SLC6A3-related progressive infantile-onset parkinsonism-dystonia that mimics cerebral palsy. Ng et al. describe clinical features and molecular findings in a new cohort of patients. They report infants with classical disease, as well as young adults manifesting as atypical juvenile-onset parkinsonism-dystonia, thereby expanding the disease spectrum.Dopamine transporter deficiency syndrome is an  SLC6A3 mutations is the first inherited dopamine ‘transportopathy’ to be described, with a classical presentation of early infantile-onset progressive parkinsonism dystonia. In this study we have identified a new cohort of patients with dopamine transporter deficiency syndrome, including, most significantly, atypical presentation later in childhood with a milder disease course. We report the detailed clinical features, molecular genetic findings and in vitro functional investigations undertaken for adult and paediatric cases. Patients presenting with parkinsonism dystonia or a neurotransmitter profile characteristic of dopamine transporter deficiency syndrome were recruited for study. SLC6A3 mutational analysis was undertaken in all patients. The functional consequences of missense variants on the dopamine transporter were evaluated by determining the effect of mutant dopamine transporter on dopamine uptake, protein expression and amphetamine-mediated dopamine efflux using an in vitro cellular heterologous expression system. We identified eight new patients from five unrelated families with dopamine transporter deficiency syndrome. The median age at diagnosis was 13 years (range 1.5–34 years). Most significantly, the case series included three adolescent males with atypical dopamine transporter deficiency syndrome of juvenile onset (outside infancy) and progressive parkinsonism dystonia. The other five patients in the cohort presented with classical infantile-onset parkinsonism dystonia, with one surviving into adulthood (currently aged 34 years) and labelled as having ‘juvenile parkinsonism’. All eight patients harboured homozygous or compound heterozygous mutations in SLC6A3, of which the majority are previously unreported variants. In vitro studies of mutant dopamine transporter demonstrated multifaceted loss of dopamine transporter function. Impaired dopamine uptake was universally present, and more severely impacted in dopamine transporter mutants causing infantile-onset rather than juvenile-onset disease. Dopamine transporter mutants also showed diminished dopamine binding affinity, reduced cell surface transporter, loss of post-translational dopamine transporter glycosylation and failure of amphetamine-mediated dopamine efflux. Our data series expands the clinical phenotypic continuum of dopamine transporter deficiency syndrome and indicates that there is a phenotypic spectrum from infancy  to childhood/adolescence and adulthood . Genotype–phenotype analysis in this cohort suggests that higher residual dopamine transporter activity is likely to contribute to postponing disease presentation in these later-onset adult cases. Dopamine transporter deficiency syndrome remains under-recognized and our data highlights that dopamine transporter deficiency syndrome should be considered as a differential diagnosis for both infantile- and juvenile-onset movement disorders, including cerebral palsy and juvenile parkinsonism.Dopamine transporter deficiency syndrome due to  All patients underwent neurological examination and video footage of the movement disorder was undertaken with written informed consent obtained from participant families. Three cases did not consent to video recording because of cultural beliefs but were thoroughly clinically assessed to document movement disorder and neurological features. Five cases consented to video recording and a detailed video was taken to demonstrate the general phenotype, gross motor features at rest, fine motor tasks and eye movements. Four child neurologists with specialist interest in movement disorders  reviewed the videos available to obtain consensus opinion. Videos on Cases 1–3 and 6 are available in the Supplementary material.Patients presenting with childhood onset parkinsonism dystonia and, where available, a neurotransmitter profile characteristic for DTDS [raised CSF homovanillic acid: 5-hydroxyindoleacetic acid (HVA: 5-HIAA) ratio >4.0] a were idSLC6A3 was undertaken using gene primers covering all coding exons and flanking intronic regions.For Cases 1–3 (three siblings from a consanguineous Pakistani family) we performed whole exome sequencing on one affected individual (Case 3), using a HiSeq 2000 sequencer with a paired-end 2 × 100 bp protocol after enrichment of exonic and adjacent splice site sequences with the SeqCap EZ Human Exome Library v.3.0 enrichment kit. This resulted in a mean coverage of 97;79% of target sequences were covered at least 30×. Data analysis and filtering was performed as previously described  were performed after preparation of mutant constructs of human DAT from wild-type pCIN4-hDAT, as previously described (primers available on request) (1 cells with mutant and wild-type human DAT was performed by use of Lipofectamine® 2000 (Invitrogen) as previously described  into suspended cells expressing human DAT was measured for 5 min at 21°C, and to monitor cocaine analogue binding, cells were incubated with 4 nM 3H-CFT  for 20 min at 21°C; for saturation analysis, 0.1–100 nM non-radioactive CFT was also present. Briefly, uptake and binding assays used a high sodium, low potassium buffer containing glucose and tropolone, and the non-specific binding was defined with 1 μM CFT as in our previous work. . Half maximal inhibitory concentration was estimated by logistic fitting of data by the ORIGIN software (Origin Lab Co.); this value was then entered into the Cheng- Prusoff equation to calculate the potency (Ki) of dopamine in inhibition of 3H-CFT binding. Km and Vmax values for uptake were calculated with the KELL program as in previous work  and eluted with SDS-PAGE sample buffer. The total lysates and biotinylated proteins were resolved on 8% Tris-glycine mini gels and probed with polyclonal anti-DAT antibody against the C-terminal of DAT (Millipore), before horseradish peroxidase-conjugated goat anti-rabbit antibody. Polyclonal anti-β-actin antibody (Sigma-Aldrich) was used as an internal control for loading. The transporter signal was visualized using Thermo Scientific SuperSignal® West Pico Chemiluminescent Substrate solution (Thermo Scientific).Cells transiently expressing wild-type or mutant human DAT were washed with cold PBS and incubated with sulpho-NHS-SS-biotin  for 60 min at 4°C, before incubation with 100 mM glycine in PBS for 20 min and extensive washing. The washed cells were lysed in mammalian protein extraction reagents (Thermo Scientific) supplemented with a protease inhibitor cocktail (Thermo Scientific) for 10 min at 21°C and transferred into eppendorf vials. The vials were incubated for 60 min on ice with vortexing every 5 min. The lysate was then centrifuged at 14 000 3H-CFT binding data. The latter binding results for wild-type and mutant human DAT were first compared by one-way ANOVA. If this test indicated significant differences between groups, the Dunnett multiple comparisons test was used to compare each mutant value with wild-type. For statistical analysis of 3H-dopamine uptake by mutants, Ki values for wild-type human DAT (controls) were used from the same set; thus values for Cases 1–3 constituted one set and values for Case 8 another set . The mutant Ki values were expressed as % Control with Control = wild-type, set to 100%; the mutant values were then subjected to a one-sample Student’s t-test (Bonferroni-corrected for multiple comparisons). Km and Vmax values were obtained for one mutant and compared with wild-type values obtained in the same set by unpaired Student’s t-test. Indicated as n is the number of independent experiments (each assayed in triplicate), for which mean ± standard error (SE) was calculated. We regarded P-values of 0.05 or lower as statistically significant.Each case was assessed for transporter properties with corresponding wild-type controls. Wild-type values were combined in the overall statistical analysis of 2, 2 mM MgCl2, 1.1 mM EGTA, 30 mM d-glucose, pH 7.35). Cells were washed with external bath solution . Upon gaining whole-cell access to the cell, dopamine-containing internal solution was allowed to diffuse into the cell under current clamp for 10 min before amphetamine or cocaine application.Amperometry studies were possible for mutant Arg445Cys as there was appreciable expression on the cell surface. HEK293 cells were cultured and transiently transfected as previously described (n = 5) presented <1 year of age (akin to previously reported children with DTDS) but there were others who presented beyond the infantile period (n = 3).The clinical features of all eight cases are summarized in We identified three brothers  from a Pakistani family with a clinical diagnosis of juvenile-onset parkinsonism presenting with tremor and progressive parkinsonian symptoms. The parents are consanguineous first cousins sharing common paternal grandparents . There iAll three brothers had a normal birth history, neonatal course, and achieved normal early developmental milestones in infancy. Age appropriate cognitive, motor and speech and language skills were reported in the first decade of life with no neurodevelopmental concerns. At 10–11 years of age, they all developed tremor affecting the head, with progression of symptoms in their 20 s, clearly observed in the elder brothers.Supplementary Video 1). He has normal speech, good balance and normal gait. He also reports intermittent tremor affecting hands and lower limbs. The second brother  now 26 years old, presented in the same manner as his younger brother with mild tremor affecting the head and hands. His movement disorder has evolved to develop cervical dystonia resulting in cervical antecollis with hypertrophy of trapezius muscles. He has frontalis hyperactivity with an associated intermittent low amplitude fine head tremor. He has normal eye movements with hypomimic facies (Supplementary Video 2). He also reports intermittent fine hand tremor and deterioration in speech and now speaks in single words only. The eldest brother is aged 28 years  and presented with the same symptoms as his two brothers initially. Over the past 20 years his symptoms have progressed and he is the most severely affected of the three. He now has prominent head tremor, a coarse resting hand tremor with ocular flutter and hypomimia (Supplementary Video 3). He experiences recurrent falls associated with poor balance and difficulty in initiating movements. His speech deteriorated initially to the use of single words from 20 