A proportion of individuals who start antiretroviral therapy (ART) fail to achieve adequate CD4 cell reconstitution despite sustained viral suppression. We determined the frequency and clinical significance of suboptimal CD4 reconstitution despite viral suppression (SO-CD4) in an urban HIV research cohort in Kampala, UgandaWe analyzed data from a prospective research cohort of 559 patients initiating ART between 04/04–04/05. We described the patterns of SO-CD4 both in terms of:- I) magnitude of CD4 cell increase  and II) failure to achieve a CD4 cell count above 200 cells/μl at 6,12 and 24 months of ART. Using criteria I) we used logistic regression to determine the predictors of SO-CD4. We compared the cumulative risk of clinical events (death and/or recurrent or new AIDS-defining illnesses) among patients with and without SO-CD4.Of 559 patients initiating ART, 386 (69%) were female. Median (IQR) age and baseline CD4 counts were 38 yrs (33–44) and 98 cells/μl (21–163) respectively; 414 (74%) started a d4T-based regimen (D4T+3TC+NVP) and 145 (26%) a ZDV-based regimen (ZDV+3TC+EFV). After 6, 12 and 24 months of ART, 380 (68%), 339 (61%) and 309 (55%) had attained and sustained HIV-RNA viral suppression. Of these, 78 (21%), 151 (45%) and 166 (54%) respectively had SO-CD4 based on criteria I), and 165(43%), 143(42%) and 58(19%) respectively based on criteria II). With both criteria combined, 56 (15%) and 129 (38%) had SO-CD4 at 6 and 12 months respectively. A high proportion (82% and 58%) of those that had SO-CD4 at 6 months (using criteria I) maintained SO-CD4 at 12 and 24 months respectively. There were no statistically significant differences in the incidence of clinical events among patients with [19/100PYO (12–29)] and without SO-CD4 [23/100PYO (19–28)].Using criteria I), the frequency of SO-CD4 was 21% at 6 months. Majority of patients with SO-CD4 at 6 months maintained SO-CD4 up to 2 years. We recommend studies of CD4 T-cell functional recovery among patients with SO-CD4. There is considerable variability in the magnitude and rate of CD4 T cell count recovery in Human immunodeficiency virus type 1 (HIV-1)-infected individuals, receiving antiretroviral therapy (ART). Most patients show a progressive rise in CD4 T cell counts after initiation of ART , howeverThe Infectious Diseases Institute (IDI) is a private-public partnership institution that is a center of excellence in HIV care, training, and research at Makerere University Medical School and Mulago Teaching Hospital in Kampala, Uganda. Since 2004, the IDI clinic (IDC) has provided free care to HIV positive patients, and by December, 2007, IDC had enrolled 20,000 patients into HIV care, of whom 13,000 are in active follow-up and 4700 have initiated ART according to WHO and Uganda Ministry of Health guidelines. The clinic has 15 exam rooms and is staffed by 20 physicians, 30 nurses, and 10 counselors and patients are reviewed monthly. The drugs are provided by the Global Fund . Daily co-trimoxazole prophylaxis was provided and patients allergic to co-trimoxazole were given dapsone. Adherence to ART was encouraged by at least 3 individual and group counseling sessions. Patients are reviewed monthly by the general clinic physicians that evaluated among others; adherence to medication, toxicities and acute infections. Patients are evaluated by the study physicians every 3 months or earlier if they develop any illness. HIV RNA viral loads, complete blood counts and CD4 lymphocyte counts are tested at 6 monthly intervals.In this study, we used (i) previously used definitions of SO-CD4 in terms of the magnitude of the CD4 cell increase [a CD4 count increase of < 50 CD4 cells/μl after 6 months of ART [3-6]; <100 cells/μl increase after 12 months ; and <20Patients were included in the analysis if they had attained and sustained HIV-RNA viral load ≤ 400 copies per ml at 6, 12 and 24 months. The chi square test was used to compare the baseline clinical characteristics of patients with and without SO-CD4 and the level of significance was 0.05. Proportions of patients with SO-CD4 were calculated using the two criteria independently and with the two criteria combined. The combination of the two criteria was the intersection of patients with SO-CD4 on both criteria I) and II). Logistic regression by stepwise model selection was used to analyze predictors of SO-CD4. The independent variables included age, sex, baseline CD4 cell counts, body mass index (BMI), baseline hemoglobin, initial ART regimen, magnitude of CD4 increase in first 6 months and Hepatitis B surface Antigen sero-status. Variables were included in the multivariate model if they had a p value ≤ 0.25 on bivariate analysis. The proportions of clinical events were examined among patients with and without SO-CD4. In addition, the cumulative risk of development of AIDS-related clinical events was estimated by Kaplan-Meier analysis. Patients were censored on the occurrence of an AIDS-related clinical event (the primary outcome) as required by the survival analysis technique. Differences between the survival curves were tested using the log-rank test.Of 559 patients initiating ART, 386 (69%) were female, with a median age of 38 yrs (IQR 33–44), and a median CD4 count of 98 cells/μl IQR 21–163). Half 283(51%) of the patients had severe immune suppression with CD4 counts below 100 cells/μl at initiation of ART. Majority of patients, 414 (74%) started a d4T-based regimen (D4T+3TC+NVP) and 145 (26%) a ZDV-based regimen (ZDV+3TC+EFV). Baseline characteristics were comparable among optimal and sub-optimal responders apart from the baseline CD4 count that was significantly higher among sub-optimal than optimal responders. Patients with a lower BMI at initiation of therapy were more likely to have SO-CD4 after 12 months although the difference was no longer significant after 24 months of ART. Similarly, patients that initiated a ZDV-based regimen were more likely to have SO-CD4 at 6 and 12 months although the difference was no longer significant after 24 months of ART , 339 (61%) and 309 (55%) had attained and sustained HIV-RNA viral suppression. Of these, 78 (21%), 151 (45%) and 166 (54%) respectively had SO-CD4 using the CD4 increase criteria (described in the methods section). Of the patients with SO-CD4 at 6 months, 64/78 (82%) and 45/78 (58%) still had SO-CD4 after 12 months and 24 months respectively. By the end of 2 years on ART the overall median change in CD4 cell count and percentage was 193(104–273) and 11.5% (IQR 8.6–14.6) respectively though it was 77 [IQR 25–127] cells/μl and 7.2% [IQR 4.1–9.6] respectively among patients with SO-CD4 , 143/339(42%) and 58/309 (19%) had SO-CD4 at 6, 12 and 24 months of ART respectively. Of the patients with SO-CD4 at 6 months, 112/165 (68%) and 46/165 (41%) still had SO-CD4 at 12 and 24 months respectively. By 2 years on ART the median change in CD4 cell count and percentage was 54 [IQR 22–99] cells/μl and 6.4% [IQR 3.5–8.1] among patients with SO-CD4 based on threshold definition , 129/339 (38%) and 3/309 (1%) had SO-CD4 after 6, 12 and 24 months of ART respectively. Of the patients with SO-CD4 at 6 months, 42/56 (75%) still had SO-CD4 after 12 months of therapy.Patients with baseline CD4 counts of 50–199 cells/μl were more likely to have SO-CD4 than those with baseline CD4 counts of 0–49 cells/μl at 6 months [OR 2.5(1.1–5.5) P = 0.03] and at 12 months [OR 2.9(1.6–5.4) P = 0.001]. In addition, patients who initiated zidovudine-containing ART regimen were more likely to have SO-CD4 than patients on stavudine-containing ART at 6 months [OR 4.5(2.4–8.3) P < 0.001] and at 12 months [3.6(2.0–6.4) P < 0.001]. Other factors like age, sex, body mass index and hemoglobin level were not significant predictors of SO-CD4.Overall, there were 22 clinical events/100 PYO (18–26) among patients with sustained viral suppression. There were no statistically significant differences in the clinical events among patients with [19/100PYO (12–29)] and without SO-CD4 (using the CD4 increase criteria) [23/100PYO (19–28) p = 0.43] see Figure In this population with good rates of viral suppression as was previously reported , the freWe found that patients with baseline CD4 counts of 50–199 cells/μl were about 3 times more likely to have SO-CD4 than those with baseline CD4 counts of 0–49 cells/μl. Our results are similar to reports from South Africa where patients in the lower CD4 stratum had a higher gradient of CD4 increase ,15. We aPatients that initiated therapy with a zidovudine-containing regimen were 3.6 times more likely to develop SO-CD4 than patients on a d4T-containing regimen and we attribute this to the myelosuppressive effects of zidovudine . We inteAge was not a significant predictor of SO-CD 4 in our study and this is consistent with what was reported in a US cohort . HoweverMajority of the patients with SO-CD4 after 6 months, using either of the criteria, still had SO-CD4 at 12 months despite sustained HIV-RNA viral suppression. Since patients with SO-CD4 at 6 months are likely to maintain the phenomenon, they may need evaluation of the recovery of CD4 cell function, more so in Africa where there is an increased background risk of opportunistic infections. It is possible that the CD4 cells do not recover both in absolute numbers and function because of the high levels of T-cell activation in Africans due to frequent infections by the various pathogens endemic in the region ,25,26.It is also likely that these patients may require extended periods of prophylaxis against opportunistic infections. Our analysis was however limited to recovery of peripheral CD4 T cell counts and not CD4 T cell function. We recommend studies to examine other markers of recovery of immunological function among patients with SO-CD4.We found that about a third of the opportunistic infections occurred among patients with SO-CD4 reconstitution as defined by either the CD4 increase or the threshold criteria. Similar to what has been reported in other cohorts, most of the AIDS-related events occurred in the first 6 months -29 and tThe findings in this study are strengthened by the relatively homogenous study population of ART-naive individuals receiving ART at a single facility using standardized clinical protocols. Our patients used NNRTI-based ART regimen that are used in most HIV care facilities in Africa so our results can be generalized to most patients in Africa however they are limited to patients with sustained HIV-RNA viral suppression which, among others, is the ultimate goal of ART. We need to design studies of interventions for patients on ART with poor immune reconstitution and minimize the time spent with CD4 counts below the 200 cells/μl critical threshold. It is important to note that adherence to ART and previous exposure to ART were not considered to contribute to SO-CD4 in our study since all patients were naïve to ART and patients were included in the analysis only if they had HIV-RNA viral load < 400 copies/ml which we used as a proxy for good adherence.The frequency of SO-CD4 is high in SSA and many of the patients with SO-CD4 at 6 months maintain the phenomenon up to 2 years of therapy. However, the rates of AIDS-related clinical events were no higher in those with SO-CD4. We recommend studies of CD4 T-cell functional recovery among patients with SO-CD4.The authors declare that they have no competing interests.DN conceived of the study, and participated in the design, data analysis, interpretation of data, drafting and revising the paper. AK participated in the study design and statistical analysis. FI participated in the statistical analysis. BC participated in the acquisition of data, coordination of the study and in revising the paper. MRK made substantial contribution to the conception, design and coordination of the study. PJE made substantial contribution study design, statistical analysis and revision of the paper. All authors read and approved the final manuscript.


