Human T-cell leukemia virus (HTLV) -1 and -2 are deltaretroviruses that infect a wide range of cells. Glut1, the major vertebrate glucose transporter, has been shown to be the HTLV Env receptor. While it is well established that the extracellular surface component (SU) of the HTLV envelope glycoprotein (Env) harbors all of the determinants of interaction with the receptor, identification of SU subdomains that are necessary and sufficient for interaction with the receptor, as well as critical amino acids therein, remain to be precisely defined. Although highly divergent in the rest of their genomes, HTLV and murine leukemia virus (MLV) Env appear to be related and based on homologous motifs between the HTLV and MLV SU, we derived chimeric HTLV/MLV Env and soluble HTLV-1 and -2 truncated amino terminal SU subdomains.bona fide interaction with the HTLV receptor as isolated SU subdomains specifically interfered with HTLV Env-mediated binding, cell fusion, and cell-free as well as cell-to-cell infection. Therefore, the HTLV receptor-binding domain (RBD) lies in the amino terminus of the SU, immediately upstream of a central immunodominant proline rich region (Env residues 180 to 205), that we show to be dispensible for receptor-binding and interference. Moreover, we identified a highly conserved tyrosine residue at position 114 of HTLV-1 Env, Tyr114, as critical for receptor-binding and subsequent interference to cell-to-cell fusion and infection. Finally, we observed that residues in the vicinity of Tyr114 have lesser impact on receptor binding and had various efficiency in interference to post-binding events.Using these SU constructs, we found that the 183 and 178 amino terminal residues of the HTLV-1 and -2 Env, respectively, were sufficient to efficiently bind target cells of different species. Binding resulted from The first 160 residues of the HTLV-1 and -2 mature cleaved SU fold as autonomous domains that contain all the determinants required for binding the HTLV receptor. Indeeiquitous . A cell iquitous .The corresponding domain in MLV Env SU – located upstream of a conserved K/R L L T/N L V Q motif in the SU of the HTLV-1 and F-MLV Env ,10 – is per se, as well as interference to receptor-binding, cell-to-cell fusion, syncytium formation, and infection involve several distinct cell surface-associated parameters [Several studies using synthetic peptides and neutralizing antibodies against the HTLV Env have shown that determinants within this proline rich region homologue (PRRH) are involved in interference to Env-mediated syncytium formation -21. The rameters -29. In tAs shown in Figure 183FEnv, comprises the 183 amino terminal residues of the HTLV-1 SU ending with the PSQL residues fused to the PIGP sequence of the F-MLV PRR  or rabbit immunoglobulin Fc (rFc) carboxy terminal tag Figure . The H12y Figure . The H11e Figure .215SU and H2211SU, containing the PRRH sequence, and H2178SU lacking this PRRH were all efficiently expressed in transfected cells . In contrast, no significant cleavage was observed with the other soluble domains released in the medium (not shown) . Immunoblots of cell extracts revealed two forms of intracellular H1215SU and H2211SU  Figure . As expeU Figure ; this waU Figure .397SU) and used as a heterologous control. A single form of this truncated SU was efficiently expressed in transfected cells  [178SU was similar to H2211SU, demonstrating that the first 158 residues of the mature HTLV-2 SU, without the 20 amino acids of the amino terminal signal peptide, are sufficient for cell surface binding, and therefore that the PRRH is not required for receptor binding  , HeLa (hoblasts) , none ofoblasts)  and datag Figure .bona fide binding to the HTLV receptor, we performed an Env-specific binding interference assay. In this assay, transfection of the above described chimeric Env and SU subdomains into 293T cells resulted in interference to cell surface binding by the soluble HA-tagged H2178SU subdomain   Figure . This efn Figure . Therefo183FEnv and the HTLV SU amino terminal subdomains to interfere with HTLV Env-mediated cell fusion. Interference to cell fusion was measured using a quantitative HTLV envelope cell fusion interference assay (CFIA), as previously described [Viral envelope interference occurs when cell surface receptors are occupied by receptor-interacting Env components -35. Sincescribed .215SU subdomain in target cells, 12% ± 2% of control fusion (P < 0.001), consistent with previous observations using the H1215FEnv chimera [183FEnv chimera, which lacked a PRRH, down to 26% ± 4% of control fusion (P < 0.001)   , although unnecessary for receptor binding, modulates the efficiency of interference to HTLV Env-induced cell-to-cell fusion (P < 0.03).HTLV-1 Env-induced cell fusion was significantly diminished upon expression of the H1 chimera . SignifiInterference, as described above, was based on the inhibition of cell-to-cell fusion induced by fusogenic Env expressed in the absence of other viral proteins. We further evaluated the abilities of the Env chimeras and soluble subdomains to specifically interfere with HTLV Env-mediated infection. HTLV Env-pseudotyped MLV virions, MLV(HTLV), were produced to infect 293T target cells. Because these recombinant cell-free virions are not competent for replication, this viral pseudotype infection assay tests a single round of infection, and does not measure replication and subsequent exponential viral dissemination. Therefore, relative infection values are expressed in linear rather than logarithmic scales.397SU  infection to 324 ± 98 iu/ml, 3.3% ± 1% of control infection, and to 307 ± 129 iu/ml, 3.1% ± 1.3% of control infection, respectively  infection to 191 ± 56 iu/ml and 215 ± 122 iu/ml, 1.9% ± 0.6% and 2.2% ± 1.3% of control infection, respectively , and this was taken as 100% control infection Figure . SimilarU Figure . ExpressBecause HTLV dissemination appears to occur mostly via cell-to-cell contact, we also tested envelope interference to infection by HTLV-1 SU amino terminal domains using a cell-to-cell transmission interference assay. In this assay, cells harboring interfering chimeric Env and soluble subdomains were cocultured with cells producing MLV(HTLV) virions. Transfection of either chimeric Env or soluble subdomains into HeLa target cells decreased MLV(HTLV) infection to levels similar to those observed in the cell fusion interference assay presented in figure 94) and serine 101 (Ser101) for HTLV-1 Env which correspond to Arg90and Ser97 in HTLV-2 Env, have been shown to alter cell-to-cell fusion and infection when mutated [215SU subdomains with either Arg94 or Ser101 mutated to Ala, yielding the mutant H1(R94A)SU and H1(S101A)SU subdomains, respectively. We also evaluated mutations of Asp106, mutant H1(D106A)SU, and Tyr114, mutant H1(Y114A)SU, both residues found to be highly conserved between all human and simian T cell leukemia viruses (unpublished observations). Surprisingly, cell surface binding profiles of H1(R94A)SU and H1(S101A)SU mutants were not significantly altered when compared to binding by the parental H1215SU, whereas the H1(D106A)SU mutant presented reduced binding to HTLV receptor-bearing cells and the H1(Y114A)SU mutant showed a nearly complete abrogation of cell surface binding . Altogether, these experiments demonstrated that Tyr114, and to a lesser extent Asp106, are key residues involved in HTLV Env receptor binding.Two key residues contained in the HTLV SU RBD and conserved between HTLV-1 and -2, arginine 94 . Thus, overexpression of mutant HTLV SU fragments with highly decreased receptor binding abilities can still exert, albeit to a significantly lesser extent, interference to HTLV Env-mediated cell fusion and infection.We next tested the abilities of these mutants to interfere with HTLV Env-mediated cell fusion and infection, using the assays described above. As mentioned above, all wild-type and mutant HTLV SU subdomains were produced and secreted with a similar efficiency Figure . Express215SU or the mutant H1(S101A)SU were expressed in target cells SU mutant inhibited cell fusion and infection, its effects were significantly lower than those of the wild-type H1215SU   Figure . Thus, a114 of the HTLV-1 Env, which corresponds to Tyr110 of the HTLV-2 Env, as a key residue in HTLV Env binding and for all the aforementioned HTLV Env-mediated functional assays. We could not detect binding of H1(Y114A)SU by flow cytometry, while this mutant exerted residual, albeit significantly decreased, interference to HTLV Env-mediated cell fusion and infection. Altered folding outside of the binding domain per se, rather than direct alteration of the receptor-binding site, could also account for the lack of binding of this mutant. However, we favor the latter hypothesis, since the H1(Y114A)SU mutant was properly folded and transported to the plasma membrane and secreted in the medium as efficiently as wild type RBD, thus arguing against gross misfolding of this mutant. Accordingly, Tyr114 appears to be conserved in all known human and simian T cell leukemia viruses strains, which share the same receptor.Here, we report the generation of MLV Env with chimeric HTLV/MLV SU and truncated HTLV-1 and -2 amino terminal SU subdomains that can be expressed in and secreted from eukaryotic cell lines in functional, soluble form. Using these constructs, we demonstrated that the amino terminal 163 and 158 residues  of the mature HTLV-1 and -2 Env SU, respectively, were sufficient to exert both HTLV receptor binding and