A new lineage of Galapagos giant tortoises identified from museum samples.

The Galapagos Archipelago is recognized as a natural laboratory for studying evolutionary processes. San Cristóbal was one of the first islands colonized by tortoises, which radiated from there across the archipelago to inhabit 10 islands. Here, we sequenced the mitochondrial control region from six historical giant tortoises from San Cristóbal (five long deceased individuals found in a cave and one found alive during an expedition in 1906) and discovered that the five from the cave are from a clade that is distinct among known Galapagos giant tortoises but closely related to the species from Española and Pinta Islands. The haplotype of the individual collected alive in 1906 is in the same clade as the haplotype in the contemporary population. To search for traces of a second lineage in the contemporary population on San Cristóbal, we closely examined the population by sequencing the mitochondrial control region for 129 individuals and genotyping 70 of these for both 21 microsatellite loci and >12,000 genome-wide single nucleotide polymorphisms [SNPs]. Only a single mitochondrial haplotype was found, with no evidence to suggest substructure based on the nuclear markers. Given the geographic and temporal proximity of the two deeply divergent mitochondrial lineages in the historical samples, they were likely sympatric, raising the possibility that the lineages coexisted. Without the museum samples, this important discovery of an additional lineage of Galapagos giant tortoise would not have been possible, underscoring the value of such collections and providing insights into the early evolution of this iconic radiation.

Temporal Mitogenomics of the Galapagos Giant Tortoise from Pinzón Reveals Potential Biases in Population Genetic Inference.

Empirical population genetic studies generally rely on sampling subsets of the population(s) of interest and of the nuclear or organellar genome targeted, assuming each is representative of the whole. Violations of these assumptions may impact population-level parameter estimation and lead to spurious inferences. Here, we used targeted capture to sequence the full mitochondrial genome from 123 individuals of the Galapagos giant tortoise endemic to Pinzón Island (Chelonoidis duncanensis) sampled at 2 time points pre- and postbottleneck (circa 1906 and 2014) to explicitly assess differences in diversity estimates and demographic reconstructions based on subsets of the mitochondrial genome versus the full sequences and to evaluate potential biases associated with diversity estimates and demographic reconstructions from postbottlenecked samples alone. Haplotypic diversities were equal between the temporal samples based on the full mitochondrial genome, but single gene estimates suggested either decreases or increases in diversity depending upon the region. Demographic reconstructions based on the full sequence were more similar between the temporal samples than those based on the control region alone, or a subset of 3 regions, where the trends in population size changes shifted in magnitude and direction between the temporal samples. In all cases, the estimated coalescent point was more distant for the historical than contemporary sample. In summary, our results empirically demonstrate the influence of sampling bias when interpreting population genetic patterns and punctuate the need for careful consideration of potentially conflicting evolutionary signal across the mitochondrial genome.

Reliability of Echocardiographic Indicators of Pulmonary Vascular Disease in Preterm Infants at Risk for Bronchopulmonary Dysplasia.

OBJECTIVES: To determine the assessment and inter-rater reliability of echocardiographic evaluations of pulmonary vascular disease (PVD) in preterm infants at risk for bronchopulmonary dysplasia. STUDY DESIGN: We prospectively studied echocardiograms from preterm infants (birthweights 500-1250 g) at 7 days of age and 36 weeks postmenstrual age (PMA). Echocardiograms were assessed by both a cardiologist on clinical service and a single research cardiologist. Interpretations were reviewed for inclusion of determinants of PVD and assessed for inter-rater reliability using the Prevalence Adjusted Bias Adjusted Kappa Score (PABAK). RESULTS: One hundred eighty and 188 matching research and clinical echocardiogram reports were available for the 7-day and 36-week PMA studies. At least one of the specific qualitative measures of PVD was missing from 54% of the clinical reports. PVD was diagnosed at 7 days in 31% and 20% of research and clinical interpretations, respectively (PABAK score of 0.54). At 36 weeks, PH was diagnosed in 15.6% and 17.8% of research and clinical interpretations, respectively (PABAK score of 0.80). CONCLUSIONS: Although all qualitative variables of PVD are not consistently provided in echocardiogram reports, the inter-rater reliability of cardiologists evaluating measures of PVD revealed strong agreement, especially at 36 weeks PMA. We speculate that establishment of a protocol for echocardiographic evaluation may improve the identification of PVD in preterm infants.