Triticum monococcum (2n = 2x = 14) is an ancient diploid wheat with many useful traits and is used as a model for wheat gene discovery. DArT (Diversity Arrays Technology) employs a hybridisation-based approach to type thousands of genomic loci in parallel. DArT markers were developed for T. monococcum to assess genetic diversity, compare relationships with hexaploid genomes, and construct a genetic linkage map integrating DArT and microsatellite markers.T. monococcum as well as 1536 T. boeoticum representative genomic clones, was used to fingerprint 16 T. monococcum accessions of diverse geographical origins. In total, 846 polymorphic DArT markers were identified, of which 317 were of T. monococcum origin, 246 of hexaploid, 157 of tetraploid, and 126 of T. boeoticum genomes. The fingerprinting data indicated that the geographic origin of T. monococcum accessions was partially correlated with their genetic variation. DArT markers could also well distinguish the genetic differences amongst a panel of 23 hexaploid wheat and nine T. monococcum genomes. For the first time, 274 DArT markers were integrated with 82 simple sequence repeat (SSR) and two morphological trait loci in a genetic map spanning 1062.72 cM in T. monococcum. Six chromosomes were represented by single linkage groups, and chromosome 4Am was formed by three linkage groups. The DArT and SSR genetic loci tended to form independent clusters along the chromosomes. Segregation distortion was observed for one third of the DArT loci. The Ba (black awn) locus was refined to a 23.2 cM region between the DArT marker locus wPt-2584 and the microsatellite locus Xgwmd33 on 1Am; and the Hl (hairy leaf) locus to a 4.0 cM region between DArT loci 376589 and 469591 on 5Am.A DArT array, consisting of 2304 hexaploid wheat, 1536 tetraploid wheat, 1536 T. monococcum. The constructed genetic linkage map will facilitate localisation and map-based cloning of genes of interest, comparative mapping as well as genome organisation and evolution studies between this ancient diploid species and other crops.DArT is a rapid and efficient approach to develop many new molecular markers for genetic studies in  Triticum monococcum (2n = 2x = 14), generally known as einkorn wheat, is an ancient diploid species domesticated in the Fertile Crescent ~10,000 years ago  is closely related to T. urartu (AuAu), the donor of the A-genome of hexaploid wheat [T. monococcum has gradually been recognised as an attractive diploid model for exploitation of useful traits, discovery of novel genes and variant alleles, and functional genomics. Many traits have been examined in T. monococcum which can be useful for modern wheat breeding [T. monococcum has also been successfully used for gene discovery in a subgenome map-based cloning approach, as exemplified by the cloning of the leaf rust resistance gene Lr10 [VRN1 and VRN2 [Q [NAC gene family [T. monococcum populations have been made available and were used to identify and map genes of agronomic importance [; ). Thus, in the foreseeable future T. monococcum is expected to play an important role in wheat genetic and genomic studies.Wild relatives of hexaploid wheat are known to be important sources of traits for wheat genetic improvement. id wheat -6. Recenbreeding -9. T. moene Lr10 , the verand VRN2 ,13, the  VRN2 [Q ,15, and e family . Naturalportance ,18. FurtT. monococcum have been collected and retained in major germplasm stock centres. In order to use T. monococcum resources efficiently in programmes on genetic improvement of hexaploid wheat, it is necessary to assess diversity of this species at the genome level. For this, high-throughput molecular marker technologies are needed. T. monococcum has been shown to possess a high level of polymorphism at DNA marker loci[T. monococcum domestication [T. monococcum in comparison with hexaploid wheat and its A-genome donor diploid species T. urartu. This information has been used to produce a genetic map integrating RFLPs and SSRs [Globally, thousands of accessions of rker loci. RFLP  has been developed as a sequence-independent and micro-array hybridisation-based marker system . DArT geT. monococcum diversity array (DArT) for high-throughput genome-wide genotyping, (2) assess the utility of the DArT technology for analysis of genetic diversity in a representative collection of T. monococcum accessions, (3) compare the relationships between the Am-genome and other Triticum genomes using DArT markers, (4) produce a genetic linkage map for T. monococcum integrating DArT and SSR markers, and (5) refine the genome locations of two morphological trait loci.We report here the results of a study aimed to (1) develop a PstI/TaqI representation generated from a mixture of DNA of two T. monococcum accessions MDR002 and MDR308 [Triticum species with different ploidy levels . We combined clones from all projects to assemble a custom-designed array containing 1536 clones derived from the two T. monococcum accessions, 2304 clones derived from hexaploid wheats (including the Triticarte Wheat 2.3 array), 1536 clones derived from tetraploid durum wheat (including the Triticarte Durum 2.0 array), and 1536 clones derived from 15 Iranian accessions of T. boeoticum Boiss., which is the wild relative of T. monococcum  were in the 90-100% quality category with an average PIC value of 0.34 and call rate of 99.8%, respectively  values for these markers were relatively low, with only 35.5% of DArTs having PIC values of 0.4-0.5 and 34.6% with PIC values < 0.2  of Iranian origin was fairly distantly related to other accessions including most of the accessions of European origin. This accession is unique in its ability to produce fertile F1 hybrids with hexaploid wheat [T. monococcum and Triticum sinskajae [Triticum boeoticum and Triticum urartu .This set of polymorphic DArT markers was used to assess the genetic diversity of the 16 id wheat . MDR650 id wheat  and provid wheat . Furtherinskajae , whereasT. monococcum genome and clones derived from the genomes of other Triticum species. This allowed us to assess (1) the degree of genetic similarity/diversity between different T. monococcum accessions, and (2) the relationships between the genome of T. monococcum and the A-genomes of other closely related Triticum species. Analysis of the 16 T. monococcum accessions revealed that 317 of the 1536 of DArT markers (~20%) developed from T. monococcum accessions MDR002 and MDR308 were polymorphic . Overall, the DArT markers developed from T. monococcum genome were found to be more informative than those developed from the genomes of other related Triticum species.As described above, the custom-designed DArT array contained clones developed from the ic Table . In thisT. monococcum relationships, the 846 polymorphic DArT markers were split according to their genome origins and principal coordinate analyses were carried out using the four subset data. The percentages of total data variance explained by the first two coordinates were 27.75% in hexaploid wheat, 23.96% in tetraploid wheat, 35.26% in genomes of T. boeoticum, and 35.77% in T. monococcum, respectively .To test the power of the DArT markers of different origins in resolving the T. monococcum and those of hexaploid wheat, we simultaneously hybridised the genomes of nine T. monococcum accessions and 23 hexaploid wheat varieties of European, American and Chinese origins to a customised Triticarte DArT array , CFA and CFD - 39.3% (11 out of 28), BARC markers - 38.2% (21 out of 55), GDM and WMS - 33.7% (28 out of 83), and DuPw - 13.6% (3 out of 22). The data for DArT and microsatellite markers were merged for construction of a genetic linkage map for T. monococcum.To confirm that DArT markers were inherited in a Mendelian manner, we constructed a linkage map for a cross between ly Table . The samid wheat . Out of T. monococcum mapping population. Therefore, these traits are thought to be controlled by single genes Ba (black awn) and Hl (hairy leaf) and were also included in the linkage analysis (see below). The linkage map derived from the combined data set spanned 1062.72 cM, with an average length of 151.82 cM per chromosome and an average density of one marker per 2.97 cM. Each of the seven chromosomes contained both DArT and SSR markers. Six of the linkage groups corresponded to six T. monococcum chromosomes, but the chromosome 4Am was formed by three linkage groups.In total, 356 (274 DArTs and 82 SSRs) molecular markers were mapped and formed nine linkage groups  of the molecular markers, while the highest numbers (n = 68) were found on chromosome 7 Am. Kolmogorov-Smirnov tests were performed to compare nonparametrically the equality of the distributions of the DArT and SSR markers along individual chromosomes. The results showed that chromosomes 1Am, 3Am, 5Am, 6Am and 7Am had p-values smaller than 0.05 but chromosomes 2Am and 4Am had larger p-values. These results suggest that DArT and SSR tended to form independent clusters on chromosomes in T. monococcum.Various numbers of DArT and SSR markers were mapped to individual chromosome linkage groups Table . ChromosT. aestivum, T. durum, T. monococcum and T. boeoticum. Figure T. monococcum linkage groups showed that the distribution of DArT markers arising from different genomes and SSR markers was at random across chromosomes . However, in some cases, DArT markers of certain origins were either over- or under-numbered. For instance, the related A genome DArT markers were under-represented on chromosomes 3Am, but over-represented on 7Am.The mapped 274 polymorphic DArT markers were derived from Triticum species could provide substantial polymorphic information in the Am genome of T. monococcum. Each of the seven T. monococcum chromosomes carried loci of the DArT markers that were previously mapped to homoeologous chromosomes in the B- or D-genomes of Triticum species ; only 33 are of A-genome origins Table , suggests Figure . Also, tP < 0.05). These were more or less equally distributed across the genome , 