Biomarker Changes Associated with Tuberculin Skin Test (TST) Conversion: A Two-Year Longitudinal Follow-Up Study in Exposed Household Contacts

Background

A high prevalence (50–80%) of Tuberculin Skin Test Positivity (TST+ ≥10 mm indurations) has been reported in TB endemic countries. This pool forms a huge reservoir for new incident TB cases. However, immune biomarkers associated with TST conversion are largely unknown. The objective of this study was to identify immune biomarkers associated with TST conversion after acute Mycobacterium tuberculosis (MTB) exposure.

Methodology/Principal Findings

A 24 month longitudinal study was carried out in a recently MTB exposed cohort of household contacts (HC = 93; 75% TST+). Control group consisted of unexposed community controls (EC = 59; 46%TST+). Cytokine secretion was assessed in whole blood cultures in response to either mycobacterial culture filtrate (CF) antigens or mitogens (PHA or LPS) using Elisa methodology. Compared to the EC group, the HC group at recruitment (Kruskal-Wallis Test) showed significantly suppressed IFN γ (p = 0.0001), raised IL-10 (p = 0.0005) and raised TNF α (p = 0.001) in response to CF irrespective of their TST status. Seventeen TST-HC, showed TST conversion when retested at 6 months. Post TST conversion (paired t tests) significant increases were observed for CF induced IFN γ (p = 0.038), IL-10 (p = 0.001) and IL-6 (p = 0.006). Cytokine responses were also compared in the exposed HC group with either recent infection [(TST converters (N = 17)] or previous infection [TST+ HC (N = 54)] at 0, 6, 12 and 24 months using ANOVA on repeated measures. Significant differences between the exposed HC groups were noted only at 6 months. CF induced IFN γ was higher in previously infected HC group (p = 0.038) while IL-10 was higher in recently infected HC group (p = 0.041). Mitogen induced cytokine secretion showed similar differences for different group.

Conclusions/Significance

Our results suggest that TST conversion is associated with early increases in IFN γ and IL-10 responses and precedes latency by several months post exposure.     

Breadth of Neutralizing Antibody Response to Human Immunodeficiency Virus Type 1 Is Affected by Factors Early in Infection but Does Not Influence Disease Progression