years old onwards and now he is non-verbal. There is no evidence of cerebellar or pyramidal signs on neurological examination of any of these patients to date. Unfortunately CSF neurotransmitter studies and neuroimaging studies are not available to this family who are from a rural area of Pakistan, and medication trials with l-DOPA, dopamine agonists or others have not been undertaken.Currently at 16 years old, the youngest brother  has normal eye movements and facial expression, with predominant head tremor/titubation  were identified with a clinical phenotype and CSF neurotransmitter profile (undertaken in 4/5 cases) compatible with DTDS. They all presented during infancy with a complex movement disorder and motor developmental delay at a median age of 6 months (range 2–9 months). All reported a preceding normal perinatal history, birth weight and head circumference. There was no history of a movement disorder, Parkinson’s disease or psychiatric symptoms in either parents or siblings. On review, there was also an associated early history of irritability and feeding difficulties from the neonatal period to age 3 months in all these patients.Of particular interest was the clinical progress of Case 4 who is the oldest surviving adult patient with classical DTDS—infantile parkinsonism dystonia (now aged 34 years). She presented at 3 months with feeding difficulties and irritability and progressed to develop dystonia and rigidity in infancy associated with motor delay. During early childhood she was able to mobilize with a Kay-walker and had functional use of her upper limbs to steer her electric wheelchair. During adolescence, her movement disorder evolved to progressive bradykinesia and currently at 34 years, she has an asymmetric resting tremor with bradykinesia and minimal purposeful movements in her upper limbs with akinetic lower limbs, muscle wasting and osteopenia. She is hypomimic and non-verbal, requiring gastrointestinal tube feeding. In contrast, her younger sister (Case 5) presented with an early history of feeding difficulties and irritability, then developed a progressive hyperkinetic dystonic phenotype (without parkinsonian features) and died at age 10 years from pneumonia. Both siblings were only recently genetically diagnosed with DTDS in 2010, after discovery of the disease-causing gene. Indeed before diagnosis, Case 4 had a diagnostic label of ‘juvenile parkinsonism’.Supplementary Video 4; section 1). Three children were noted to have axial hypotonia at presentation and with time all five cases developed a movement disorder characterized by axial hypotonia, hypomimia, ridigity, generalized bradykinesia, generalized dystonia with dystonic tremor . Two children developed chorea of their lower limbs and had paroxysmal dystonic storms. Two cases had eye movement abnormalities with saccadic intrusion on smooth pursuit in one and recurrent oculogyric crises in the other. These two were also noted to have orolingual dyskinesia as well as dystonic tremor of tongue and limbs. All patients developed bulbar dysfunction and required artificial feeding through the nasogastric/gastrostomy route.Case 6 represents the most classical clinical phenotype  response to l-DOPA with improvement in her irritability and dyskinesia and also showed some marginal temporary improvement in her bradykinesia with selegiline therapy. Overall this group of patients with classical DTDS experience a progressive parkinsonism dystonia movement disorder that is medically refractory.Medical treatments tried in Cases 4–8 included SLC6A3 gene (SLC6A3. Retrospective single nucleotide polymorphism (SNP) array analysis confirmed that this SLC6A3 variant was contained within a 1.4 Mb region of homozygosity shared by the three affected siblings (Cases 1–3), with a different haplotype combination evident in both parents and the unaffected sisters (All eight patients in this cohort were found to harbour either homozygous or compound heterozygous missense or splice site mutations in the 6A3 gene . In Case sisters .Table 3SLC6A3. The mutations in Cases 4 and 5 were briefly reported previously (www.1000genomes.org/), dbSNP137 and the NHLBI Exome Sequencing project. Sequence alignment for novel missense mutations showed Ala314, Gly386, Tyr470, Arg85 and Arg445 amino acid residues to be highly conserved throughout mammalian species. The splice site mutations were predicted to cause deleterious splicing leading to either nonsense-mediated decay or a truncated protein product .For Cases 4–8, mutations were identified by direct Sanger sequencing of 3H-dopamine uptake at 8.83 + 0.71% of wild-type and this enabled us to monitor its uptake characteristics in more detail revealing unchanged Km, but significantly reduced Vmax . All other mutant proteins showed absent transporter activity  or severely reduced activity at ∼0.5–5.65% of normal  compared with wild-type human DAT. Activity was not further lowered in co-expressed Arg445Cys–Arg85Leu (1.82%).For all identified missense mutations , the transporter activity of mutant human DAT proteins was compared to that of wild-type human DAT after transient expression of mutant human DAT in LLC-PK1 cells . Wild-tyd of 3H-CFT binding in wild-type human DAT (14.1 nM), the Kd was decreased for Ala314Val (5.31 nM) and increased (i.e. reduced affinity) in the mutants Arg445Cys and co-expressed Arg445Cys–Arg85Leu (90.1–128 nM). The values for Gly386Arg, Tyr470Ser and Arg85Leu (5.78–34.4 nM) were not statistically different from the wild-type Kd.Compared with the Ki of dopamine inhibiting cocaine-analogue binding was decreased for Ala314Val (1.1 µM) but increased (i.e. reduced affinity) in most other mutants compared with that of wild-type human DAT with the most marked affinity reduction in Arg85Leu (Ki = 1574 μM versus 4.22 µM in wild-type human DAT). Again, co-expression of Arg445Cys–Arg85Leu did not lead to further lowered dopamine affinity.The K3H-CFT to cells mainly represent surface binding . The DAT inhibitor cocaine (10 µM) did not alter the amperometric current signal in human DAT nor human DATArg445Cys (n = 3), indicating that there was no constitutive DAT-mediated dopamine efflux  by a whole-cell patch clamp pipette. Amphetamine (10 µM) induced a robust dopamine release from wild-type human DAT cells (quantified by amperometric current). Amphetamine failed to induce dopamine release from mutant human DAT Arg445Cys cells  , presented differently, with a hyperkinetic dystonic movement disorder who died at age 10 years. We postulate that such intrafamilial variation in clinical disease severity and presentation may be due to other currently undetermined genetic influences and/or environmental factors.In this report, we additionally describe the first patient with infantile onset DTDS surviving into adulthood who was only genetically confirmed at 34 years of age. At this stage in her 30 s, she had features of parkinsonism with prominent bradykinesia, and was clinically labelled as having ‘juvenile parkinsonism’. Although she showed many of the early features of classical infantile DTDS  a her rel+ and Cl−, this results in a conformational change from outward-facing to inward-facing  is an intriguing mutant and distinct among the mutants studied here, retaining the highest residual dopamine uptake capability , amphetamine uptake, and the hydrophobicity of amphetamine likely permits some translocation across the plasma at high concentrations  loss of the primary function of DAT as indicated by absent or severely reduced dopamine uptake; (ii) reduced DAT cell surface binding; (iii) in most cases reduced affinity of DAT for dopamine; (iv) generally reduced dopamine recognition by DAT; and (v) decreased expression of mature DAT with an predominance of excess unglycosylated DAT, which is known to negatively impact both the transport function of DAT and also trafficking of DAT to the cell surface . Our datSLC6A3 genotypes may therefore have a differential impact on DAT dysfunction. Our eldest atypical DTDS patient at age 28 years appears to have a more severe disease progression than that of the patient described by As reported in this study and by our group previously, the majority of infantile cases the mutant human DAT retain 0–5% residual DAT activity whereas the human DAT mutants Ala314Val  retain higher residual dopamine. Furthermore human DAT mutants of atypical juvenile DTDS cases demonstrate some residual and even up to normal levels of expression of mature transporter protein at the cell surface a. These SLC6A3 lead to a continuum of DTDS phenotypes whereby the clinical phenotype appears to be related to residual DAT function, which determines the onset and severity of symptoms. Higher residual DAT activity may postpone the age of disease onset. Similar phenotypic continuums  are observed in other neurotransmitter disorders such as tyrosine hydroxylase deficiency   (In conclusion, we report a cohort of patients with DTDS identified in infants and adolescents and provide data corroborating loss of DAT function in this condition. DTDS is now not only described in children with infantile-onset parkinsonism dystonia, but also in adults with juvenile-onset parkinsonism and a new case report of early onset Parkinson’s disease (