43.5% (30/69), 46.2% (24/52), 38.2% (13/34), 30.4% (17/56), 30.9% (13/42) and 14.7% (10/68) of markers displaying strong allelic frequency distortion, respectively. For some markers the segregation distortion was in favour of alleles originating from the male parent MDR002, whereas for other markers the segregation distortion was in favour of alleles originating from the female parent MDR308  of the DArT markers developed from the two T. monococcum accessions were polymorphic. DArT markers developed from genomes of other Triticum species also displayed good polymorphism frequencies  in T. monococcum. In hexaploid wheat, 15.3% (788 out of 5137) of DArT markers were found to be polymorphic when assessing the genetic diversity of 13 Australian cultivars [T. monococcum accession genotyped in this study appeared to be more genetically diverse than the bread and durum wheat collections assayed in those studies. Furthermore, most of the DArT markers whose genome locations have been determined in previous studies were located on homoeologous chromosomes of T. monococcum. Thus, DArT markers from related genomes were also useful in probing genetic diversity in T. monococcum. The custom-designed DArT array developed here can be used in studies focusing on comparison of the T. monococcum genome with genomes of other Triticum species.The inclusion of DArT clones from various genomes meant the average PIC value of the data set (0.31) was lower than in comparable studies. For example, for barley, sorghum and cassava the values obtained were 0.38, 0.41 and 0.42, respectively ,33,34. Hultivars . Similarultivars ,36. ThusT. monococcum accession revealed that the site of collection is only partially correlated with genetic diversity. Most accessions used in this study have been genotyped with SSR markers in a previous study, and a similar pattern had been observed [T. monococcum as non-DArT based markers. One of the accessions, MDR650 (PI 355520) was found to be genetically distantly related to other T. monococcum accessions, which raised a question whether it is a true T. monococcum. However, MDR650 (PI 355520) possesses the three major traits of the domesticated einkorn wheat: larger and plumper seeds, a tough rachis preventing spikelets falling apart at maturity, and relatively easy threshing. These traits are absent in the wild species T. boeoticum. We have crossed MDR650 (PI 355520) with many other T. monococcum accessions and discovered that the crossability could reach 100% (data not shown). Furthermore, MDR650 (PI 355520) did not cluster with hexaploid wheat and was closer to the T. monococcum cluster. Thus, we still consider MDR650 (PI 355520) an accession of T. monococcum.Principal coordinate analysis of the 16 observed . Thus, Dm-genome of T. monococcum and those of hexaploid wheat T. aestivum and its sub-genome donor, T. urartu have been studied in different context. For instance, chromosome pairing and recombination were studied between homoeologous chromosomes 1A and 1Am, 3A and 3Am as well as 5A and 5Am and were shown to be collinear and differentiated at sub-structural level [T. monococcum [T. monococcum were either conserved or absent in hexaploid genomes, and vice versa, indicative of divergence of the Am-genome from the A-genome [T. monococcum genomes and provide the polymorphism information in T. monococcum. Thus, at the homoeologous genomes level, there is a complex relationship between T. monococcum and hexaploid wheat.The relationships between the Aal level ,46. Goodal level ,48. Othenococcum ,25. HoweT. monococcum genetic map has been previously constructed using MDR308 (DV92) as one of parental accessions by Dubcovsky et al (1996) [T. boeoticum × T. monococcum RIL population to construct another genetic linkage map integrating 177 SSR and RFLP markers, and two morphological trait loci. The total length of this map is 1262 cM, the average chromosome length is 180.3 cM, and the average marker density is one marker per 7.05 cM. In comparison, our current genetic map derived from linkage analysis of an F2 population from a cross between MDR308 and MDR002 integrates 274 DArT markers, 82 SSR markers and two morphological trait loci. This map spans over 1062.7 cM, with six chromosomes represented by single linkage groups and chromosome 4Am by three groups of linked markers. The average chromosome length is 151.8 cM, and the average marker density in the genome is one marker per 2.97 cM. Observed differences in lengths and marker densities for the three T. monococcum linkage maps may well be related to differences in the mapping populations, the types and error-rates of the genetic marker systems used, and/or the algorithms and mapping functions used to compute genetic distances. For example, Singh et al (2007) [A l (1996) . This mal (1996)  used a Tl (2007)  used theThe current genetic linkage is also very similar in length and in marker densities to the A-genome maps of hexaploid wheat constructed using the Kosambi's map function. In one case, 369 SSR markers were mapped onto a 944 cM A-genome with the marker density of one marker per 2.56 cM . Recentlm-genome of T. monococcum is closely related to the Au-genome of T. urartu and to the A-genome of the hexaploid wheat [T. monococcum and the overall good colinearity of SSR arrangement along the chromosomes [m, 4Am and 7Am in T. monococcum. In contrast, in our current linkage map for T. monococcum there was only two gaps on chromosome 4Am. This suggests that DArT markers can be used to fill the gaps and help generate higher resolution genetic linkage maps.The Aid wheat -6. This omosomes ,24. Gapsomosomes -52. SimiT. monococcum. Over one third of the marker loci across the seven chromosomes displayed allele frequencies skewed from their Mendelian expectations. This was observed for both DArT and SSR markers, with preferences for both parental alleles. Segregation distortion has also been observed by Dubcovsky et al (1996) [m and 7Am, while in other study  [m. Strong segregation distortion has also been noted during the construction of durum wheat and bread wheat linkage maps integrating SSR and DArT markers [Strong segregation distortion was observed during construction of our current genetic linkage map for l (1996)  and Singl (1996)  during cl (1996)  15% of tal 2007)  two majo markers .T. monococcum lines, MDR308 and MDR002, used for developing the F2 mapping population had a number of contrasting traits including awn colour, leaf pubescence, grain hardness, salt tolerance, as well as resistance to the fungus Mycosphaerella graminicola and to soil-borne cereal mosaic viruses [Ba and Hl trait loci. Also, this map was recently used to refine the TmStb1 locus conferring resistance to M. graminicola isolate IPO323 on chromosome 7Am, and to identify and map new QTLs conferring salt tolerance .The two parental  viruses ,53. We d2 mapping population used in this study, has previously been used for developing a range of molecular tools including a genetic linkage map integrating RFLPs, AFLPs and seed storage proteins [. The availability of DArT marker information for MDR308 (DV92) further increases the utility of this accession.MDR308 , one of the parental lines of the Fproteins , a BAC lproteins , and sevT. monococcum thereby creating a frame work consensus map for wider applications in molecular genetics and genomics studies.In the near future it should be possible to link various available genetic linkage maps of T. monococcum. The constructed genetic linkage map will facilitate localisation and map-based cloning of genes of interest, comparative mapping as well as genome organisation and evolution studies between this ancient diploid species and other crops.DArT is a rapid and efficient approach to develop many new molecular markers for genetic studies in T. monococcum accessions and an F2 population of 98 individuals derived from a cross between accessions MDR002 and MDR308 [This study used 16 PstI/TaqI representation of the MDR002 and MDR308 accessions as described previously [T. boeoticum  according to manufacturer's instructions. A set of 1536 new DArT clones were generated from a eviously ,36. The protocol ,36.T. monococcum accessions MDR002 and MDR308. These SSRs originated from 5 groups: 57 BARC markers from the Beltsville Agricultural Research Centre, USA [A total of 279 SSR markers mapped to the A-genome of hexaploid wheat were tested for polymorphism between the tre, USA ,57, 29 Ctre, USA ,58,59, 2tre, USA  et al. 2tre, USA ,62, and tre, USA . For eacT. monococcum accessions were genotyped using the custom-built DArT array as described previously [T. monococcum accessions and 23 hexaploid wheat varieties were hybridised to a newly built DArT array and scored the same way. The ploidy levels did influence the scoring as envisaged in the raw data of the hybridisation intensity. Therefore, only the DArT markers which were not affected by this context-dependent scoring were scored. The nine T. monococcum accessions and 23 hexaploid wheat varieties were hybridised in duplicate.A group of 16 eviously . The polT. monococcum accessions MDR308, MDR002 and 94 F2 individuals derived from a cross between them were genotyped with the custom-built array as described previously [Ba, black awn; Hl, hairy leaf) were used for genetic linkage map construction using JoinMap® 4.0 . Because of the inherent dominant nature of DArT markers which separates the female and male markers in the repulsion phase, the linkage maps for maternal DArT markers  and for parental DArT markers  were initially constructed independently with SSR markers. The two maps for individual chromosomal linkage groups were then merged together using SSR markers as bridges. Assignment of markers to linkage groups was achieved using logarithm of the odds (LOD) threshold values ranging from 3.0 to 10.0. The Kosambi's map function was used to estimate genetic distances. In total, 358 markers including 274 DArT markers, 82 SSR markers and 2 morphological trait locus markers were integrated