The determinants of a broad neutralizing antibody (NAb) response and its effect on human immunodeficiency virus type 1 (HIV-1) disease progression are not well defined, partly because most prior studies of a broad NAb response were cross-sectional. We examined correlates of NAb response breadth among 70 HIV-infected, antiretroviral-naïve Kenyan women from a longitudinal seroincident cohort. NAb response breadth was measured 5 years after infection against five subtype A viruses and one subtype B virus. Greater NAb response breadth was associated with a higher viral load set point and greater HIV-1 env diversity early in infection. However, greater NAb response breadth was not associated with a delayed time to a CD4⁺ T-cell count of <200, antiretroviral therapy, or death. Thus, a broad NAb response results from a high level of antigenic stimulation early in infection, which likely accounts for prior observations that greater NAb response breadth is associated with a higher viral load later in infection.Some human immunodeficiency virus (HIV)-infected individuals develop broad neutralizing antibody (NAb) responses, but the factors that lead to NAb response breadth remain elusive. Several cross-sectional studies have found that individuals with greater NAb response breadth have higher contemporaneous viral loads, suggesting that the presence of a greater amount of viral antigen may promote a greater NAb response breadth (9, 10, 25, 30, 32). However, because viral load and NAb response breadth were measured at the same time after HIV type 1 (HIV-1) acquisition in prior studies, it is difficult to discern cause and effect. There is also evidence that NAbs adapt in response to the evolving HIV-1 population throughout infection (11, 29, 35), which may contribute to a greater overall response breadth. Together, these studies support a model in which a greater NAb response breadth is driven by a higher level of antigenic stimulation, in terms of both the absolute level of virus and viral diversity. Confirmation of this model requires an assessment of the temporal relationship of viral load, HIV-1 diversity, and NAb response breadth.In addition to uncertainty regarding the determinants of NAb response breadth, the consequences of a broad NAb response for HIV-1 disease progression remains controversial. Broad NAb responses have been found in long-term nonprogressors (LTNPs) in some studies, suggesting that NAbs may contribute to control of infection in these individuals (6-8, 22, 27, 37). Other studies have found no evidence for NAb control in LTNPs (1, 2, 14, 18), including studies in which NAb response breadth was lower in LTNPs (10) or elite controllers (15, 25) than in viremic individuals. A detailed analysis of NAb response breadth versus clinical outcome has not yet been conducted, particularly for individuals with typical HIV-1 disease progression.To investigate the determinants and consequences of NAb response breadth in HIV-1 infection, we examined NAb responses in women in a seroincident cohort in Mombasa, Kenya, that began in 1993 (19-21). For each woman, the time of infection was defined by both HIV-1 serology and RNA testing (17). Women who had a banked plasma sample ∼5 years after the estimated time of HIV-1 infection were included in this study. This time period was chosen to maximize the chances for the NAb response to broaden while generally testing prior to the beginning of clinical immunodeficiency. We only included samples prior to the initiation of antiretroviral therapy (ART), which in this cohort began in March 2004, according to the WHO and Kenyan National guidelines. Plasma samples meeting these criteria were identified from 70 women and came from a median of 5.0 (range, 4.5 to 6.8) years postinfection (ypi). This subset of women was representative of the entire cohort in terms of their behavioral, clinical, and demographic characteristics (data not shown).HIV-1 subtype A accounts for most of the infections in this cohort (28), including 72% of the 53 women in this study for whom env subtype information was available (Fig. ​(Fig.1).1). Therefore, to test neutralization of viruses relevant to women in this population, we measured NAb response breadth against a panel of five recently transmitted subtype A viruses from other individuals in this cohort, which represented a spectrum of neutralization sensitivities (4). We also included one commonly studied, easy-to-neutralize subtype B virus (SF162) for comparison to other studies. The TZM-bl neutralization assay, using pseudoviruses prepared with these six envelope variants and TZM-bl indicator cells, was performed as described previously (4, 36). The median inhibitory concentration (IC₅₀) was defined as the reciprocal dilution of plasma that resulted in 50% inhibition. Figure ​Figure11 shows the IC₅₀ for each plasma-virus pair, averaged across three independent experiments that included duplicate testing of each pair.Open in a separate windowFIG. 1.Summary of the IC₅₀s and NAb response breadth scores of 70 plasma samples. The first column indicates the subject identifier of each plasma sample, and the next three columns indicate the env V1 to V5 subtype (available for 53/70 women), the set point viral load (available for 64 women), and the viral load at ∼5 ypi, when the NAb response breadth was measured. Data not available are indicated by a period. Each subsequent column shows the results with one panel virus (indicated at the top of the column). Results are the average of three experiments in which each plasma-virus pair was tested in duplicate. In the case of Q769 and Q259, two closely related viruses from the same individual were used in one (Q769.h5, Q259.d217) and two (Q769.b9, Q259.d226) of the three experiments. The IC₅₀ for each plasma-virus pair is the reciprocal dilution of plasma that led to a 50% reduction in infectivity, averaged across the three experiments. IC₅₀s are shown in gray scale to represent increasing neutralization sensitivity, with white for values of <100, light gray for values of >101 and <1,000, and dark gray for values of >1,001. Plasma-virus pairs in which 50% neutralization was not detected at the highest plasma dilution (1:50) are indicated by a pair of dashes. The NAb response breadth score for each plasma sample was calculated as follows. For each experiment, the median IC₅₀ for each virus (across all 70 plasma samples) was determined. Plasma samples were assigned a score of 1 for every virus against which their IC₅₀ was greater than the median IC₅₀, and the score was summed across all six viruses. The NAb response breadth scores that are shown here (and which were used for analysis) were calculated by taking the average response breadth score across the three independent experiments; they were not calculated from the average IC₅₀s shown.In general, we found that the viruses that had been easily neutralized in prior screening with pooled plasma, {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1 and Q168b23 (4), were the most readily neutralized by individual plasma samples from women in this study (Fig. ​(Fig.1).1). Of the 70 plasma samples tested, 68 (97%) showed detectable neutralization activity (IC₅₀, >50) against {"type":"entrez-protein","attrs":{"text":"Q461d1","term_id":"123852094","term_text":"Q461D1"}}Q461d1 and 60 (86%) showed activity against Q168b23. Most (76%) of the plasma samples also neutralized variant Q842d16 at detectable levels, although generally with lower IC₅₀s. By contrast, only about half of the plasma samples neutralized envelope variants Q769b9 and {"type":"entrez-protein","attrs":{"text":"Q259d2","term_id":"122195760","term_text":"Q259D2"}}Q259d2.26 (51% and 46%, respectively). Almost all (93%) of the plasma samples neutralized SF162.Given the different neutralization sensitivities of these viruses, we quantified the NAb responses in these individuals by using a previously described NAb response breadth score that takes into consideration the neutralization sensitivity of each virus (5). Briefly, the NAb response breadth score represents the number of viruses (out of six) that a given plasma sample neutralized at an IC₅₀ that was higher than the median IC₅₀ for that virus (across all 70 plasma samples). The response breadth score was calculated independently for each of three experiments, and the average scores are listed in Fig. ​Fig.1.1. Among all of the individuals, the median response breadth score was 2 and the response breadth scores ranged from 0 to 5.3. A potential limitation of this approach is that response breadth was calculated by using a relatively small number of viruses. However, we found that NAb response breadth measured against this 6-virus panel was highly correlated with the NAb response breadth measured against an expanded 17-virus panel (including these 6 viruses plus an additional 11 viruses representing subtypes A, C, D, A/D, and B; J. Overbaugh et al., unpublished data), for a subset of 29 women whose plasma samples were tested against the expanded panel (Spearman''s rho = 0.62, P < 0.001). Furthermore, the NAb response breadth score measured against this six-virus panel was highly correlated with NAb potency (Spearman''s rho = 0.81, P < 0.001), a measure we have used in prior studies that takes into consideration the magnitude of the IC₅₀ for each plasma-virus pair (5). These findings suggest that the NAb response breadth score measured against the six-virus panel is representative of the overall NAb response breadth.We investigated whether NAb response breadth was associated with the contemporaneous plasma viral load, which was measured at the same time as NAb response breadth (4.5 to 6.8 ypi). Viral loads ranged from 1.7 to 6.7 log₁₀ copies/ml among all of the individuals, with a median of 4.7 log₁₀ copies/ml. As shown in Fig. ​Fig.2a,2a, individuals with higher viral loads had greater NAb response breadth (Spearman''s rho = 0.31, P = 0.009), consistent with prior studies (9, 10, 30, 32). A similar relationship was observed between viral load set point and NAb potency, a measure that takes into account the magnitude of neutralization (data not shown). There was no association between NAb response breadth and CD4⁺ T-cell count (Spearman''s rho = −0.15, P = 0.2) among the 64 women with contemporaneous CD4⁺ T-cell counts available.Open in a separate windowFIG. 2.Associations between NAb response breadth and viral load. In each plot, the NAb response breadth score is indicated on the y axis and the contemporaneous (∼5 ypi) viral load (a) or viral load set point (b) is indicated on the x axis. Each point represents one individual. The results of Spearman correlation analysis are shown above the plots.To further assess whether the viral load may drive NAb response breadth, we examined the relationship between the viral load set point and NAb response breadth. For each individual, the viral load set point was defined as the first available viral load measurement 4 to 24 months after infection (16), and this ranged from 2.1 to 6.2 log₁₀ copies/ml (median, 4.6 log₁₀ copies/ml) among the 64 individuals for whom this measurement was available. As shown in Fig. ​Fig.2b,2b, individuals with higher viral load set points had greater NAb response breadth at ∼5 ypi (Spearman''s rho = 0.35, P = 0.005). The viral load set point was also highly correlated with the viral load measured at ∼5 ypi (Spearman''s rho = 0.42, P = 0.001). Therefore, we investigated whether the relationship between NAb response breadth and the contemporaneous (∼5 ypi) viral load could be explained by the viral load set point. In multivariate linear regression analysis, NAb response breadth was significantly associated with the viral load set point (coefficient of variation = 0.55, P = 0.02) but not with the contemporaneous viral load (coefficient of variation = 0.25, P = 0.3). Thus, the relationship between the contemporaneous viral load and NAb response breadth appeared to be driven by the viral load set point, with each 1-log increase in the viral load set point associated with an increase in the response breadth score of 0.55.Given this association between the viral load set point and NAb response breadth, we wondered whether another factor in early infection—HIV-1 sequence diversity—might influence the development of NAb response breadth. Proviral HIV-1 sequences were available from 26 individuals and had been sampled a median of 87 (range, 17 to 299) days postinfection. For each individual, gag and env V1 to V5 diversity was calculated from a median of seven single-copy sequences per gene as described previously (26). Across all 26 individuals, the median env diversity was 0.28% (range, 0 to 4.0%) and the median gag diversity was 0.19% (range, 0 to 1.28%). Individuals with greater env diversity early in infection had greater NAb response breadth at ∼5 ypi (Spearman''s rho = 0.51, P = 0.008). However, there was no association between early gag diversity and NAb response breadth (Spearman''s rho = 0.10, P = 0.6). Although both early env diversity and the viral load set point were associated with NAb response breadth, there was no association between these factors among the women in this study (Spearman''s rho = 0.21, P = 0.3). However, in a larger study of 156 women in this cohort, women with greater early env heterogeneity (as measured by heteroduplex mobility assay) had higher viral load set points (31). Further work is needed to clarify whether early env diversity and the viral load set point are independent determinants of NAb response breadth or whether early env diversity may drive both the viral load and NAb response breadth.Because the viral load set point and early env diversity have also been shown to be associated with HIV-1 disease progression in this cohort (17, 31), we explored the relationship of NAb response breadth, the viral load set point, and disease progression. We performed Cox proportional hazard analysis by using a composite survival outcome of time to the first occurrence of a CD4⁺ T-cell count of <200, ART initiation, or death. Among all 70 women, 45 reached this composite outcome over a median of 6.8 years of follow-up after HIV-1 infection (range, 1.2 to 14.2 years). In univariate analysis, a greater NAb response breadth was associated with an increased risk of HIV-1 disease progression (Table ​(Table1,1, hazard ratio [HR], 1.27 per unit increase in breadth, P = 0.03). However, this association was attenuated, and no longer statistically significant, in a multivariate analysis adjusting for the viral load set point (HR = 1.06, P = 0.6). In this multivariate model, a higher viral load set point was associated with a greater risk of HIV-1 disease progression (HR = 2.02, P = 0.003), as expected. In a second multivariate analysis considering only those outcome events that occurred after NAb response measurement (n = 25 events among 50 women), there was an association between NAb response breadth and HIV-1 disease outcomes (HR = 1.39, P = 0.03) but again this did not persist after adjustment for the viral load (HR = 1.17, P = 0.4). Thus, we found no evidence that NAb response breadth affected HIV-1 disease progression independently of the viral load set point.