Vitamin B-12 treatment of asymptomatic, deficient, elderly Chileans improves conductivity in myelinated peripheral nerves, but high serum folate impairs vitamin B-12 status response assessed by the combined indicator of vitamin B-12 status.

BACKGROUND: It is uncertain whether vitamin B-12 supplementation can improve neurophysiologic function in asymptomatic elderly with low vitamin B-12 status or whether folate status affects responses to vitamin B-12 supplementation. OBJECTIVE: We assessed the effects of a single intramuscular injection of 10 mg vitamin B-12 (which also contained 100 mg vitamin B-6 and 100 mg vitamin B-1) on vitamin B-12 status and neurophysiologic function in elderly community-dwelling Chileans with low serum vitamin B-12 concentrations who were consuming bread fortified with folic acid. DESIGN: A pretreatment and posttreatment study was conducted in 51 participants (median ± SD age: 73 ± 3 y; women: 47%) with serum vitamin B-12 concentrations <120 pmol/L at screening. Vitamin B-12 status was defined by combining vitamin B-12, plasma total homocysteine (tHcy), methylmalonic acid (MMA), and holotranscobalamin into one variable [combined indicator of vitamin B-12 status (cB-12)]. The response to treatment was assessed by measuring cB-12 and neurophysiologic variables at baseline and 4 mo after treatment. RESULTS: Treatment increased serum vitamin B-12, holotranscobalamin, and cB-12 (P < 0.001) and reduced plasma tHcy and serum MMA (P < 0.001). Treatment produced consistent improvements in conduction in myelinated peripheral nerves; the sensory latency of both the left and right sural nerves improved on the basis of faster median conduction times of 3.1 and 3.0 ms and 3.3 and 3.4 ms, respectively (P < 0.0001). A total of 10 sensory potentials were newly observed in sural nerves after treatment. Participants with high serum folate at baseline (above the median, ≥33.9 nmol/L) had less improvement in cB-12 (P < 0.001) than did individuals whose serum folate was less than the median concentration (i.e., with a concentration <33.9 nmol/L). CONCLUSION: Asymptomatic Chilean elderly with poor vitamin B-12 status displayed improved conductivity in myelinated peripheral nerves after vitamin B-12 treatment and an interaction with folate status, which was detected only with the use of cB-12. This trial was registered at www.controlled-trials.com as ISRCTN02694183.

A comparison of characteristics and outcomes in severe human metapneumovirus and respiratory syncytial virus infections in children treated in an intensive care unit.

BACKGROUND: Human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are among the leading causes of respiratory tract infections requiring admission to the pediatric intensive care unit (PICU). We evaluated the risk factors, clinical courses and outcomes of severe HMPV disease relative to severe RSV in children admitted to the PICU. METHODS: Retrospective chart review of children ≤18 years old admitted to a tertiary PICU between October 2008 through July 2010 with acute respiratory tract infection and positive direct antigen stain or polymerase chain reaction for RSV or HMPV. RESULTS: One hundred thirty-three patients met inclusion criteria: 107 (80.5%) with RSV and 26 (19.5%) with HMPV. HMPV-infected patients were older than RSV children (3.4 vs. 1.5 years, P = 0.002) and more likely to have congenital heart disease (34.6% vs. 10.3%, P = 0.002). Although HMPV children required longer duration of mechanical ventilation (11 vs. 7 days, P = 0.01), there were no other differences in hospital course. HMPV patients were more likely to be discharged receiving inhaled steroids (53.8% vs. 30.8%, P = 0.03), but there were no differences in other outcome assessments. CONCLUSIONS: Children admitted to the PICU with HMPV are significantly older and more likely to have congenital heart disease than those with RSV. The course of illness was similar between the 2 groups, but HMPV-infected children were more likely to be discharged with inhaled steroid therapy.

Vitamin B-12 supplementation of rural Mexican women changes biochemical vitamin B-12 status indicators but does not affect hematology or a bone turnover marker.