into nine linkage groups. Goodness of fit for all the loci to an expected 1:2:1 or 1:3 segregation ratio was tested using chi-square (χ2) analysis. The graphical representation of the map was drawn using GGT2.2 software [eviously . In totasoftware .th edition, VSN International, UK). For principal coordinate analysis Jaccard similarity matrices were generated using the DArT markers. Two-dimensional scores were calculated and used to generate scatter plot matrices of scores. For the Mantel test which looks for association between the off-diagonal values of two similarity (or distance) matrices, the correlation of the matrices were evaluated. The DArT markers were subdivided into four different categories depending on their genome origins and similarity matrices calculated. The similarity coefficients from these matrices were then compared pair by pair to form a new correlation matrix. The significance of each correlation was assessed using a randomisation test (with 1000 random permutations). The p-values were calculated and were always < 0.001 (where the null hypothesis is one of zero association). For the Kolmogorov-Smirnov test, the map position data of the DArT and SSR markers on individual chromosomes were fed into GenStat and the p-values were calculated. The Chi-square goodness-of-fit test was carried out to examine the random distribution of DArT markers of different origins across the genome, by calculating the association between the numbers of the markers of different origins the chromosomes.All statistical analyses were carried out using GenStat  provide DArT array commercial genotyping services for a range of crops and may benefit financially from this work.Triticum monococcum. PW, EH and AK developed the DArT array for T. monococcum, mapped DArT markers, and edited the manuscript. KHK raised the funds, initiated and supervised the whole project.All authors read and approved the final manuscript. KHK, HCJ and AK designed the study and coordinated the research activities. HCJ, KHK and KK drafted the manuscript. HCJ, CB, KK and SB developed SSR markers for Triticum boeoticum used to generate the customised DArT array used in this study.The 15 Iranian accessions of Click here for fileHexaploid wheat varieties used in comparative analysis using DArT markers.Click here for file


While female mate preference is very well studied, male preference has only recently begun to receive significant attention. Its existence is found in numerous taxa, but empirical research has mostly been limited to a descriptive level and does not fully address the factors influencing its evolution. We attempted to address this issue using preference functions by comparing the strength of male preference for females of different sizes in nine populations of four poeciliid species. Due to environmental constraints (water toxicity and surface versus cave habitat), females from these populations vary in the degree to which their size is correlated to their fecundity. Hence, they vary in how their size signals their quality as mates. Since female size is strongly correlated with fecundity in this subfamily, males were sequentially presented with conspecific females of three different size categories and the strength of their preference for each was measured. Males preferred larger females in all populations, as predicted. However, the degree to which males preferred each size category, as measured by association time, was not correlated with its fecundity. In addition, cave males discriminated against smaller females more than surface males. Assuming that male preference is correlated with female fitness, these results suggest that factors other than fecundity have a strong influence on female fitness in these species. These different relationships evolved as a response to living in different habitats  to test whether male preferences can be detected using preference functions, and (2) to see if male preference tracks changes in female fecundity in these populations. Our prediction was that male preference for larger females would be stronger in populations from nontoxic environments, where the relative increase in female fecundity is higher as compared to populations from toxic environments.Gambusia eurystoma is a surface species endemic to the sulfidic Baños del Azufre in Tabasco, Mexico . The mature females were then sorted into roughly equally sized groups according to size , and were then placed in different 38 L stock tanks. Males were randomly assigned an ID number that determined the order in which they would be tested, and they were housed in individual 5 L tanks that were out of sight of the females.Fish from the stock tanks were caught with a small seine and segregated by sex. Mature females were then selected using minimum standard length  as the criterion for sexual maturity. Due to natural size differences between species, the exact criterion used varied  made out of clear plexiglass were located in the center of each section of the tank. The cylinder in the center of the tank had solid walls, while the two outer cylinders were perforated with seven circular holes 6 mm in diameter to allow for chemical and mechanosensory signals. Chemical and mechanosensory signals have been found to be important factors in poeciliid mating behavior, influencing the repeatability of individual preferences as well as the overall preference . All thrA randomly selected female from the predetermined size category  was placed in the cylinder in one of the outer preference zones. The order of the females which each male would be presented with, as well as the side in which each female would be placed, were randomly determined. A male was then placed in the central cylinder for 5 min. After the 5 min of acclimation, the cylinder around the male was gently removed. Using two stopwatches, the amount of time the male spent in the preference zone containing the female was measured by the observer. This was done for 5 min, after which the fish were removed from the tank. The water was then stirred to homogenize any lingering chemical signals from affecting the results of future trials. Another pair of fish was then placed in their corresponding cylinders to acclimate. Every three pairs, a partial water change was also made previous to the acclimation period of the next fish. Male weight and standard length were also measured and used as covariates, but were not included in the final model because neither was significant.After checking the assumptions, a mixed between-within subjects ANOVA was performed to determine the effect of two habitat variables on male preference for small, medium, and large females. The two habitat variables used were “cave habitat”, whether the population originated from a cave or from a surface stream, and “toxicity”, whether the population originated from a toxic or non-toxic stream. Because the raw results were not normally distributed, the male preference variables were reflected and square root transformed to meet the normality assumption.Experiments were approved by the University of Oklahoma IACUC (R09-030).p = 0.026). However, this violation was not deemed to be severe enough to invalidate the ANOVA. As expected, there was a significant main effect for time spent with larger females, regardless of the habitat of origin  = 5.99, p = 0.003, F = 0.047, p = 0.829, F = 1.043, p = 0.309, F = 0.011, p = 0.917, All statistical assumptions were met after the data transformation, with the exception of homogeneity of variances for the time males spent with medium females  = 4.36, p = 0.015, F = 0.26, p = 0.77, F = 8.86, p = 0.06, There was also a significant interaction between cave habitat and the time males spent with females from different size categories , and epigean or hypogean habitat (“cave habitat”). Previous studies have shown that in P. mexicana  and nontoxic  surface populations.This result suggests that the change in female fecundity experienced from benign to toxic habitats is only weakly correlated with the change female quality. The reason for this is unclear and could be due to a combination of factors. It is possible that female size in nontoxic habitats is correlated with increased female mortality and/or with a decrease in offspring quality. An alternative possibility is that female size and quality are highly correlated, but that there has been insufficient time for male preference to change as a response to female adaptation. It is currently unknown how long the populations in toxic habitats have been adapting to their environments, and how recently the changes in female fecundity have evolved. Since male preference is likely under weaker selection than other traits, it is possible that males from toxic populations have either not had a sufficient amount of time to adapt, or that the amount of gene flow from nontoxic populations has been able to counteract the effects of selection. It is currently unknown how much gene flow there is between toxic  and those in the surface  Cave habitat, independent of water toxicity, does affect male preference. Cave males had a relative lack of preference for small females. We suggest that this could be a result of differences in predation pressure which could lead to relatively increased mortality for small females in the caves, relatively increased mortality for medium and large females in the surface, or both. If true, this would highlight the role that predators play in the evolution of male mate choice.In summary, we have shown that (1) male preference for larger female size exists. This is consistent with previous research, indicating that absolute preference functions are a valid approach in this system. (2) Hydrogen sulphide does not affect the shape of male preference function for female size in this system. Since H10.7717/peerj.140/supp-1Supplemental Information 1w⋯ columns) and transformed data (RefSqr.. columns) from all of the individual trials. Females were presented in a random order.Raw (TimeClick here for additional data file.