Kaposi sarcoma (KS) is a complex multifocal neoplasm and is the major cause of death for about 50% of acquired immunodeficiency syndrome (AIDS) patients. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus with a causal role in the development of all types of KS. KS is prevalent among the Uygur people in Xinjiang, especially in south area. Here we carried out a cross-sectional study among 1534 general Uygur individuals from south and north region of Xinjiang to assess the seroprevalence of KSHV and to identify the potential correlation between KSHV seroprevalence and KS incidence.Seroprevalence of KSHV in South and North Xinjiang was 23.1% and 25.9%, respectively. Older age was independently associated with higher KSHV seroprevalence. In subjects from South Xinjiang, lower educational level and reported drinking were each independently associated with higher KSHV seroprevalence. Furthermore, the antibody titer was significantly lower in both south and north KSHV seropositive individuals compared with KS patients, as analyzed by gradient dilution (P < 0.001).KSHV is highly prevalent in the general Uygur population in both South and North Xinjiang. Interestingly, the infection rate of KSHV in these two geographical areas did not correlate well with KS incidence. Perhaps unknown factors exist that promote the progression of KSHV infection to KS development in the local minority groups. Kaposi sarcoma (KS) is a mesenchymal tumour involving blood and lymphatic vessels . KS can KSHV prevalence exhibits considerable variation in different geographical regions and populations. Several studies have demonstrated that KSHV seroprevalence correlates with the occurrence of KS -17. In mXinjiang is located at the middle point of the Silk Road that used to extend from Rome to China. Many ethnicities, such as the Uygur (48%), Han (38%) and Kazakh people (7%), mix in this area. Classical KS cases occur most frequently in two of these minority groups: the Uygur and Kazakh groups. Furthermore, about 90% of KS cases have been reported in the Uygur group, which reside in the south region of Xinjiang . HoweverOf the 1534 Uygur people included in the study, 1008 (65.7%) were from the southern part of Xinjiang and 526 (34.3%) were from the northern part of Xinjiang. The seroprevalence of KSHV among the general Uygur population in the southern and northern parts of Xinjiang was 23.1% and 25.9%, respectively. Table A multivariate logistic regression analysis was used to identify independent risk factors for KSHV infection in total study population Table . In thisThe characteristics of the 1008 Uygur village participants from Southern Xinjiang and the KSHV seroprevalence rates are presented in Table To further identify independent risk factors, variables that were significant in univariate analysis at P = 0.100 were included in multivariate logistic regression analysis and the data are presented in Table 2 = 33.74, P < 0.001).These results indicated that compared with KS patients, KSHV-infected individuals in both south and north presented a lower antibody titer against KSHV.The distribution pattern of KSHV antibody titers among high seropositive individuals from both north and south and KS patients were compared and the results are displayed in Figure More than 95% of KS cases in China, including all epidemiological forms, were observed in minority groups in Xinjiang, particularly in the Uygur group . PreviouThe seroprevalence of KSHV in the general Uygur population in Xinjiang was significantly more prevalent in individuals aged ≥55 years compared to those aged < 55 years. These results are consistent with those of several other studies, which described an increase in seroprevalence of KSHV with age in different populations -29. ThisFurther analyses were then carried out to focus on the 1008 rural Uygur people from South Xinjiang. This is the first report regarding KSHV seroprevalence and risk factors in this area, a region endemic for KS. The seroprevalence of KSHV in this study population was independently associated with educational level. It was apparent that with improved educational level (from illiterate/semiliterate to Junior high school level education), KSHV seroprevalence decreased from 28.3% to 17.7%, suggesting that a higher education level is a protection factor for KSHV infection. Notably, the educational level of this study population was usually below the high school level, and among the older age group (≥55) the percentage of illiterate/semiliterate individuals was 43.7%. Therefore, knowledge about health issues is far more limited in the Uygur population in this region, especially in the elder Uygur population. Furthermore, the mobility of the population in this region is relatively low. The traditional life style and behavior of the Uygur people has remained unchanged over the past two decades , with thWe identified a high seroprevalence of KSHV infection in the general Uygur population in both South and North Xinjiang, and the distribution of KSHV seroprevalence in these two geographical areas did not show a clear correlation with KS incidence. Perhaps other unknown factors have promoted the association between KSHV infection and KS development in the local minority groups. Understanding the epidemiology of KSHV infection in rural areas of Uygur is critical for designing effective intervention strategies to decrease the transmission of this virus and to prevent malignancies associated with KSHV infection.2) values were also calculated for all subjects. Serum and plasma samples were collected, separated immediately and stored at -80°C prior to testing.South Xinjiang was represented by Moyu Country of Hotan City which is located in the southernmost region of Xinjiang surrounding the Tarim Basin and where more than 95% of the population were rural Uygur people. North Xinjiang was represented by Urumqi, which is the most highly developed and industrialized region in Xinjiang. A total of 1543 research subjects were randomly selected from this two regions of Xinjiang by using a stratified, multistage sampling. Among them, residents (age > 18years) from 15 villages in 3 rural town of Moyu Country and employees from top 10 developed enterprises of Urumqi were 1008 and 526, respectively. Trained staff obtained the informed consent of all participants and carried out interviews. A questionnaire on age, gender, ethnicity, education level and residence was completed for all subjects. Other characteristics, such as family number, drinking, cigarette smoking and anthropometric measurements, including height, weight, waistline and hip circumference, were also recorded for all of the subjects in the southern part of Xinjiang. All data were analyzed anonymously. Body mass index (BMI) , to compare antibody titers with KSHV infected persons.The study protocol was approved by the local research ethics committee of the First Teaching Hospital of Xinjiang Medical University. Written informed consents were obtained from all study subjects before data collection.2SO4 and absorbance was measured at a wavelength of 450 nm. Serum samples from patients with classical-KS in Xinjiang and with AIDS-KS or skin carcinoma in France which were obtained from the Medical center of Besancon University France-Comte were used to assay the sensitivity and specificity in this study. Based on the surveys of above groups, the combined antigens ELISA had a sensitivity of 81.8% and specificity of 97.9%, respectively. In the following screening, serum from an AIDS-KS patient that had high antibody titers to both KSHV latent and lytic antigens, and serum from three healthy children in Sichuan province without any specific antibodies to KSHV, were used as positive and negative controls, respectively, in all assays. Both positive and negative controls were used in three wells in each plate in this study. Each sample was also tested three times. Based on the results of assays with the control groups, a serum sample with an absorbance value above the mean plus three standard deviations of the negative control wells in an assay was considered as positive. A highly seropositive result for KSHV was set considered as a value greater than the mean absorbance value plus five standard deviations of the negative control wells. All assays were examined by a single observer.Three KSHV specific viral genes were expressed and purified as GST-fusion proteins used to construct the combined antigens enzyme-linked immunosorbent assay(ELISA) including latent antigen ORF73 and lytic antigens ORF K8.1 and ORF65. Briefly, Viral antigens were diluted in coating buffer to a final concentration of 10 μg/ml and used to coat ELISA plates  overnight at 4°C. After washing with phosphate-buffered saline (PBS) containing 0.05% Tween 20(PBST), 1:100 diluted serum samples were added and plates were incubated for 60 min at 37°C. This was followed by incubation with goat anti-human IgG conjugated with peroxidase  for 60 min at 37°C. The color reaction was developed for 15 min at RT with tetramethyl benzidine after PBST washing. Reactions were stopped with 50 μl of 1 M HSerum samples that were highly positive for antibodies against KSHV in the ELISA assay and the sera of 51 KS patients were analyzed to determine their antibodiy titers using gradient serum dilutions . The positive standard followed the criterion above. Antibody titers lower than 1:400, between 1:400~1:800 and higher than 1:800 were classified into the low group, medium and high group, respectively.The data were entered in duplicate and analyzed using the SPSS11.0 software .Univariate analysis was performed to examine associations between the KSHV serostatus and all of the questionnaire variables. Factors that were significant in univariate analyses at P = 0.100 were included in a multivariate logistic regression analysis, to identify which factors, if any, were independently associated with KSHV infection. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to quantify the relationships, while P-values were calculated to indicate the statistical significance. P-values < 0.050 were considered significant. The chi-square test was also performed when appropriate to compare antibody titers between KSHV -infected individuals and KS patients.KS: Kaposi sarcoma; KSHV: Kaposi's sarcoma-associated herpesvirus; HHV-8: Human herpesvirus 8; AIDS: acquired immunodeficiency syndrome; PEL : primary effusion lymphoma; MCD: multicentric Castleman's disease; OR: Odds ratios; 95%CI: 95% confidence intervals; HBV: hepatitis B viruses; HCV: hepatitis C viruses; BMI: Body mass index; ELISA: enzyme-linked immunosorbent assay; RT: room temperature; ORF: open reading frameThe authors declare that they have no competing interests.HW carried out study design, sample collection, and statistical analyses performance and participated in the antibody detection. JL and LL participated in sample collection. Dilimulati and ZH R wrote and collected the questionnaire. HW participated in the design of the study and carried out statistical analyses. XW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing). The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators.Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while  Much of plant growth and development is shaped by sequence-specific transcription factors, proteins that act in response to external and internal cues to modulate gene expression. The MYB family is the largest family of plant sequence-specific transcription factors, with greater than 100 family members in individual plant species While members of the R2R3-MYB family are being characterised in increasing numbers, these investigations largely focus on the involvement of a particular MYB in the manifestation of a specific plant phenotype. That is, most of these analyses do not extend to a more detailed examination of MYB function at the molecular level. Nevertheless, some general themes with respect to R2R3-MYB function at the molecular level are emerging AtMYB61, a member of the Arabidopsis thaliana R2R3-MYB family of transcription factors, illustrates the involvement of R2R3-MYB family members in the regulation of plant-specific processes. AtMYB61 is a pleiotropic regulator of three major facets of the plant transpiration system: xylem cell differentiation; lateral root outgrowth; and, stomatal aperture AtMYB61 modifies gene expression in response to diurnal cues so as to appropriately modify the aperture of stomata AtMYB61 plays a role in modifying the capacity to take up carbon dioxide for photosynthesis, while limiting the loss of water from the plant body. AtMYB61 also alters gene expression in response to sugars, resulting in modification of plant architecture and cell wall structure AtMYB61 to bring about important changes in plant function are unknown. Furthermore, although AtMYB61 has been shown to bind to certain consensus motifs AtMYB61 has not yet been determined quantitatively.AtMYB61 and its DNA target sequences. Cyclic amplification and selection of targets (CASTing), which enables identification of a transcription factor’s DNA-binding sites from a pool of random oligonucleotides, was used to identify target DNA-binding sites for AtMYB61 AtMYB61 sequence-specific binding, and to hypotheses about the roles these may play in shaping AtMYB61 function in vivo.Given that R2R3-MYB proteins are involved in a rich variety of plant-specific processes Antibody generation was carried out in strict accordance with the Province of Ontario’s Animals for Research Act, and the requirements of the federal Canadian Council on Animal Care. The protocol was approved at the University of Toronto, which involved full committee review by the Local Animal Care Committee (LACC), followed by approval by the University of Toronto Office of Research Ethics, the University Veterinarian, and finally the University of Toronto Animal Care Committee (UACC) . All efforts were made to minimise suffering.AtMYB61 protein was produced in E. coli using the coding sequence cloned in frame into the NdeI and BamHI sites of the pET15b vector (Novagen). Recombinant AtMYB61 protein was produced, extracted and affinity purified as described previously for pine MYB proteins Recombinant AtMYB61 polyclonal antibodies were produced against the recombinant fusion protein in rabbits as described previously Anti-For western blot analysis, total soluble protein extracts were separated by SDS-PAGE and transferred to Bio-Rad Laboratories Nitrocellulose Trans-Blot Transfer Medium (0.45 µm) by electrophoretic transfer . Chemiluminescent western blot analysis was performed on the filters with Invitrogen’s Western Breeze Chemiluminescent kit as described by the manufacturer . Primary antibody dilutions were done at a final dilution of 1/20000.et alAtMYB61 full length recombinant protein. This complex was added to a Protein G Dynabead  plus post-injection AtMYB61 antibody complex, causing the complex to immunoprecipitate. The immunoprecipitated complex was then washed 3 times, resuspended in 100 µL PCR buffer, boiled and then PCR amplified for 30 cycles with 15 pmol of forward and reverse primers. 10 µl of the amplified selected targets were kept for analysis and 90 µL were used to continue with the next cycle. This cycle was repeated four more times to select for AtMYB61 consensus DNA target sequences. The selected targets were then cloned into Invitrogen’s pCR4 TOPO vector and sequenced .The CASTing assay was completed according to Wright MEME was conducted as described previously AtMYB61 protein and passed through BioRad nitrocellulose filters (0.2 µm) . The relative binding of recombinant AtMYB61 protein to the CASTing motifs and mutated AC-I sequences were recorded. The dissociation constants (Kd) of the CASTing targets to AtMYB61 were determined by GRAFIT program which linearised the nonlinear regression via scatchard plots to calculate the point at which half of the ligand was bound to AtMYB61.The nitrocellulose filter-binding assay was conducted as described by Hall and Kranz AtMYB61 protein was produced, extracted and affinity purified as described previously for pine MYB proteins AtMYB61 protein in place of pine MYB protein.Recombinant AtMYB61 was predicted using the tool PHYRE www.sbg.bio.ic.ac.uk/phyre/html/index.html). PHYRE proposed that the resolved structure that shared the most homology to AtMYB61 was the animal c-MYB DNA-binding domain, which was resolved previously with its DNA consensus motif (AACNG) by heteronuclear multidimensional NMR AtMYB61 with an E-value of 3.8e-13 and an estimated precision of 100%. The two protein sequences were 44% alike using amino acid sequence alignment. The PBD (protein data bank) file recovered from the PHYRE analysis (PBD ID = c1msfC) was used to superimpose the predicted AtMYB61 crystal structure with the c-MYB crystal structure using DaliLite AtMYB61 using homology. The PBD files for the AC-I and NBS nucleotide motifs were created from the http://structure.usc.edu/make-na/server.html server. Using Pymol The tertiary structure of AtMYB61 coding sequence in place of pine MYB sequences. Transcriptional activation assays were conducted with three biologically independent replicates per condition.Transcriptional activation assays using yeast were as described previously cyclic amplification and selection of targets (CASTing) assay, antibodies were raised against a non-conserved region in the AtMYB61 C-terminus (14 DNA molecules) pool of “randomers” was incubated with AtMYB61 full-length recombinant protein  and sequenced. Following enrichment, 89 CASTing-derived oligonucleotides were sequenced. Sequences were subjected to analysis to discover over-represented motifs using MEME (Multiple Em for Motif Elicitation) Five CASTing cycles were undertaken to enrich the pool of oligonucleotides in DNA binding-sites bound by in plantaPinus taeda) MYB1 Eucalyptus grandis) MYB2 Pinus taeda) MYB1 and MYB4 bound AC elements present in the gene regulatory sequences of a pine gene encoding glutamate synthetase (GS1b) AtMYB61 Arabidopsis thaliana.AC elements, also known as PAL boxes or H-boxes, play key roles in regulating transcription for a variety of genes, particularly those encoding enzymes implicated in phenylpropanoid metabolism AtMYB61 protein to the CASTing-derived sequences were determined (−09 M) was ACCTAC (AC-I) ; however, when a mutation is present in the last three nucleotides of the AC-I motif, the binding is reduced but not completely abolished  . Since t binding . A guaniI motifs . Bindingbolished . The relby EMSAs . EMSAs wng assay . This enAtMYB61 bound its preferred target AC-I (ACCTAC) with a binding constant of 9.12E−09 M (−09 M±28% ) et al. found that AACG nucleotides in the MYBSI binding site were critical for binding AtMYB61 had a set of core recognition nucleotides – ACC – that could not be mutated without abolishing binding  was solved by heteronuclear multidimensional NMR AtMYB61. The AC-I (ACCTAC) and NBS (GAGACC) nucleotide models were then docked into the predicted binding sites of the AtMYB61 model . For example, there were more hydrogen bonds shared between AtMYB61 DNA-binding domain and AC-I compared to NBS . Based on the model of AtMYB61 bound to AC-I, several specific intermolecular interactions are predicted to create binding specificity. These include hydrogen bonds between the following residues: asparagine-59 (R3 helix) of AtMYB61 with adenine-1 nitrogen of AC-I; asparagine-106 (R3 helix) oxygen of AtMYB61 with adenine-1 hydrogen of AC-I; asparagine-59 (R3 helix) oxygen of AtMYB61 with cytosine-2 hydrogen of AC-I; asparagine-102 (R3 helix) oxygen of AtMYB61 with cytosine-3 hydrogen of AC-I; arginine-56 (R2 helix) oxygen of AtMYB61 with cytosine-3 hydrogen of AC-I; arginine-54 (R2 helix) hydrogen of AtMYB61 with thymidine-4 oxygen of AC-I; and, lysine-51 (R2 helix) of AtMYB61 with adenine-5 nitrogen of AC-I. The leucine-55 (R2 helix) methyl group of AtMYB61 is predicted to form a non-polar bond with thymidine-4 methyl group of AC-I. Cytosine-6 remained unbound in the model. In comparison, the NBS model had only one hydrogen bond present, involving asparagine-59 (R3 helix) oxygen of AtMYB61 with adenine-2 hydrogen of AC-I.Based on the model of AtMYB61 protein is sufficient to drive transcription in yeast from promoter sequences that contain AC elements AtMYB61 affinity to specific DNA sequences and its capacity to drive transcription ; a Caffeoyl-CoA 3-O-methyltransferase ; and a pectin-methylesterase . In keeping with their role as putative AtMYB61 targets, loss-of-function mutants corresponding to each of the genes phenocopy aspects of the myb61 loss-of-function mutant phenotype.Previous experiments predicted three putative direct downstream target genes of AtMYB61 bound to the 5′ gene regulatory sequences of all three putative direct target genes in an AC dependent manner AtMYB61 binds to AC elements in a distinct set of target genes to modify gene expression.The CASTing targets were identified in the 1000 bp 5′ non-coding regions of the three putative direct target genes and were determined by algorithm-based screening to be statistically over-represented . AtMYB61AtMYB61 is recruited to target genes via its interactions with a set of unique sequences, and thereby modifies gene expression. Surprisingly, the affinity of AtMYB61 to specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences, suggesting that the conformation of AtMYB61 may be altered allosterically when bound to specific target sequences. These findings point to additional complexities in the regulation of plant gene expression, and argue for the need for greater exploration of the molecular intricacies involved in the interactions between plant transcription factors and their DNA targets.Despite the size and importance of the plant R2R3-MYB family of transcriptional regulators, little is known about the molecular functioning of given family members. The work described herein casts greater light on the interaction between an R2R3-MYB family member and its cognate DNA targets. The findings support the hypothesis that Figure S1AtMYB61 antibody generation and validation. (A) Amino acid sequence similarity of AtMYB61 along with its closest family member AtMYB50. The two proteins have conserved N-terminal amino acid sequences but unique C-terminal domains, which was the domain selected to generate AtMYB61 antibodies against (highlighted region). (B) A chemiluminescent western blot validate anti-AtMYB61 antibody specificity. Lanes correspond to full-length recombinant AtMYB61 protein (Lane 1), of antibody alone (Lane 2), and AtMYB61 recombinant protein immunoprecipitated with pre-immune serum (Lane 3) and with AtMYB61-specific antiserum (Lane 4). Western blot was done with 1∶20000 dilution of immune serum. Western blot shows greater quantities of AtMYB61 protein eluted from the Magnetic Dynabeads Protein G antibody complex compared to the Magnetic Dynabeads Protein G pre-immune serum complex, showing that the immunoprecipitation was successful.(TIF)Click here for additional data file.Table S1Relative binding of CASTing targets and mutated AC-I sequences to AtMYB61.(DOC)Click here for additional data file.