A high prevalence of low serum vitamin B-12 concentrations has been reported in studies and surveys in Latin America including Mexico, but the functional consequences are unknown. This randomized controlled trial assessed the response to a high-dose vitamin B-12 supplementation of women in rural Querétaro, Mexico. Participants aged 20-59 y were stratified at baseline to deficient, marginal, and adequate status groups (serum vitamin B-12, 75-148, 149-220, and >220 pmol/L, respectively), and each group was randomized to vitamin B-12 treatment (single dose of 1 mg i.m. then 500 µg/d orally for 3 mo, n = 70) or placebo (n = 62). Measures at baseline and 3 mo included: complete blood count, serum vitamin B-12, holotranscobalamin (holoTC), folate, ferritin, C-reactive protein (CRP), bone alkaline phosphatase, and methylmalonic acid (MMA) and plasma total homocysteine (tHcy). At baseline, 11% of the women were vitamin B-12 deficient and 22% had marginal status. HoloTC was low (<35 pmol/L) in 23% and correlated with serum vitamin B-12 (r = 0.7; P < 0.001). Elevated MMA (>271 nmol/L) and tHcy (>12 µmol/L) occurred in 21 and 31%, respectively, and correlated with serum vitamin B-12 (r = -0.28, P < 0.0007 and r = -0.20, P < 0.01, respectively). Supplementation increased serum vitamin B-12 and holoTC and lowered MMA and tHcy, normalizing all values except for elevated tHcy in 21% of the women. Supplementation did not affect hematology or bone-specific alkaline phosphatase. Vitamin B-12 supplementation normalized biochemical indicators of vitamin B-12 status in the treatment group but did not affect the functional outcomes measured.

Long-term isolation of a highly mobile seabird on the Galapagos.

The Galapagos Islands are renowned for their high degree of endemism. Marine taxa inhabiting the archipelago might be expected to be an exception, because of their utilization of pelagic habitats-the dispersal barrier for terrestrial taxa-as foraging grounds. Magnificent frigatebirds (Fregata magnificens) have a highly vagile lifestyle and wide geographical distribution around the South and Central American coasts. Given the potentially high levels of gene flow among populations, the species provides a good test of the effectiveness of the Galapagos ecosystem in isolating populations of highly dispersive marine species. We studied patterns of genetic (mitochondrial DNA, microsatellites and nuclear introns) and morphological variation across the distribution of magnificent frigatebirds. Concordant with predictions from life-history traits, we found signatures of extensive gene flow over most of the range, even across the Isthmus of Panama, which is a major barrier to gene flow in other tropical seabirds. In contrast, individuals from the Galapagos were strongly differentiated from all conspecifics, and have probably been isolated for several hundred thousand years. Our finding is a powerful testimony to the evolutionary uniqueness of the taxa inhabiting the Galapagos archipelago and its associated marine ecosystems.

Graft versus host disease in a rat small bowel transplant model after T-cell depleted donor specific bone marrow infusion

Low cytoreductive regimen of irradiation associated to unmodified bone marrow infusion (UBM) does not prevent the occurrence of graft versus host disease (GVHD) after transplant. PURPOSE: In this study we evaluated the potential advantages of a long-term immunossupression and T-cell depleted bone marrow infusion (TCDBMI) in preventing the occurrence of GVHD after small bowel transplantation (SBTx). METHODS: Heterotopic SBTX was performed with Lewis rats as recipients and DA as donors and distributed into 5 groups according to the irradiation, duration of immunossupression and the use of UBM or TCDBMI: G1 (n=6), without irradiation and G2 (n=9), G3 (n=4), G4 (n=5) and G5 (n=6) was given 250 rd of irradiation. Groups 1,2,4 and G3 and 5 were infused with 100 x 106 UBM and TCDBM respectively. Animals in G1, 2, 3 were immunossupressed with 1mg/ FK506/Kg/IM for 5 days and G4 and G5 for 15 days. Anti CD3 monoclonal antibodies and immunomagnetic beads were used for T-cell depletion.Animals were examined for rejection, GVHD, chimerism characterization and ileal and skin biopsies. RESULTS: Minimal to mild rejection was observed in all groups; however, GVHD were present only in irradiated groups. Long-term immunossupression changed the severity of GVHD in G4 and G5. Rejection was the cause of death in G1 while GVHD in G2, 3, 4 and 5, not avoided by the use of TCDBMI. Total chimerism and T-cell chimerism was statistically higher in irradiated groups when compared to G1. CONCLUSION: Extended immunossupression associated to low dose of irradiation decrease the severity of GVHD, not avoided by the use of TCDBMI.