This paper describes methods to analyze the brain's electric fields recorded with multichannel Electroencephalogram (EEG) and demonstrates their implementation in the software CARTOOL. It focuses on the analysis of the spatial properties of these fields and on quantitative assessment of changes of field topographies across time, experimental conditions, or populations. Topographic analyses are advantageous because they are reference independents and thus render statistically unambiguous results. Neurophysiologically, differences in topography directly indicate changes in the configuration of the active neuronal sources in the brain. We describe global measures of field strength and field similarities, temporal segmentation based on topographic variations, topographic analysis in the frequency domain, topographic statistical analysis, and source imaging based on distributed inverse solutions. All analysis methods are implemented in a freely available academic software package called CARTOOL. Besides providing these analysis tools, CARTOOL is particularly designed to visualize the data and the analysis results using 3-dimensional display routines that allow rapid manipulation and animation of 3D images. CARTOOL therefore is a helpful tool for researchers as well as for clinicians to interpret multichannel EEG and evoked potentials in a global, comprehensive, and unambiguous way. The traditional analysis of the electroencephalogram (EEG) and event-related potentials (ERPs) focuses on waveform morphology over time at certain electrode positions. Scalp sites of interest are selected and the time course of the potential recorded at any site is analyzed using a variety of signal processing tools in the time and frequency domains. While this approach has provided many important insights into normal and pathological neuronal activity, it disregards another important dimension that multichannel EEG offers: the spatial characteristics of the electric fields at the scalp and the temporal dynamics of these fields. Any distribution and orientation of the active neurons at a given moment in time will generate a certain electric field on the scalp surface due to volume conduction . While d With the program CARTOOL, which exists now since over 14 years with constantly increasing capabilities, we wanted to provide an analysis tool for those researchers and clinicians who are interested in such reference-free EEG mapping techniques. It started with dynamic displays of EEG maps and calculation of some basic quantitative topographic measures. Soon after, standard data preprocessing tools were implemented, such as interpolation of electrodes, filtering, averaging, rereferencing. The next versions implemented spatial analysis methods for spontaneous and event-related EEG, most importantly the spatial microstate segmentation, initially proposed by Lehmann and collaborators , and subIn the following we will describe some of the main methods for the spatial analysis of the scalp electric fields and how they are implemented in CARTOOL. Not all methods will be covered here, but they will give an impression of how multichannel EEG and ERP data can be analyzed in a comprehensive way. More details regarding the different spatial analysis methods can be found in the book “Electrical Neuroimaging” .Topographic analysis of the electric field at the scalp very crucially depends on the quality of the data at each channel. Contamination by bad or noisy electrodes can lead to steep local gradients that have no neurophysiologic basis, which can in turn obfuscate interpretability of results . Artifacts on one particular channel are not always easy to detect if only EEG waveforms are displayed. In contrast, contaminated channels are readily seen on time series of EEG maps because they behave differently from the neighboring channels and appear as isolated “spots” in the maps ,exporting single or multiple tracks before or after applying the above preprocessing steps.For evoked potential analysis, single or averaged epochs can be calculated for any combination of triggers or markers with or without baseline correction. Automatic artifact detection and epoch rejection using amplitude windows is available. CARTOOL puts a lot of emphasis in a flexible visualization of the EEG tracks during evoked potential analysis for manual determination of artifacts. During the evoked potential analysis, a trigger validation file is generated that can later be used to more rapidly redo the averaging with the same epochs.In addition to the epoching according to defined triggers, CARTOOL allows to set markers according to specific characteristics in certain channels. This allows, for example, one to set markers at the onset of a motor response recorded with the EMG or at the peak of epileptic spikes. Finally, a file calculator tool has been implemented in CARTOOL that allows applying preset as well as user-defined mathematical operations to different files in a batch-mode processing.In the default display mode, CARTOOL always shows two global topographic measures together with the waveforms and the maps . These tui is the voltage of the map u at the electrode i, u and N is the number of electrodes of the map u. Scalp potential fields with pronounced peaks and troughs and steep gradients will result in high GFP, while GFP is low in maps which have a “flat” appearance with shallow gradients. GFP is a one-number measure of the map at each moment in time. Displaying this measure over time allows to identify moments of high signal-to-noise ratio, corresponding to moments of high global neuronal synchronization [nization , 16. GFPThe Global Map Dissimilarity measure (GMD) is a measure of topographic differences of scalp potential maps. It is defined asui is the voltage of map u at the electrode i, vi is the voltage of map v at the electrode i, u, v, and N is the total number of electrodes. In order to assure that only topography differences are taken into account, the two maps that are compared are first normalized by dividing the potential values at each electrode of a given map by its GFP. The GMD is 0 when two maps are equal, and maximally reaches 2 for the case where the two maps have the same topography with reversed polarity. Figure 2 in [ure 2 in  illustraThe GMD is equivalent to the spatial Pearson's product-moment correlation coefficient between the potentials of the two maps to compare . The calfunctional microstates first described by Lehmann et al. [The display of the GMD across time has a very characteristic behavior which is similar for spontaneous EEG and for evoked potentials: the topography of the maps remains stable for several tens of milliseconds and then abruptly switches to a new configuration in which it remains stable again. This leads to periods of low GMD interrupted by sharp GMD peaks . This hin et al. , 20. Then et al. . This con et al. –25. Withn et al. . While sn et al. .In light of this interpretation of the observed sequential periods of map stability, different methods have been proposed to objectively and automatically define the different microstates and to statistically evaluate the specificity of certain microstates under given experimental conditions. CARTOOL has implemented these methods in the “microstate segmentation” and “map fitting” modules. The microstate segmentation is based on cluster analysis using either a modified k-means cluster analysis  or an atThe cluster analysis can be applied to one data file or to different files of different experimental conditions and/or populations . The resThe cluster maps are finally fitted back to the original data and each time point is labeled with the cluster maps it correlated best with (in terms of GMD). In order to ensure a certain degree of continuity in time of the different a final relabeling step is performed which satisfies two requirements: (1) the correlation between the measurement and the cluster map should be high, and (2) the majority of the neighboring measurements should belong to the same microstate. Standard smoothing techniques, well-known in statistics, are used to fulfill this compromise between goodness of fit and smoothness . CARTOOLAnother method that has been proposed to define the most dominant evoked component topographies in a dataset is based on an independent component analysis . It hasThe cluster analysis of ERPs is usually applied to the group-averaged files. All experimental conditions/populations are entered into the cluster analysis, and the optimal number of clusters for the whole data set is determined , 32. TheIt is important to emphasize that the microstate segmentation on the grand mean data allows hypothesis generation and is not the final result. Changing the number of clusters might change the results at this level by proposing more or less map differences across time or between conditions. A second statistical step is needed to confirm these hypotheses and define those microstates that remain statistically significant. The “microstate fitting” module of CARTOOL allows to perform this test. The fitting procedure is the same as for the grand mean, but now the cluster maps are fitted to the individual ERPs of each subject and each condition/population  Figure . SeveralThe microstate segmentation using the cluster analysis can also be applied to spontaneous EEG. It leads to a reduction of the data to a stream of microstates of certain durations, on average around 80–100 msec . It is it-tests. At present, only univariate statistics are implemented in CARTOOL, but multivariate analysis procedures are currently under evaluation before formal inclusion. The univariate analysis can be applied to the potential at each electrode and each time point as a comprehensive exploratory analysis of the data [CARTOOL offers a variety of parametric and non-parametric statistical EEG mapping analysis procedures . Non-parthe data , 37, 38.the data . In orden participants is 2n. The GMD value from the actual group-average ERPs is then compared with the values from the empirical distribution to determine the likelihood that the empirical distribution has a value higher than the GMD from the actual group-average ERPs. In a within-subject design, the permutation of the maps is done within the subjects, while the permutation is done across subjects in group comparisons. In the example shown in Testing for differences in GFP at each time point is straightforward using the parametric or nonparametric tests. In order to test for differences in topography, CARTOOL implemented what has been called a “topographic ANOVA”, or TANOVA , 40. SinThe statistical analysis can also be performed on the values that result from the microstate fitting procedure. Again, only univariate statistics are currently implemented. For multivariate analysis the spreadsheets have to be read into other statistical packages. Ongoing developments are underway for the implementation of multivariate statistics , 42 as wQuantitative analysis of spontaneous EEG has traditionally relied on Fourier-transformation based spectral analysis. Thereby, the power of the different frequencies or frequency bands is compared between different conditions/populations. In multichannel data, power maps are often used and statistical maps of power differences are calculated. This approach has been very successful and helped to characterize vigilance changes, sleep stages, drug effects and various neurological and psychiatric disorders . More reHowever, frequency power maps have two important problems. First, they are reference-dependent. In contrast to potential maps in the time