We showed that this effect was mediated through D1-like and not D2-like dopamine receptors, and we found no evidence that tonic dopaminergic transmission affected UP states in our model.Cortical networks display persistent activity in the form of periods of sustained synchronous depolarizations (‘UP states’) punctuated by periods of relative hyperpolarization (‘DOWN states’), which together form the slow oscillation. UP states are known to be synaptically generated and are sustained by a dynamic balance of excitation and inhibition, with fast ionotropic glutamatergic excitatory and GABAergic inhibitory conductances increasing during the UP state. Previously, work from our group demonstrated that slow metabotropic GABA receptors also play an important role in terminating the UP state, but the effects of other neuromodulators on this network phenomenon have received little attention. Given that persistent activity is a neural correlate of working memory and that signalling through dopamine receptors has been shown to be critical for working memory tasks, we examined whether dopaminergic neurotransmission affected the slow oscillation. Here, using an  Wistar rats  were used in all experiments.m): 126 NaCl, 3–3.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2 and 26 NaHCO3, and were incubated at room temperature (22 − 26 °C) for 1 h in interface conditions with standard artificial cerebrospinal fluid, before being transferred to modified artificial cerebrospinal fluid with reduced MgSO4 (1 mm) and CaCl2 (1.2 mm). Slices were maintained in interface conditions prior to recording; they were then mounted on a coverslip (coated with 0.1% poly-l-lysine in ultrapure H2O) and transferred to a submerged-style recording chamber where they were superfused with modified artificial cerebrospinal fluid at 4–5 mL/min at 32–34 °C. The maintenance of slices in interface conditions was critical for the development of spontaneous UDS, and the use of poly-l-lysine coverslips allowed slices to be mechanically stable under high perfusion rates and also improved laminar flow across the slice; these conditions are known to promote spontaneous network activity in submerged slices  containing the mEC were prepared from postnatal day 14–21 rats of both sexes after decapitation under deep isoflurane-induced anaesthesia. Slices were cut in ice-cold (< 4 °C) standard artificial cerebrospinal fluid containing (in mm): 110 K gluconate, 40 HEPES, 2 ATP-Mg, 0.3 GTP, 4 NaCl and 2–4 mg/mL biocytin . Membrane potential values were not corrected for the liquid junction potential.Whole-cell current-clamp recordings were made from principal cells in layer 3 of the mEC, using glass pipettes pulled from standard borosilicate glass (6–8 MΩ) containing .Data were recorded using an Axon Multiclamp 700B amplifier (Molecular Devices) and low-pass filtered at 2 kHz. The signal was digitized at 5 kHz using an Instrutech ITC-18 on a PC running Igor Pro  using procedures written in-house. UDS transitions were monitored automatically using an algorithm that detected changes in DC membrane potential and membrane potential fluctuations using a moving average window method Craig, . All detRaclopride and SCH23390 were purchased from Tocris Bioscience. All other chemicals were purchased from Sigma-Aldrich.n = 37 slices, m dopamine reduced the incidence of UP states . We next asked which type of dopamine receptor, D1-like or D2-like, mediates the effects of dopamine on UDS.Whole-cell current-clamp recordings made from principal cells in layer 3 of the mEC were used to monitor UDS. UP states occurred spontaneously with an incidence of 2.9 ± 0.3/min and a mean duration of 5.2 ± 0.4 s , dopamine and raclopride  or dopamine and SCH23390 . We conclude that dopamine-induced suppression of persistent activity is mediated through D1-like receptors.To investigate the dopamine receptor subtype involved in suppressing persistent activity, we repeated the previous experiment in the presence of either the DP states A, with n1-like receptor antagonist SCH23390 could prevent dopamine from suppressing UP states, we next sought to determine whether this suppression was reversible. After recording spontaneous UDS for 10 min, 10 μm dopamine was bath-applied. Subsequently, we then applied either 10 μm SCH23390 or 200 nm raclopride. Application of SCH23390 significantly reversed the suppression of UP states, whereas raclopride had no effect .Given that the Dm raclopride appeared to cause a small but significant increase in UP state incidence  that was at the lower end of those reported in the literature, but that should still have activated both D1-like and D2-like receptors. There are a number of potential mechanisms that could explain why dopamine suppresses persistent activity. First, an early study in the entorhinal cortex found that dopamine could inhibit layer 5 pyramidal cells by acting via D1-like receptors to increase IH (Rosenkranz & Johnston, 1-like receptors also depolarize neocortical fast-spiking interneurons (Towers & Hestrin, 1-like receptors can actually reduce evoked inhibitory postsynaptic currents in the prefrontal cortex (Gonzalez-Islas & Hablitz, 1/D5 receptors have also been shown to increase the N-methyl-d-aspartate component of excitatory postsynaptic currents in prefrontal cortex pyramidal cells (Seamans et al., 1-like vs. D2-like receptors. An additional degree of variability may arise due to differential expression of the intracellular targets of activated dopamine receptors between different areas of the cortex.Although it has been shown that, in the striatal spiny neurons, activation of D1-like dopamine receptor activation (Bandyopadhyay & Hablitz, 1-like dopamine receptors (Weiss et al., 1-like receptors. Further questions remain, e.g. are the actions of dopamine mediated through a specific type of interneuron, or do they come from a general increase in inhibitory tone? These questions have functional implications; understanding the effects of dopamine on the network, as opposed to neuronal level, may help give insights into the progression and treatment of brain disorders.A more recent study reported that, in prefrontal cortex slices, bath application of dopamine selectively increased inhibitory over excitatory currents and, using calcium imaging, dopamine was observed to inhibit the spread of local activity via DGiven the diversity of neuronal functions that can be attributed to dopamine, which vary depending on the location and time course of release Schultz, , in addi


Cerebral cortex is generally thought to provide the neural basis for higher cognitive and perceptual functions  and via an iPad or iPhone “app.” From the results, the authors were able to propose how particular groups of neural pathways centered around cerebral cortex might subserve specific cognitive functions. This article illustrates the importance of a comprehensive neuroanatomical assessment of a complete brain.A solution to this Neuroinformatics challenge is important for progressing the discovery of cortical wiring principles. In an ambitious study (Solari and Stoner, Overall, we think this Research Topic demonstrates the complexity and diversity of cortical organization and the wide variety of approaches that can and have been made to understand how we think and perceive. We hope the reader will find something here to stimulate their curiosity concerning a topic of considerable importance to the individual and society.


Chemistry Central Journal (CCJ) is meant to serve at least two functions. One, consistent with the long-term goal of this journal, is to bring to its audience the full span of topics and areas in which chemistry plays a key role, even if it is not the only key contributing subject. Obviously energy is very much such a subject; chemistry plays an integral role in the acquisition, generation, distribution and efficient use of energy. But its role is only fully meaningful when chemistry’s contributions are integrated with those of other sciences such as climatology, and, every bit as important, with economics, sociology and political decision-making. This brings us to the second function of this supplement. It consists of a set of six papers developed in a course at the University of Chicago in the Fall term of 2010, and then later revised a bit for CCJ. Each paper was the work of an interdisciplinary team, consisting of three to five students; each team had to include at least one student in a natural science and at least one in a social science such as economics or public policy. A primary goal of the course was educating the students, all fourth-year undergraduates or graduate students, in working together across interdisciplinary lines, learning to convey the relevant concepts of their own field to others in very different fields and to integrate the concepts from very different areas to come to a coherent analysis of their chosen topic.This supplement to the Each team made a progress report to the class midway through the term, and then made a final presentation in one of the last sessions. Students were encouraged to ask questions and make critical comments about papers prepared by other teams.Each team could choose a topic from a list of suggestions or request approval of a topic of their own invention. The papers selected for this supplement were ones deemed particularly relevant by the two people giving the course, Professor George Tolley, an economist, and myself. Both of us have worked for many years in various aspects of energy, and a good fraction of that work was done together. It might be relevant here, to give a sense of the environment in which the papers were written, to point out that Professor Tolley and I agreed on some things and openly disagreed and argued in class about some other things. So these papers were written in a context in which questioning, challenging and testing ideas was pervasive. And the course continues!The author has no competing interests in this material.


Eradication of bovine tuberculosis (bTB) through the application of test-and-cull programs is a declared goal of developed countries in which the disease is still endemic. Here, longitudinal data from more than 1,700 cattle herds tested during a 12 year-period in the eradication program in the region of Madrid, Spain, were analyzed to quantify the within-herd transmission coefficient (β) depending on the herd-type (beef/dairy/bullfighting). In addition, the probability to recover the officially bTB free (OTF) status in infected herds depending on the type of herd and the diagnostic strategy implemented was assessed using Cox proportional hazard models.Overall, dairy herds showed higher β (median 4.7) than beef or bullfighting herds (2.3 and 2.2 respectively). Introduction of interferon-gamma (IFN-γ) as an ancillary test produced an apparent increase in the β coefficient regardless of production type, likely due to an increase in diagnostic sensitivity. Time to recover OTF status was also significantly lower in dairy herds, and length of bTB episodes was significantly reduced when the IFN-γ was implemented to manage the outbreak.Our results suggest that bTB spreads more rapidly in dairy herds compared to other herd types, a likely cause being management and demographic-related factors. However, outbreaks in dairy herds can be controlled more rapidly than in typically extensive herd types. Finally, IFN-γ proved its usefulness to rapidly eradicate bTB at a herd-level. Mycobacterium tuberculosis complex (mainly M. bovis and to a lesser extent M. caprae), is a zoonotic disease affecting cattle worldwide [Bovine tuberculosis (bTB), caused by members of the orldwide . Test anorldwide . Howeverorldwide ,4. Certaorldwide . Herd tyorldwide ,7, althoorldwide . Studiesorldwide ,10 and dorldwide . HoweverIn Spain, herd prevalence has decreased since the implementation of the National Eradication Program from 11.4% in 1986 to 1.65% in 2009 . HoweverThe study here was aimed at assessing the association between herd type and metrics of evolution of the bTB control program in ACM, including time to recover bTB free status and transmission rate, using data collected over >10 years. The influence that use of new diagnostic tests for identification of positive animals has had in such metrics has been also assessed. Results will help to clarify the dynamics for disease transmission in ACM and to estimate the impact of alternative control strategies in one of the regions of Europe in which the disease is yet to be eradicated.Through the twelve-year period 1997–2009) under study, 18,056 herd tests and 990,643 individual tests were performed in 1,734 farms of ACM. Most of the tested herds were beef herds , whereas the remaining herds were categorized as dairy  and bullfighting . Median number of tested animals per round was 29, although it varied with the herd type , 144 (27.96%) and 41 (7.96%) outbreaks were reported in 308 (59.81%), 100 (22.52%) and 36 (8.11%) beef, dairy and bullfighting herds, respectively  