Chimerism induction by nonmyeloablactive preconditioning and bone marrow infusion in rat small bowel transplantation

ABSTRACT: In our previous work we demonstrated that the use of donor specific bone marrow infusions ( DSBMI ) after small bowel transplantation did not improve the graft survival after a short course of immunossupression. PURPOSE: In the current study, we evaluated whether recipient preconditioning with different regimens of radiation combined with DSBMI may enhance small bowel allograft survival with minimum recipient morbidity. METHODS: Heterotopic small bowel transplantation (SBTx) was performed with Lewis rats as recipients and DA rats as donors, which were immunossupressed with a short course of tacrolimus (FK 506 ) at 1mg/Kg/day for 5 days and distributed in 4 groups: group 1 (n= 4) without both irradiation and DSBMI; Groups 2 (n= 6), 3 (n= 9) and 4 (n= 6) received 100 x 106 DSBM cells at the time of the transplant. Groups 3 and 4 were irradiated with 250 and 400 rd respectively. Animals were examined daily for clinical signs of rejection or GVHD. Blood samples were taken weekly for chimeric studies by FC and intestinal biopsies were performed every 2 weeks. RESULTS: Animals in G1 and G2 had minimal rejection at day 15 after SBTx while GVHD was clinically and histologically characterized in G 3 and G 4. Total chimerism and T-cell chimerism was higher in irradiated groups when compared to non-irradiated groups. With exception of G1 and 2 where rejection was the cause of death, all animals in G3 and 4 died of GVHD. CONCLUSION:We concluded that low cytoreductive of irradiation can successfully decrease the graft rejection but not prevent the occurrence of GVHD.

High prevalence of cobalamin deficiency in Guatemalan schoolchildren: associations with low plasma holotranscobalamin II and elevated serum methylmalonic acid and plasma homocysteine concentrations.

BACKGROUND: Studies conducted in Guatemala, Mexico, and Venezuela have found high prevalences of low plasma cobalamin (vitamin B-12) concentrations in infants and children. It is not known whether these low cobalamin concentrations are accompanied by altered metabolic functions. OBJECTIVE: We sought to assess the prevalence of cobalamin deficiency in Guatemalan children by using sensitive and specific markers of deficiency. DESIGN: Children (n = 553) were screened for low plasma cobalamin. Those with low plasma cobalamin (< 162 pmol/L) were matched by age, grade, and sex to those with marginal (162-221 pmol/L) and adequate (> 221 pmol/L) concentrations. In this matched subset (n = 180), additional biochemical indicators of cobalamin deficiency were measured. RESULTS: Of the 553 children screened, 11% had low plasma cobalamin and an additional 22% had marginal concentrations. The prevalences of elevated serum methylmalonic acid (MMA), plasma homocysteine, or both were significantly higher in children with low and marginal plasma cobalamin than in children with adequate plasma cobalamin. Mean serum MMA was high in all groups compared with values reported in other populations. Mean plasma holotranscobalamin II concentrations were significantly lower in children with low rather than marginal or adequate plasma cobalamin. However, holotranscobalamin II was a less sensitive indicator of cobalamin depletion than was MMA. CONCLUSION: Biochemical markers of cobalamin deficiency confirmed that the cobalamin status of children with low and marginal plasma cobalamin is inadequate to support normal metabolic function.

Predictors of cobalamin deficiency in Guatemalan school children: diet, Helicobacter pylori, or bacterial overgrowth?