domain, power maps in the frequency domain change when the position of the recording reference changes , 46. SecWhile the analysis of the scalp potential maps as described up to now has the advantage, as compared to waveform analysis, to be reference independent and considers the whole brain electrical activity, it does not provide any direct conclusions about the number, location and orientation of the intracranial generators . InverseA major breakthrough in the spatial analysis of multichannel EEG/MEG was the development of distributed inverse solution methods that allow the estimation of the 3-dimensional distribution of neuronal activity in the whole brain at each moment in time , 52. TheThe results of the inverse solutions (norm or vectors) are displayed 3-dimensionally in the real  MRI . Slices It is worthwhile to note that the segmentation of the brain surface and the grey matter is implemented in CARTOOL including manual correction tools to exclude incorrect classification of grey matter or exclude structures as brainstem and cerebellum if desired. Alternatively, already segmented brains can be read into CARTOOL if preferred, such as standard template brains. Also the warping transformations as well as the distribution of the solution points are done within CARTOOL. Thus, the whole source localization process can be performed within CARTOOL, starting with the original EEG and the original MRI.Besides source reconstruction in the individual MRI, CARTOOL can also use template brains such as the MNI brain. In this case the solution space remains the same for all subjects, allowing group studies on the source level. In the case of the MNI brain, all solution points are labeled with their Talairach coordinates as well as their anatomical labels. Time periods of interest  or regions of interest (ROI) can be created and the mean current density within these segments or ROI can be stored for further statistical analysis . FinallySeveral other analysis tools are available in CARTOOL. In addition to the analysis of scalp EEG, CARTOOL also allows to visualize and analyze intracranial EEG, to read and fusion 3D images of different imaging modalities, to create and work with 3D regions of interest, and to convert and manipulate MR images.The analysis of intracranial recordings includes the color-coded mapping of grids as well as along depth electrodes . IntracrCARTOOL can read any type of 3D volumes in Analyze format. In case of functional images (fMRI or PET) the activation areas are displayed as colored blobs within the MRI. Several different volumes can be overlapped and thus results of different imaging modalities (including the inverse solutions) can be displayed within the same MRI. MRIs can also be manipulated and converted in various ways and stored as new volumes.A large palette of 3D display tools is available in CARTOOL that allows flexible visualization of the data. Any 3D object can be inserted into another one by a single click, allowing the merging of different information instantly, while retaining the spatial coherence of the objects. Different windows can easily be synchronized, allowing the user to visualize the dynamic behavior of the data on traces, maps and inverse solutions simultaneously .CARTOOL is constantly implementing new analysis methods that appear as useful and have been published. Concerning the statistical analysis, multivariate methods will soon be implemented. The TANOVA described above can easily be extended to multivariate measures . Also teN-dimensional space, where N is the number of the electrodes. The computation is initialized by a K-means algorithm, which iteratively improves the estimation of means, covariances and priors of the Q Gaussians until the likelihood reaches a plateau. For each time point and trial it then provides Q conditional probabilities that relate the topographies to the clusters. Like in the labeling procedure described above, each time point and trial is then labeled with the cluster with which it has highest conditional probability. The method has been shown to reliably identify specific component maps in the single trial ERPs despite the clear dominance of the ongoing spontaneous EEG activity.Concerning microstate segmentation, De Lucia et al. , 62 propAn important new development in the field of EEG/MEG analysis is the measure of connectivity between different brain areas as a way to understand the organized behavior of different brain regions . The useMethods and Acknowledgements sections of their papers. Currently about 650 users from all over the world have registered and downloaded CARTOOL.CARTOOL is not an Open Source project; however the program is distributed freely to any nonprofit research group. Users need to register only once and will then be informed of any updates of the software. They are asked to cite the use of CARTOOL in the OpenGL, so it needs an OpenGL accelerated graphic card, and as much memory as possible for the most demanding operations. Matlab is not needed to run CARTOOL.CARTOOL runs only on Windows platforms (ranging from Windows 95 up to Windows 7) as a standalone compiled executable program, included with its complete documentation within a single installation program. It is fully written in C++ in order to attain the highest speed and compactness in memory use. The advanced display is completely done in Drag & Drop in CARTOOL, the landing of the drop usually conditioning the actions to be undertaken on the files.Interoperability is achieved mainly by exchanging intermediate results through files. Considerable efforts have been put in reading and writing standard files with a maximum of convenience for the user. For example, most of the operations can be done by Reference Guide, describing all the options and technical details for all processes, is included in the distribution in the form of compiled HTML (10 MB chm file). In addition a CARTOOL Community group (Google Sites) has been built in order to offer a central access for the users, which includes a User's Guide, collaboratively edited as a Wiki, a Discussion Forum for all questions and announcements, some FAQs, and a few shared files. Finally, it is worth noting that CARTOOL updates, including Beta releases, can be easily assessed online through a Google Docs repository.Help is provided at different levels. A thorough Here are the main internet addresses for CARTOOL:http://brainmapping.unige.ch/cartoolhttp://cartoolcommunity.unige.ch Files formats read by CARTOOL include formats produced by the EEG systems form the companies Biologic, Biosemi, Brain Products, Deltamed, EGI, and Neuroscan. Also EDF format and standard text files can be read. Concerning MRI, Analyze and AVS formats are read. A complete list of file formats is included in the Reference Guide of Cartrool.


The gold standard management for ureter transitional cell carcinoma (UTCC) is radical nephroureterectomy with excision of the bladder cuff. However, some patients cannot undergo this procedure for several reasons. In the case reports described herein, we performed stereotactic body radiotherapy (SBRT) on three patients with inoperable or surgery-rejected localized UTCC. Two out of the three patients did not develop local recurrence or distant metastasis during the observation period. However, recurrence was detected in the bladder of one patient 22 months after the treatment. No acute or late adverse events occurred in any of the three patients. SBRT may become one of the treatment options for inoperable or surgery-rejected UTCC patients. Ureter transitional cell carcinoma (UTCC) is rare, accounting for only 1–3% of urinary transitional cell carcinoma cases . HoweverAn 87-year-old woman with rheumatism presented with gross hematuria and dysuria. A cystoscopic examination showed a papillary tumor at the left ureteral orifice protuberating into the bladder. A histological examination confirmed transitional cell carcinoma grade 2. A CT scan revealed the mass at the distal ends of the left ureter with no lymphatic or distant metastasis. Therefore, this patient was diagnosed with ureter cancer T2N0M0. Radical nephroureterectomy was considered to be nonadaptive because of comorbidities of chronic kidney disease (CKD) and rheumatism; therefore, she was referred to our department. SBRT was performed by coplanar dynamic conformal radiotherapy with a linear accelerator (LINAC) . A total dose of 60 Gy was delivered in 10 fractions at the center of planning target volume (PTV) using 10 MV photons . No acutAn 87-year-old man presented with occult hematuria. A cystoscopic examination showed no abnormal findings. The result of urine cytology was class V, transitional cell carcinoma. A CT scan showed a small tumor at the distal one-third of the left ureter with dilation of the upper ureter and ipsilateral renal pelvis. There was no evidence of lymphatic or distant metastasis. Therefore, the patient was diagnosed with ureter cancer T2N0M0. The patient refused surgery and was referred to our department. SBRT was performed by noncoplanar dynamic arch radiotherapy with a LINAC. A total dose of 50 Gy was delivered in 10 fractions at the center of PTV using 10 MV photons . No acutAn 85-year-old woman presented with gross hematuria. A cystoscopic examination revealed a tumor in the left ureter. A histological examination confirmed transitional cell carcinoma grade 3. A CT scan showed the tumor at the distal ends of the left ureter with no lymphatic or distant metastasis . Therefo10 [The standard treatment of UTCC is surgical resection. In the present study, summarized in 10 . Based o10). In contrast, in case 2, the tumor was localized at the distal one-third of the ureter, and the small intestine was in close proximity to the tumor. Therefore, the tumor was treated with 50 Gy in 10 fractions, equal to 75 Gy on BED10, in order to suppress the exposure dose to the small intestine. However, there were no decisive differences of local control in all three cases.In the present study, the prescribed doses differed between cases. In cases 1 and 3, the tumor was localized at the distal end or orifice of the ureter, and 60 Gy was delivered in 10 fractions, which was equal to 96 Gy on a biological effective dose of 10 (BEDThere are various techniques of SBRT in sight of irradiation and image guidance. The most commonly used one is a linear accelerator (LINAC), and we performed SBRT with a CT-LINAC system in all three cases. Incidentally, SBRT with dedicated machines, including cyberknife, is also often performed especially for localized prostate cancer in genitourinary organs .The sizes of the tumors in all three cases had decreased within 3 months of the treatment. Furthermore, there was no evidence of recurrence in the observation period spanning between 13 and 24 months. Therefore, SBRT was considered sufficiently effective for local control. Cases 1 and 2 showed no evidence of local recurrence, lymph node relapse, or distant metastasis in the observation period . However, bladder relapse was detected in case 3 22 months after the treatment. Hall et al. examined 252 patients treated surgically for upper urinary tract TCC and reported 12 months as the median time of disease relapse in 67 patients (27%) . These fNo acute or late adverse events were observed in any of the three cases presented herein. However, a longer observation period may be needed to more accurately estimate late adverse events.In conclusion, SBRT may become one of the treatment choices for inoperable localized UTCC. Further studies including a larger number of cases and longer observation periods are needed.