in dairy cattle  compared to beef  and bullfighting  cattle . At the test level, the impact of the introduction of IFN-γ assay in positive herds yielded a statistically significant  increase in the values of βt for every herd type compared to those calculated when only the SIT test was performed, although this increase was less evident in dairy herds  than in beef  and bullfighting  herds. The value of βds Table .f values computed for 17  of the 298 positive herds in which this parameter could be estimated were identified as outliers and therefore excluded from the analysis. Nevertheless, their inclusion did not affect the results presented here (data not shown). The computed values of βf fitted log-normal distributions for each herd type .The βEighteen and 34 out of the 515 outbreaks detected in the period of study were not included in the survival analysis due to anomalous length values (outliers) or because they were resolved by stamping out respectively. From the remaining 463 outbreaks used for fitting the two univariable Cox proportional hazards models, OTF status had been obtained in 438 (95%) of the affected herds and bTB outbreaks were still ongoing in the remaining 25 (5%) herds at the time of the analysis.Herd type was significantly associated with time to recover OTF status, with outbreaks in dairy herds showing significantly shorter lengths median 349 days) compared to beef and bullfighting herds , and an increased probability of resolving the outbreak faster, as indicated by the hazard rates Figure , Table 349 days cOutbreaks managed after implementation of IFN-γ assay in infected herds in 2006 had also significantly shorter duration (median duration 307 days) and increased probability of recovery than those declared before 2006 (median 517 days)  in the multivariable Cox model, whereas hazard ratio for the other two significant variables (herd type and use of IFN-γ assay) did not substantially differ (<15%), compared to the coefficients obtained in the bivariate analyses (data not shown).Schoenfeld residuals indicated that the assumption of proportional hazards was violated for outbreaks in which time to recover OTF status was shorter than 240 days. Cox models were re-run after elimination of those outbreaks; subsequently, the proportional hazards assumption was met (P = 0.3) and results of the hazards ratios remain unchanged as indicated by the overlapping 95% CI.Eradication of bTB at a herd level, which is prerequisite for disease eradication at a region and country level, has been impaired by the chronic nature of the disease that can lead to relatively long periods of “silent infection” in infected animals, especially at low infectious doses . This fet, estimated here was similar to the values reported in Argentina [t was significantly higher for dairies compared with other herd types. Dairy farms were at higher risk for bTB than beef and bullfighting herds, as indicated by the larger values of βt, proportion of outbreaks, and proportion of recurrently infected farms observed in dairy herds compared to other herd types. This finding may be related to a combination of factors that favor disease spread in these herds including, for example, high contact rates, high density, and presence of stressors associated with the intensive management of animals, as previously suggested [The median value of βrgentina , New Zeargentina  and the rgentina , suggestuggested ,7. Arguat  including, for example, recording bias, introduction of infected animals, or participation of wildlife reservoirs in the maintenance and transmission of the disease (whose effect on bTB transmission would be included in the same βf estimate). Still, from the decision making perspective, identification and further investigation of such outliers is of interest, because they may represent farms with particular epidemiological conditions that truly increase the risk for the disease, or, alternatively, misreporting of data related with disease identification. Nevertheless, if outliers were not excluded from the analyses the results obtained did not change significantly as shown by the overlapping 95% CI in all estimates (data not shown).Outliers were removed from the dataset before the survival analysis (10% outbreaks) and before identifying the theoretical distribution that best fitted the within-herd transmission data (6% herds) because of the uncertainty related to the accuracy of such estimations. There are a number of factors that could lead to unusual lengths of outbreaks and high values of βArguably, the formulation used here to compute the transmission rate is relatively simple, because certain factors that may affect transmission, such as absence of random contact among susceptible and infected animals, management practices, or the potential role that certain wildlife species may play in disease transmission, were not considered in the formulation. Increasing model complexity would require quantitative knowledge on the value that certain epidemiological factors took in the study population that was not available to the authors including, for example, number and distribution of wildlife species or rate of contact between cattle per herd. Most important, it is uncertain whether inclusion of such information in the model formulation would result in significantly different estimates for the transmission rate compared to those presented here. In the absence of such information, estimates presented here may be useful approximations of the true value values of the transmission rate of bovine tuberculosis in Spain that allow to compare the estimates between different herd types and other epidemiological factors. This could aid in the evaluation of the impact of control strategies for different production systems. In any case, the methodological approach presented here may help the administration of control programs to identify and further investigate those farms in which unusually high transmission rates suggest the occurrence of an anomaly in the pattern of disease transmission.Results presented here show that herd type influenced the likelihood of success of a bTB control program in an infected region of Spain and that use of the IFN-γ contributed to the success of the control program as a complementary tool in infected herds. Estimates and analytical approaches presented here will help in the design, implementation and evaluation of bTB control programs in ACM and other infected regions of Spain.Data from every farm located in ACM and included in the Spanish bTB Eradication Program between 1997 and 2009  were analyzed. Recorded information included herd type , herd size, number of animals tested, and number of positive animals at every round of testing. Management and handling of animals was performed according to Spanish Legislation. No data from experimental research has been included in the study.TB diagnosis was performed according to specifications provided in the Spanish bTB Eradication Program. Briefly, single intradermal tuberculin (SIT) test was used as the screening technique following European and Spanish legislation (EU Council Directive 64/432/CEE and RD 2611/1996). The SIT test was performed by bovine field practitioners using 0.1 mL  of the official bovine PPD , whose potency was periodically assessed according to the OIE standards by the Spanish National Reference Laboratory using in-vivo assays, and every >6 weeks old cattle in the herd was tested. For the purpose of the analysis a bTB outbreak was declared in a farm if one or more reactors were disclosed in a herd-test, and ended when the herd obtained the official bTB-free (OTF) status. A recurrently infected herd was defined as a herd in which more than one bTB episode was declared. Although several adjustments were introduced in the eradication program in the 12 year period assessed here (1997–2009), two main stages can be differentiated based on the testing strategy used.· Before 2006, to obtain OTF status a herd had to undergo two consecutive herd-tests at least 6 months apart in which no reactors were disclosed, and subsequently, be subjected to yearly testing of all > 6 weeks of age animals to maintain the status. To re-gain OTF status in bTB positive herds, the interval between the two consecutive tests was at least 60 days. If reactors were detected in a OTF herd but post-mortem analysis could not confirm the presence of the disease and infection was not suspected based on epidemiological grounds a herd could also recover OTF status after a single negative round-test performed at least 60 days after the removal of the reactors.· In 2006 the interferon-gamma (IFN-γ) detection assay was introduced as an ancillary test for detection of the maximum number of infected animals in positive herds  to increase the sensitivity of the detection system ; the test was performed using the same bovine PPD used for skin testing and 0.05 as the cut-off value as described previously , and onlCt) – ln (StIt/N), as described elsewhere for bTB [t, the number of bTB-infectious animals, was estimated as a function of Ct and Ct+1. Ct and Ct+1 are the number of bTB cases detected in time and t + 1, respectively, and St is the number of susceptible (non infected) cattle in time t, and time t and t + 1 indicate, respectively, two consecutive herd tests. The values of Ct and Ct+1 were computed considering the number of bTB-positive results and assuming that the sensitivity and the specificity of the diagnostic test ranged from 63.2 to 100% and 75.5 to 99% respectively, with 83.9% and 96.8% as most probable values for each of them [t and Ct+1 were computed following a stochastic process. Because it is uncertain how many of the bTB-infected animals were infectious and because bTB-positive reactors were immediately removed from the herd, It was assumed to follow a uniform distribution with parameters 1 and Ct, which represent the extreme scenarios in which only one and every infected animal were infectious, respectively. If over the course of an outbreak there was a herd test with no positive reaction, followed by a herd test with positive results, a value of It = 1 was used, i.e., assuming that there was one infected animal in the farm that was not detected by the test. Values of β were computed after 1000 simulations using @RISK 5.5 . Median β values were estimated for each period between consecutive herd tests (βt) and for each farm (βf) when time since the last herd test performed was available. Outliers in the distribution of βt and βf per herd type were detected as described below and removed because authors suspect that were associated with information bias. The theoretical distribution that best fitted (P > 0.05) the observed distribution of βf was identified for each herd type using a Chi-square test implemented in the @RISK software. Association between βf and herd type was explored using a Kruskal-Wallis test, which was implemented in the SPSS software .The transmission coefficient, β, was defined as the average number of animals infected from an infectious individual per unit of time . For the for bTB , where I of them . Thus, aThe association between the probability of suffering more than one bTB outbreak and herd type was quantified using a Chi-square test.Life tables were created to display the probability of OTF recovery by productive type in infected herds. Start of the at-risk period was set by the first positive herd-test in a farm, and test date at which the farm recovered OTF status was used as the failure time. Censoring time was fixed at the last herd-test recorded for those farms in which outbreaks could not be solved.The association between herd type and time for recovery of OTF status after detection of an outbreak was explored using a Cox proportional hazards model after removal of outbreaks showing anomalous length values, which were identified as outliers if lying more than 1.5*interquartile range (IQR) from the first or third quartile. Another Cox proportional hazards model was fitted to compare official bTB-free status recovery times before and after the implementation of the IFN-γ in 2006. Additionally, a multivariable Cox proportional hazards model was also fitted using herd type, use of IFN-γ assay , number of reactors disclosed in the initial test, and β coefficient as predictor variables. The assumption of proportional hazards in the Cox tests was assessed by computing Schoenfeld residuals .Proportional hazards models were implemented using the R software ,24.JA performed the statistical analyses and drafted the manuscript, AP participated in the data analyses and manuscript preparation and editing, JB and CC gathered the data from the official database systems and performed descriptive analyses, BR and SR carried out the IFN-γ tests, JLS, RD and JC coordinated the eradication campaigns, designed the databases and revised critically the manuscript, LdJ and LD designed the study and coordinated the units involved in it. All authors read and approved the final manuscript.