TABLE 1.

Association between NAb response breadth and risk of HIV-1 disease progression^a

The FYVE Domain of Smad Anchor for Receptor Activation (SARA) Is Required to Prevent Skin Carcinogenesis,but Not in Mouse Development

Smad Anchor for Receptor Activation (SARA) has been reported as a critical role in TGF-β signal transduction by recruiting non-activated Smad2/3 to the TGF-β receptor and ensuring appropriate subcellular localization of the activated receptor-bound complex. However, controversies still exist in previous reports. In this study, we describe the expression of two SARA isoforms, SARA₁ and SARA₂, in mice and report the generation and characterization of SARA mutant mice with FYVE domain deletion. SARA mutant mice developed normally and showed no gross abnormalities. Further examination showed that the TGF-β signaling pathway was indeed altered in SARA mutant mice, with the downregulation of Smad2 protein expression. The decreasing expression of Smad2 was caused by enhancing Smurf2-mediated proteasome degradation pathway. However, the internalization of TGF-β receptors into the early endosome was not affected in SARA mutant mouse embryonic fibroblasts (MEFs). Moreover, the downregulation of Smad2 in SARA mutant MEFs was not sufficient to disrupt the diverse cellular biological functions of TGF-β signaling, including growth inhibition, apoptosis, senescence, and the epithelial-to-mesenchymal transition. Our results indicate that SARA is not involved in the activation process of TGF-β signal transduction. Using a two-stage skin chemical carcinogenesis assay, we found that the loss of SARA promoted skin tumor formation and malignant progression. Our data suggest a protective role of SARA in skin carcinogenesis.     