Radiative cooling technology utilizes the atmospheric transparency window (8–13 μm) to passively dissipate heat from Earth into outer space (3 K). This technology has attracted broad interests from both fundamental sciences and real world applications, ranging from passive building cooling, renewable energy harvesting and passive refrigeration in arid regions. However, the temperature reduction experimentally demonstrated, thus far, has been relatively modest. Here we theoretically show that ultra-large temperature reduction for as much as 60 °C from ambient is achievable by using a selective thermal emitter and by eliminating parasitic thermal load, and experimentally demonstrate a temperature reduction that far exceeds previous works. In a populous area at sea level, we have achieved an average temperature reduction of 37 °C from the ambient air temperature through a 24-h day–night cycle, with a maximal reduction of 42 °C that occurs when the experimental set-up enclosing the emitter is exposed to peak solar irradiance. Radiative cooling relies on the atmosphere's transparency window. Here the authors achieve up to 42 °C drops in temperature for low thermal loads under diffuse sunlight by improving the selectivity of the emissivity and the thermal management of their devices. From fundamental thermodynamics considerations, high-efficiency conversion from heat to work requires both a high-temperature heat source and a low-temperature heat sink. The vast majority of energy conversion processes at present use our ambient surrounding here on Earth as the heat sink. On the other hand, outer space, at a temperature of 3 K, provides a much colder heat sink. Moreover, Earth's atmosphere has a transparency window in the wavelength range from 8 to 13 μm that coincides with the peak of the blackbody spectrum of typical terrestrial temperatures ∼300 K, enabling the process of radiative cooling, that is, radiative ejection of heat from Earth to outer space, and hence the direct radiative access to this colder heat sink. Exploitation of radiative cooling therefore has the potential to drastically improve a wide range of energy conversion and utilization processes on Earth.The study of radiative cooling has a long history12345678910111213141516171819203456789In this paper, we first theoretically show that ultra-large temperature reductions up to 60 °C below ambient can be achieved. The key to such ultra-large temperature reduction is to use highly selective thermal emitter matched to the atmospheric transparency window, and to minimize parasitic heat losses. Experimentally, we demonstrate an apparatus, which exhibits continuous passive cooling throughout both day and night. In a 24 h day–night cycle in winter, the cooler is maintained at a temperature that is at least 33 °C below ambient air temperature, with a maximal temperature reduction of 42 °C, which occurs when the apparatus enclosing the cooler is exposed to peak solar irradiance.To illustrate the pathway towards achieving ultra-large temperature reduction, we first consider the ideal case, where the atmosphere is 100% transparent at a particular wavelength range outside the solar spectrum. In such a case, an emitter that has non-zero emissivity within this wavelength range, and zero emissivity outside, will reach the temperature of outer space of 3 K in the absence of parasitic heat loss, since in such a case the emitter is undergoing thermal exchange only with outer space.For a more realistic case, we perform the theoretical analysis as illustrated in Qsample), the absorbed thermal radiation from the atmosphere (Qatm) and the parasitic heat losses (Qparasitic) characterized by a heat transfer coefficient h. We consider three different emitters: a black emitter, a near-ideal selective emitter  reaches zero. Here we fix the ambient temperature (Tambient) to be 20 °C, and use a typical atmospheric transmittance at Stanford in winter .as a function of the temperature of the sample, n winter . For each=8 Wm−2K−1), the difference in performance between the black and the selective emitter is relatively small. Both the black emitter and the near-ideal selective emitter are restricted to a temperature reduction |ΔT|∼10 °C. Second, when the parasitic heat loss is completely eliminated (h=0 Wm−2K−1), there is a very large difference in terms of performance between the black and the selective emitter. Whereas the temperature reduction of the black emitter is limited to |ΔT|∼20 °C, the near-ideal selective emitter achieves a far higher temperature reduction |ΔT|∼60 °C. Thus, to approach the fundamental limit on radiative cooling, both selective emitter and ultra-low parasitic heat loss are essential. These considerations, together with the need to suppress solar irradiance during the daytime, motivate our design of the experimental apparatus and the selective emitter.−6 Torr), which encloses 10 concentric reflective radiation shields, and 4 long-hollow ceramic pegs in addition to the thermal emitter. These pegs provide mechanical support, while minimizing conduction loss to the emitter  is well within the resolution of the thermocouple.3N4), amorphous silicon (Si) and aluminium (Al), with thickness of 70, 700 and 150 nm, respectively  appears, when the apparatus enclosing the cooler is exposed to peak solar irradiance. This seemingly counter-intuitive observation points to the effectiveness of the sun-shade/mirror-cone for blocking sunlight, and arises from the high contrast between the ambient air temperature and the dew point, when the solar irradiance reaches its peak.We performed measurements by exposing the experimental apparatus to a clear sky throughout a 24-h day–night cycle at Stanford, California in winter see  shows thhttps://www.wunderground.com/us/ca/stanford). In general, the experiment agrees well with the theory. The few outliers in Qatm) and the parasitic heat loss (Qparasitic). On the other hand, the use of the selective emitter and the vacuum chamber significantly reduces these two terms, and as a result the dependence of the steady-state temperature of the sample on the ambient air temperature becomes weaker. Thus, although the lowest sample temperature occurs when the ambient temperature is low, the maximal temperature reduction occurs at the maximal ambient temperature. To conclude from In summary, the experiments here provide a record-setting performance in radiative cooling during both day and night. The demonstrated steady-state temperature is far below the freezing point even though the apparatus enclosing the cooler is exposed to peak sunlight. Our work demonstrates the possibility of reaching the fundamental limit of radiative cooling by combining photonic and thermal design. From a practical point of view, radiative cooling is becoming important in a number of areas, including passive building coolingWe end by briefly commenting on the prospect for scaling up the present system. Commercial evacuated solar water collectorsThe data that support the findings of this study are available from the corresponding author on request.How to cite this article: Chen, Z. et al. Radiative cooling to deep sub-freezing temperatures through a 24-h day–night cycle. Nat. Commun.7, 13729 doi: 10.1038/ncomms13729 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes, Supplementary References.


