Structure-function analysis of the C-terminal domain of CNM67, a core component of the Saccharomyces cerevisiae spindle pole body

The spindle pole body of the budding yeast Saccharomyces cerevisiae has served as a model system for understanding microtubule organizing centers, yet very little is known about the molecular structure of its components. We report here the structure of the C-terminal domain of the core component Cnm67 at 2.3 Å resolution. The structure determination was aided by a novel approach to crystallization of proteins containing coiled-coils that utilizes globular domains to stabilize the coiled-coils. This enhances their solubility in Escherichia coli and improves their crystallization. The Cnm67 C-terminal domain (residues Asn-429—Lys-581) exhibits a previously unseen dimeric, interdigitated, all α-helical fold. In vivo studies demonstrate that this domain alone is able to localize to the spindle pole body. In addition, the structure reveals a large functionally indispensable positively charged surface patch that is implicated in spindle pole body localization. Finally, the C-terminal eight residues are disordered but are critical for protein folding and structural stability.     

Insights Gained From 50 Years of Studying the Evolution of Self-Compatibility in <Emphasis Type="Italic">Leavenworthia</Emphasis> (Brassicaceae)

The shift from outcrossing to selfing is one of the most common evolutionary trends in plants, and there is intense interest in why this is so. The genus Leavenworthia has been the focus of research on this question for half of a century, with particular attention paid to the evolution of self-compatibility from self-incompatibility. In this review, we discuss the last 50 years of research concerning this evolutionary transition in Leavenworthia. Selfing appears to have evolved independently at minimum three times within this genus of eight species. Work on the ecological basis of mating system evolution in Leavenworthia has clarified that selection among individuals is likely a major force behind the recurrent evolution of selfing. Although inadequate pollination is appreciated as a factor favoring selfing, definitive ecological mechanisms that act to favor selfing are still not known and future work on the efficacy of pollinating bees and the effects of climate change is needed. Recent research has likely identified the SRK ortholog at the S-locus controlling self-incompatibility in Leavenworthia alabamica. Analyses of S-locus variation have revealed substantial S-allele diversity in outcrossing populations, with the recurrent fixation of mutations at the S-locus permitting the parallel evolution of selfing in this species. Although we appreciate some of the factors that may explain the evolution of selfing in this group, there is less known about the mechanisms underlying the widespread maintenance of outcrossing at the population and species levels. Studies in Leavenworthia have revealed that genetic diversity is lost over the long-term within selfing populations and leads to elevated population subdivision, but work is needed to determine why these genetic consequences of selfing cause lineages to become evolutionary dead ends.     

On unsatisfied hydrogen bonds in the N-terminal subdomain of villin headpiece

Villin headpiece is a small autonomously folding protein that has emerged as a model system for understanding the fundamental tenets governing protein folding. In this communication, we employ NMR and X-ray crystallography to characterize a point mutant, H41F, which retains actin-binding activity, is more thermostable but, interestingly, does not exhibit the partially folded intermediate observed of either wild-type or other similar point mutants.     

Folivory, frugivory, and postcanine size in the cercopithecoidea revisited

At a given body mass, folivorous colobines have smaller postcanine teeth than frugivorous cercopithecines. This distinction is a notable exception to the general tendency for folivorous primates to have relatively larger postcanine tooth rows than closely related frugivores. The reason for this anomalous pattern is unclear, but one potential explanation is that the difference in facial size between these two subfamilies confounds the comparison-i.e., it may be that the large postcanine teeth of cercopithecines are a consequence of their large faces. The goal of this study was to test this hypothesis. Phylogenetic comparative methods were used to examine the relationships among postcanine area, facial size, and body mass in 29 anthropoid primates, including eight colobines and eight cercopithecines. Results indicate that there is a strong and highly significant partial correlation between postcanine area and facial size when body mass is held constant, which supports the hypothesis that facial size has an important influence on postcanine size. Moreover, colobines have larger postcanine teeth relative to facial size than cercopithecines. Surprisingly, when facial size is held constant, the partial correlation between postcanine area and body mass is weak and nonsignificant. These results suggest that facial size may be more appropriate than body mass for size-adjusting postcanine measurements in some contexts. A phylogenetic comparative test of the association between diet and relative postcanine size (scaled using facial size) confirms that folivorous anthropoids are characterized by relatively large postcanine teeth in comparison to closely related nonfolivores.     

