Impact of T Cell Repertoire Diversity on Mortality Following Cord Blood Transplantation.

INTRODUCTION: Cord blood transplantation (CBT) recipients are at increased risk of mortality due to delayed immune recovery (IR). Prior studies in CBT patients have shown that recovery of absolute lymphocyte count is predictive of survival after transplant. However, there are no data on the association of T-cell receptor (TCR) and clinical outcomes after CBT. Here we retrospectively performed TCR beta chain sequencing on peripheral blood (PB) samples of 34 CBT patients. METHODS: All patients received a total body irradiation based conditioning regimen and cyclosporine and MMF were used for graft versus host disease (GvHD) prophylaxis. PB was collected pretransplant on days 28, 56, 80, 180, and 1-year posttransplant for retrospective analysis of IR utilizing high-throughput sequencing of TCRß rearrangements from genomic DNA extracted from PB mononuclear cells. To test the association between TCR repertoire diversity and patient outcomes, we conducted a permutation test on median TCR repertoire diversity for patients who died within the first year posttransplant versus those who survived. RESULTS: Median age was 27 (range 1-58 years) and most of the patients (n = 27) had acute leukemias. There were 15 deaths occurring between 34 to 335 days after transplant. Seven deaths were due to relapse. Rapid turnover of T cell clones was observed at each time point, with TCR repertoires stabilizing by 1-year posttransplant. TCR diversity values at day 100 for patients who died between 100 and 365 days posttransplant were significantly lower than those of the surviving patients (p = 0.01). CONCLUSIONS: Using a fast high-throughput TCR sequencing assay we have demonstrated that high TCR diversity is associated with better patient outcomes following CBT. Importantly, this assay is easily performed on posttransplant PB samples, even as early as day 28 posttransplant, making it an excellent candidate for early identification of patients at high risk of death.

Cord blood transplantation: rewind to fast forward.

The utilization of cord blood as a source of stem cells for transplantation has decreased in recent years. Although cord blood transplantation (CBT) is an established practice for the treatment of adult and pediatric patients with hematological malignancies, the high acquisition cost of CB units along with high transplant-related mortality due to delayed hematopoietic recovery and immune reconstitution have contributed to the slowing in widespread adoption of CBT. Strategies aimed to enhance speed of engraftment and ongoing clinical trials are investigating ways to make CBT more widely available. Meanwhile, the recent clinical data suggest that the choice of CBT might be preferable for patients with pre-transplant minimal residual disease. We review here the background data on the utilization of CB for the treatment of hematological malignancies, and discuss the current challenges and future directions in the field of CBT.

Roots and associated fungi drive long-term carbon sequestration in boreal forest.

Boreal forest soils function as a terrestrial net sink in the global carbon cycle. The prevailing dogma has focused on aboveground plant litter as a principal source of soil organic matter. Using (14)C bomb-carbon modeling, we show that 50 to 70% of stored carbon in a chronosequence of boreal forested islands derives from roots and root-associated microorganisms. Fungal biomarkers indicate impaired degradation and preservation of fungal residues in late successional forests. Furthermore, 454 pyrosequencing of molecular barcodes, in conjunction with stable isotope analyses, highlights root-associated fungi as important regulators of ecosystem carbon dynamics. Our results suggest an alternative mechanism for the accumulation of organic matter in boreal forests during succession in the long-term absence of disturbance.

137Cs in fungal sporocarps in relation to vegetation in a bog, pine swamp and forest along a transect.