efficient interference to diverse HTLV Env-mediated functions, including binding, cell-to-cell fusion and cell-free as well as cell-to-cell infection. Although the PRRH sequence comprising amino acid residues 180 to 215 of the HTLV-1 Env and 176 to 211 of the HTLV-2 Env was previously thought to be a receptor binding site, our data preclude a major role for this region in the binding properties described above. Indeed, whereas a synthetic peptide composed of amino acids 197 to 216 and located within the HTLV-1 PRRH, has been reported to interfere with HTLV Env-induced syncytia formation , this pe114 as a key HTLV-1 RBD residue points at this determinant as a very likely receptor-binding core. This, together with previous works relying on syncytia formation and cell-to-cell transmission [bona fide receptor binding determinants and determinants involved at a post-binding level.The receptor-binding site in MLV RBD is composed of a combination of several cysteine loops located upstream of the PRR ,39 whichsmission ,37, willAnother recently identified determinant, the Pro-His-Gln SU motif conserved among gammaretroviruses such as MLV and feline leukemia viruses (FeLV), has been determined to play a major role in viral entry during post-binding events . The mecThe recent identification of Glut1, the ubiquitous glucose transporter of vertebrates , as a reWe have generated truncated domains of the HTLV Env amino terminus, upstream of residues 183 and 178 of the HTLV-1 and -2 Env, respectively, that were sufficient to bind target cells of different species through interaction with the HTLV Env receptor. We also identified a tyrosine at position 114 and 110 in HTLV-1 and -2 Env, respectively, as a key determinant for this binding. In addition to their use for further exploration of the mechanisms involved in HTLV entry, the tagged HTLV-1 and -2 RBD subdomains described here are novel tools for the detection of Glut1 cell surface expression and intracellular trafficking. Indeed, we tracked intracellular expression of EGFP-tagged HTLV SU subdomains by time-lapse microscopy, and found that they are preferentially routed toward cell-cell contact areas (unpublished observations), where Glut1 is particularly abundant  and our MfeI restriction site in the HTLV-1 and F-MLV Env. Introduction of this site in F-MLV resulted in the substitution of a glutamine and leucine (QL) dipeptide for the parental arginine and valine (RV) residues of the GPRVPIGP motif, at the start of the MLV Env PRR. Introduction of the MfeI site in the PSQL motif of the HTLV-1 SU maintained the parental QL residues, at the start of the HTLV Env PRRH. By exchanging domains at the MfeI sites, we derived the H1183FEnv chimera containing the amino terminal 183 residues of the HTLV Env followed by the F-MLV PRR. In this chimera, the PSQL/PIGP hybrid sequence is generated at the exchange border, and the PRRH of HTLV is replaced by the F-MLV PRR  [To exchange the PRR and PRRH regions, we introduced an allelic R Figure . In cont HHproFc . The H11n vector . The HTL intron)  at the H215SU, H2211SU, H1179SU, and H2178SU subdomains, corresponding to the HTLV-1 and -2 SU amino terminus with and without their respective PRRH, were generated by PCR and subcloned into the pCSI expression vector as fusion proteins harboring a carboxy terminal rFc or HA tag  transfection protocol. Transfection medium was replaced with 3 ml of fresh culture medium twenty hours post-transfection. Forty-eight hours post-transfection cell culture medium (supernatant) was recovered and filtered through a 0.45 μm pore-size membrane to remove cell debris. Twenty μl were directly analyzed by SDS-PAGE (15% polyacrylamide gel), and the rest was aliquoted and stored at -20°C for later use in binding assays (see below). Cell extracts were collected 48 h post-transfection in 1 ml of cell lysis buffer  and clarified by two successive centrifugations at 13,000 rpm for 10 min at 4°C in a microcentrifuge. Approximately 20 μl of each extract, adjusted after normalization for protein concentration using the Bradford assay (Sigma), were subjected to electrophoresis on SDS-15% acrylamide gels, followed by transfer onto nitrocellulose . Membranes were blocked in phosphate-buffered saline (PBS) containing 5% powdered milk and 0.5% Tween 20, probed with a 1:1000 dilution of a goat anti-RLV gp70 polyclonal antibody (Viromed) followed by a horseradish peroxidase-conjugated anti-goat immunoglobulin (for detection of chimeric Env), or goat anti-rabbit-IgG-horseradish peroxidase-conjugated immunoglobulins (for detection of rFc-tagged SU subdomains). Immunoblots were subsequently washed three times with PBS-0.1% Tween 20 and revealed by chemiluminescence .Approximately 5 × 105 target cells were detached with a PBS-EDTA solution, collected by centrifugation, incubated for 30' at 37°C with 300 μl of rabbit Fc-tagged soluble HTLV-1, HTLV-2, or Ampho-MLV truncated SU, washed, labeled with an anti-rabbit-IgG FITC-conjugated antibody, and analyzed on a FACSCalibur (Becton Dickinson). Data analysis was performed using the CellQuest software (Becton Dickinson). For interference studies, 293T cells were transfected with 4 μg of Env or Env SU subdomain expression vectors  using the calcium-phosphate-HBS method. Under these conditions, transfection efficiencies ranged from approximately 80 to 90% of the target cells. Twenty-four and 48 hours post-transfection, cells were collected and transfected 293T cells expressing the different interfering HTLV or Ampho-MLV domains were incubated with a challenging HA-tagged soluble HTLV-2 SU amino terminal subdomain (H2178SU-HA). Cells were stained using a primary 12CA5 anti HA antibody followed by an anti-mouse-IgG FITC-conjugated antibody before detection by flow cytometry.Binding assays were performed as previously described . Briefly183FEnv, was used to interfere with challenging HTLV Env. The interfering non-fusogenic H1183FEnv and truncated HTLV SU subdomains were transiently transfected into HeLaCD4LTRLacZ, a cell line highly susceptible to HTLV Env-induced fusion that contains a stably integrated Tat-dependent LacZ expression vector [183FEnv Env and truncated HTLV SU subdomains plasmid DNA (2 to 3 μg) was transfected into HeLaCD4LTRLacZ cells, while challenging, fusogenic HTLV-1 Env plasmid (1 μg) was transfected into NIH3T3(TK-)Tat. The interfering Env or SU subdomain-presenting cells were detached 24 hours post-transfection and 1–2 × 105 cells were cocultured for 24 hours with 1–2 × 105 challenging HTLV-1 Env-presenting NIH3T3(TK-)Tat cells. Subsequently, the cocultured cells were fixed and stained for β-galactosidase expression as described previously [t test.Briefly, the HTLV/MLV Env chimera, H1n vector . These tn vector ,29. H118eviously . Transfe6 293T cells with 5 μg pCSI/Ampho or pCSIX/H2, respectively, 5 μg pCL/Gag-Pol [LacZ gene marker. pCSI/Ampho is an expression vector encoding the Ampho-MLV Env, and the HTLV-2 Env expression vector, pCSIX/H2, is described above.MLV(Ampho) and MLV(HTLV) pseudotyped virions were produced after transfection of 10/Gag-Pol , using aVirion-containing supernatants were used to infect target 293T cells expressing the chimeric Env or HTLV RBD subdomains. Transfection efficiencies of target 293T cells were >80% in all experiments. Infections were performed 36–48 hours post-transfection on cultures grown in 12 well plates (Costar) at 37°C, medium was changed 24 hours later, and confluent cell monolayers were fixed, stained for β-galactosidase activity before counting blue foci. Interference to infection was determined by infecting transfected target cells with approximately 100 and 1000 iu. Infection was evaluated as described above, and the number of LacZ-positive blue colonies counted was normalized by multiplying by the appropriate dilution factor. The resulting infection values were analyzed as iu/ml of virus containing supernatant. Subsequently the relative infection levels in cells expressing the HTLV SU domains were compared to those of mock transfected cells and were expressed as percentages of control infection (% control).HTLV Human T-cell leukemia virusSU envelope extracellular surface componentEnv envelope glycoproteinMLV murine leukemia virusF-MLV Friend-MLVRBD receptor-binding domainPRR proline-rich regionPRRH proline rich region homologueAmpho amphotropicHA influenza hemagglutininrFc rabbit immunoglobulin constant fragment397SU Ampho-MLV Env fused to a carboxy terminal rFc tagACFIA cell fusion interference assayiu/ml infectious units per ml94arginine 94Arg101serine 101Ser114tyrosine 114TyrFeLV feline leukemia virusesHBS Hepes buffered salinePBS phosphate-buffered salineSDS sodium dodecyl sulfateThe authors declare that they have no competing interests.FJK designed and realized or supervised most of the experiments and co-wrote the manuscript. NM participated to some molecular constructions, set up, realized and analyzed most binding assays and FACS analyses and participated to the redaction of the manuscript. ENG set up and performed the cell-to-cell transmission assay and performed the corresponding experiments, CV constructed some of the RBD point mutants and tested them, MS initiated the project, co-participated in the design of the study, co-coordinated its realization and co-wrote the manuscript, and JLB realized some of the molecular constructs, performed some of the experiments, co-participated in the design of the study, co-coordinated its realization and co-wrote the manuscript. All authors read and approved the final manuscript.


