of esophageal adenocarcinoma in obesity.

Cell permeabilization by electric pulses (EPs), or electroporation, has been well established as a tool to indiscriminately increase membrane flows of water solutes down the concentration and voltage gradients. However, we found that EPs of nanosecond duration (nsEPs) trigger formation of voltage-sensitive and inward-rectifying membrane pores. NsEP-treated cells remain mostly impermeable to propidium, suggesting that the maximum pore size is approximately 1nm. The ion-channel-like properties of nsEP-opened nanopores vanish if they break into larger, propidium-permeable "conventional" pores. However, nanopores can be stable for many minutes and significantly impact cell electrolyte and water balance. Multiple nsEPs cause fast cell swelling and blebbing, whereas opening of larger pores with digitonin abolishes swelling and causes blebs to implode. The lipid nature of nsEP-opened nanopores is confirmed by fast externalization of phosphatidylserine residues. Nanopores constitute a previously unexplored ion transport pathway that supplements classic ion channels but is distinctly different from them.

Dental plaque has the properties of a biofilm, similar to other biofilms found in the body and the environment. Modern molecular biological techniques have identified about 1000 different bacterial species in the dental biofilm, twice as many as can be cultured. Oral biofilms are very heterogeneous in structure. Dense mushroom-like structures originate from the enamel surface, interspersed with bacteria-free channels used as diffusion pathways. The channels are probably filled with an extracellular polysaccharide (EPS) matrix produced by the bacteria. Bacteria in biofilms communicate through signaling molecules, and use this "quorum-sensing" system to optimize their virulence factors and survival. Bacteria in a biofilm have a physiology different from that of planktonic cells. They generally live under nutrient limitation and often in a dormant state. Such "sleepy" bacteria respond differently to antibiotics and antimicrobials, because these agents were generally selected in experiments with metabolically active bacteria. This is one of the explanations as to why antibiotics and antimicrobials are not as successful in the clinic as could be expected from laboratory studies. In addition, it has been found that many therapeutic agents bind to the biofilm EPS matrix before they even reach the bacteria, and are thereby inactivated. Taken together, these fundings highlight why the study of bacteria in the oral cavity is now taken on by studying the biofilms rather than individual species.

Dyspepsia refers to a heterogeneous group of symptoms that are localized in the epigastric region. Typical dyspeptic symptoms include postprandial fullness, early satiation, epigastric pain and epigastric burning, but other upper gastrointestinal symptoms such as nausea, belching or abdominal bloating often occur. Functional dyspepsia is defined as the presence of dyspeptic symptoms in the absence of an organic cause that readily explains them. The Rome III consensus proposed the subdivision of functional dyspepsia into postprandial distress syndrome (PDS), characterized by postprandial fullness and early satiation, and epigastric pain syndrome (EPS), characterized by epigastric pain or burning. Epidemiological studies in the USA and Europe confirmed the presence of both subgroups, with good separation between EPS and PDS. By contrast, other studies have found major overlap between EPS and PDS in patients with functional dyspepsia in specialist care centres in Europe and Asia. Preliminary pathophysiological studies suggest that PDS might be characterized by a higher prevalence of impaired gastric accommodation than EPS and raised duodenal eosinophil counts. Whether different treatment approaches are needed for EPS and PDS is currently unclear; only acotiamide, a new drug for the treatment of functional dyspepsia, has been found to be efficacious in PDS but not in EPS. Further randomized controlled trials testing treatment response by subgroup are urgently needed.

Little is known about optimal application and behavior of exposure propensity scores (EPS) in small studies. In a cohort of 103,133 elderly Medicaid beneficiaries in New Jersey, the effect of nonsteroidal antiinflammatory drug use on 1-year all-cause mortality was assessed (1995-1997) based on the assumption that there is no protective effect and that the preponderance of any observed effect would be confounded. To study the comparative behavior of EPS, disease risk scores, and "conventional" disease models, the authors randomly resampled 1,000 subcohorts of 10,000, 1,000, and 500 persons. The number of variables was limited in disease models, but not EPS and disease risk scores. Estimated EPS were used to adjust for confounding by matching, inverse probability of treatment weighting, stratification, and modeling. The crude rate ratio of death was 0.68 for users of nonsteroidal antiinflammatory drugs. "Conventional" adjustment resulted in a rate ratio of 0.80 (95% confidence interval: 0.77, 0.84). The rate ratio closest to 1 (0.85) was achieved by inverse probability of treatment weighting (95% confidence interval: 0.82, 0.88). With decreasing study size, estimates remained further from the null value, which was most pronounced for inverse probability of treatment weighting (n = 500: rate ratio = 0.72, 95% confidence interval: 0.26, 1.68). In this setting, analytic strategies using EPS or disease risk scores were not generally superior to "conventional" models. Various ways to use EPS and disease risk scores behaved differently with smaller study size.

This final rule implements the provisions of the American Recovery and Reinvestment Act of 2009 (ARRA) (Pub. L. 111-5) that provide incentive payments to eligible professionals (EPs), eligible hospitals and critical access hospitals (CAHs) participating in Medicare and Medicaid programs that adopt and successfully demonstrate meaningful use of certified electronic health record (EHR) technology. This final rule specifies--the initial criteria EPs, eligible hospitals, and CAHs must meet in order to qualify for an incentive payment; calculation of the incentive payment amounts; payment adjustments under Medicare for covered professional services and inpatient hospital services provided by EPs, eligible hospitals and CAHs failing to demonstrate meaningful use of certified EHR technology; and other program participation requirements. Also, the Office of the National Coordinator for Health Information Technology (ONC) will be issuing a closely related final rule that specifies the Secretary's adoption of an initial set of standards, implementation, specifications, and certification criteria for electronic health records. ONC has also issued a separate final rule on the establishment of certification programs for health information technology.

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log(10) CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 microg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12 degrees C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.