In this study, we estimated the relative importance of vegetation and fungi for radiocesium uptake and biological retention in adjacent bog, pine swamp, and forest. The measurements for (137)Cs activity concentration in sporocarps (i.e. fruitbodies of fungi) and vegetation along a bog to forest transect were combined with complementary published data to calculate estimates. Aboveground vegetation comprised 17.7% of the total fallout-derived radiocesium in the system in bog, 16.5% in pine swamp, and 40.6% in forest. In fungal sporocarps grown along a gradient, (137)Cs activity comprised <0.001% of the total radiocesium for peat bog, <0.02% for pine swamp, and 0.11% for forest. Total (137)Cs activity in sporocarps increased along the gradient due to increased production of sporocarps in the presence of trees from 0.006 (bog), 0.097 (pine swamp) and 0.67 (forest) g dwt m(-2). Based on calculation of the total vegetation biomass and through relationships between fungal biomass in sporocarps and as mycelia in soil, the total (137)Cs activity located in fungi was estimated as 0.1% in bog, 2% in pine swamp, and 11% in forest. An analysis of the time-dependency of (137)Cs in the sporocarps in forest between 1990 and 2011 suggested an ecological half-life for (137)Cs between 8 and 13 years. Although fungi comprised a relatively small fraction of the total radiocesium in the systems, its activity decreased slowly with time, and ecological residence time for (137)Cs in sporocarps of fungi was long, suggesting they will continue to contribute to the accumulation and cycling of this radionuclide in forest.

Patterns of fungal communities among and within decaying logs, revealed by 454 sequencing.

Owing to previous methodological limitations, knowledge about the fine-scale distribution of fungal mycelia in decaying logs is limited. We investigated fungal communities in decaying Norway spruce logs at various spatial scales at two environmentally different locations in Sweden. On the basis of 454 pyrosequencing of the ITS2 region of rDNA, 1914 operational taxonomic units (OTUs) were detected in 353 samples. The communities differed significantly among logs, but the physical distance between logs was not found to have a significant effect on whether fungal communities had any resemblance to each other. Within a log, samples that were closer together generally had communities that showed more resemblance to each other than those that were further apart. OTUs characteristic for particular positions on the logs could be identified. In general, these OTUs did not overlap with the most abundant OTUs, and their ecological role was often unknown. Only a few OTUs were detected in the majority of logs, whereas numerous OTUs were rare and present in only one or a few logs. Wood-decaying Basidiomycetes were often represented by higher sequence reads in individual logs than Ascomycete OTUs, suggesting that Basidiomycete mycelia spread out more rapidly when established. OTU richness tended to increase with the decay stage of the sample; however, the known wood decayers were most abundant in less-decomposed samples. The fungi identified in the logs represented different ecological strategies. Our findings differ from previously published sporocarp studies, indicating that the highly abundant fruiting species may respond to environment in different ways than the rest of the fungal community.

Correlations between potassium, rubidium and cesium ((133)Cs and (137)Cs) in sporocarps of Suillus variegatus in a Swedish boreal forest.

An analysis of sporocarps of ectomycorrhizal fungi Suillus variegatus assessed whether cesium ((133)Cs and (137)Cs) uptake was correlated with potassium (K) or rubidium (Rb) uptake. The question was whether intraspecific correlations of Rb, K and (133)Cs mass concentrations with (137)Cs activity concentrations in sporocarps were higher within, rather than among, different fungal species, and if genotypic origin of sporocarps within a population affected uptake and correlation. Sporocarps (n = 51) from a Swedish forest population affected by the fallout after the Chernobyl accident were studied. The concentrations were 31.9 ± 6.79 g kg(-1) for K (mean ± SD, dwt), 0.40 ± 0.09 g kg(-1) for Rb, 8.7 ± 4.36 mg kg(-1) for (133)Cs and 63.7 ± 24.2 kBq kg(-1) for (137)Cs. The mass concentrations of (133)Cs correlated with (137)Cs activity concentrations (r = 0.61). There was correlation between both (133)Cs concentrations (r = 0.75) and (137)Cs activity concentrations (r = 0.44) and Rb, but the (137)Cs/(133)Cs isotopic ratio negatively correlated with Rb concentration. Concentrations of K and Rb were weakly correlated (r = 0.51). The (133)Cs mass concentrations, (137)Cs activity concentrations and (137)Cs/(133)Cs isotopic ratios did not correlate with K concentrations. No differences between, within or, among genotypes in S. variegatus were found. This suggested the relationships between K, Rb, (133)Cs and (137)Cs in sporocarps of S. variegatus is similar to other fungal species.