With effective treatment strategies, the focus of tuberculosis (TB) management has shifted from the prevention of mortality to the avoidance of morbidity. As such, there should be an increased focus on quality of life (QoL) experienced by individuals being treated for TB. The objective of our study was to identify areas of QoL that are affected by active TB using focus groups and individual interviews.English, Cantonese, and Punjabi-speaking subjects with active TB who were receiving treatment were eligible for recruitment into the study. Gender-based focus group sessions were conducted for the inner city participants but individual interviews were conducted for those who came to the main TB clinic or were hospitalized. Facilitators used open-ended questions and participants were asked to discuss their experiences of being diagnosed with tuberculosis, what impact it had on their lives, issues around adherence to anti-TB medications and information pertaining to their experience with side effects to these medications. All data were audio-recorded, transcribed verbatim, and analyzed using constant comparative analysis.39 patients with active TB participated. The mean age was 46.2 years (SD 18.4) and 62% were male. Most were Canadian-born being either Caucasian or Aboriginal. Four themes emerged from the focus groups and interviews. The first describes issues related to the diagnosis of tuberculosis and sub-themes were identified as 'symptoms', 'health care provision', and 'emotional impact'. The second theme discusses TB medication factors and the sub-themes identified were 'adverse effects', 'ease of administration', and 'adherence'. The third theme describes social support and functioning issues for the individuals with TB. The fourth theme describes health behavior issues for the individuals with TB and the identified sub-themes were "behavior modification" and "TB knowledge."Despite the ability to cure TB, there remains a significant impact on QOL. Since much attention is spent on preventative or curative mechanisms, the impact of this condition on QoL is often not considered. Attention to the issues experienced by patients being treated for TB may optimize adherence and treatment success. Mycobacterium tuberculosis  will bring it to me". However, despite high compliance in these patients, several identified alcohol (n = 13) or other illicit drug use (n = 8) as being the reason why they had missed doses.Those who came to the TB treatment clinic expressed high compliance due to the perceived gravity of the diagnosis. For example, one woman patient stated, "It is easy to remember because it is at the fore-front of mind. I want to get rid of it". Another person attributed compliance to the law: "I never forget to take my pills because I don't want to go to jail". Representative quotes from these participants are included in Table This theme describes social support and functioning issues for the individuals with TB Figure . SpecifiMost participants expressed that their family and friends were aware of their TB diagnosis (n = 18), while 11 stated that only their friends knew and 10 stated that only their immediate family knew. Of those who stated that only their friends were aware of their diagnosis, most of these persons residing in the inner city noted that they did not have family with whom they communicated. Representative quotes from these participants are included in Table However, one individual stated that she was "secretive because other people will feel that I am contagious". Another male participant stated a reluctance to tell his friends because "I do not want go cause mass hysteria". A school-age boy did not tell his friends out of fear of being shunned. In addition, he missed eight weeks of school and had to retake several courses. In one instance, fear of being shunned in a Punjabi speaking participant was instilled by the treating physician , although others had negative feelings such as fear (n = 7), shock or disbelief (n = 8), and anger (n = 3). One participant stated "my mom was really concerned but my friends did not believe it...they encouraged me to get the right diagnosis". Another stated that her partner had increased his reading on TB and was receiving regular skin tests although her mother and brother would not talk about the TB diagnosis or the clinic visits. Another stated that since his partner was not understanding about modifying his lifestyle, he was forced to end the relationship with her and move out. Representative quotes from these participants are included in Table This theme describes health behavior issues for the individuals with TB Figure . Sub-theWhen asked if they had done anything else beyond medications to help manage their TB, about half of the participants stated that they had done nothing in particular (n = 16). For example, a Cantonese speaking female stated "I do nothing special as TB is very common". However, of the 12 participants who stated that they had changed their health behavior, seven said that they consumed a healthier diet, four stated that they exercised more, three took vitamin supplements specifically to help their TB, and two used less illicit drugs and alcohol. Representative quotes from these participants are included in Table In response to the question "do you believe that you will be cured of TB?", most participants (n = 33) stated that they believed that they would be eventually cured. However, some individuals believed that their TB would never be cured ("I believe that I can keep it in remission but it can't be cured") while others were not sure if it could be cured. Two participants denied having TB despite being informed by health care providers and taking medication. There were comments regarding the participants' impressions of provider knowledge of which a representative sample have been included in Table This qualitative study has revealed that TB has a large impact on affected individuals' QoL through issues related to its diagnosis, treatment, social support and functioning, and health behavior. Specifically, we found that the domains of QoL that were affected by TB included those that are typically affected by most illnesses such as physical functioning and emotional/mental well-being. However, TB patients' social functioning was also affected through isolation, variable social support by family and friends, and the ability to continue with social and leisure activities. Also, the process of getting treatment for TB from the initial hospitalization to the daily medication schedules adversely affected the lives of our participants, although, almost all recognized the need for appropriate treatment.Although other studies ,23 have However, there were some important differences between our two studies. For example, these investigators included only 10 English speaking patients from the Baltimore city area and 13 health care providers whereas ours included non-English participants through the use of interpreters, a much large sample of patients (n = 39), but no health care providers. In addition, we included hospitalized and ambulatory patients from both inner city and public health clinic environments in order to assess the full spectrum of patients afflicted with this disease. Finally, in our study, all patients had active TB and were receiving treatment at the time of the interviews unlike the Baltimore study who recruited patients who were already cured and had completed treatment. We believe that our methodology of interviewing currently afflicted patients might have minimized recall bias although one potential advantage of the Baltimore approach was determining long-lasting influences of TB on patients lives (the investigators received 17 comments in this regard). Also, the use of health providers in the Baltimore study added an interesting perspective with the provision of comments that were, at times, in direct contrast to those stated by patients with respect to the effects of TB on health related quality of life. For example, most physicians underestimated the impact that TB had on the QoL of patients assuming that, because it was curable, its detrimental effects would be minimal.These differences in design between the two studies might have accounted for some different findings. For example, the Baltimore study found that the financial well-being of some of the participants was adversely affected through loss of income and health care expenses whereas participants in our study did not report this issue . Also, some of the male participants in the Baltimore study reported sexual dysfunction whereas this concern was not reported during our interviews. Finally, patients in the Baltimore study reported spirituality as an important domain which we did not identify as an important theme, perhaps due to the different ethnic/religious make-up of our sample.One surprising aspect of our results was the negative feelings associated with TB diagnosis and the initial hospitalization. Some participants expressed frustration with their primary care physicians for the lack of a prompt diagnosis or inappropriate management. There was a common perception among many of the participants that health care providers needed more extensive education regarding TB. We have recently commented on the need to consider TB as a diagnosis and in the appropriate setting, consider the initiation of empiric TB treatment . In addiDespite several negative comments regarding the size, dosing schedule and adverse effects of the anti-TB medications, most patients specified that they understood the need for treatment. As such, self-reported compliance was very high and participants reported a variety of different strategies to help manage adverse events. Our inner-city participants expressed gratitude for the street nurses who delivered their medications to them on a regular basis and did not report the intrusiveness and imposition on lifestyle that has been associated with similar programs (such as directly observed therapy or DOT) in other studies ,26.Although most comments were related to adverse impacts of TB on QOL, some participants stated that acquiring TB had resulted in positive health behavior modification. Many participants took the development of TB to be a "wake-up" call to change their lifestyle and improve their health behavior by either eliminating or reducing drug and alcohol intake, increasing exercise, or eating better. These findings were also reported by the Baltimore study group suggesting that the positive health behavior impacts of this disease might be widespread throughout those afflicted with TB in North America. Because many of those afflicted with TB in North America engage in other high-risk behaviors such as use of illicit drugs, the overall effects of this health behavior modification might be significant. Future studies should attempt to quantify this impact on the downstream development of other conditions. Although hospitalization for management of TB has negative aspects we have noted that this interlude in subjects with a history of substance abuse allows access to chemical dependency treatment resources while away from their usual chaotic environments.Although some studies in other countries have shown that TB can result in job loss, participants did not report that this had occurred in our sample ,28. One Our study had some limitations. We examined a self-selected group of TB patients who may not be representative of the entire population in Canada affected by TB. For example, in British Columbia, foreign-born persons account for close to 70% of all TB cases in the province. Despite this, we feel that we were able to get a representative sample of foreign-born persons  as well as a good cross section of marginalized inner city patients . In factOur study indicates that despite the ability to cure TB with medical therapy, there still remains a sizeable impact on the lives of afflicted patients. Since much of the current attention on TB is spent on preventative or curative mechanisms such as drug therapy, the impact of this condition on QoL is either underestimated or rarely considered. In order to fully evaluate the outcomes that are achieved through TB prevention and treatment, QoL of these patients must be considered. Further studies need to build upon these observations and instruments need to be developed to better characterize QoL in patients with this disease. This process will not only provide an added parameter to evaluate the effectiveness of a given program, but will also focus care providers to be attentive to the non-medication aspects of TB management.CAM conceived of the study, participated in the design, analysis and co-wrote the initial version of the manuscript. FM conceived of the study, obtained funding, participated in the interviews and focus groups, participated in the analysis, coordinated research staff, and co-wrote the initial version of the manuscript. VC participated in the interviews and focus groups and participated in the analysis. AP participated in the design of the study, and the interviews and focus groups. JMF participated in the design and analysis of the study. All authors read and approved the final manuscript.