Plasma Nesfatin-1 Is Not Affected by Long-Term Food Restriction and Does Not Predict Rematuration among Iteroparous Female Rainbow Trout (Oncorhynchus mykiss)

The metabolic peptide hormone nesfatin-1 has been linked to the reproductive axis in fishes. The purpose of this study was to determine how energy availability after spawning affects plasma levels of nesfatin-1, the metabolic peptide hormone ghrelin, and sex steroid hormones in rematuring female rainbow trout (Oncorhynchus mykiss). To limit reproductive maturation, a group of female trout was food-restricted after spawning and compared with a control group that was fed a standard broodstock ration. The experiment was conducted twice, once using two-year-old trout (second-time spawners) and once using three-year-old trout (third-time spawners). During monthly sampling, blood was collected from all fish, and a subset of fish from each treatment was sacrificed for pituitaries. Pituitary follicle-stimulating hormone-beta (fsh-β) mRNA expression was analyzed with q-RT-PCR; plasma hormone levels were quantified by radioimmunoassay (17β-estradiol and ghrelin) and enzyme-linked immunosorbent assay (11-keto-testosterone and nesfatin-1). Although plasma nesfatin-1 levels increased significantly in the months immediately after spawning within both feeding treatments, plasma nesfatin-1 did not differ significantly between the two treatments at any point. Similarly, plasma ghrelin levels did not differ significantly between the two treatments at any point. Food restriction arrested ovarian development by 15–20 weeks after spawning, shown by significantly lower plasma E2 levels among restricted-ration fish. Pituitary fsh-β mRNA levels were higher among control-ration fish than restricted-ration fish starting at 20 weeks, but did not differ significantly between treatment groups until 30 weeks after spawning. Within both treatment groups, plasma 11-KT was elevated immediately after spawning and rapidly decreased to and persisted at low levels; starting between 20 and 25 weeks after spawning, plasma 11-KT was higher among control-ration fish than restricted-ration fish. The results from these experiments do not provide support for plasma nesfatin-1 as a signal for the initiation of reproductive development in rematuring female rainbow trout.     

B Lymphocyte Stimulator (BLyS) Is Expressed in Human Adipocytes In Vivo and Is Related to Obesity but Not to Insulin Resistance

Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS) is increased in human obesity. In contrast to several pro-inflammatory factors, we found the source of BLyS in human adipose tissue to be the adipocytes rather than immune cells. In grade 3 obese human subjects, expression of BLyS in vivo in adipose tissue is significantly increased (p<0.001). Furthermore, BLyS serum levels are elevated in grade 3 human obesity (862.5+222.0 pg/ml vs. 543.7+60.7 pg/ml in lean controls, p<0.001) and are positively correlated to the BMI (r = 0.43, p<0.0002). In the present study, bariatric surgery significantly altered serum BLyS concentrations. In contrast, weight loss due to a very-low-calorie-formula-diet (800 kcal/d) had no such effect. To examine metabolic activity of BLyS, in a translational research approach, insulin sensitivity was measured in human subjects in vivo before and after treatment with the human recombinant anti-BLyS antibody belimumab. Since BLyS is known to promote B-cell proliferation and immunoglobulin secretion, the present data suggest that adipocytes of grade 3 obese human subjects are able to activate the adaptive immune system, suggesting that in metabolic inflammation in humans both, innate and adaptive immunity, are of pathophysiological relevance.     

Immune Investment Is Explained by Sexual Selection and Pace-of-Life,but Not Longevity in Parrots (Psittaciformes)

Investment in current reproduction should come at the expense of traits promoting future reproduction, such as immunity and longevity. To date, comparative studies of pace-of-life traits have provided some support for this, with slower paced species having greater immune function. Another means of investment in current reproduction is through secondary sexual characters (SSC). Investment in SSC''s is considered costly, both in terms of immunity and longevity, with greater costs being borne by species with more elaborate traits. Yet within species, females prefer more ornate males and those males are typically immunologically superior. Because of this, predictions about the relationship between immunity and SSC''s across species are not clear. If traits are costly, brighter species should have reduced immune function, but the opposite is true if SSC''s arise from selection for more immunocompetent individuals. My approach was to investigate immune investment in relation to SSC''s, pace-of-life and longevity while considering potentially confounding ecological factors. To do so I assessed leukocyte counts from in a novel group, the Psittaciformes. Investment in SSC''s best explained investment in immunity: species with brighter plumage had higher leukocyte counts and those with a greater degree of sexual dichromatism had fewer. Ecological variables and pace-of-life models tended to be poor predictors of immune investment. However, shorter incubation periods were associated with lower leukocyte counts supporting the notion that species with a fast pace-of-life invest less in immunity. These results suggest that investment in reproduction in terms of fast pace-of-life and sexual dichromatism results in reduced immunity; however, investment in plumage colour per se does not impose a cost on immunity across species.     