OBJECTIVES: The authors investigated whether low vitamin B12 intake, impaired gastric function, infection, and bacterial overgrowth were risk factors for the high prevalence of cobalamin deficiency observed in Guatemalan children. METHODS: The plasma cobalamin concentration of 556 school children was measured and classified as low, marginal, or adequate. In 60 children from each of these three groups, concentrations of serum methylmalonic acid (MMA), plasma homocysteine, and plasma holotranscobalamin II were measured, and usual dietary B12 intake was estimated. Serum gastrin and pepsinogen I concentrations were measured, and and bacterial overgrowth were diagnosed using C-urea and C-xylose breath tests, respectively. RESULTS: infection was present in 83% (144 of 174) of children, and bacterial overgrowth was found in 25% (28 of 113). Children with infection had higher serum gastrin and pepsinogen I. There were no significant differences among the plasma cobalamin groups in the prevalence of infection, bacterial overgrowth, serum gastrin, or pepsinogen I concentrations. However, there was a significant positive correlation between serum MMA and gastrin concentrations. The average daily consumption of dietary B12 was 5.5 +/- 5.2 microg/day, but intakes for 23% of children were <1.8 micro g/day. B12 intake from fortified snacks added an additional 0.3 +/- 0.2 microg/day. B12 intake was not significantly different among the plasma cobalamin groups, but it was significantly correlated with plasma cobalamin. CONCLUSIONS: The specific cause of cobalamin deficiency in this population remains unclear, but these results suggest that low dietary B12 intake is a risk factor and alterations in gastric secretions may also play a role.

Graft versus host disease in a rat small bowel transplant model after T-cell depleted donor specific bone marrow infusion

Low cytoreductive regimen of irradiation associated to unmodified bone marrow infusion (UBM) does not prevent the occurrence of graft versus host disease (GVHD) after transplant. PURPOSE: In this study we evaluated the potential advantages of a long-term immunossupression and T-cell depleted bone marrow infusion (TCDBMI) in preventing the occurrence of GVHD after small bowel transplantation (SBTx). METHODS: Heterotopic SBTX was performed with Lewis rats as recipients and DA as donors and distributed into 5 groups according to the irradiation, duration of immunossupression and the use of UBM or TCDBMI: G1 (n=6), without irradiation and G2 (n=9), G3 (n=4), G4 (n=5) and G5 (n=6) was given 250 rd of irradiation. Groups 1,2,4 and G3 and 5 were infused with 100 x 10(6) UBM and TCDBM respectively. Animals in G1, 2, 3 were immunossupressed with 1mg/ FK506/Kg/IM for 5 days and G4 and G5 for 15 days. Anti CD3 monoclonal antibodies and immunomagnetic beads were used for T-cell depletion.Animals were examined for rejection, GVHD, chimerism characterization and ileal and skin biopsies. RESULTS: Minimal to mild rejection was observed in all groups; however, GVHD were present only in irradiated groups. Long-term immunossupression changed the severity of GVHD in G4 and G5. Rejection was the cause of death in G1 while GVHD in G2, 3, 4 and 5, not avoided by the use of TCDBMI. Total chimerism and T-cell chimerism was statistically higher in irradiated groups when compared to G1. CONCLUSION: Extended immunossupression associated to low dose of irradiation decrease the severity of GVHD, not avoided by the use of TCDBMI.

Baixas doses de irradiação associadas à infusão de células da medula óssea não previnem a ocorrência da reação do enxerto versus hospedeiro após o transplante intestinal. OBJETIVO: Neste estudo foi avaliado a potencial vantagem em estender o regime imunossupressor associado a infusão de células de medula óssea do doador depletadas de células T na prevenção da reação do enxerto versus hospedeiro após o transplante intestinal. MÉTODOS: Transplante heterotópico de intestino delgado foi realizado em ratos Lewis como receptores e DA como doadores, distribuídos em cinco grupos de acordo com a duração da imunossupressão, irradiação e do uso de medula óssea normal ou depletada: G1 (n=6), sem irradiação e G2 (n=9), G3 (n=4), G4 (n=5) e G5 (n=6) foram irradiados com 250 rd. Grupos1, 2, 4 e G3 e 5 foram infundidos com 100 x 10(6) células da medula normal e depletada respectivamente. Animais no G1,2,3 foram imunossuprimidos com 1mg/kg/FK506/ IM por cinco dias e G4 e cinco por 15 dias. Anticorpos monoclonais contra células CD3 e colunas magnéticas foram utilizadas para a depleção da medula óssea. Os animais foram examinados para a presença de rejeição, reação do enxerto versus hospedeiro, chimerismo e biópsias intestinais e da pele. RESULTADOS: Rejeição mínima foi observada em todos os grupos; entretanto, a reação do enxerto versus hospedeiro somente nos animais irradiados. Extensão da imunossupressão alterou a gravidade da reação nos animais dos G4 e 5. Rejeição foi a causa mortis no G1 e a reação do enxerto versus hospedeiro nos Grupos 2,3,4 e 5, não controlada com a infusão de medula óssea depletada. O chimerismo total e de células T do doador foi estatisticamente maior nos grupos irradiados em comparação ao G1. CONCLUSÃO: A extensão do regime de imunossupressão associado a baixas doses de irradiação diminui a gravidade da reação do enxerto versus hospedeiro, não abolida pelo uso de medula óssea depletada.