The recent progress on radiative cooling reveals its potential for applications in highly efficient passive cooling. This approach utilizes the maximized emission of infrared thermal radiation through the atmospheric window for releasing heat and minimized absorption of incoming atmospheric radiation. These simultaneous processes can lead to a device temperature substantially below the ambient temperature. Although the application of radiative cooling for nighttime cooling was demonstrated a few decades ago, significant cooling under direct sunlight has been achieved only recently, indicating its potential as a practical passive cooler during the day. In this article, the basic principles of radiative cooling and its performance characteristics for nonradiative contributions, solar radiation, and atmospheric conditions are discussed. The recent advancements over the traditional approaches and their material and structural characteristics are outlined. The key characteristics of the thermal radiators and solar reflectors of the current state‐of‐the‐art radiative coolers are evaluated and their benchmarks are remarked for the peak cooling ability. The scopes for further improvements on radiative cooling efficiency for optimized device characteristics are also theoretically estimated. Radiative cooling is such a promising cooling method with their ability to offer a cooling power of more than 100 W mIn this article, we seek to review the detailed characteristics, recent advancements, and the scope for further performance improvements of radiative coolers. In Section 2FigureThe Earth's atmosphere has a highly transparent window in the infrared (IR) wavelength range between 8 and13 μm, i.e., the atmosphere's radiative emission is very weak in that window. Outside the atmospheric window, the Earth's atmosphere is highly emissive. Coincidentally, the atmospheric window falls within the peak thermal radiation of a black body defined by Plank's law at the ambient temperature (at around 300 K). This feature permits for a passive cooling mechanism for a terrestrial body at the ambient temperature by eliminating heat via radiative emission through the atmospheric window. The emitted radiation escapes through the Earth's atmosphere to the outer space which acts as a gigantic thermal reservoir. The atmospheric window allows the terrestrial body's outgoing radiative emission to exceed its absorbed incoming atmospheric radiation and thus to passively cool below the ambient temperature. Along with the effect of the incoming atmospheric radiation, the cooling performance of a radiator depends on other factors, such as, the nonradiative (conductive and convective) heat gain from the surrounding media and the incoming solar radiation during the daytime. In addition, the cooling efficiency can heavily be affected by the emission profile of the radiator in use, i.e., a broadband radiator or a selective radiator. The IR window transmittance for certain atmospheric conditions can also significantly influence the cooing potential. The following subsections discuss the theoretical aspects of a radiative cooler and the associated factors affecting the cooling efficiency.2.1Taking into account all the heat exchange processes, the net cooling power of a radiative cooler can be defined asUB=2hc2λ51ehc/λkBT−1 is the spectral radiance of a black body defined by Planck's law at any temperature T, where h is Planck's constant, kB is the Boltzmann constant, c is the speed of light in vacuum, and λ is the wavelength. The emissivity of the radiator can be defined by its absorptivity er according to Kirchhoff's law. The angle dependent emissivity of the atmosphere is given byea=1−t(λ)1/cosθ, where t(λ) is the atmospheric transmittance in the zenith direction. Tr is the temperature of the radiator and Ta is the ambient temperature.Here, q=qcond+qconv is the combined nonradiative heat coefficient stemming from the conductive and convective heat exchange of the radiator with the surrounding air. Without any insulation for the nonradiative heat gain, the cooling power of the radiator is limited. In most of the experimental demonstrations, the radiators were insulated by polystyrene foam to suppress the conductive heat gain from the back surface and side walls and highly IR transparent low‐density polyethylene film was used as convection cover on top of the radiator with sufficient air gap.−2 °C−1 were estimated in several reports for radiative cooling which indicate effective suppression of nonradiative heat gain.The third term of the right hand side of Equation −2 and a significant absorption of this amount of power can easily diminish any cooling effect of the radiator. To avoid the adverse effects of solar radiation, IR transparent solar reflectors can either be integrated on the top of the thermal radiators or suitable cooling devices can be designed to serve as a simultaneous solar reflector and an IR radiator. The recent demonstrations of the daytime radiative coolers utilized the latter, where the cooling device reflected 97% of the incident solar radiation while providing significant IR emission within the 8–13 μm wavelengths range.The last term on the right hand side of Equation Pnet at the ambient temperature Ta in Equation Tr drops below Ta and reaches a steady state temperature Tss, (Tr = Tss < Ta) where the outgoing power balances the absorbed incoming power, i.e., Pnet (Tss) = 0. Thus, to achieve a state where Tss is significantly below Ta, the radiative emission within the 8–13 μm wavelength region must be maximized and the absorbed power from the incoming atmospheric radiation, nonradiative contributions, and absorption of solar power must be minimized. FigureA radiator can provide with practical cooling only when the radiated output power exceeds the net absorbed power. With a positive value of 2.2Ta–Tr) of selective and broadband radiators with and without the presence of solar and nonradiative contributions are shown in Figure Ta = 25 °C. A practical cooling device with 50 W m−2 cooling power at the ambient temperature and covering only a surface area of 50 m2 on the rooftop of a residential building would allow removing heat equivalent to a net cooling load of 15 kWh for a peak daytime between 11 am and 5 pm. An estimate of energy savings (in kWh) by radiative coolers on rooftop operating over the course of a year has also been reported in ref. [3].The performance of radiative coolers is highly related to the spectral emissivity profiles of the different kinds of radiators. The first one is a broadband radiator which has emissivity like a blackbody within the entire emission band of the atmosphere except the main solar spectral band from 300 nm to 2.5 μm. The second one is a selective radiator comprising unity emissions only within the wavelengths of 8–13 μm, selectively spanning over the atmospheric window. The ability of the selective radiator for cooling below the ambient temperature is vastly superior to that of a broadband radiator. As a comparison, the cooling power and the achievable cooling temperatures (Ta–Tr) in practice. The reason behind is that a broadband radiator operating considerably below the ambient temperature, may emit the less radiative power than it receives from the incoming atmospheric radiation at the ambient temperature inhibiting it to cool further without any net cooling power. An ideal emitter thus should selectively and strongly emit within the entire 8–13 μm wavelengths range. This has important consequences. The emitter does not absorb radiation outside the 8–13 μm window where the atmosphere is highly emissive which allows a minimal radiative heat exchange with the atmosphere. The impact of this becomes highly important to achieve cooling significantly below the ambient temperature. In addition, a strong emission within the 8–13 μm window ensures a high emitted power to overcome the nonradiative and solar heat contributions to reach a steady state temperature significantly below the ambient temperature. To understand this further we consider the cooling powers of broadband and selective radiators in Figure FigureTa = 25 °C (integrated over the entire hemisphere) by the broadband and selective radiators  and thus has a net cooling power. However, it may not be able to provide with a cooling temperature well below the ambient temperature  and using IR transparent polyethylene cover, the nonradiative heat transfer can be significantly minimized.Catalanotti et al. and Bartoli et al. utilized a commercially available polyvinyl‐fluoride polymer film (TEDLAR) as the radiative emitter which possesses somewhat selective and high IR absorption from 9 to 13 μm wavelengths range.2) was coated on aluminum plates and was used as the IR radiator. Although realistic cooling was not achieved directly under the sun at mid noon, a steady cooling of around 10 °C below the ambient temperature under clear sky conditions was reported during the night. On a relatively low humid day a maximum cooling 15 °C below the ambient was also demonstrated. In later works, B. Orel et al. reported that mixing barium sulfate (BaSO4) with TiO2 as pigments in the white paint can improve the cooling performances even further, which is due to the enhanced absorption in the 8–13 μm wavelengths range by the SO4 stretching vibrations of the BaSO4 content.3N4) possessing similar selective emission properties as SiO films were also radiative for potential radiative cooling.2 nanoparticles together in polyethylene films deposited on Al can yield high IR emission with better selectivity.Apart from the polymer films, pigmented paints, naturally available inorganic compounds and gases with IR emission were also investigated for nocturnal radiative cooling applications.2H2) gas to demonstrate a maximum cooling of 10 °C below the ambient temperature at daytime but avoiding direct exposure to sunlight. Further reports on radiative cooling by other potential gases such as, ammonia (NH3) and ethylene oxide (C2H4O) were also investigated.In addition to solid materials, radiative cooling with gases containing selective IR emission was also reported.3.22) white pigments and carbon black particles as a cover material for the IR radiative emitter.To achieve daytime radiative cooling, several kinds of solar reflecting materials were investigated in combination with IR emitters.Pr − Pa) is higher than the absorbed solar power , the device can yield a temperature well below the ambient temperature provided that convective heat gain is suppressed. On the other hand, if the absorbed solar power is higher than the net radiated power, the device temperature can be higher than the ambient air temperature. In this circumstance, the use of convective covers is undesirable as natural convection can aid to release heat from the cooling device. In the absence of direct solar radiation convection covers can greatly improve the performances for maximum cooling.Apart from solar reflectors, convection covers can also play important role to optimize the performance of the radiative cooler. For daytime applications, if the cooling device's net radiated power  and SiO2 with varying thickness. The top three thick layers mainly contributed to the IR emissivity including the 8–13 μm wavelengths range and the four thinner bottom layers deposited on Al coated Si wafer worked as chirped 1D photonic crystal for a maximum of 97% solar reflection ,Ta−Tr > 15 °C) than a broadband radiator. For increasing bandwidth of the radiator, the cooling performance decreases and becomes almost stable for bandwidth >11 μm, where it starts to behave like a blackbody radiator. There is a sudden small increase of the cooling performance for a radiator emission bandwidth >12 μm where the radiator starts to interact with the weak secondary atmospheric window from 16 to 23 μm wavelengths range. For decreasing emission bandwidths, i.e., for bandwidths <|8–13| μm, Ta−Tr rapidly decreases and does not provide any effective cooling for a bandwidth <1.25 μm. As discussed in Section From the analysis in the previous sections, it is clear that the radiative cooling performance depends on the maximized thermal radiation within the atmospheric window, the radiator emissivity profile, the nonradiative heat gains, solar power absorption, and atmospheric conditions. To further estimate the variation of cooling performance on the emissivity profile of the radiator, we consider a selective radiator with a varying emission bandwidth where the center emission band is at 10.5 μm fitting within the mid of 8–13 μm window range. The atmospheric model for Perth with transmittance within atmospheric window Figure a is adop−2 °C−1),Ta−Tr for both the ideal selective and broadband radiator for varying nonradiative heat gains with a practical 3% solar power absorptionq = 10 W m−2 °C−1, the selective radiator does not provide any substantial cooling advantage and the radiator temperature falls only a few degrees below the ambient temperature. Indeed, this points to the recent demonstration of daytime radiative cooling without any nonradiative insulations,In the presence of significant nonradiative heat gain (7 W m6Radiative cooling promises a vital impact with its highly efficient passive cooling potential. In particular, the recent demonstrations for daytime radiative cooling below the ambient temperature