The role of the crural diaphragm during increased intra-abdominal pressure is not exactly known. We investigated the hypothesis that the crural diaphragm undergoes reflex phasic contraction on elevation of the intra-abdominal pressure with a resulting increase of the lower esophageal pressure and prevention of gastro-esophageal reflux.The esophageal pressure and crural diaphragm electromyographic responses to straining were recorded in 16 subjects  during abdominal hernia repair. The electromyogram of crural diaphragm was recorded by needle electrode inserted into the crural diaphragm, and the lower esophageal pressure by a saline-perfused catheter. The study was repeated after crural anesthetization and after crural infiltration with saline.The crural diaphragm exhibited resting electromyographic activity which showed a significant increase on sudden  or slow sustained (p < 0.01) straining with a mean latency of 29.6 ± 4.7 and 31.4 ± 4.5 ms, respectively. Straining led to elevation of the lower esophageal pressure which was coupled with the increased electromyographic activity of the crural diaphragm. The crural response to straining did not occur during crural diaphragm anesthetization, while was not affected by saline infiltration. The lower esophageal pressure declined on crural diaphragm anesthetization.Straining effected an increase of the electromyographic activity of the crural diaphragm and of the lower esophageal pressure. This effect is suggested to be reflex in nature and to be mediated through the "straining-crural reflex". The crural diaphragm seems to play a role in the lower esophageal competence mechanism. Further studies are required to assess the clinical significance of the current results in gastro-esophageal reflux disease and hiatus hernia. The LES contributes to the prevention of gastric reflux into the esophagus [Swallowing is a physiologic process by which the food bolus is transmitted from the pharynx to the stomach without esophagopharyngeal or gastro-esophageal reflux . A sphinculature . The LESculature ,6. Cholisophagus ; howeverThe diaphragm is believed to play a contributory role in the barrier function of the lower esophagus. This auxiliary function seems to be carried out by the crural and not the costal diaphragm. The latter contracts and relaxes with respiration. Crural diaphragm (CD) contraction effects LES pressure increase which is directly proportional to the depth of inspiration at the force of diaphragmatic contraction . PressurThe role of the CD during increased intra-abdominal pressure is not completely understood. We hypothesized that the CD, upon increase in intra-abdominal pressure by coughing, sneezing or straining, undergoes reflex phasic contraction with a resulting augmentation of the lower esophageal pressure and inhibition of stress reflux of the gastric contents into the esophagus. This hypothesis was investigated in the current communication.Sixteen subjects were enrolled in the study. Ten were men and six women with a mean age of 36.6 ± 11.2 SD years, (range 27–43). The tests were performed during operative repair of an upper abdominal ventral hernia in 9 patients and of incisional hernia after cholecystectomy for calculous cholecystitis in 7 patients. The patients did not complain of swallowing problems in the past or at the time of enrollment. They gave an informed consent after having been fully informed about the nature of the tests to be done and their role in the study.Physical examination results, including neurologic assessment, were normal. Also barium swallow studies and upper gut endoscopy yielded normal findings. The results of laboratory work including blood count, renal and hepatic function tests as well as electrocardiography were unremarkable.The study was approved by the Review Board and Ethics Committee of the Cairo University Faculty of Medicine.The EMG activity of the CD was recorded during coughing and during straining. The subjects had received general anesthesia using 5% halothane/ 95% oxygen for their above mentioned hernia operations.A concentric electromyographic needle electrode of 40 mm in length and 0.65 mm in diameter  was introduced into the CD as it encircled the lower end of the esophagus. A ground electrode was applied to the thigh.A standard electromyographic (EMG) apparatus  was used to amplify and display the potentials recorded. Films of the potentials were taken on light-sensitive paper  from which measurements of the motor unit action potentials' duration were obtained. The electromyopraphic signals were also stored on an FM tape recorder  for further analysis as required.Before performing the experiment, the normality of the EMG activity of the CD was tested by stimulating it with a needle electrode introduced into the CD and registering the motor unit action potentials from the already inserted needle electrode. The CD had normal EMG activity in all examined subjects.2O/s. During pressure measurements, the catheter was rotated so as to record anteroposterior and lateral pressures.A manometric 6-F catheter was introduced into the esophagus to lie in the high pressure zone at its lower end. The catheter with 2 side ports and a metallic clip applied to its distal closed end for fluoroscopic control was connected to a pneumohydraulic capillary infusion system . The pump delivered saline solution continuously via the capillary tube at a rate of 0.6 ml / min. The transducer outputs were registered on a rectilinear recorder . Occlusion of the recording orifice produced a pressure elevation rate that was greater than 250 cm HNear the end of the operation when the effect of muscle relaxant had waned, the anesthetist was asked to induce coughing and straining via laryngeal and tracheal stimulation by moving the endotracheal tube while lying in the trachea. The EMG response of the CD to increased intra-abdominal pressure was registered. Readings were recorded during two types of straining: the sudden forcible straining as that induced by coughing, and the slow sustained straining which simulates that occurring during defecation or micturition. The latency of the crural response was measured from the stimulus (straining) to the first deflection of the muscle action potential complex. The millisecond latencies were calculated when the movement artifact associated with straining appeared on the crural EMG and then the time to the first muscle action potential was measured as an index of latency.To define whether the effect of coughing or straining on the crural diaphragm was direct or reflex action, the following lest was done. In 8 subjects (5 men and 3 women), the CD was infiltrated with 5 ml of 2% lidocaine to anesthetize the crura around the needle electrode. The crural response to sudden and slow sustained straining was recorded after 10 minutes and after 2 hours when the anesthetic effect had waned. Similarly, normal saline was injected and the crural response to straining was registered.The results were analyzed statistically using the Student's t test and values were given as the mean ± standard deviation. Differences assumed significance at p < 0.05.The CD in all of the subjects showed a basal activity with a mean of 112.3 ± 16.3 μV  was reproducible in all studied subjects. It was weaker in women than men, and in the elderly than in the young subjects, though the difference was insignificant (p > 0.05). The CD response disappeared when straining was sustained for more than 15–18 seconds (mean 16.8 ± 1.2) and was not evoked after frequent successive straining. The latency of the response recorded a mean of 29.6 ± 4.7 ms . Saline injection of the crura did not affect the crural response to straining.2O  which are responsible for pain and reflex activity ,16. The The mechanism of gastroesopageal competence is vague and incompletely understood -7. ThereMeanwhile, it is highly probable in the light of the findings of our study that the prevention of gastro-esophageal reflux is a "reflex process" rather than an anatomical entity. We have previously demonstrated that gastric distension by food or an increase in the intra-abdominal pressure would evoke the "gastroesophageal reflex" which acts to tighten the LES . The morThe current study presumably denotes that the CD shares reflexly in the competence mechanism of the gastroesophageal junction. Thus, upon increase of the intra-abdominal pressure, the straining-crural reflex seems to be evoked effecting crural contraction and increase of the lower esophageal pressure.In view of the aforementioned results and discussion, we believe that the "reflex theory" plays a more important role in gastroesophageal competence than the diaphragm pinchock, the flap valve mechanism or other possible anatomical factors.The CD appears to play a role in the lower esophageal competent mechanism. Straining effected an increase in the EMG activity of the CD and in the lower esophageal pressure. This effect is suggested to be reflex in nature and to be mediated through the "straining-crural reflex". Further studies are needed to evaluate the clinical significance of the current results in the pathogenesis and treatment of gastresophageal disease and hiatus hernia.lower esophageal sphincter (LES)crural diaphragm (CD)electromyographic (EMG)The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


Single photon emission computed tomography (SPECT) following injection of radiotracer during a seizure is known as ictal SPECT. Comparison of an ictal SPECT study to a baseline or interictal study can aid identification of a seizure focus.A young woman with encephalitis and refractory seizures underwent brain SPECT during a period of frequent seizure-like episodes, and during a seizure-free period. A focal area of increased radiotracer uptake present only when she was experiencing frequent seizure-like episodes was originally localized to the brainstem, but with later computerized co-registration of SPECT to MRI, was found to lie outside the brain, in the region of the sphenoid sinus.Low-resolution SPECT images present difficulties in interpretation, which can be overcome through co-registration to higher-resolution structural images. Radiotracers used for brain single photon emitted computed tomography (SPECT) pass the blood-brain barrier and bind intracellularly on their first pass through the circulation, providing a "snapshot" of cerebral perfusion at a particular timepoint. When injected during a focal epileptic seizure, an area of significantly increased radiotracer uptake typically corresponds to the region of maximal abnormal activity, often the seizure focus. This ictal pattern of cerebral blood flow can be compared to an interictal/baseline pattern obtained when the patient is not having a seizure, to provide unique information about the nature and location of a patient's epileptic focus, which can be used to guide therapy [See  for a reA previously-healthy young woman developed behavioral changes followed by seizures and refractory status epilepticus. She was diagnosed with encephalitis and treated with antiviral and multiple antiepileptic agents. She required nasotracheal intubation and mechanical ventilation for respiratory support. She experienced persistent episodes of facial twitching resembling seizures. These episodes were not however associated with an ictal EEG pattern on continuous video/EEG monitoring. To clarify the nature of these episodes, 99mTc-HMPAO was injected during a period of frequent twitching. Brain SPECT showed a prominent focus of increased uptake interpreted by the radiologists and clinical team as arising in the upper brainstem Figure . A repeaWith later co-registration of SPECT data to the patient's MRI using a computerized medical image registration and visualization program,  the areaThe nature of this patient's twitching episodes remains uncertain, though they were likely due to epileptic activity involving too small a volume of brain to be detectable by either EEG or SPECT. Her case is described here to illustrate a previously-unreported pitfall in the interpretation of brain SPECT studies, with the goal of emphasizing the importance of image co-registration in nuclear medicine. Diagnostic limitations inherent to low-resolution images can be overcome through use of computer software to accurately co-register brain images of different modalities acquired separately (as described here) or hybrid medical devices such as PET-CT scanners to automatically fuse simultaneously-acquired images. Both methods have been shown to be superior to visual inspection and mental integration of information by experienced radiologists ,8.SPECT: single photon emitted computed tomography; MRI: magnetic resonance imaging; EEG: electroencephalographyThe author(s) declare that they have