The Early Innate Response of Chickens to Salmonella enterica Is Dependent on the Presence of O-Antigen but Not on Serovar Classification

Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonella serovar Enteritidis or Typhimurium which results in a decrease in S. Enteritidis and S. Typhimurium but may allow other Salmonella serovars to fill an empty ecological niche. In this study we were therefore interested in the early interactions of chicken immune system with S. Infantis compared to S. Enteritidis and S. Typhimurium, and a role of O-antigen in these interactions. To reach this aim, we orally infected newly hatched chickens with 7 wild type strains of Salmonella serovars Enteritidis, Typhimurium and Infantis as well as with their rfaL mutants and characterized the early Salmonella-chicken interactions. Inflammation was characterized in the cecum 4 days post-infection by measuring expression of 43 different genes. All wild type strains stimulated a greater inflammatory response than any of the rfaL mutants. However, there were large differences in chicken responses to different wild type strains not reflecting their serovar classification. The initial interaction between newly-hatched chickens and Salmonella was found to be dependent on the presence of O-antigen but not on its structure, i.e. not on serovar classification. In addition, we observed that the expression of calbindin or aquaporin 8 in the cecum did not change if inflammatory gene expression remained within a 10 fold fluctuation, indicating the buffering capacity of the cecum, preserving normal gut functions even in the presence of minor inflammatory stimuli.     

Learning to Obtain Reward,but Not Avoid Punishment,Is Affected by Presence of PTSD Symptoms in Male Veterans: Empirical Data and Computational Model

Post-traumatic stress disorder (PTSD) symptoms include behavioral avoidance which is acquired and tends to increase with time. This avoidance may represent a general learning bias; indeed, individuals with PTSD are often faster than controls on acquiring conditioned responses based on physiologically-aversive feedback. However, it is not clear whether this learning bias extends to cognitive feedback, or to learning from both reward and punishment. Here, male veterans with self-reported current, severe PTSD symptoms (PTSS group) or with few or no PTSD symptoms (control group) completed a probabilistic classification task that included both reward-based and punishment-based trials, where feedback could take the form of reward, punishment, or an ambiguous “no-feedback” outcome that could signal either successful avoidance of punishment or failure to obtain reward. The PTSS group outperformed the control group in total points obtained; the PTSS group specifically performed better than the control group on reward-based trials, with no difference on punishment-based trials. To better understand possible mechanisms underlying observed performance, we used a reinforcement learning model of the task, and applied maximum likelihood estimation techniques to derive estimated parameters describing individual participants’ behavior. Estimations of the reinforcement value of the no-feedback outcome were significantly greater in the control group than the PTSS group, suggesting that the control group was more likely to value this outcome as positively reinforcing (i.e., signaling successful avoidance of punishment). This is consistent with the control group’s generally poorer performance on reward trials, where reward feedback was to be obtained in preference to the no-feedback outcome. Differences in the interpretation of ambiguous feedback may contribute to the facilitated reinforcement learning often observed in PTSD patients, and may in turn provide new insight into how pathological behaviors are acquired and maintained in PTSD.     

Modulation of Dehydration Tolerance in Soybean Seedlings (Dehydrin Mat1 Is Induced by Dehydration but Not by Abscisic Acid)

Germinated soybean (Glycine max L. cv Williams 82) seedlings subjected to rapid dehydration begin to lose the ability to recover when the relative water content of the plant decreases below 60%. The expanded cells of the hypocotyl appear more susceptible to dehydration-induced damage than do cells in the hypocotyl zone of cell growth. Pretreatment of seedlings prior to rapid dehydration with nonlethal water deficit or exogenous abscisic acid (ABA) shifts this viability threshold to progressively lower relative water contents, indicating the acquisition of increased dehydration tolerance. Increased tolerance is associated with osmotic adjustment in the hypocotyl zone of cell growth and with increases in soybean dehydrin Mat1 mRNA levels. The accumulation of Mat1 mRNA is dehydration dependent but insensitive to ABA. Induction of Mat1 mRNA accumulation by dehydration but not by ABA makes it an unusual member of the dehydrin family.     