Microsatellite markers for the ectomycorrhizal basidiomycete Lactarius mammosus.

We report the characterization of polymorphic microsatellite markers in the ectomycorrhizal fungus Lactarius mammosus Fr. Two enrichment protocols were used to isolate microsatellite loci and polymorphism was explored within 31 sporocarps originating from a forest site in northern Sweden. We found nine variable microsatellite loci with the number of alleles per locus varying between 2 and 5, and expected heterozygosities ranging from 0 to 0.84. These loci are available for the analysis of genetic structure and gene flow in L. mammosus populations.

Respondent burden and patient-perceived validity of the PDQ-39.

OBJECTIVES: To evaluate the respondent burden and patient-perceived content validity of the Parkinson's disease (PD)-specific health status questionnaire PDQ-39, and the linguistic validity of its revised Swedish version. MATERIALS AND METHODS: Eighteen PD patients completed the revised Swedish version of the PDQ-39. Respondent burden was assessed by recording the time taken to complete the questionnaire. Content and linguistic validity was evaluated qualitatively. RESULTS: Patients with mild, moderate and advanced PD needed a mean time of 9.5, 11.3 and 20.1 min, respectively, to complete the PDQ-39. One-third of the patients identified irrelevant items and 50% identified important health-related areas that were missing. Revisions had eliminated previous linguistic problems with the Swedish PDQ-39. CONCLUSIONS: Undue respondent burden challenged the appropriateness of the PDQ-39 among patients with more advanced disease. Overall content validity was acceptable but compromised by lack of important content areas. Observations supported the linguistic validity of the revised Swedish PDQ-39.

Prevalence of spinal cord injury in Helsinki.

STUDY DESIGN: Retrospective register-based epidemiological study. OBJECTIVE: To estimate the prevalence rate of persons with spinal cord injury (SCI) with special reference to ASIA Impairment Grade A-D. SETTING: Helsinki, Finland. METHODS: Cases were identified using the registers of the Kapyla Rehabilitation Centre, Helsinki University Central Hospital and the local organization for the disabled. Local health centres were informed about the study, residential service houses were contacted, and announcements were published in patient magazines. RESULTS: A regional population was found to have a prevalence rate of 28/100,000 inhabitants with SCI (ASIA Impairment Scale A-D). CONCLUSION: The prevalence rate in this study is consistent with the data published in other Nordic countries. SPONSORSHIP: The Finnish Cultural Foundation.

Bladder management in persons with spinal cord lesion.

STUDY DESIGN: Cross-sectional clinical descriptive prevalence study. OBJECTIVE: To evaluate the methods of self-reported bladder management, the frequency of urinary tract infection (UTI) and subjective disturbance of bladder problems of all those individuals with traumatic spinal cord lesion (SCL) living in Helsinki area. SETTING: : Helsinki, Finland. METHODS: A total of 152 persons with SCL were found in the Helsinki area (546 000 inhabitants). A structured questionnaire was sent to all subjects and they were invited to a clinical visit. RESULTS: The final study-group consisted of 129 (85%) subjects. They were defined into seven specific subgroups of bladder management: 14 (11%) subjects in the normal voiding group, 15 (12%) in the controlled voiding group, 16 (12%) in the clean intermittent catheterization (CIC) group, 30 (23%) in the mixed group, 31 (24%) in the suprapubic tapping group, 16 (12%) in the compression or straining group and seven (5%) in the catheter or conduit group. The frequency of UTI was highest in the mixed group. The bladder management was a biggest bother to the subjects in the compression or straining group. CONCLUSIONS: This prevalence study assesses the self-reported bladder management methods in all the persons with traumatic SCL in the Helsinki area. The subjects who used CIC and other methods for bladder management had more problems than others. These subjects might manage better by using either CIC or suprapubic tapping as the only method for bladder emptying.

Life situation of spinal cord-injured persons in Central Finland.