no competing interests.TB performed image analysis and co-registration and drafted the manuscript. LJH identified the patient, conceived of the study, and drafted a preliminary version of the manuscript. JC participated in data collection and analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Persistent atrial fibrillation (AF) is difficult to treat. In the absence of class I or III antiarrhythmic drugs sinus rhythm is maintained in only 30% of patients during the first year after electrical cardioversion (ECV). One of the remodeling processes induced by AF is fibrosis, which relates to inducibility and maintenance of AF. The renin-angiotensin system may play a important role in this. The aim of this study was to investigate the role of angiotensin-converting enzyme (ACE) inhibitor use on efficacy of ECV, and occurrence of subacute recurrences.One hundred-seven consecutive patients with persistent AF underwent ECV. In twenty-eight (26%) patients ACE inhibitors had been started before initiation of the present episode of AF ('pre-treated' patients).ECV was successful in 96% of patients who were on ACE inhibitors before start of the present episode of AF compared to 80% of the patients not pre-treated (p = 0.04). After 1 month of follow-up 49% of the pre-treated patients and 50% of those not pre-treated with ACE inhibition were still in sinus rhythm (p=ns). Multivariate analysis showed that pre-treatment with ACE inhibitors and a smaller left atrial size were independent predictors of successful ECV .Pre-treatment with ACE inhibitors may improve acute success of ECV but does not prevend AF recurrences. Persistent atrial fibrillation (AF) is difficult to treat. In the absence of class I or III antiarrhythmic drugs sinus rhythm is maintained in only 30–50% of patients during the first year after Direct Current electrical cardioversion (ECV),2. FurthIn the present study we report on the effects of ACE inhibition on the outcome of ECV and the prevention of early recurrences after ECV of persistent AF.onset of the current AF episode. Most patients on ACE inhibitors used these drugs for hypertension or congestive heart failure. To make sure that all patients were not completely remodeled at the very moment of start of the current episode of AF , only patients with at least 1 month sinus rhythm before the current episode of AF were included in this study. Duration of AF was determined as precisely as possible by previous electrocardiograms, 24-hours Holter registrations, and by the patient's history. None of the patients were on class I or III antiarrhythmic drugs neither at the moment of ECV nor during follow-up.One hundred-seven consecutive patients with persistent AF, defined as the presence of AF for at least 24 hours were included in this study. ECV wasQuantitative variables were compared between groups using a two-tailed t-test for normally distributed variables or a Wilcoxon two-sample test for skewed distributed variables. For qualitative variables , group differences were evaluated using a Fisher's exact test or a Chi-square test. Accordingly, baseline characteristics are given in mean ± SD, median and range (min-max) or percentages.To determine the predictive factors for successful ECV, an univariate logistic regression analysis was performed using the relevant baseline predictors. Variables with a p-value < 0.20 were selected for the multiple logistic regression analysis to derive a model with statistically significant predictors, by using a backward selection method. All p-values are two-sided and a p-value of < 0.05 was considered statistically significant. SAS version 6.12  was used for all statistical evaluations.before start of the current episode of AF (pre-treated patients). In the latter group, ECV was successful in 96% while in the group of patients not pre-treated with ACE inhibitors before start of the current AF episode only 80% had a successful ECV (p = 0.04).Baseline characteristics are listed in table In general, patients treated with ACE inhibitors before start of the current episode of AF had more evidence for heart disease than those who were not pre-treated. Prevalence of hypertension , and left ventricular dysfunction  was significantly higher in the pre-treated group in comparison to those who were not pre-treated with ACE inhibitors. . Use of beta adrenergic receptor blockers, calcium channel blockers or digoxin, started before the present episode of AF or not, did not influence success of ECV.An additional 21 patients received ACE inhibitors After 1 month of follow-up 49% of the pre-treated patients compared to 50% of those who were not pre-treated with ACE inhibition were still in sinus rhythm .This post-hoc retrospective analysis shows that use of ACE inhibitors before the onset of AF enhances acute ECV outcome but it does not improve maintenance of sinus rhythm. Furthermore, when ACE inhibitiors are instituted later, i.e. after the start of AF, it does no longer improve success of ECV.ACE inhibitors reduce the incidence of AF in patients with left ventricular dysfunction,6. This In 1995 it was shown that AF induces several electrophysiological changes, called electrical remodeling. Experimafter the start of AF. No differences were found in electrical cardioversion outcome between the two treatment groups which is in contrast to the present study. This may relate to the fact that all patients were in AF at the very moment of start of irbesartan. Two months after cardioversion it appeared that patients treated with irbesartan and amiodarone had a significantly lower AF recurrence rate compared to amiodarone alone treated patients, 15% versus 37%, respectively (p = 0.007). This difference was maintained during a median follow-up of 254 days. According to their figure 2, the benefit of irbesartan (in combination with amiodarone) was mainly achieved by reduction of recurrences after the first two weeks after cardioversion. An earlier study on the role of ACE inhibitors in patients with heart failure and AF showed a trend towards more patients maintaining sinus rhythm after ECV when instituted on lisinopril in comparison to patients not treated with lisinopril[Madrid et al. investigated the role of the angiotensin II type 1 receptor antagonist irbesartan in amiodarone treated patients. Persistisinopril. In the This was a non-randomized and post-hoc analysis. This implies that all findings can only be used to generate hypotheses. However, this is the first clinical study showing that ACE-inhibition initiated before start of AF enhances direct ECV outcome.Only a very small amount of patients was on angiotensin receptor blockers. Whether the effect of pretreatment with angiotensin receptor blockers on cardioversion outcome would be the same as the effect of pretreatment with ACE inhibitors could not be investigated in this study. However, in this context the results of the study of Madrid et al. are encouraging .Pretreatment with ACE-inhibitors significantly improves acute outcome of ECV when initiated before the onset of AF but it does not lead to better maintenance of sinus rhythm. When ACE inhibitiors are, however, instituted later, i.e. after the start of AF, it does no longer improve success of ECV.The author(s) declare that they have no competing interests.TVN carried out the study and drafted the manuscript, HC designed the study, and interpreted the data, MB participated in the draft of the manuscript and interpreted the data, DVV participated in the draft of the manuscript and interpreted the data, IVG participated in the design of the study and interpreted the data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Behavioral recovery , encompassing 1 week after unilateral labyrinthectomy, has been termed vestibular compensation. Evidence suggesting that the histamine H3 receptor  as well as three isoforms which display variable lengths of the third intracellular loop of the receptor were analyzed using in situ hybridization on brain sections containing the rat medial vestibular nucleus after unilateral labyrinthectomy. We compared these expression levels to H3 receptor binding densities.Expression levels for histamine H3 receptor mRNA levels (detected by oligo probe H3X) as well as mRNA levels of the three receptor isoforms studied  showed a pattern of increase, which was bilaterally significant at 24 h post-lesion for both H3X and H3C, followed by significant bilateral decreases in medial vestibular nuclei occurring 48 h (H3X and H3B) and 1 week post-lesion . Expression levels of H3B was an exception to the forementioned pattern with significant decreases already detected at 24 h post-lesion. Coinciding with the decreasing trends in H3 receptor mRNA levels was an observed increase in H3 receptor binding densities occurring in the ipsilateral medial vestibular nuclei 48 h post-lesion.Total H3 receptor may be an essential part of pre-synaptic mechanisms required for reestablishing resting activities 48 h after unilateral labyrinthectomy.Progressive recovery of the resting discharge of the deafferentated medial vestibular nuclei neurons results in functional restoration of the static postural and occulomotor deficits, usually occurring within a time frame of 48 hours in rats. Our data suggests that the H In rat, deafferentation of one labyrinth  results in a characteristic syndrome of ocular motor and postural disorders. These disorders have been divided into two categories : One category of symptoms, called static, includes head rotation in both the frontal and horizontal planes and ocular nystagmus . The oth3 receptor that was initially characterized as an autoreceptor controlling histamine synthesis and release iodoproxyfan . Non-specific binding was determined by incubating consecutive sections in the presence of 1 μM (R)α-methylhistamine . At the completion of incubation, the tissues were washed four times (4 min each) in the same fresh ice-cold buffer, dipped into ice-cold water, and dried under a current of air. Brain sections from control animals and animals 4 h, 24 h, 48 h, and 1 week post-lesion were treated simultaneously. Dried sections, along with standards, were exposed to Kodak BioMax X-ray films  for 2 days.Autoradiographic localization of [d before . BrieflyAutoradiographic films were quantified by digitizing the film images with a computer based MCID 5+ image analysis system  and by measuring gray scale pixel values. The relative optic density was converted to integrated optic density (IOD) based on a standard curve derived from standards exposed to films. Gray scale values were determined from four sections for each animal, measurements from white matter of each respective section were subtracted to obtain the final values, and the data were analyzed using either a paired t-test or one-way ANOVA combined with Bonferroni's Multiple Comparison Test as a post-hoc test. Significance was determined when p < 0.05.in situ hybridization and binding studies, performed all of the image and statistical analyses, and participated in the design of the study. AAA performed the surgeries, assisted with tissue collection, assisted with image analyses, and participated in the design of the study. KK established the method of in situ hybridization in the lab, designed the oligonucleotide probes, assisted with surgeries and tissue collection, and participated in the design of the study. HS synthesized and supplied rare chemicals required for the binding study. PP acquired funding, coordinated the study, and participated in its design. All authors read and approved the final manuscript.AL assisted with the surgeries and tissue collection, sectioned all of the brains, carried out the