Predicting the future is a dangerous undertaking at best, and not meant for the faint-hearted. However, viewing the advances in molecular medicine, genomics and proteomics, it is easy to comprehend those who believe that molecular imaging methods will open up new vistas for medical imaging. The knock on effect will impact our capacity to diagnose and treat diseases. Anatomically detectable abnormalities, which have historically been the basis of the practice of radiology, will soon be replaced by molecular imaging methods that will reflect the under expression or over expression of certain genes which occur in almost every disease. Molecular imaging can then be resorted to so that early diagnosis and characterisation of disease can offer improved specificity. Given the growing importance of molecular medicine, imagers will find it profitable to educate themselves on molecular targeting, molecular therapeutics and the role of imaging in both areas. And this has been primarily catalysed by two concurrent developments, namely the understanding of the molecular basis of disease, and the development of drugs directed at these molecular targets.While medicine has taken on a molecular character and the Human Genome Project has yielded a wealth of information for mapping the human body, nothing really explains how the body works. Molecular imaging will play a key role in delivering molecular medicine since it defines many cellular and biochemical mechanisms . It mustin vivo. The pathways targeted play a key role in disease processes. Molecular imaging holds the unique potential of being able to find, diagnose and treat disease in vivo simultaneously , as well as depict how well a particular treatment is working i.e., theragnostics. This is in contrast to “classical” diagnostic imaging where most of diseases are diagnosed based on the manifestation of signs and symptoms following which remedial therapy is then applied to modify or ameliorate those symptoms. In most diseases, since the origins are often unknown and even in those instances where it is known, the treatments are meant at best to keep things under control.Molecular imaging is a growing research discipline  aimed at developing and testing novel tools, reagents, and methods to image specific molecular pathways with whole body imaging instruments Molecular imaging exploits specific molecules as the source of image contrast. This paradigm shift from non-specific physical to specific molecular sources underlies many of the current molecular imaging research efforts. It is this search for the “holy grail” of modern medicine that may make it possible eventually to manipulate an individual's genetic constitution appropriately, to get rid of disease processes.Molecular imaging is to a certain extent technology driven since molecular information can be obtained with some “high-end” imaging technologies. To do more would require newer equipment. However, on a conceptual level, molecular imaging is neither about technology nor about a change in the practice but essentially another revolution in the way medicine is viewed. One underlying premise of molecular imaging is that this emerging field is not defined by the imaging technologies that underpin acquisition of the final image per se but rather by the underlying biological questions of medicine. The ramifications will be felt in every nook and cranny of medical practice and then extend into the legal, social and ethical aspects of society. No concept and basis of medicine, currently practiced will be spared.As a consequence, a new approach to wellness, disease prevention and treatment needs to be developed to counter the effects of poor health and illness, especially in the world’s less developed countries. To enhance such an approach, the potential contributions of biomedical imaging, bioengineering, and bioinformatics to emerging research areas, such as functional genomics, proteomics, molecular biomechanics and drug delivery systems, tissue and cell engineering, quantitative biology and computer modelling, molecular and computational imaging, computer-aided diagnosis, metabolic imaging, ultra fast and integrated imaging systems are of prime importance. In the current phase of the increasingly complex and ever changing health care environment, governments, medical speciality organisations, researchers, vendors, and providers are finding it hard to cope with these challenges alone. Partnerships are critical to face any new concept. It is therefore imperative that new relationships sharing the risk of adopting new technological innovation, programmes, services and processes designed to improve overall patient care be worked at.in vivo, researchers have already developed and characterised methods for imaging endogenous proteins, such as receptors, transporters, or enzymes and their respective functions. More recently, methods have also been validated to analyse specific expression of transgenes of interest. This is typically performed by quantifying the activity of reporter genes delivered by a viral vector, as used in gene therapy, or through use of exogenous reporter genes in genetically engineered cells [Increasingly, the power of molecular imaging comes from the capacity to harvest vast amounts of knowledge gained from molecular biology. Currently we can theoretically design a molecular imaging agent to target each step in the sequence from DNA replication to protein synthesis and subsequently all the steps of protein metabolism. To begin investigating molecular signals ed cells  and traned cells .in vivo. If we could improve the analysis of metabolic reactions in living organisms, it would lead to immense savings for the pharmaceutical companies and open a promising market. Methods that produce visual images are being used with increasing frequency to study metabolic processes, e.g., to find out when individual genes become active (are expressed). Here, highly specific molecules (probes) are employed, which often are customised with genetic engineering. These special molecules search for a specific substance and, when they have found it, link with it and emit signals that can be depicted visually to provide a diagnosis of changes to a patient's metabolism. The key elements to sampling molecular information are [Processes in the human body are extremely complex and depend on a multitude of factors. As such, they are much more difficult to analyse than a laboratory sample. In fact, metabolic processes can only be truly understood tion are :The use of special imaging probes with high specificity. In fact due to the advances in chemistry and screening it is possible to have increased specificity without sacrificing sensitivity.The availability of appropriate amplification strategiesHigh resolution images from systems with increased sensitivity, andCapability of overcoming biological barriers to delivery of probesWith regard to the strategy for imaging these probes:Direct imaging uses a probe specific for cell surface receptors, intracellular molecules or gene expression, which interacts directly with the target providing an image intensity correlating to the amount of target actively present.Indirect imaging is more complicated as it often uses both a reporter gene and a reporter probe which interacts within specifically targeted cells to produce a metabolite trapped in the cells that is visualised when scanned.Surrogate imaging detects downstream effects of endogenous molecular-genetic processes using established radiopharmaceuticals and clinical imaging protocols.Regarding the imaging probes there are three basic types :i.e., flow and perfusion) and as mentioned above the probe does not directly image the molecular process but a surrogate.The compartmental probe typically assesses physiological parameters , high-spatial-resolution MR and nuclear imaging techniques , fusion imaging and micro imaging systems  play an important role in the field. Each of these techniques has its particular advantages and disadvantages, and the use of one or the other technique is mostly dependent on the specific research question and hypothesis to be tested .Radionuclide imaging devices visualise very low concentrations of radionuclide probes (nano- to femtomolar) in real-time  and provMagnetic Resonance Imaging's (MRI) ability was enhanced significantly with the development of functional MRI. The revival of interest in molecular imaging has expanded the frontiers of MRI even further. MR imaging has two particular advantages over techniques that involved the use of isotopes, namely the higher spatial resolution , and the simultaneous extraction of physiologic and anatomic information.In comparison with isotope techniques, however, MR imaging is several magnitudes less sensitive (millimolar rather than picomolar), which is why reliable signal amplification strategies must be developed. MR techniques in cell imaging are also maturing where cells are induced to take-up superparamagnetic iron oxide formulations .in vivo. Even though CT is often not recognised as a modality for molecular imaging, it has a role especially for the study of soft tissue tumours in bone and lung because of its low cost, reasonable resolution and fast scanning times.Ultrasound in molecular imaging allows real-time imaging with resolution of less than 50 µm. With the developments of microbubble technology  and harmin vivo surface imaging [Finally, optical imaging techniques have already been developed for applications in molecular and cellular biology  and  imaging . One of Another notable advantage of optical techniques is multiple probes with different spectral characteristics can be used for multichannel imaging, similar to in vitro karyotyping . In addiToday's scientists are not just looking at specific molecules, but are also evaluating the components within the nucleus of their cells. They are learning what causes the cells to turn on and off, what makes them do what they do and how their fundamental function can be boosted or shut down. While molecular imaging has significantly advanced oncology, cardiology, neurology, infectious disease detection and therapy, drug development, and disease treatment, even more is expected. Here is what molecular imaging promises:in vivo using deoxyglucose, a metabolic substance that is voraciously glycolised and trapped by targeted cancer cells. By labelling deoxyglucose with a radioactive agent and injecting the resulting molecular imaging agent into patients, scientists can make nuclear images of the primary tumour as well as metastatic sites throughout the body.In the future, advances in molecular imaging will lead to the development of a broader array of imaging probes that will cover all the body's major systems and associated disease types, making even earlier detection of disease possible. Our capacity to image these molecular changes will directly affect patient care by allowing much earlier detection of diseases, e.g. image molecular changes that we currently define as “predisease states”. If such a situation came to pass, then we would allow intervention when the outcome can be significantly altered. Because of the greater specificity of the imaging probes and techniques we would be able to look for specific cell types, e.g., cancer cells have an increase in metabolic activity in comparison with normal cells. This fact makes it possible to image cancer cells When a cell is dying (apoptosis), it turns inside out, presenting an otherwise unexposed protein binding site. The body responds by producing a protein called annexin, which seeks out and connects to the binding site of these dying cells to “tag” them for destruction by the immune system. By creating a human annexin, attaching it to the imaging agent technetium and injecting it into the patient, scientists can “seek and illuminate” dying cells. Physicians can use this information to decide whether to change or keep a patient's therapy regimen. For example, when chemotherapy or radiotherapy is used to kill cancer cells, apoptosis occurs. Apoptosis is the necessary death of cells to make way for new cells and to remove cells where DNA has been damaged to the point at which cancerous change is likely to occur. If the chosen therapy is effective, apoptosis can be demonstrated within 24 to 48 hours. Physicians can use molecular imaging to determine whether apoptosis has occurred and can change the therapy if it does not. Not only does this ability mean that the treatments used will be more effective, but further costs associated with ineffective therapy can be avoided. This can be done using SPECT (single photon emission computed tomography) or MRI. In the example quoted above if therapy is proving effective, then, annexin could be used as a delivery vehicle to further enhance cell death. Physicians would add a payload of radioactive toxin to annexin. When injected, the “loaded” annexin will deliver the toxic agent to the site of the dying cancer cells to cause even more cells to die. This process creates a cycle of cell death because the more cells that die, the more toxin-loaded annexin will be attracted to the cancer site. This molecular chain of events helps accelerate the therapy's effectiveness.With the US Food and Drug Administration's (FDA) approval of Avastin  as a first-line treatment for patients with metastatic colorectal cancer, it was a major landmark in the field of angiogenesis. With the development of an image based angiogenetic marker, it would help select patients for appropriate therapeutic regimens including the most appropriate combination of angiogenic and other therapeutic agents, help to identify the optimal time window and the appropriate dosage of the different therapeutic agents, assist in monitoring the effects of such treatments, and provide functional information for adjusting the therapeutic regimens over time in an interactive basis . Since