STUDY DESIGN: Individual interview and questionnaire of a regional spinal cord-injured (SCI) population. OBJECTIVE: Local health care centres are mainly responsible for the SCI patients health care and rehabilitation after initial hospitalization in Finland. The purpose of the present study was to study aspects of the SCI patients life situation and their opinion of the health care services after 1 year since the injury. SETTING: Regional study in Central Finland. METHODS: Subjects were individually interviewed in their homes using a semi-structured questionnaire, the Functional Independence Measure (FIM) and the Beck Depression Inventory (BDI). RESULTS: The female/male ratio was 1:4. The mean age at the time of injury was 34 years in female and 40 in male. Traffic accidents accounted for 46% of the injuries. In all, 48 subjects (63%) used electrical or manual wheelchair or both. Most subjects regarded their living conditions as good having enough personal assistance in their everyday life. Only 10% of the subjects were employed. Half of the subjects had had out-patient physiotherapy, but no other therapies after 1 year since the injury. More than half of the subjects were dissatisfied with the current health care services. Many had experienced complications after the initial hospitalization, especially urinary tract infections, decubitus ulcers and neurogenic-type pain. FIM classified well according to the disability groups. The motor complete tetraplegic group had the lowest mean scores (63) and the recovered group the highest ones (122). There was no association between the FIM and the BDI results. There was a correlation between the BDI and the subject's age at the time of the injury and the year of the injury. The older the subjects were when injured, the higher were the BDI scores, that is, they had more depressive symptoms. Those injured in the 1990s had the highest BDI scores. Nearly one-third of the subjects had mild, moderate or severe depression. CONCLUSIONS: The reported medical complications, depression and dissatisfaction with the health care services support a life-long care for SCI patients in Jyväskylä Central hospital, not in the local health care centres. The psychological services, for example, prevention and treatment of depression, in particular, require more attention.

Parental tracking in the postfire wood decay ascomycete Daldinia loculata using highly variable nuclear gene loci.

The origin of the male and female gametes involved in fertilization events within a local population of the postfire wood decay ascomycete Daldinia loculata was investigated by genotyping the mycelia growing in the wood and the sexual ascospores, using three highly variable nuclear gene loci. The study was conducted in a geographically isolated burned forest site in southern Sweden. An intensive sampling was performed by collecting stromata containing ascospores and wood samples containing mycelia. In total, from 32 mapped burned birches, cultures of 22 haploid genets from decayed wood and six ascospores from each of 19 stromata were isolated and analysed. In 80% of the investigated burned branches, only one genet was found. From the analysis of the ascospore genotypes, we detected 30 fertilization events and 60% of them were the result of mating between conidia (clonal propagules) acting as male gametes and the genets in the branches representing the female gametes. The male parents producing the conidia were detected within the same local population as the female parents in 27% of the fertilization events and originated either from the same branch or from different trees located at 0.5-36 m away from the female parents. In 33% of the fertilization events, conidia originated from three male parents that were not found within the local population sampled. These parents could be anywhere inside or outside the sampled area. For the remaining fertilization events, we could not rule out the ascospores or the conidia as fertilizing propagules. No strong evidence for fertilization by recombinant propagules (ascospores) was detected in this study. The pyrophilous insect species associated with conidia of D. loculata are suggested to be essential vectors for the realization of the sexual cycle of this fungal species. By feeding on the conidia and flying between nearby trees inhabiting wood decay mycelia, these insects allow the transfer of conidia and therefore the opposite mating types to meet within a localized burned forest site.

Diversion of water flow from a floodplain wetland stream: an analysis of geomorphological setting and hydrological and ecological consequences.