Demography, pollination, and Baker's Law

Baker's Law states that selfing should commonly be selected during dispersal because bottlenecks during colonization limit the availability of mates. Although this truism has broad intuitive appeal, a recent body of theory (Cheptou and Massol 2009; Massol and Cheptou 2011) casts doubt on whether adaptation favors both selfing and dispersal when both parameters are free to evolve. In these models, the joint evolution of dispersal and the selfing rate are considered in a metapopulation, with a spatially and temporally variable pollination environment. Under these conditions, adaptation favors one of two strategies: the "dispersal/outcrosser" syndrome and the "no dispersal/selfing" syndrome. These results appear to contradict the prediction of Baker's Law. These models clarify how variation in the pollination environment per se cannot generate an association between selfing and dispersal. That being said, demographic factors during dispersal episodes are likely to be important in generating patterns consistent with Baker's law. Determining whether Baker's law maintains its predictive utility requires determining whether seed banks, the perennial habit, multiple introductions, or the simultaneous arrival of many founders weaken selection for selfing during the bottleneck associated with a dispersal event. These issues highlight the many assumptions that are necessary for Baker's law to hold.     

Skeletal adaptation to intramedullary pressure-induced interstitial fluid flow is enhanced in mice subjected to targeted osteocyte ablation

Interstitial fluid flow (IFF) is a potent regulatory signal in bone. During mechanical loading, IFF is generated through two distinct mechanisms that result in spatially distinct flow profiles: poroelastic interactions within the lacunar-canalicular system, and intramedullary pressurization. While the former generates IFF primarily within the lacunar-canalicular network, the latter generates significant flow at the endosteal surface as well as within the tissue. This gives rise to the intriguing possibility that loading-induced IFF may differentially activate osteocytes or surface-residing cells depending on the generating mechanism, and that sensation of IFF generated via intramedullary pressurization may be mediated by a non-osteocytic bone cell population. To begin to explore this possibility, we used the Dmp1-HBEGF inducible osteocyte ablation mouse model and a microfluidic system for modulating intramedullary pressure (ImP) to assess whether structural adaptation to ImP-driven IFF is altered by partial osteocyte depletion. Canalicular convective velocities during pressurization were estimated through the use of fluorescence recovery after photobleaching and computational modeling. Following osteocyte ablation, transgenic mice exhibited severe losses in bone structure and altered responses to hindlimb suspension in a compartment-specific manner. In pressure-loaded limbs, transgenic mice displayed similar or significantly enhanced structural adaptation to Imp-driven IFF, particularly in the trabecular compartment, despite up to ～50% of trabecular lacunae being uninhabited following ablation. Interestingly, regression analysis revealed relative gains in bone structure in pressure-loaded limbs were correlated with reductions in bone structure in unpressurized control limbs, suggesting that adaptation to ImP-driven IFF was potentiated by increases in osteoclastic activity and/or reductions in osteoblastic activity incurred independently of pressure loading. Collectively, these studies indicate that structural adaptation to ImP-driven IFF can proceed unimpeded following a significant depletion in osteocytes, consistent with the potential existence of a non-osteocytic bone cell population that senses ImP-driven IFF independently and potentially parallel to osteocytic sensation of poroelasticity-derived IFF.     

Esophageal cancer metabolite biomarkers detected by LC-MS and NMR methods

Background

Esophageal adenocarcinoma (EAC) is a rarely curable disease and is rapidly rising worldwide in incidence. Barret''s esophagus (BE) and high-grade dysplasia (HGD) are considered major risk factors for invasive adenocarcinoma. In the current study, unbiased global metabolic profiling methods were applied to serum samples from patients with EAC, BE and HGD, and healthy individuals, in order to identify metabolite based biomarkers associated with the early stages of EAC with the goal of improving prognostication.

Methodology/Principal Findings

Serum metabolite profiles from patients with EAC (n = 67), BE (n = 3), HGD (n = 9) and healthy volunteers (n = 34) were obtained using high performance liquid chromatography-mass spectrometry (LC-MS) methods. Twelve metabolites differed significantly (p<0.05) between EAC patients and healthy controls. A partial least-squares discriminant analysis (PLS-DA) model had good accuracy with the area under the receiver operative characteristic curve (AUROC) of 0.82. However, when the results of LC-MS were combined with 8 metabolites detected by nuclear magnetic resonance (NMR) in a previous study, the combination of NMR and MS detected metabolites provided a much superior performance, with AUROC = 0.95. Further, mean values of 12 of these metabolites varied consistently from healthy controls to the high-risk individuals (BE and HGD patients) and EAC subjects. Altered metabolic pathways including a number of amino acid pathways and energy metabolism were identified based on altered levels of numerous metabolites.

Conclusions/Significance

Metabolic profiles derived from the combination of LC-MS and NMR methods readily distinguish EAC patients and potentially promise important routes to understanding the carcinogenesis and detecting the cancer. Differences in the metabolic profiles between high-risk individuals and the EAC indicate the possibility of identifying the patients at risk much earlier to the development of the cancer.     