iFurther developments are occurring where imaging and therapeutic agents are tagged with a targeted molecular agent are being developed and this may enhance treatment e.g. Zevalin , a therapeutic regimen for treatment of relapsed or refractory low grade, follicular or transformed B-cell non-Hodgkin's lymphoma. Indium-111 labelled Zevalin scanning allows visualisation of disease and calculation of dose to be delivered by Yittrium-90 labelled anti-CD 20 monoclonal antibodies .Current evidence suggests that one course of anti-CD20 radio immunotherapy is as efficacious as six to eight cycles of combination chemotherapy.2 or a variety of lung disorders, the commonest cause is ischemia due to an oxygen demand greater than the local oxygen supply. In tumours, low tissue p02 is often observed, most often due to a blood supply inadequate to meet the tumour's demands. Decreased tissue oxygen tension is a component of many diseases. In the heart tissue hypoxia is often observed in persistent low-flow states, such as hibernating myocardium. In patients with stroke, hypoxia has been associated with the penumbral region, where an intervention could preserve function. In some tumours the efficacy of conventional radiotherapy is limited by the presence of a hypoxic, radioresistant, and repair-proficient subset of tumour cells.Decreased tissue oxygen tension is a component of many diseases. Although hypoxia can be secondary to a low inspired P0in vivo has limited its role in clinical decision making. This has led to the development and testing of hypoxic imaging techniques and agents. An ideal hypoxia imaging agent should have high membrane permeability for easy access to intracellular mitochondria and low redox potential to confer stability in normal tissue, but it should be able to be reduced by mitochondria with abnormally high electron concentrations in hypoxic cells. Imaging with some of these agents can provide direct evidence of tissue with low oxygen levels that is viable. In the experimental setting this information is useful to plan a more aggressive approach to treating tumours, or revascularise a heart suffering ischemic dysfunction. Oxygen electrode measurements in animal experiments have demonstrated a strong correlation between low tumour pO2 and excess 60Cu-diacetyl-bis(N(4)-methylthiosemicarbazone (60Cu-ATSM) accumulation. Some studies have suggested that pretreatment imaging with 18F-2-fluoro-2-deoxy-D-glucose(FDG) and 64Cu-ATSM would allow stratification of tumour phenotypes such that those tumours that are most susceptible would be selected for treatment [Despite the potential importance of oxygen levels in tissue, difficulty in making this measurement reatment .Currently treatment instituted for most diseases is based on “one size fits all”. This is based on our inability to separate the responders from those who are either not likely to respond or those that may actually develop more complications from the treatment offered. But increasingly many new-generation therapeutic drugs are designed with highly specific molecular targeting capabilities and delivery mechanisms. By directly imaging the underlying alterations of diseases, we have the potential to be able to directly image the effects of therapy. This would provide an opportunity to play a direct role in determining the efficacy of treatment. Further, response to therapy could be assessed shortly after therapy has been initiated and not in the many months subsequently as required today to determine whether pharmacological or biological intervention has been beneficial.In addition the toxic effects of treatment on the patient's healthy tissue can be avoided. In the future, molecular imaging is expected to aid in identifying the presence of drug-resistant genes that will enable clinicians to pre-determine which treatment regimens will be most effective within an hour of initiating treatment and avoid delays in optimizing a patient's therapy.Furthermore, molecular tracers, or radiopharmaceuticals, will be used to create diagnostic images which visually indicate whether cancer patients are susceptible to multi-drug resistance, a condition in which the defensive response to one type of chemotherapy also diminishes the potency of other chemo agents. With this information, physicians can make accurate and fairly rapid diagnoses and provide patients with more precise treatment.Others are working on nanoparticle delivery platforms that can potentially deliver both imaging and therapeutic agents to endothelial targets. Using a vascular targeted imaging agent for selecting patients to be treated and for monitoring response with the therapeutic agents delivered via the same delivery vehicle satisfies the requirements for “personalised treatment” -18.It may seem like the movie, Osmosis Jones (Warner Bros) where researchers are working to integrate molecular imaging with nanotechnologies  to detect disease and enable even more precise therapy in many instances eliminating if not minimising the need for surgery entirely. Some of envisioned scenarios may include the development of biosensors providing the exact location of disease, and new nanotechnologies which can be dispatched into the body that can deliver drug therapies directly to cancerous cells by affixing themselves to those specific cells and releasing cancer-killing agents.Just like nano-robots, these nanotechnologies shall be designed to self-assemble themselves at the appropriate place and time following which they are able to repair bones or tears and if necessary stimulate the growth of new blood vessels or tissue. There would additionally be imaging technologies which could monitor the process, ensure that it is working properly, and measure the results, the ultimate of image guided minimally invasive surgery!Molecular imaging will facilitate the development of new drugs, by providing early stage chemical compounds that will enable researchers in the public and private sectors to validate new drug targets, which could then move into the drug-development pipeline. This is particularly true for rare diseases, which may not be attractive for development by the private sector. Three key technological advances drive NIH's effort to build small molecule libraries.The successful completion of the Human Genome Project has provided an enormous cache of human biology to be studied and potential drug targets to be discovered.Developments in chemistry have given researchers in the public sector the ability to synthesise large numbers of related molecules, a capability previously available only to researchers in pharmaceutical and biotechnology companies.Advances in robotic technology and informatics now allow scientists to screen hundreds of thousands of compounds in a single day, an orders of magnitude greater capacity than was available a decade ago .Molecular imaging is a powerful concept that envisions the promise of disease characterisation / phenotyping and early assessment of therapeutic efficacy. From this, it is a logical step to envisaging “personalised medicine,” where disease phenotyping will be used to tailor the most optimum therapeutic regimen patient by patient and not the usual one size fits all. This is already happening but is not directly related. Imaging entails the use of Her-2/neu gene expression as an indicator of whether breast cancer patients will respond to the drug Herceptin, a monoclonal antibody.Today the power of imaging is so sophisticated that we now have the capacity to identify unresolved biological and clinical questions and focus on how imaging techniques might be used to solve these problems. Emphasis continues to be on minimising invasiveness, reducing image processing time, lower cost, less radiation dose but at the same time maximising resolution and contrast as well as easy interpretation of data. Future advancements lie in not only in new modalities e.g. optical imaging but also in improved imaging modalities as well in producing displays that intelligently combine structural, chemical, electrical, magnetic, acoustic, and motion information.However though in the past, diagnostic imaging tests were designed to be stand alone investigations with specialised image acquisition, analysis and display, it is important that we do not continue to utilise diagnostic imaging tests in this same manner, but rather exploit the synergies between each individual i.e. the sum of the parts is larger than each alone! For example, the development of PET/CT systems allows the synthesis of the rich metabolic and functional information gained from PET with the morphologic information provided by CT, new information can be gained that cannot be obtained with each modality alone. These multi-modality systems (together or image fusion from two separate systems) is only the beginning of what may prove to be a significant paradigm shift in medical imaging device design and manufacturing .Molecular imaging promises to become a powerful addition to the ammunition of medical imaging. “Who controls and gives direction to that growth remains arguably an open question. Today’s imaging research defines the practice of the future” . LeadersThe 21st century will witness further innovation, growth and clinical utility imaging and therapy. These challenges are already formidable enough for imaging community in the developed world but be daunting for those in the developing countries where meeting the current imaging needs is at most times, barely sufficient. Despite these challenges, we in the Southeast Asian region must make a concerted and co-ordinated effort to ensure that we are not left behind. The following are some of the actions that will help ensure that the imaging community here does not lag behind.Firstly, leaders in imaging generally should recognise the importance of this new and rapidly expanding field. They need to encourage acquisition of knowledge necessary to incorporate these new methods into their practices, including knowledge of fundamental principles in molecular biology. They should also promote education and research that will undoubtedly impact the future of imaging.Although we may not be able to change the current line of thinking of the current practitioners; the future generations however, must be aware of the developments in molecular imaging and so there must be changes in the curriculum with introduction of subjects like biochemistry, immunology, as well as oncology. This is being incorporated in the new integrated curriculum implemented in some institutions.Thirdly, there should be greater co-operation as well as coordination between the regional societies, e.g., ASEAN Association of Radiology and Asian-Oceania Society of Radiology, in promoting greater awareness of biomolecular imaging. This is being done by the introduction of specific seminars and conferences , and wouFinally, the setting up of local, national or regional centres of excellence in biomolecular imaging would be another initiative to ensure and maintain the lead. These centres are also important since the disease patterns and demographics are very different from that in the West. However, setting up such centres is intensive: both financially and in terms of human resource and is probably beyond the reach of many countries but Singapore , Thailanwww.biij.org) is also essential in putting all these different aspects together. It also provides a forum for publication, dissemination, and discussion as well as networking between the different groups and organisations in the region.This potential requires interdisciplinary partnerships and collaborative efforts between physicians, medical physicists, biomedical engineers and computer scientists. We will all need to learn each others' technical language to move forward. The setting up of a regional journal such as biij (In view of these changes, as well as the increasing emphasis on imaging at the cellular level or even at the genetic level, research in the field of biomedical imaging is set to explode.Impossible is nothing!


E. coli and K. pneumoniae with ESBLs isolated from hospitalized patients.To implement antimicrobial reasonable use and evaluate its impact on nd/3rd generation cephalosprines (GCPs) use from 2006.1 to 2012.12 was converted to DDDs per 1,000 patient-days in a tertiary teaching infectious disease hospital in Tianjin, China. Multi-strategy, including contextually appropriate antibiotic use guidelines, active audit, as well as performance feedback/education in-field, were conducted by antimicrobial stewardship in the hospital since 2006. E. coli and K. pneumoniae isolated from all identified and suspected infections were selected at the same time. For ESBLs status, agar diffusion test with clavulanic acid plus or minus cefotaxime and ceftazidime was used. Linear correlation was carried out to analyze the relationship between 2nd/3rd GCPs use and major Enterobacteriaceae producing ESBLs.The 2nd GCPs consumption was 12.488, 12.563, 12.637, 20.221, 48.598, 6.647, and 8.129 DDDs per 1,000 patient-days during 2006 to 2012 respectively. The 3rd GCPs consumption was 35.248, 32.135, 31.720, 23.661, 31.696, 20.284 and 15.722 DDDs per 1,000 patient-days respectively at the same time. ESBL-producing strains rates of E. coli and K. pneumoniae were 27.42% (17/62), 26.79% (15/56), 29.09% (16/55), 23.53% (24/102), 39.29% (66/168), 25.58% (33/129) and 11.46% (18/157) from 2006 to 2012 respectively. Lower 2nd/3rd GCPs annually use was associated with a lower rate of E. coli and K. pneumoniae with ESBLs isolated from contemporary hospitalized patients. .The 2nd/3rd GCPs in the hospital of Tianjin, China. The decrease of 2nd/3rd GCPs represented a significant driver in the reduction in rate of E. coli and K. pneumoniae with ESBLs from identified and suspected infections.Multi-strategy intervention by antimicrobial stewardship was effective on commonly used 2None declared