The ars Detoxification System Is Advantageous but Not Required for As(V) Respiration by the Genetically Tractable Shewanella Species Strain ANA-3

Arsenate [As(V); HAsO₄²⁻] respiration by bacteria is poorly understood at the molecular level largely due to a paucity of genetically tractable organisms with this metabolic capability. We report here the isolation of a new As(V)-respiring strain (ANA-3) that is phylogenetically related to members of the genus Shewanella and that also provides a useful model system with which to explore the molecular basis of As(V) respiration. This gram-negative strain stoichiometrically couples the oxidation of lactate to acetate with the reduction of As(V) to arsenite [As(III); HAsO₂]. The generation time and lactate molar growth yield (Y_lactate) are 2.8 h and 10.0 g of cells mol of lactate⁻¹, respectively, when it is grown anaerobically on lactate and As(V). ANA-3 uses a wide variety of terminal electron acceptors, including oxygen, soluble ferric iron, oxides of iron and manganese, nitrate, fumarate, the humic acid functional analog 2,6-anthraquinone disulfonate, and thiosulfate. ANA-3 also reduces As(V) to As(III) in the presence of oxygen and resists high concentrations of As(III) (up to 10 mM) when grown under either aerobic or anaerobic conditions. ANA-3 possesses an ars operon (arsDABC) that allows it to resist high levels of As(III); this operon also confers resistance to the As-sensitive strains Shewanella oneidensis MR-1 and Escherichia coli AW3110. When the gene encoding the As(III) efflux pump, arsB, is inactivated in ANA-3 by a polar mutation that also eliminates the expression of arsC, which encodes an As(V) reductase, the resulting As(III)-sensitive strain still respires As(V); however, the generation time and the Y_lactate value are two- and threefold lower, respectively, than those of the wild type. These results suggest that ArsB and ArsC may be useful for As(V)-respiring bacteria in environments where As concentrations are high, but that neither is required for respiration.     

Olomoucine II,but Not Purvalanol A,Is Transported by Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1)

Purine cyclin-dependent kinase inhibitors have been recognized as promising candidates for the treatment of various cancers; nevertheless, data regarding interaction of these substances with drug efflux transporters is still lacking. Recently, we have demonstrated inhibition of breast cancer resistance protein (ABCG2) by olomoucine II and purvalanol A and shown that these compounds are able to synergistically potentiate the antiproliferative effect of mitoxantrone, an ABCG2 substrate. In this follow up study, we investigated whether olomoucine II and purvalanol A are transported by ABCG2 and ABCB1 (P-glycoprotein). Using monolayers of MDCKII cells stably expressing human ABCB1 or ABCG2, we demonstrated that olomoucine II, but not purvalanol A, is a dual substrate of both ABCG2 and ABCB1. We, therefore, assume that pharmacokinetics of olomoucine II will be affected by both ABCB1 and ABCG2 transport proteins, which might potentially result in limited accumulation of the compound in tumor tissues or lead to drug-drug interactions. Pharmacokinetic behavior of purvalanol A, on the other hand, does not seem to be affected by either ABCG2 or ABCB1, theoretically favoring this drug in the potential treatment of efflux transporter-based multidrug resistant tumors. In addition, we observed intensive sulfatation of olomoucine II in MDCKII cell lines with subsequent active efflux of the metabolite out of the cells. Therefore, care should be taken when performing pharmacokinetic studies in MDCKII cells, especially if radiolabeled substrates are used; the generated sulfated conjugate may largely contaminate pharmacokinetic analysis and result in misleading interpretation. With regard to chemical structures of olomoucine II and purvalanol A, our data emphasize that even drugs with remarkable structure similarity may show different pharmacokinetic behavior such as interactions with ABC transporters or biotransformation enzymes.     

Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G₂ checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.     