Arabidopsis miRNAs have been predicted. Putative target mRNAs have been identified for most of the candidate genes.Using bioinformatic methods, 83 novel  Arabidopsis thaliana microRNAs and their target mRNAs. This method uses characteristic features of known plant miRNAs as criteria to search for miRNAs conserved between Arabidopsis and Oryza sativa. Extensive sequence complementarity between miRNAs and their target mRNAs is used to predict miRNA-regulated Arabidopsis transcripts.A class of eukaryotic non-coding RNAs termed microRNAs (miRNAs) interact with target mRNAs by sequence complementarity to regulate their expression. The low abundance of some miRNAs and their time- and tissue-specific expression patterns make experimental miRNA identification difficult. We present here a computational method for genome-wide prediction of Arabidopsis miRNAs and identified 83 new miRNAs. Evidence for the expression of 25 predicted miRNAs came from northern blots, their presence in the Arabidopsis Small RNA Project database, and massively parallel signature sequencing (MPSS) data. Putative targets functionally conserved between Arabidopsis and O. sativa were identified for most newly identified miRNAs. Independent microarray data showed that the expression levels of some mRNA targets anti-correlated with the accumulation pattern of their corresponding regulatory miRNAs. The cleavage of three target mRNAs by miRNA binding was validated in 5' RACE experiments.Our prediction covered 63% of known Arabidopsis and O. sativa and report a wide range of transcripts as potential miRNA targets. Because MPSS data are generated from polyadenylated RNA molecules, our results suggest that at least some miRNA precursors are polyadenylated at certain stages. The broad range of putative miRNA targets indicates that miRNAs participate in the regulation of a variety of biological processes.We identified new plant miRNAs conserved between  Arabidopsis thaliana and Caenorhabditis elegans to mouse and human  are non-coding RNA molecules with important regulatory functions in eukaryotic gene expression. The majority of known mature miRNAs are about 21-23 nucleotides long and have been found in a wide range of eukaryotes, from iewed in . Experimd so far ,13,15-17C. elegans let-7 is sufficient for its specific expression at different developmental stages ATP and T4 polynucleotide kinase (New England Biolabs) to generate high specific activity probes. Hybridization was carried out using the ULTRAHyb-Oligo solution according to the manufacturer's directions (Ambion), and signals were detected by autoradiography.Two-day-old seedlings, 4-week-old adult plants, root-regenerated calluses and mixed-stage flowers of Arabidopsis miRNAs. If an intergenic region encoding a miRNA had fewer than 500 nucleotides on either side of the miRNA locus, sequences were extracted up to the neighboring gene.To obtain genomic regions corresponding to miRNA precursors, we extracted 500 nucleotides upstream and downstream of every genomic locus of all known and predicted Arabidopsis MPSS datasets were used in this study: a MPSS database from abscisic acid (ABA)-treated plants, and plants with elevated levels of endogenous cytokinin [Arabidopsis tissues at different developmental stages - around 10-week-old active growing calluses initiated from seedlings, mixed-stage buds and immature flowers, 14-day-old leaves, 14-day-old roots and 24- to 48-h post-fertilization siliques [Two ytokinin ,44 and asiliques .Arabidopsis miRNAs and their O. sativa orthologs were used as query datasets. mRNA sequences of the Arabidopsis and O. sativa annotated genes were used as target datasets. Gaps were allowed in the pairing of miRNA and their target mRNAs. Mismatches were preferred over gaps by assigning higher penalties to gaps in the alignment algorithm. Consecutive gaps were preferred over scattered individual gaps by assigning higher penalties to gap opening than to gap extension. The top 500 putative hits from Arabidopsis miRNA target list and their O. sativa ortholog target list were compared. For each mRNA hit of an Arabidopsis miRNA, if a rice ortholog from the same gene family was also found among the top 500 rice miRNA hits, the Arabidopsis mRNA hit was selected as a putative miRNA target.miRNA target gene prediction was performed by aligning miRNA sequences with target mRNA sequences using the TimeLogic implementation of the Smith-Waterman nucleotide-alignment algorithm. Sequences of known and predicted Tissue comparison (reference vs flower) microarray data used for target gene validation were downloaded from TAIR . mRNA saTo identify the products of miRNA-directed cleavage we used the First Choice RLM-RACE Kit (Ambion) in 5' RACE experiments, except that we used total RNA (2 μg) for direct ligation to the RNA adaptor without further processing of the RNA sample. Subsequent steps were according to manufacturer's directions. Oligonucleotide sequences for PCR amplification of At3g26810, At1g12820 and TIR1 (At3g62980) are available upon request.Arabidopsis miRNAs using the MEME motif-searching software and Smith-Waterman gapped local alignment to identify homologs of known miRNAs. Pairs of aligned sequences were grouped by transitive closure, and multiple alignments were generated with ClustalW [Predicted miRNAs were compared to known ClustalW -54. The We used mfold program to calculate the free energy (ΔG) of predicted miRNA::mRNA pairs. For each miRNA::mRNA pair, the miRNA sequence was linked by 'LLL' to the target mRNA sequence. The 'LLL' linker sequence tells the mfold program to treat the miRNA and target sequence as two separate RNA sequences for energy calculation .Arabidopsis miRNAs, some of which are included among the predicted miRNAs in this work, confirming the validity of our approach.During revision of this manuscript, three groups -57 reporArabidopsis miRNA candidates and their rice orthologs (Additional data file Arabidopsis miRNAs (Additional data file The following additional data are available with the online version of this paper: the complete list of predicted miRNAs (Additional data file The complete list of predicted miRNAsClick here for additional data fileArabidopsis miRNA candidates and their rice orthologsA full list of the secondary structures of predicted precursors of Click here for additional data fileArabidopsis miRNAsMPSS evidence for known and predicted Click here for additional data filea complete list of predicted target mRNAs and their pairing with miRNA sequencesClick here for additional data file


The Cold Spring Harbor Laboratory Oral History Collection presents interviews with over 80 leading scientists, drawn from the fields of molecular biology and genetics That there's going to be a great comfort with people standing up and challenging a particular perspective. That young people are welcome. That old codgers are welcome as well. This is sort of the idyllic way that science is supposed to happen. And Cold Spring Harbor [Laboratory] provides that opportunity.”—Francis Collins, Cold Spring Harbor Laboratory Oral History CollectionCold Spring Harbor Laboratory (CSHL) has played a significant role in the development of genetics, a role that began at the Laboratory in 1904, just a few years after the discovery of Mendel's work. In 1908, George Shull's development of hybrid corn (maize) shaped the future of modern agricultural genetics; 55 years ago, Barbara McClintock worked on maize cytogenetics and discovered “jumping genes”; and in 1977, Richard Roberts was a codiscoverer of “split genes.” Throughout the 1950s, the Phage Course at CSHL trained many of the leaders of the then new field of molecular genetics, and the Laboratory's courses continue to train young scientists.CSHL Archives is a unique repository of rare books, manuscripts, photos, and scientific reprints that document the institution's significant contributions to molecular genetics research over the past 100 years. In 2000, the Archives received James Watson's personal collection of original materials, including manuscripts, correspondence, photographs, lab notes, lectures, memorabilia and reprints, for processing and preservation. This is an invaluable documentary resource for the history of molecular biology. While organizing over 60 years of Watson's correspondence, we realized that many of his correspondents have passed through CSHL as researchers, participants in the laboratory's leading scientific meetings, or instructors of our courses. We decided to talk to these scientists and record their commentaries, stories, and personal memories. These oral history interviews will enhance our collection by adding the voices of participants in significant scientific events.The project began in 2000. In 2002, we received a grant from the Gladys Brook Foundation that enabled us to purchase state-of-the-art recording and editing equipment for carrying out this project, and created a web site exhibiting taped conversations. During the last three years, we have interviewed over 80 scientists from several generations working in different fields of molecular biology and genetics, and have developed a video collection at CSHL's Carnegie Library. (A customized DVD of this collection is available.)The unique environment of the CSHL facilitated our task. Throughout the year, researchers from all over the world attend our scientific meetings to exchange information and ideas about their research, so most of the interviews took place at the Laboratory during these meetings. The scientists were always very pleasant and receptive to our requests for interviews. Other interviews were conducted in Cambridge and Boston, Massachusetts; La Jolla, California; Washington, DC; and abroad, in Oxford, England, and Sydney and Melbourne, Australia.Interviewing scientists was very exciting. We often wanted to come back for an additional interview because one or two hours was not enough time to collect all the memories that they wanted to share with us. It is well known that Sydney Brenner is a superb storyteller. After spending a few hours listening to Sydney in La Jolla, California, I received a call confirming my meeting with Francis Crick, which was to take place in the next 30 minutes. Despite looking forward so much to this meeting, I was torn between my present interview, in which I was deeply involved, and my next, which I was gladly anticipating.library.cshl.edu) provides an opportunity to “meet and get to know” the leading minds of molecular biology and genetics. We hope that our site makes the history of science come to life. These interviews can be helpful to all categories of users: students, scientists, historians, writers, journalists, and others.The CSHL Oral History Collection web site (The site is searchable and allows visitors to cross-reference people and information, and links them to the world of narratives and anecdotes, as told in the voices of those interviewed. Selected interview clips are organized into five topics: CSHL, James D. Watson, prominent scientists, scientific experience, and genome research. Each topic is divided into subtopics; for example, “Genome Research” includes: involvement in genomics, mechanism of the Human Genome Project, challenges of the Human Genome Project, dangers of genomic research, the future of genomics, and others. “Woman in Science”  is one oOur Web site adds a new dimension to the papers and books written by leading scientists—now you can listen to them discuss their work in their own voices! On this site you will meet a remarkable range of scientists, from friends and students of Barbara McClintock and Jim Watson, to the evolutionist Ernst Mayr, to the genome bioinformaticist James Kent. You'll hear Nancy Hopkins, Tom Maniatis, Matt Ridley, and Joan Steitz, to name just a few.We remain committed to our objective of providing an unprecedented and exciting approach for scientists, historians, scholars, and students to gain a firsthand account of the history of molecular biology and the stories behind the people who contributed to it. We have many more interviews planned, so in the future you can expect to meet even more of the fascinating figures who illuminate the glory days of the world of molecular biology.


Sleep duration may be an important regulator of body weight and metabolism. An association between short habitual sleep time and increased body mass index (BMI) has been reported in large population samples. The potential role of metabolic hormones in this association is unknown.Study participants were 1,024 volunteers from the Wisconsin Sleep Cohort Study, a population-based longitudinal study of sleep disorders. Participants underwent nocturnal polysomnography and reported on their sleep habits through questionnaires and sleep diaries. Following polysomnography, morning, fasted blood samples were evaluated for serum leptin and ghrelin (two key opposing hormones in appetite regulation), adiponectin, insulin, glucose, and lipid profile. Relationships among these measures, BMI, and sleep duration  were examined using multiple variable regressions with control for confounding factors.p for slope = 0.01), with a predicted 15.5% lower leptin for habitual sleep of 5 h versus 8 h, and high ghrelin (p for slope = 0.008), with a predicted 14.9% higher ghrelin for nocturnal (polysomnographic) sleep of 5 h versus 8 h, independent of BMI.A U-shaped curvilinear association between sleep duration and BMI was observed. In persons sleeping less than 8 h (74.4% of the sample), increased BMI was proportional to decreased sleep. Short sleep was associated with low leptin (Participants with short sleep had reduced leptin and elevated ghrelin. These differences in leptin and ghrelin are likely to increase appetite, possibly explaining the increased BMI observed with short sleep duration. In Western societies, where chronic sleep restriction is common and food is widely available, changes in appetite regulatory hormones with sleep curtailment may contribute to obesity. The less people sleep the higher their risk for being overweight. This study shows that sleep duration affects appetite-regulating hormones In population studies, a dose-response relationship between short sleep duration and high body mass index (BMI) has been reported across all age groups –7. In thA number of