Diversion of water has been ongoing in the Mkuze Wetland for several decades. Two canals form the focus of this study; the Mpempe-Demazane Canal and the Tshanetshe Canal. The former involved an ambitious excavation over a distance of 13.5 km in the lower part of the wetland, while the latter was a minor excavation over a distance of approximately 100 m in the upper part of the wetland. Although ambitious and costly, the Mpempe-Demazane Canal resulted in little downward or headward erosion, and there was minor diversion of flow. However, the minor excavation of the Tshanetshe Canal resulted in erosion downstream of the excavation (the Tshanetshe Stream), downward and lateral erosion of the excavated section, and headward erosion that has propagated almost 4 km upstream along the Mkuze River. Most of the flow of the Mkuze River has been captured by the Tshanetshe Canal and Stream. The impact of canalisation on floodplain wetlands is thus more dependent on the location than the scale of activity. The avulsion of the Mkuze River into the Tshanetshe Canal and Stream is due to a large difference in elevation between the Mkuze River and floodplain into which it was diverted, and the fact that in this region the river typically has high discharges. This avulsion may have been inevitable as a result of natural processes of sedimentation. In contrast, the difference in elevation between the Mkuze River and the basin into which it was diverted via the Mpempe Canal was small as is discharge of the Mkuze River in this part of the wetland. Thus, the diversion was unsuccessful. The presence of hippos that create hydraulically efficient pathways that are oriented parallel to the regional hydraulic slope, may accelerate avulsion in large African wetlands. Overall, it is argued that the environmental consequences of excavation need to be viewed against the background that wetlands are dynamic features within the landscape.

Staphylococcus aureus domain V functions in Escherichia coli ribosomes provided a conserved interaction with domain IV is restored.

Domain V of Escherichia coli 23 S rRNA (residues 2023-2630) was replaced by that from Staphylococcus aureus, thereby introducing 132 changes in the rRNA sequence. The resulting ribosomal mutant was unable to support cell growth. The mutant was rescued, however, by restoring an interaction between domains IV and V (residues 1782 and 2586). Although the importance of this interaction, U/U in E. coli, C/C in S. aureus, is therefore demonstrated, it cannot be the only tertiary interaction important for ribosomal function as the rescued hybrid grew more slowly than the wild type. Additionally, although the single-site mutations U1782C and U2586C in E. coli are viable, the double mutant is lethal.

Initiation factor IF 2 binds to the alpha-sarcin loop and helix 89 of Escherichia coli 23S ribosomal RNA.

During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2.

The polypeptide tunnel system in the ribosome and its gating in erythromycin resistance mutants of L4 and L22.

Variations in the inner ribosomal landscape determining the topology of nascent protein transport have been studied by three-dimensional cryo-electron microscopy of erythromycin-resistant Escherichia coli 70S ribosomes. Significant differences in the mouth of the 50S subunit tunnel system visualized in the present study support a simple steric-hindrance explanation for the action of the drug. Examination of ribosomes in different functional states suggests that opening and closing of the main tunnel are dynamic features of the large subunit, possibly accompanied by changes in the L7/L12 stalk region. The existence and dynamic behavior of side tunnels suggest that ribosomal proteins L4 and L22 might be involved in the regulation of a multiple exit system facilitating cotranslational processing (or folding or directing) of nascent proteins.

A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition.

The ribosome is a dynamic particle that undergoes many structural changes during translation. We show through chemical probing with dimethyl sulfate (DMS) that conformational changes occur at several nucleotides in the peptidyl transferase center upon alterations in pH, temperature, and monovalent ion concentration, consistent with observations made by Elson and coworkers over 30 years ago. Moreover, we have found that the pH-dependent DMS reactivity of A2451 in the center of the 23S rRNA peptidyl transferase region, ascribed to a perturbed pKa of this base, occurs only in inactive 50S and 70S ribosomes. The degree of DMS reactivity of this base in the inactive ribosomes depends on both the identity and amount of monovalent ion present. Furthermore, G2447, a residue proposed to be critical for the hypothesized pKa perturbation, is not essential for the conditional DMS reactivity at A2451. Given that the pH-dependent change in DMS reactivity at A2451 occurs only in inactive ribosomes, and that this DMS reactivity can increase with increasing salt (independently of pH), we conclude that this observation cannot be used as supporting evidence for a recently proposed model of acid/base catalyzed ribosomal transpeptidation.