Performance of the 2007 WHO Algorithm to Diagnose Smear-Negative Pulmonary Tuberculosis in a HIV Prevalent Setting

Background

The 2007 WHO algorithm for diagnosis of smear-negative pulmonary tuberculosis (PTB) including Mycobacterium tuberculosis (MTB) culture was evaluated in a HIV prevalent area of Kenya.

Methods

PTB smear-negative adult suspects were included in a prospective diagnostic study (2009–2011). In addition, program data (2008–2009) were retrospectively analysed. At the first consultation, clinical examination, chest X-ray, and sputum culture (Thin-Layer-Agar and Lowenstein-Jensen) were performed. Patients not started on TB treatment were clinically re-assessed after antibiotic course. The algorithm performance was calculated using culture as reference standard.

Results

380 patients were included prospectively and 406 analyzed retrospectively. Culture was positive for MTB in 17.5% (61/348) and 21.8% (72/330) of cases. Sensitivity of the clinical-radiological algorithm was 55.0% and 31.9% in the prospective study and the program data analysis, respectively. Specificity, positive and negative predictive values were 72.9%, 29.7% and 88.6% in the prospective study and 79.8%, 30.7% and 80.8% in the program data analysis. Performing culture increased the number of confirmed TB patients started on treatment by 43.3% in the prospective study and by 44.4% in the program data analysis. Median time to treatment of confirmed TB patients was 6 days in the prospective study and 27 days in the retrospective study. Inter-reader agreement for X-ray interpretation between the study clinician and a radiologist was low (Kappa coefficient = 0.11, 95%CI: 0.09–0.12). In a multivariate logistic analysis, past TB history, number of symptoms and signs at the clinical exam were independently associated with risk of overtreatment.

Conclusion

The clinical-radiological algorithm is suboptimal to diagnose smear-negative PTB. Culture increases significantly the proportion of confirmed TB cases started on treatment. Better access to rapid MTB culture and development of new diagnostic tests is necessary.     

The burden of trachoma in South Sudan: assessing the health losses from a condition of graded severity

Introduction

Trachoma is a disease that can lead to visual impairment and ultimately blindness. Previous estimates of health losses from trachoma using the Global Burden of Disease methodology have not, however, included the stage prior to visual impairment. We estimated the burden of all stages of trachoma in South Sudan and assessed the uncertainty associated with the severity and duration of stages of trachoma prior to full blindness.

Methods

The prevalence of trachoma with normal vision, low vision and blindness in the Republic of South Sudan has been estimated previously. These estimates were used to model the incidence and duration of the different stages employing DISMOD II. Different assumptions about disability weights and duration were used to estimate the Years Lived with Disability (YLD).

Results

We have estimated the total burden of trachoma in South Sudan to be between 136,562 and 163,695 YLD and trichiasis with normal vision contributes between 5% and 21% of the total depending on the disability weight applied. Women experience more of this burden than men. The sensitivity of the results to different assumptions about the disability weights is partly dependent upon the assumed duration of the different disease states.

Interpretation

A better understanding of the natural history of trachoma is critical for a more accurate burden estimate.     

Morbillivirus infection in live stranded, injured, trapped, and captive cetaceans in southeastern Queensland and northern New South Wales, Australia

We report serologic evidence of cetacean morbillivirus (CMV) infection in five of eight cetacean species found live stranded, injured, or trapped along the coast of southeastern Queensland and northern New South Wales, Australia between December 2005 and January 2011. Antibody to CMV was detected in 13 of 27 (48%) wild cetaceans sampled. Antibody prevalence was significantly higher in clinically diseased (69%) compared to nondiseased (18%) animals (P=0.018). There was high antibody prevalence (83%, n=6) in melon-headed whales (Peponocephala electra). Two of 13 (15%) captive cetaceans sampled between November 2005 and January 2011 had CMV antibodies and, as infection was unlikely to have occurred while in captivity, CMV infection appears to have been present in Australian wild cetaceans since at least 1985. These results indicate that morbillivirus infection is occurring without widespread cetacean mortality in this region. However, as the deaths of two immature Australian offshore bottlenose dolphins (Tursiops truncatus) were attributed to CMV infection, morbillivirus infection should be included in the differential diagnosis of disease in cetaceans in Australia. Captive cetacean populations may be prone to significant mortality as a result of CMV introduction, so strict quarantine procedures should be enforced when injured or stranded cetaceans are hospitalized and rehabilitated at Australian zoos and marine parks.