hormones may mediate the interactions between short sleep duration, metabolism, and high BMI. We hypothesized that the two key opposing hormones in appetite regulation, leptin and ghrelin ,9, play The institutional review board of the University of Wisconsin Medical School approved all protocols for the study, and informed consent was obtained from all participants. The Wisconsin Sleep Cohort Study is an ongoing longitudinal study of sleep habits and disorders in the general population . BrieflyThis investigation is based on Wisconsin Sleep Cohort Study data collected from the mailed sleep surveys, overnight studies, and 6-d sleep diaries. Overnight studies were conducted in laboratory bedrooms, with participants setting their own sleep and rise times. After informed consent was obtained, questionnaires on lifestyle and health history were administered, and height and weight measured. A blood sample was collected shortly after awakening from overnight polysomnography.An 18-channel polysomnographic system was used to assess sleep states, and respiratory and cardiac variables . Sleep wPolysomnographic measures of sleep duration just prior to blood sampling were used to evaluate degree of “acute sleep restriction.” “Total sleep time” was total hours of polysomnographically defined sleep. “Wake after sleep onset” (WASO) was hours of wake time after three epochs of sleep had occurred. “Sleep efficiency” was total sleep time divided by time from lights out until arising in the morning.Two variables were used to evaluate degree of “chronic sleep restriction” by estimating average nightly sleep duration: (i) “usual sleep” (from questionnaires) and (ii) “average nightly sleep” (from sleep diaries).Usual sleep was estimated from the following questions: how many hours of sleep do you usually get in (a) a workday night? (b) a weekend or non-work night? These questions were included in all mailed surveys and were added to questionnaires completed at the overnight study in 1998. For participants studied after 1998, data from questionnaires administered at the overnight study were used (58%); for the remainder of the sample, data from the mailed survey closest in time to the overnight study were used. Usual sleep was calculated as (5 × workday sleep + 2 × weekend sleep)/7.Average sleep duration was also estimated using a 6-d sleep diary, kept by 721 participants as part of an added protocol to measure daytime sleepiness. The median time between the blood collection and completion of the diary was 18 d. Almost all diaries (97%) were completed within 6 mo of blood sampling. In diaries, participants recorded the time they went to bed and arose each day, and the duration of any naps. “Average nightly sleep” was calculated as the sum of the hours between bedtime and arising divided by six. “Average nightly sleep plus naps” added naps to the above sum.For the analysis of the association of BMI and sleep duration, the sample comprised 1,040 participants with at least one 6-d sleep diary. Of these, 4-, 8-, and 12-y follow-up studies had been completed by 397, 179, and 11 participants respectively , providing repeated measures data for greater analytic efficiency and precision.I) + log (G)), where I is fasting insulin and G is fasting glucose [Following overnight fasting, serum was collected soon after awakening and stored at −70 °C. All samples were assayed in duplicate. It was not possible to assay samples from all participants in all assays because of the volume of serum available; this particularly affected the ghrelin assay, which required the most volume. Leptin and insulin were determined using enzyme-linked immunoassays . Total ghrelin and adiponectin were measured by radioimmunoassay (Linco Research). Sensitivity for the leptin and insulin enzyme-linked immunoassays was 0.5 ng/ml and 2 μU/ml, respectively. Sensitivity for the ghrelin and adiponectin radioimmunoassays was 10 pg/ml and 1 ng/ml, respectively. Intra- and inter-assay variations were all less than 5% and less than 10%, respectively. The quantitative insulin sensitivity check index (QUICKI) was 1/ from 1,040 participants with sleep diary data available. Mixed modeling techniques were used to account for within-subject correlation for participants with multiple visits. SAS procedure mixed was used for modeling and hypothesis testing using robust standard errors and a compound symmetric within-subject correlation structure. All reported p values are two-sided. For illustrative purposes, changes in leptin, ghrelin, and BMI for different sleep amounts were calculated at the average values and sex distribution of the relevant sample.All analyses were cross-sectional and performed using SAS/STAT 8.2. Leptin, ghrelin, and adiponectin were square-root transformed and insulin log transformed based on the distribution of residuals from the multivariate regression models. We evaluated the relationship of age, sex, BMI, and time of storage of blood sample on hormones using multiple regression. Partial correlations adjusted for age, sex, and BMI were calculated for hormones and QUICKI, with and without control of other potential confounders. The relationships between hormones and sleep were evaluated using multiple linear regression after control for potential confounders including age, sex, BMI, SDB, and morningness tendencies . In all analyses involving insulin, glucose, and QUICKI , participants with diabetes  were excluded. Participants with SDB were not removed from the analyses shown. When controlling for AHI in models, participants who used continuous positive airway pressure or who had inadequate sleep were excluded. Because controlling for AHI did not significantly change the sleep-hormone regression coefficients, these analyses are not shown. The relationship of BMI with average nightly sleep was evaluated using a quadratic fit. This was examined using multiple visits 2 coefficient = 0.156, p = 0.008; the two coefficients define a curve). The minimum BMI was predicted at 7.7 h of average nightly sleep. The most striking portion of the curve was for persons sleeping less than 8 h (74.4% of the sample), where increased BMI was proportional to decreased sleep. An increase in BMI from 31.3 to 32.4 (+3.6%) corresponded approximately to an average nightly sleep duration decrease from 8 h to 5 h, as estimated at the mean age (53.1 y) and sex distribution  of the sample with available sleep diary data.p = 0.01). When evaluated at the average values and sex distribution of our sample, a decrease from 8 to 5 h in average nightly sleep was associated with a predicted 15.5% decrease in leptin. p = 0.008). When evaluated at the average values and sex distribution of our sample, a decrease from 8 to 5 h of polysomnographically defined total sleep time was associated with a predicted 14.9% increase in ghrelin. There was no significant correlation between sleep duration (acute or chronic) and serum adiponectin, insulin, glucose, or QUICKI. Results of our analyses were unchanged after adjusting for the weighted sampling scheme.Using polysomnography to measure objective sleep immediately prior to blood sampling, ghrelin correlated significantly with total sleep time, sleep efficiency, and WASO . Using qWe found that habitual sleep duration below 7.7 h was associated with increased BMI, similar to findings in other studies including children ,17, adolPrevious studies have shown that both acute sleep deprivation  and chroLevels of ghrelin, a potent stimulator of appetite ,24,25, wr = 0.55, p < 0.001). One-night polysomnographically defined total sleep time had a similar intra-person mean difference (0.10 h), with a somewhat larger standard deviation (0.68) for 713 participants with at least three sleep studies.Our findings are strengthened by the large and well-characterized population-based sample, attention to bias and confounding factors, and in-laboratory polysomnographic data. The changes in hormones with sleep duration were consistent and of significant magnitude. They also represent the first demonstration of a correlation between peripheral hormone levels and both self-reported (questionnaire and diary data) and polysomnographically determined sleep amounts in a general population sample. While these data are more comprehensive than previous studies on this topic, some misclassification error may exist because of intra-person variability or limitations of polysomnographic measurement. Little is known about the stability of self-reported sleep duration and polysomnographic measures of sleep duration over time. We examined the stability of the self-reported sleep duration data, and found these measures to be stable. For 860 participants who completed three surveys, the mean (standard deviation) of intra-person differences in usual sleep for two 5-y periods was 0.10 (0.47) h. For 190 participants with at least three sleep diaries, the mean (standard deviation) of intra-person differences in average nightly sleep for two 4-y intervals was 0.09 (0.41) h. Furthermore, the subjectively reported hours of usual sleep and the diary-derived average nightly sleep values were highly correlated , supporting the concept that these measures reflect long-term and short-term changes in sleep amounts, respectively. Our findings are in agreement with the current view that leptin is important in signaling long-term nutritional status while ghrelin has a more significant role in acute hunger. The changes in leptin and ghrelin with sleep restriction could, therefore, provide a powerful dual stimulus to food intake that may culminate in obesity.Elevated ghrelin mainly correlated with acute sleep loss as measured by polysomnography immediately prior to blood sampling see B, while Longitudinal and intervention studies will be necessary to define further the link between sleep curtailment and increased BMI. Only total ghrelin was measured, since active octanoylated ghrelin is unstable. Although both total and active ghrelin appear to be regulated in a similar and parallel manner, future studies will need to focus on measurement of the biologically active form. Other potentially important appetite regulatory hormones, such as PYY 3–36 , were noHormone measurements were all performed on a single fasted, morning sample and may not reflect the 24-h profile. It is possible that participants with shorter sleep woke up earlier and that hormone differences may be partially related to circadian time. Leptin and ghrelin levels rise slightly during the night , and thiAnimal studies have suggested a link between sleep and metabolism ,32. In rSleep loss from a baseline of 7.7 h was associated with a dose-dependent increase in BMI. This was the predominant effect in a population increasingly curtailing sleep . Sleep gInsulin resistance with sleep deprivation has been reported in a laboratory study of young, healthy volunteers . When coOur results demonstrate an important relationship between sleep and metabolic hormones. The direct effect of chronic partial sleep loss on food intake, energy expenditure, and obesity now needs to be explored. Altering sleep duration may prove to be an important adjunct to preventing and treating obesity.Recent studies have shown that there is a link between sleeping less and gaining weight. It isn't clear why there is this link—perhaps, for example, those who are awake in the middle of the night tend to head for the refrigerator for a snack. Another possibility is that the amount of sleep that we have might affect the hormones that control our appetite. We know that extreme sleep deprivation affects the level of leptin, a hormone that controls appetite. Here, the researchers wanted to study levels of leptin and other appetite hormones under more normal conditions, in people with a range of sleeping habits.The researchers studied participants in a large sleep study that has been going on in Wisconsin for over 15 years. These participants have been filling out questionnaires about their sleep habits and their health in general, have kept sleep diaries, and have occasionally spent a night in the laboratory, where researchers studied their sleep in more detail. After sleeping overnight in the laboratory, the participants gave blood samples, which were tested for hormones.The researchers found that people who slept less were on average heavier. And people who slept less had lower levels of leptin and higher levels of ghrelin, another hormone that controls food intake.The combination of low leptin and high ghrelin is likely to increase appetite. In other words, short sleep might stimulate appetite, which increases weight.Future studies need to examine the effect of regular short sleeping hours on appetite, food intake, and obesity. These studies could help to answer the question of whether the rise in obesity in many societies is partly due to the fact that people are sleeping less. And it seems well worth testing whether increasing sleep to seven or eight hours per night could help people to lose weight.http://www.sleepfoundation.org/features/obesity.cfmNational Sleep Foundation Web page on obesity and sleep: http://www.wfsrs.org/homepage.htmlWorld Federation of Sleep Research Societies: http://www.rems.com.ar/Red en Medicina del Sueño: http://www.esrs.org/European Sleep Research Society:


In the present study we tested the hypothesis that progression of streptozotocin (STZ)-induced diabetes (14-days to 28-days) would produce renal and vascular dysfunction that correlate with altered p38- mitogen-activated protein kinase (p38-MAPK) phosphorylation in kidneys and thoracic aorta.Male Sprague Dawley rats (350–400 g) were randomized into three groups: sham (N = 6), 14-days diabetic (N = 6) and 28-days diabetic rats (N = 6). Diabetes was induced using a single tail vein injection of STZ  on the first day. Rats were monitored for 28 days and food, water intake and plasma glucose levels were noted. At both 14-days and 28-days post diabetes blood samples were collected and kidney cortex, medulla and aorta were harvested from each rat.The diabetic rats lost body weight at both 14-days (-10%) and 28-days (-13%) more significantly as compared to sham (+10%) group. Glucose levels were significantly elevated in the diabetic rats at both 14-days and 28-days post-STZ administration. Renal dysfunction as evidenced by renal hypertrophy, increased plasma creatinine concentration and reduced renal blood flow was observed in 14-days and 28-days diabetes. Vascular dysfunction as evidenced by decreased carotid blood flow was observed in 14-days and 28-days diabetes. We observed an up-regulation of inducible nitric oxide synthase (iNOS), prepro endothelin-1 (preproET-1) and phosphorylated p38-MAPK in thoracic aorta and kidney cortex but not in kidney medulla in 28-days diabetes group.The study provides evidence that diabetes produces vascular and renal dysfunction with a profound effect on signaling mechanisms at later stage of diabetes. Diabetes is a complex and multifarious group of disorders characterized by hyperglycemia that has reached epidemic proportions in the present century. Infection is a leading cause of morbidity and mortality among the diabetic population . DiabeteMitogen-activated protein kinases (MAPKs) are implicated in the etiology of diabetes ,5. MAPKstype 1 and type 2, is associated with decreased NO bioavailability  would produce renal and vascular dysfunction that correlate with altered p38-MAPK phosphorylation in kidneys and thoracic aorta. Therefore, the specific aims of the study are to characterize the progression of STZ-induced renal and vascular dysfunction at 14- and 28-days; and to examine if the progression of STZ-induced diabetes would alter the biosynthesis and activation of ET-1, NO and the phosphorylation of p38-MAPK and ERK1/2 in thoracic aorta, kidney cortex and kidney medulla.NIH Guide for the Care and Use of Experimental Animals, and were approved by the Institutional Animal Care and Use Committee of North Dakota State University.Male Sprague-Dawley rats  weighing 350–400 g were used in the study. The rats were acclimatized to the laboratory conditions for at least 7 days following their arrival. All animal experiments were conducted in compliance with humane animal care standards outlined in the All animals were age-matched at the onset of the study. The rats were randomly divided into three groups: control, 14-days and 28-days diabetic rats (n = 6 for each group).®, Bayer Corporation, IN) 2 days after STZ-injection. Diabetes was confirmed if blood glucose > 200 mg/dL. The animals were not given insulin supplementation. Food intake and water intake were examined everyday after STZ-injection.Diabetes was produced by a single tail vein injection of streptozotocin  . DiabeteThe non-diabetic control rats received an injection of 1 mL/kg saline. 14-days and 28-days post-STZ administration recordings of systemic hemodynamics, carotid blood flow, and renal arterial blood were done. Arterial blood was collected in plastic tubes containing EDTA (1 mg/mL) and heparin (5 units/mL) to determine the plasma concentration of ET-1, NO by-products (NOx) and creatinine. The animals were euthanitized by pentobarbital , the thoracic aorta and kidney were harvested. The concentration of ET-1, NOx, creatinine and protein expression of preproET-1, iNOS, eNOS, phosphorylated p38-MAPK (pp38-MAPK), total p38-MAPK, phosphorylated ERK, total ERK were determined in the thoracic aorta, kidney cortex and kidney medulla obtained from each animal.®, Abbott; 50 mg/kg).Separate group of animals (N = 6) were used for hemodynamic study. On the day of experiment, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium  was placed carefully around the artery. The probe was manually positioned so that the artery was centered within its window and then probe was held in position. Through a sterile 10 CC syringe loaded with sterile K-Y brand lubricating jelly , the acoustical window within the flow probe was filled while avoiding any air bubble.Under pentobarbital (50 mg/kg I.P.) anesthesia, rats were placed in dorsal recumbency and through a midline cervical incision, the left common carotid artery was identified and carefully separated from adhering connective material. The carotid artery was cleared from the vagal nerve and a Transonic® flow probe was placed around it. The probe was manually positioned so that the artery is centered within the window and the probe was held in position. The acoustical window within the flow probe was filled with K-Y brand lubricating jelly . Renal and carotid artery blood flow was measured using Transonic® flow meter T206 attached to MP100 system of Biopac Systems Inc., CA via analog to digital conversion. The MP100 system was calibrated with minimum and maximum flow capacity of the individual probe connected to the flow meter. The sampling of data was carried out at 1000 Hz and recorded to a dedicated computer using AcqKnowledge™ software.Under pentobarbital anesthesia, rats were place in dorsal recumbency, and through a midline abdominal skin incision the left renal artery was carefully separated from renal vein and a 0.5 VB Transonic-1.Kg-1) normalized to individual rat body weight was calculated.The concentration of creatinine was determined in plasma using creatinine liquid reagents . The blood samples immediately after collection were spun down at 5000 rpm for 10 minutes. The plasma was then decanted and stored at -20°C until the time of creatinine determination using manufacturer's instruction. Plasma concentration of creatinine (mg.dLThe concentration of ET-1 was determined in plasma. The blood samples were collected in plastic tubes containing EDTA (1 mg/mL) before euthanasia. The sample was centrifuged at 3,000 × g for 15 min at 4°C and plasma was separated and assayed for ET-1. Plasma was acidified adding an equal volume of 20% acetic acid. ET-1 like material was extracted from plasma  using C-X (nitrate + nitrite) was determined in plasma, thoracic aorta, kidney cortex and medulla. Blood (500 μL) was collected and 40 μL of heparin was added to each sample to prevent clotting. The blood sample was then decanted and stored at -20°C until the time of nitric oxide byproducts, (NO2 + NO3) NOx, determination. To determine the NOx level of thoracic aorta, kidney cortex and kidney medulla, those tissue samples were harvested from each experimental rat and immediately homogenized with cold phosphate buffered saline (PBS) on ice, which inhibited the activity of NOS ex vivo. The homogenate was centrifuged  and the supernatant was collected. The supernatant obtained from tissues and plasma was passed through a 1.2 μm multiscreen filter plate. Plasma NOx and tissue NOx concentration was determined by using Greiss reaction  were added to each well and the plates were incubated for an additional 10 min at room temperature. Absorbance was measured at 540 nm using a plate reader and converted to NOx concentration using a nitrate standard curve and expressed as μM in plasma and μmoles/g protein in tissue. Protein in the supernatant obtained from each sample was determined using standard Lowry method.The concentration of endogenous NOreaction ,15. 6 μLPreproET-1, iNOS, eNOS, total and phosphorylated p38-MAPK and ERK protein expressions of thoracic aorta, kidney cortex and kidney medulla were determined using standard SDS-PAGE and immunoblot technique. Briefly, thoracic aorta, kidney cortex and kidney medulla tissues were homogenized in lysis buffer and centrifuged as described by Pollack et al. . The supAll the data were expressed as mean ± SEM. One-way ANOVA was performed to analyze the hemodynamic and biochemical data using SPSS software. Following a significant F value, a post hoc Students Newman Keuls test was performed for inter- and intra-group comparisons. Statistical significance was realized at p ≤ 0.05 to approve the null hypothesis for individual parameters.All control rats were freely moving in their individual cages through out the study. Although diabetic rats appeared to be lethargic and displayed restricted movements, there was no sign of infection or motor disorder in any of the rats studied. The food intake and water intake of 14-days and 28-days diabetes groups were significantly increased as compared to control group  on aortic and renal ET-1 biosynthesis we determined the concentration of ET-1 in plasma and expression of ET-1 precursor, preproET-1 in thoracic aorta, kidney cortex and kidney medulla. A significant elevation in plasma ET-1 concentration was observed in 28-days diabetes as compared to 14-days diabetes and control groups Fig . We did A significant increase in the concentration of NOx in plasma of 28-days diabetes group as compared to the control and 14-days diabetes groups was observed Fig . The NOxA significant up-regulation of eNOS and iNOS in thoracic aorta in 14-days and 28-days diabetes groups was obtained as compared to control group Fig . Also inTotal p38-MAPK and phosphorylated p38-MAPK protein expressions in thoracic aorta were significantly elevated in 14-days and 28-days diabetes groups as compared to control group Fig . ExpressA significant decrease in the expression of phosphorylated ERK 1/2 of thoracic aorta in 28-days diabetes group but not in 14-days diabetes group was observed as compared to control group. We found that total ERK 2 but not total ERK 1 was significantly increased in thoracic aorta in 14-days diabetes groups as compared to control group. There was no change in the expression of total ERK 1 in 14-days and 28-days diabetes groups and ERK 2 in 28-days diabetes group as compared to control group Fig . In kidntype 2 diabetes in various animal models. This well-established model is characterized by insulin deficiency associated with insulin resistance [et al. [STZ has long been used as a drug of choice to induce sistance . It was sistance . In the  [et al.  found thRenal hypertrophy can be detected as early as one day after the onset of diabetes and seen regularly post 60-hr of single STZ injection . It has et al. [ET-1, a potent vasoconstrictor peptide, has been implicated in diabetes and cardiovascular disorders. Elevated, unchanged and attenuated plasma ET-1 levels have been reported during diabetes -27. Althet al.  followinet al. -30. In tet al.  or lack et al. .et al. demonstrated that ET-1 via binding to ETB receptors produces activation of NOS [et al. [et al. [B receptors via elevated ET-1 mechanisms. However, further studies will be required to strengthen this speculation.Hirata n of NOS  and gene [et al.  demonstr [et al. ,35. Incr [et al. . In a pr [et al. . We alsoet al. [et al. [Hyperglycemia has been shown to phosphorylate ERK1/2 in rat glomerular and mesangial cells  and p38-et al.  and Tian [et al.  demonstrin vivo and in vitro results suggest that ERK and p38MAPK may be involved in high-glucose-induced cellular hypertrophy [et al. [et al. [Both ertrophy . In the  [et al. . We spec [et al.  where thThe results obtained in the present study characterize the STZ -induced diabetic rat model in our laboratory. The marked characteristics of diabetic rat model include weight loss, increase food and water intake, and bradycardia. STZ-induced diabetes, both at 14-days and 28-days, produced renal dysfunction and vascular dysfunction that correlated well with levels of ET-1 and NOx and expression of preproET-1 and NOS proteins in kidney cortex and thoracic aorta. The data obtained in the present study demonstrate that progression of diabetes from 14-days to 28-days caused a factorial increase in p38-MAPK phosphorylation along with NOS upregulation in kidney cortex and thoracic aorta. The study provides evidence that diabetes produces vascular and renal dysfunction with a profound effect on signaling mechanisms at later stage of diabetes. However, more studies will be required to further delineate the inherent link or interaction between p38-MAPK upregulation, ET-1 and NO mechanisms and development of renal and vascular dysfunction during diabetes.