Chimerism induction by nonmyeloablactive preconditioning and bone marrow infusion in rat small bowel transplantation

In our previous work we demonstrated that the use of donor specific bone marrow infusions ( DSBMI ) after small bowel transplantation did not improve the graft survival after a short course of immunossupression. PURPOSE: In the current study, we evaluated whether recipient preconditioning with different regimens of radiation combined with DSBMI may enhance small bowel allograft survival with minimum recipient morbidity. METHODS: Heterotopic small bowel transplantation (SBTx) was performed with Lewis rats as recipients and DA rats as donors, which were immunossupressed with a short course of tacrolimus (FK 506 ) at 1mg/Kg/day for 5 days and distributed in 4 groups: group 1 (n= 4) without both irradiation and DSBMI; Groups 2 (n= 6), 3 (n= 9) and 4 (n= 6) received 100 x 10(6) DSBM cells at the time of the transplant. Groups 3 and 4 were irradiated with 250 and 400 rd respectively. Animals were examined daily for clinical signs of rejection or GVHD. Blood samples were taken weekly for chimeric studies by FC and intestinal biopsies were performed every 2 weeks. RESULTS: Animals in G1 and G2 had minimal rejection at day 15 after SBTx while GVHD was clinically and histologically characterized in G 3 and G 4. Total chimerism and T-cell chimerism was higher in irradiated groups when compared to non-irradiated groups. With exception of G1 and 2 where rejection was the cause of death, all animals in G3 and 4 died of GVHD. CONCLUSION:We concluded that low cytoreductive of irradiation can successfully decrease the graft rejection but not prevent the occurrence of GVHD.

Em estudo recente demonstramos que a infusão de células da medula óssea do doador após o transplante intestinal não aumentou a sobrevida do enxerto quando se utilizou series curtas de drogas imunossupressoras. OBJETIVO: Neste estudo avaliamos se a utilização de diferentes regimes de irradiação em associação com a infusão de medula óssea altera a sobrevida do enxerto e a morbidade sobre receptor. MÉTODOS: Realizou-se o transplante heterotópico de intestino delgado, utilizando-se ratos Lewis como receptores e DA como doadores, imunossuprimidos com FK 506 na dose de 1mg/kg/dia por 5 dias e distribuídos em 4 grupos: G1 (n= 4), não irradiado e sem infusão de medula óssea; G2 (n= 6), G3 (n= 9) e G4 (n= 6) foram infundidos com 100 x 10(6) células de medula após o transplante. Grupos 3 e 4 foram irradiados com 250 e 400 rd respectivamente. Os animais foram examinados diariamente para a detecção de rejeição e reação do enxerto versus hospedeiro, tendo sido colhidas amostras semanais de sangue para estudos de quimerismose biopsias quinzenais da estomia. RESULTADOS: Animais nos G1 e G2 apresentaram rejeição mínima no 15º pós-operatório, enquanto a reação do enxerto versus hospedeiro foi caracterizada nos G3 e G4. Os níveis de quimerismo total e de células T foram maiores nos grupos irradiados em comparação aos não irradiados. A causa mortis nos G1 e G2 foi a rejeição enquanto que nos G3 e G4 foi a reação do enxerto versus hospedeiro. CONCLUSÃO: Concluímos que a utilização de baixas doses de irradiações retardam o aparecimento da rejeição, mas não previne a ocorrência da reação do enxerto versus hospedeiro.