The Time to Most Recent Common Ancestor Does Not (Usually) Approximate the Date of Divergence

With the advent of more sophisticated models and increase in computational power, an ever-growing amount of information can be extracted from DNA sequence data. In particular, recent advances have allowed researchers to estimate the date of historical events for a group of interest including time to most recent common ancestor (TMRCA), dates of specific nodes in a phylogeny, and the date of divergence or speciation date. Here I use coalescent simulations and re-analyze an empirical dataset to illustrate the importance of taxon sampling, in particular, on correctly estimating such dates. I show that TMRCA of representatives of a single taxon is often not the same as divergence date due to issues such as incomplete lineage sorting. Of critical importance is when estimating divergence or speciation dates a representative from a different taxonomic lineage must be included in the analysis. Without considering these issues, studies may incorrectly estimate the times at which historical events occurred, which has profound impacts within both research and applied (e.g., those related to public health) settings.     

Evidence for Activation of the Oxidative Pentose Phosphate Pathway during Photosynthetic Assimilation of NO(3) but Not NH(4) by a Green Alga

Addition of NO₃⁻ to N-limited Selenastrum minutum during photosynthesis resulted in an immediate drop in the NADPH/NADP ratio and a slower increase of the NADH/NAD ratio. These changes were accompanied by a rapid decrease in glucose-6-phosphate and increase in 6-phosphogluconate, indicating activation of glucose-6-phosphate dehydrogenase and a role for the oxidation pentose phosphate pathway during photosynthetic NO₃⁻ assimilation. In contrast, the short-term changes in pyridine nucleotides and metabolites during photosynthetic assimilation of NH₄⁺ were not consistent with a stimulation of the oxidative pentose phosphate pathway.     

Behavioral Inhibition in Rhesus Monkeys (Macaca mulatta) Is Related to the Airways Response,but Not Immune Measures,Commonly Associated with Asthma

Behavioral inhibition reflects a disposition to react warily to novel situations, and has been associated with atopic diseases such as asthma. Retrospective work established the relationship between behavioral inhibition in rhesus monkeys (Macaca mulatta) and airway hyperresponsiveness, but not atopy, and the suggestion was made that behavioral inhibition might index components of asthma that are not immune-related. In the present study, we prospectively examined the relationship between behavioral inhibition and airway hyperresponsiveness, and whether hormonal and immune measures often associated with asthma were associated with behavioral inhibition and/or airway hyperresponsiveness. In a sample of 49 yearling rhesus monkeys (mean = 1.25 years, n = 24 behaviorally inhibited animals), we measured in vitro cytokine levels (IL-4, IL-10, IL-12, IFN-γ) in response to stimulation, as well as peripheral blood cell percentages, cortisol levels, and percentage of regulatory T-cells (CD3+CD4+CD25+FOXP3+). Airway reactivity was assessed using an inhaled methacholine challenge. Bronchoalveolar lavage was performed and the proportion of immune cells was determined. Behaviorally inhibited monkeys had airway hyperresponsiveness as indicated by the methacholine challenge (p = 0.031), confirming our earlier retrospective result. Airway hyperresponsiveness was also associated with lower lymphocyte percentages in lavage fluid and marginally lower plasma cortisol concentrations. However, none of the tested measures was significantly related to both behavioral inhibition and airway hyperresponsiveness, and so could not mediate their relationship. Airway hyperresponsiveness is common to atopic and non-atopic asthma and behavioral inhibition has been related to altered autonomic activity in other studies. Our results suggest that behavioral inhibition might index an autonomically mediated reactive airway phenotype, and that a variety of stimuli (including inflammation within lung tissue that is not specifically associated with behavioral inhibition) may trigger the airways response.     

Formal Comment to Pettengill: The Time to Most Recent Common Ancestor Does Not (Usually) Approximate the Date of Divergence

In 2013 Zhou et al. concluded that Salmonella enterica serovar Agona represents a genetically monomorphic lineage of recent ancestry, whose most recent common ancestor existed in 1932, or earlier. The Abstract stated ‘Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s.’ These conclusions have now been criticized by Pettengill, who claims that the evolutionary models used to date Agona may not have been appropriate, the dating estimates were inaccurate, and the age of emergence of Agona should have been qualified by an upper limit reflecting the date of its divergence from an outgroup, serovar Soerenga. We dispute these claims. Firstly, Pettengill’s analysis of Agona is not justifiable on technical grounds. Secondly, an upper limit for divergence from an outgroup would only be meaningful if the outgroup were closely related to Agona, but close relatives of Agona are yet to be identified. Thirdly, it is not possible to reliably date the time of divergence between Agona and Soerenga. We conclude that Pettengill’s criticism is comparable to a tempest in a teapot.     

Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization

Nervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively.