Correlations Between DCE MRI and Histopathological Parameters in Head and Neck Squamous Cell Carcinoma

BACKGROUND: Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) can characterize perfusion and vascularization of tissues. DCE MRI parameters can differentiate between malignant and benign lesions and predict tumor grading. The purpose of this study was to correlate DCE MRI findings and various histopathological parameters in head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: Sixteen patients with histologically proven HNSCC (11 cases primary tumors and in 5 patients with local tumor recurrence) were included in the study. DCE imaging was performed in all cases and the following parameters were estimated: K^trans, V_e, K_ep, and iAUC. The tumor proliferation index was estimated on Ki 67 antigen stained specimens. Microvessel density parameters (stained vessel area, total vessel area, number of vessels, and mean vessel diameter) were estimated on CD31 antigen stained specimens. Spearman''s non-parametric rank sum correlation coefficients were calculated between DCE and different histopathological parameters. RESULTS: The mean values of DCE perfusion parameters were as follows: K^trans 0.189 ± 0.056 min⁻¹, K_ep 0.390 ± 0.160 min⁻¹, V_e 0.548 ± 0.119%, and iAUC 22.40 ± 12.57. Significant correlations were observed between K_ep and stained vessel areas (r = 0.51, P = .041) and total vessel areas (r = 0.5118, P = .043); between V_e and mean vessel diameter (r = −0.59, P = .017). Cell count had a tendency to correlate with V_e (r = −0.48, P = .058). In an analysis of the primary HNSCC only, a significant inverse correlation between K^trans and KI 67 was identified (r = −0.62, P = .041). Our analysis showed significant correlations between DCE parameters and histopathological findings in HNSCC.Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) has been reported as a technique which is able to characterize perfusion and vascularization of tissues [1], [2]. It has been shown that DCE MRI can be helpful to differentiate between malignant and benign lesions [1]. For example, Yuan et al. reported that lung cancer had a larger volume transfer constant (K^trans) and a lower volume of the extravascular extracellular leakage space (V_e) in comparison to benign lesions [3]. Similar results were reported by Li et al. for breast lesions [4]. Furthermore, according to Cho et al., DCE MRI parameters can be used to distinguish prostatic cancer from benign changes [5]. Moreover, DCE MRI parameters can also predict tumor grading. As reported previously, K^trans correlated well with Gleason score in prostatic cancer [5], [6]. According to other reports, K^trans and V_e correlated with glioma grade [7], [8].DCE MRI parameters were also associated with prognosis in several malignancies [9], [10]. Koo et al. showed that breast cancers with higher K^trans or lower V_e had poor prognostic factors and were often of the triple-negative subtype [10].According to the literature, DCE MRI parameters can predict response to therapy in different tumors. For instance, some authors mentioned that low pretreatment K^trans in regional lymph node metastases in head and neck cancer was associated with a poor response to concurrent chemoradiation therapy [11].Furthermore, Andersen et al. showed that DCE MR parameters obtained prior to chemoradiotherapy predicted survival of patients with cervical cancer [12].Presumably, DCE MRI parameters may be based on tissue composition, such as cellularity and vascular density. However, in this regard there are contradictory data in the literature. While some studies identified significant correlations between DCE MRI and histopathological parameters, others did not [13], [14], [15], [16].The purpose of this study was to correlate DCE MRI findings and various histopathological parameters in head and neck squamous cell carcinoma (HNSCC).     

Gene Expression of the EGF System—a Prognostic Model in Non–Small Cell Lung Cancer Patients Without Activating EGFR Mutations

OBJECTIVES: Contradicting results have been demonstrated for the expression of the epidermal growth factor receptor (EGFR) as a prognostic marker in non–small cell lung cancer (NSCLC). The complexity of the EGF system with four interacting receptors and more than a dozen activating ligands is a likely explanation. The aim of this study is to demonstrate that the combined network of receptors and ligands from the EGF system is a prognostic marker. MATERIAL AND METHODS: Gene expression of the receptors EGFR, HER2, HER3, HER4, and the ligands AREG, HB-EGF, EPI, TGF-α, and EGF was measured by quantitative polymerase chain reaction in tumor samples from 100 NSCLC patients without EGFR activating mutations. Results were dichotomized into high or low levels of target expression. Coexpression of the ligands and receptors was observed, and a score was developed based on the summed effect of receptors and ligands. Akaike’s information criteria selected the optimal score. Results were correlated with age, sex, stage, histology, performance status, and overall survival. RESULTS: Patients were randomly split 1:1 to create test and validation cohorts. In multivariate analyses, the only individual prognostic marker was EPI (hazard ratio [HR] 0.38 [0.20-0.72], P = .003). The optimal score in the test cohort was validated as a marker of inferior survival in the validation cohort and by bootstrapping. Multivariate analysis confirmed the combined score as a prognostic marker of inferior survival (HR 3.75 [2.17-6.47], P < .00001). CONCLUSION: Our study has developed a model that takes the complexity of the EGF system into account and shows that this model is a strong prognostic marker in NSCLC patients.Despite advances in the treatment, non–small cell lung cancer (NSCLC) remains the leading cause of cancer-related death in the world [1]. In particular, the overall prognosis is poor for the metastatic stages, with a median overall survival (OS) of only 8 to 10 months. Even in the early nonmetastatic stages, the 5-year survival rate is as low as 50% [2], [3]. Prognostic markers are needed to stratify patients with different risk outcome. Several biomarkers have been evaluated in NSCLC, but only a few have proven to be clinically relevant. An activating mutation in the epidermal growth factor receptor (EGFR) is both a well-described predictive marker of benefit of EGFR-targeted tyrosine kinase inhibitors but also a debated prognostic marker of better OS [4], [5], [6], [7], [8], [9]. As EGFR expression has been associated with OS in head and neck, colorectal, and esophagus cancer [10], [11], [12], attention has been directed toward the use of EGFR expression as a prognostic marker in NSCLC, but contradicting results have been demonstrated [13], [14], [15], [16]. The EGF system is complex, and the effect of ligand-receptor interaction depends on a variety of different factors, which provides a plausible explanation for the divergence observed between studies that only evaluate EGFR expression in general. EGFR is one out of four related receptors from the EGF system and is capable of forming homodimers or heterodimers with one of the three other receptors when activated by a ligand. Several ligands from the EGF system such as amphiregulin (AREG), epidermal growth factor (EGF), and transforming growth factor–α (TGF-α) only activate EGFR, whereas some have the ability to activate several combinations of the four EGF receptors like heparin-binding epidermal growth factor (HB-EGF), epiregulin (EPI), and betacellulin (BCL). Most knowledge on the role of the ligands in NSCLC is from in vitro studies or from smaller clinical studies. In vitro studies have suggested that the biological effect of EGFR activation is dependent on the specific activating ligand as well as the dimerization partner [17]. Yet, no clinical studies have evaluated the effect of the network of receptors and ligands influencing EGFR in NSCLC. Furthermore, the majority of the clinical studies exploring EGFR expression are based on immunohistochemistry which is a semiquantitative method with a great risk of interobserver variability. Quantitative gene expression analyses provide a more accurate measure and are therefore more suitable for studies comparing expression levels. Prospectively, we have collected fresh tumor samples from patients suspected of lung cancer. Accordingly, the aim of this study is to evaluate the gene expression of the network of receptors and ligands of the EGF system affecting EGFR as a prognostic markers in NSCLC.     

Archaeological Support for the Three-Stage Expansion of Modern Humans across Northeastern Eurasia and into the Americas

Background

Understanding the dynamics of the human range expansion across northeastern Eurasia during the late Pleistocene is central to establishing empirical temporal constraints on the colonization of the Americas [1]. Opinions vary widely on how and when the Americas were colonized, with advocates supporting either a pre-[2] or post-[1], [3], [4], [5], [6] last glacial maximum (LGM) colonization, via either a land bridge across Beringia [3], [4], [5], a sea-faring Pacific Rim coastal route [1], [3], a trans-Arctic route [4], or a trans-Atlantic oceanic route [5]. Here we analyze a large sample of radiocarbon dates from the northeast Eurasian Upper Paleolithic to identify the origin of this expansion, and estimate the velocity of colonization wave as it moved across northern Eurasia and into the Americas.

Methodology/Principal Findings

We use diffusion models [6], [7] to quantify these dynamics. Our results show the expansion originated in the Altai region of southern Siberia ∼46kBP , and from there expanded across northern Eurasia at an average velocity of 0.16 km per year. However, the movement of the colonizing wave was not continuous but underwent three distinct phases: 1) an initial expansion from 47-32k calBP; 2) a hiatus from ∼32-16k calBP, and 3) a second expansion after the LGM ∼16k calBP. These results provide archaeological support for the recently proposed three-stage model of the colonization of the Americas [8], [9]. Our results falsify the hypothesis of a pre-LGM terrestrial colonization of the Americas and we discuss the importance of these empirical results in the light of alternative models.

Conclusions/Significance

Our results demonstrate that the radiocarbon record of Upper Paleolithic northeastern Eurasia supports a post-LGM terrestrial colonization of the Americas falsifying the proposed pre-LGM terrestrial colonization of the Americas. We show that this expansion was not a simple process, but proceeded in three phases, consistent with genetic data, largely in response to the variable climatic conditions of late Pleistocene northeast Eurasia. Further, the constraints imposed by the spatiotemporal gradient in the empirical radiocarbon record across this entire region suggests that North America cannot have been colonized much before the existing Clovis radiocarbon record suggests.     

Community Based Case-Control Study of Rotavirus Gastroenteritis among Young Children during 2008-2010 Reveals Vast Genetic Diversity and Increased Prevalence of G9 Strains in Kolkata

Background

Group A Rotaviruses are a major etiologic agent of gastroenteritis in infants and young children (<5 years) worldwide. Although rotavirus vaccines have been successfully administered in many countries, in India the introduction of rotavirus vaccine in national immunization program was approved in 2014. Since high disease burden and large number of genetic variants have been reported from low income countries including India, monitoring of rotavirus was initiated prior to implementation of the vaccine in the region.

Methods

A total number of 3,582 stool samples were collected from an urban slum community in Kolkata, among which 1,568 samples were obtained from children of ≤5 years of age, with moderate to severe diarrhoea and 2,014 samples were collected from age-sex matched healthy neighbourhood controls. Rotavirus positive samples were typed by multiplex semi-nested PCR and nucleotide sequencing. Circulating strains were phylogenetically analyzed.

Results

Among 1,568 children with diarrhoea, 395 (25.2%), and among 2,014 asymptomatic children, 42 (2%) were rotavirus positive. G1P[8] was identified as the most common strain (32%) followed by G9P[8] (16.9%), G2P[4] (13.5%) and G9P[4] (10.75%). G12 strains with combinations of P[4], P[6] and P[8] comprised 11.9% of total positive strains. The rest (<10%) were rare and uncommon strains like G1P[4], G1P[6], G2P[8] and animal-like strains G4P[6], G6P[14] and G11P[25]. The 42 rotavirus positive samples from asymptomatic children revealed common genotypes like G1, G2 and G9.

Conclusion

This community based case-control study showed increased predominance of genotype G9 in Kolkata. It also confirmed co-circulation of a large number of genetic variants in the community. Asymptomatic rotavirus positive children though low in number can also be a source of dispersal of infection in the community. This study provides background information to the policy makers for implementation of rotavirus vaccines in this region.     

An Increasing Danger of Zoonotic Orthopoxvirus Infections

On May 8, 1980, the World Health Assembly at its 33^rd session solemnly declared that the world and all its peoples had won freedom from smallpox and recommended ceasing the vaccination of the population against smallpox. Currently, a larger part of the world population has no immunity not only against smallpox but also against other zoonotic orthopoxvirus infections. Recently, recorded outbreaks of orthopoxvirus diseases not only of domestic animals but also of humans have become more frequent. All this indicates a new situation in the ecology and evolution of zoonotic orthopoxviruses. Analysis of state-of-the-art data on the phylogenetic relationships, ecology, and host range of orthopoxviruses—etiological agents of smallpox (variola virus, VARV), monkeypox (MPXV), cowpox (CPXV), vaccinia (VACV), and camelpox (CMLV)—as well as the patterns of their evolution suggests that a VARV-like virus could emerge in the course of natural evolution of modern zoonotic orthopoxviruses. Thus, there is an insistent need for organization of the international control over the outbreaks of zoonotic orthopoxvirus infections in various countries to provide a rapid response and prevent them from developing into epidemics.The genus Orthopoxvirus of the family Poxviridae comprises the species variola (smallpox) virus (VARV), with human as its only sensitive host; zoonotic species monkeypox virus (MPXV), cowpox virus (CPXV), vaccinia virus (VACV), and camelpox virus (CMLV); and several others. These orthopoxviruses are immunologically cross-reactive and cross-protective, so that infection with any member of this genus provides protection against infection with any other member of the genus [1], [2]. Traditionally, the species of the Orthopoxvirus genus have been named primarily according to the host animal from which they were isolated and identified based on a range of biological characteristics [1]. Most frequently, zoonotic orthopoxviruses have been initially isolated from animals immediately close to humans being incidental hosts for the virus, the natural carriers of which are, as a rule, wild animals. Correspondingly, the name of an orthopoxvirus species does not reflect the actual animal that is its natural reservoir.With accumulation of the data on complete genome nucleotide sequences for various strains of orthopoxvirus species, it has been found that an interesting feature of the orthopoxvirus genomes is the presence of genes that are intact in one species but fragmented or deleted in another [3]–[8]. These data confirm the concept of a reductive evolution of orthopoxviruses, according to which the gene loss plays an important role in the evolutionary adaptation of progenitor virus to a particular environmental niche (host) and emergence of new virus species [9]. CPXV has the largest genome of all the modern representatives of the genus Orthopoxvirus, and this genome contains all the genes found in the other species of this genus [2], [4], [10]–[12]. Therefore, Cowpox virus was proposed as the closest of all the modern species to the progenitor virus for the genus Orthopoxvirus, while the remaining species, Variola virus included, had appeared as a result of multistage reductive evolution [4], [9], [13].VARV, the most pathogenic species for humans, has the smallest genome of all the orthopoxviruses [2]–[7]. This suggests a potential possibility for emergence of a VARV-like variant from the currently existing zoonotic orthopoxviruses with longer genomes in the course of natural evolution. It is known that although mutational changes are rather a rare event for the poxvirus DNA [13], characteristic of these viruses is the possibility of intermolecular and intramolecular recombinations, as well as genomic insertions and deletions [14], [15]. It has been recently found that duplication/amplification of genomic segments is typical of poxviruses, and in the case of a certain selective pressure (for example, host antiviral defenses), certain genes are able to relatively rapidly accumulate mutations that would provide the virus adaptation to new conditions, including a new host [16].The conducted analysis of the available archive data on smallpox and the history of ancient civilizations as well as the newest data on the evolutionary relationships of orthopoxviruses has allowed me to suggest the hypothesis that smallpox could have repeatedly emerged in the past via evolutionary changes of a zoonotic progenitor virus [17].Because of the cessation of the vaccination against smallpox after its eradication 35 years ago, a tremendous part of the world human population currently has no immunity not only against smallpox, but also against any other zoonotic orthopoxvirus infections. This new situation allows orthopoxviruses to circulate in the human population and, as a consequence, should alter several established concepts on the ecology and range of sensitive hosts for various orthopoxvirus species.The most intricate case is the origin of VACV. For many decades, VACV has been used for vaccinating humans against smallpox, and it was considered that this virus, variolae vaccinae, originates from zoonotic CPXV, introduced to immunization practice by Jenner as early as 1796 [1]. Only in the 20^th century was it found out that the orthopoxvirus strains used for smallpox vaccination significantly differ in their properties from both the natural CPXV isolates recovered from cows and the other orthopoxvirus species examined by that time [18]. Correspondingly, they were regarded as a separate species, Vaccinia virus [19]. Moreover, it was inferred that the VACV natural reservoir was unknown and numerous hypotheses attempted to explain the origin of this virus while passaging progenitor viruses in animals in the process of vaccine production [1], [2], [20].The issue of VACV origin was somewhat clarified after sequencing the complete genome of horsepox virus (HSPV) [21], which appeared to be closely related to the sequenced VACV strains. Only after this was attention paid to the fact that Jenner specified the origin of his vaccine from an infection of the heels of horses (“grease”) and indicated that the vaccine became more suitable for human use after passage through the cow [20]. This suggests that VACV may originate from a zoonotic HSPV, which naturally persisted concurrently with CPXV. Some facts suggest that the infectious materials not only from cow lesions but also from horse lesions were used for smallpox vaccination in the 19^th century. The vaccine lymph from the horse gave the most satisfactory results in inducing an anti-smallpox immunity as well as less side reactions [1]. By all accounts, they gradually commenced using HSPV isolates for smallpox vaccination, the future generations of which recovered decades later were ascribed to the separate species Vaccinia virus [19], rather than CPXV for smallpox vaccination everywhere.Since the 1960s, VACVs have been repeatedly isolated in Brazil [22]. The first VACV isolates were recovered from wild rodents (sentinel mice and rice rat) [23]. Since 1999, an ever-increasing number of exanthematous outbreaks affecting dairy cows and their handlers have been recorded [24]–[27], supplemented recently with outbreaks among horses [28], [29]. Several VACV strains have been isolated during these outbreaks from cows, horses, humans, and rodents [22], [27], [28], [30], [31]. The questions that arise are when and how VACV entered Brazil and the wild nature of the American continent. The more widespread point of view is that VACV strains could be transmitted from vaccinated humans to domestic animals and further to wild ones with subsequent adaptation to the rural environment [22]. My standpoint implies that HSPV/VACV could have been repeatedly accidentally imported from Europe to South America with the infected horses or rodents to be further introduced into wildlife. Possibly, the latter hypothesis more adequately reflects the actual pathway of VACV transmission to the Brazilian environment, since recent phylogenetic studies have suggested an independent origin for South American VACV isolates, distinct from the vaccine strains used on this continent during the WHO smallpox eradication campaign [22], [32]. Presumably, genome-wide sequencing of the viruses will give a more precise answer to the origin of VACV variants isolated in Brazil.In the past, the outbreaks of buffalopox had occurred frequently in various states of India as well as in Pakistan, Bangladesh, Indonesia, Egypt, and other countries [33]. The causative agent, buffalopox virus (BPXV), is closely related to VACV and affiliated with the species Vaccinia virus, genus Orthopoxvirus [2], [34]. Recently, mass outbreaks of buffalopox in domestic buffaloes along with severe zoonotic infection in milk attendants were recorded at various places in India [35], [36]. In several buffalopox outbreaks, the BPXV-caused infections were recorded in cows in the same herds [37]. An increase in BPXV transmission to different species, including buffaloes, cows, and humans, suggests the reemergence of zoonotic buffalopox infection [35], [38]. The buffalopox outbreaks recorded in different distant regions of India are likely to suggest the presence of an abundant natural BPXV reservoir represented by wild animals, most probably rodents. Correspondingly, it is of the paramount importance to perform a large-scale study of the presence of orthopoxviruses in wild animals of India.Thus, yet incomplete data on the modern ecology of VACV and BPXV allow for speculation that the orthopoxviruses belonging to the species Vaccinia virus have a wide host range, are zoonotic, are currently spread over large areas in Eurasia and South America, and that their natural carriers are several rodents.CPXV has relatively low pathogenicity for humans but has a wide range of sensitive animal hosts [2], [39]. Human cowpox is a rare sporadic disease, which develops when CPXV is transmitted from an infected animal to human [2], [40]. This disease is mainly recorded in Europe. In wildlife, CPXV carriers are asymptomatically infected rodents [41], [42]. During the last two decades, reports on an increasing number of CPXV infections in cats, rats, exotic animals, and humans have been published [43]–[47]. Comparative studies of the properties of CPXV isolates recovered from various hosts at different times and in several geographic zones have shown sufficient intraspecific variations [2], [48], [49]. A recent phylogenetic analysis of the complete genomes of 12 CPXV strains recovered from humans and several animal species suggests that they be split into two major Cowpox virus–like and Vaccinia virus–like clades [50]. This means that the criteria of the separation of orthopoxviruses into these two species should be corrected.MPXV is a zoonotic virus causing a human infection similar to smallpox in its clinical manifestations with a lethality rate of 1–8% [51]. The natural reservoir of MPXV is various species of African rodents [8], [10]. The active surveillance data in the same health zone (Democratic Republic of Congo) from the 1980s to 2006–2007 suggest a 20-fold increase in human monkeypox incidence 30 years after the cessation of the smallpox vaccination campaign [52]. This poses the question of whether MPXV can acquire the possibility of a high human-to-human transmission rate, characteristic of VARV, under conditions of a long-term absence of vaccination and considerably higher incidence of human infection. If this occurs, humankind will face a problem considerably more complex than with the smallpox eradication. First and foremost, this is determined by the fact that MPXV, unlike VARV, has its natural reservoir represented by numerous African rodents [2], [53].In its biological properties and according to the data of phylogenetic analysis of the complete virus genomic sequence, CMLV is closest to VARV, the causative agent of smallpox, as compared with the other orthopoxvirus species [1], [8]. Camelpox is recognized as one of the most important viral diseases in camels. This infection was first described in India in 1909. Subsequently, camelpox outbreaks have been reported in many countries of the Middle East, Asia, and Africa [54], [55]. Until recently, it has been commonly accepted that the host range of CMLV is confined to one animal species, camels [1], [55]. However, the first human cases of camelpox have been recently confirmed in India [56]. This suggests that camelpox could be a zoonotic disease. Since camelpox outbreaks occur irregularly in distant regions of the world and the viruses isolated during these outbreaks display different degrees of virulence [55], it is possible to postulate the presence of a wildlife animal reservoir of CMLV other than camels. Since the camelpox outbreaks are usually associated with the rainy season of the year, when rodents are actively reproducing, it is likely that rodents could be the natural carriers of CMLV.It is known that most of the emerging human pathogens originate from zoonotic pathogens [57]–[59]. Many viruses do not cause the disease in their natural reservoir hosts but can be highly pathogenic when transmitted to a new host species. Emerging and reemerging human pathogens more often are those with broad host ranges. The viruses able to infect many animal species are evolutionarily adapted to utilizing different cell mechanisms for their reproduction and, thus, can extend/change their host range with a higher probability [58].There are no fundamental prohibitions for the possible reemergence of smallpox or a similar human disease in the future as a result of natural evolution of the currently existing zoonotic orthopoxviruses. An ever-increasing sensitivity of the human population to zoonotic orthopoxviruses, resulting from cessation of the mass smallpox vaccination, elevates the probability for new variants of these viruses, potentially dangerous for humans, to emerge. However, the current situation is radically different from the ancient one, since many outbreaks of orthopoxvirus infections among domestic animals and humans are recorded and studied.Recently, the efforts of scientists under WHO control are directed to the development of state-of-the-art methods for VARV rapid identification as well as design of new generation safe smallpox vaccines and drugs against VARV and other orthopoxviruses [60]. The designed promising anti-orthopoxvirus drugs display no pronounced virus species specificity. Therefore, they are applicable in the outbreaks caused by any orthopoxvirus species. International acceptance of the designed highly efficient anti-orthopoxvirus drugs ST-246 and CMX001 [60] is of paramount importance.In the areas of high incidence of zoonotic orthopoxviral infections, it would be purposeful to vaccinate domestic and zoo animals as well as the persons closely associated with them using state-of-the-art safe vaccines based on VACV, which has a wide range of sensitive hosts. This would considerably decrease the likelihood for such infections to spread from wildlife into the human environment.In the African region endemic for monkeypox, which also displays a high rate of HIV infection, the population could be vaccinated with the VACV strain MVA, which has been recently demonstrated to be safe even for HIV-infected persons [61].Taking into account the above mentioned increased incidence of outbreaks of animal and human orthopoxvirus infections and their potential danger, it is important to accelerate organization of the international Smallpox Laboratory Network, discussed by the WHO Advisory Committee on Variola Virus Research [62], [63], and orient this network to express diagnosing not only of VARV but also of other zoonotic orthopoxviruses. This will provide constant monitoring of these infections in all parts of the world and make it possible to prevent the development of small outbreaks into expanded epidemics, thereby decreasing the risk of evolutional changes and emergence of an orthopoxvirus highly pathogenic for humans.The international system for clinical sampling and identification of infectious agents has been worked out and optimized while implementing the global smallpox eradication program under the aegis of the WHO as well as anti-epidemic measures and methods for mass vaccination [1]. The accumulated experience is of paramount importance for the establishment of international control not only over currently existing orthopoxvirus infections but also other emerging and reemerging diseases.     

Aging in a Long-Lived Clonal Tree

From bacteria to multicellular animals, most organisms exhibit declines in survivorship or reproductive performance with increasing age (“senescence”) [1],[2]. Evidence for senescence in clonal plants, however, is scant [3],[4]. During asexual growth, we expect that somatic mutations, which negatively impact sexual fitness, should accumulate and contribute to senescence, especially among long-lived clonal plants [5],[6]. We tested whether older clones of Populus tremuloides (trembling aspen) from natural stands in British Columbia exhibited significantly reduced reproductive performance. Coupling molecular-based estimates of clone age with male fertility data, we observed a significant decline in the average number of viable pollen grains per catkin per ramet with increasing clone age in trembling aspen. We found that mutations reduced relative male fertility in clonal aspen populations by about 5.8×10⁻⁵ to 1.6×10⁻³ per year, leading to an 8% reduction in the number of viable pollen grains, on average, among the clones studied. The probability that an aspen lineage ultimately goes extinct rises as its male sexual fitness declines, suggesting that even long-lived clonal organisms are vulnerable to senescence.     

MRI Features in a Canine Model of Ischemic Stroke: Correlation between Lesion Volume and Neurobehavioral Status during the Subacute Stage

The purpose of this study was to evaluate the diagnostic value of magnetic resonance imaging (MRI) and assess the correlation between the volume of the ischemic lesion and neurobehavioral status during the subacute stage of ischemic stroke. Ischemic stroke was induced in 6 healthy laboratory beagles through permanent occlusion of the middle cerebral artery (MCAO). T2-weighted and fluid-attenuated inversion recovery (FLAIR) imaging, diffusion-weighted imaging (DWI), measurement of the apparent diffusion coefficient (ADC) ratio, and neurobehavioral evaluation were performed 3 times serially by using a 1.5-T MR system: before and 3 and 10 d after MCAO. Ischemic lesions demonstrated T2 hyperintensity, FLAIR hyperintensity, and DWI hyperintensity. The ADC ratio was decreased initially but then was increased at 10 d after MCAO. Ischemic lesion volumes on T2-weighted and FLAIR imaging were not significantly different from those on DWI. The lesion volume and neurobehavioral score showed strong correlation. Our results suggest that conventional MRI may be a reliable diagnostic tool during the subacute stage of canine ischemic stroke.Abbreviations: ADC, apparent diffusion coefficient; DWI, diffusion-weighted imaging; FLAIR, fluid-attenuated inversion recovery; MCAO, middle cerebral artery occlusion; MRI, magnetic resonance imaging; PWI, perfusion-weighted imagingIn human medicine, stroke is a leading cause of adult mortality and neurologic disability worldwide.¹ Strokes previously were thought to be uncommon in small animals, but the true prevalence is unknown.⁴ These events are now recognized more frequently in dogs because of increased use of magnetic resonance imaging (MRI).^5,14,17Because the infusion of thrombolytic agents, such as urokinase or tissue plasminogen activator, within 3 to 6 h of the onset symptoms is effective in restoring blood flow and improving stroke outcome in humans,¹⁹ the detection of early ischemic changes is now thought to be necessary to improve patient outcome. Computed tomography and conventional MRI are not sufficiently sensitive to predict the presence and extent of ischemic damage during the acute stage after a stroke.^12,20 Therefore several MRI sequences, such as fluid-attenuated inversion recovery (FLAIR), diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), and MR angiography, have been developed for early diagnosis and subsequent follow-up of ischemic stroke.³ High-field magnetic strengths (at least 1.5 T) are necessary to perform these sequences.In contrast to the situation in humans, ischemic stroke in many dogs is diagnosed during the subacute stage—24 h to 6 wk after the vascular insult—due to the time lag between the onset of clinical signs to referral and to the lack of standard diagnostic protocols for ischemic stroke in dogs. In most reports of strokes in dogs, the median interval between the onset of neurologic dysfunction and performance of an MRI was more than 2 d.^5,14,17 Whereas DWI has marked sensitivity to very early ischemic changes in the brain, T2-weighted and FLAIR images gradually become more hyperintense later (that is, during the first 24 h after the insult).³ Therefore, hyperintensity on T2-weighted and FLAIR images is believed to be representative of mature lesions.¹⁵ In light of these findings, we hypothesized that conventional MR sequences, such as T2-weighted and FLAIR imaging as well as DWI would be used for the diagnosis of the subacute stage of ischemic stroke in dogs.The purpose of this study was to evaluate the diagnostic value of MRI and assess the correlation between the volume of ischemic lesions and neurobehavioral status during the subacute stage of ischemic stroke in dogs. We therefore investigated the lesion volume of T2-weighted and FLAIR images compared with that on DWI images. Furthermore, we assessed the relationship between the apparent diffusion coefficient (ADC) of the ischemic lesions and the neurobehavioral status of the dogs.     

Extinction Risk Escalates in the Tropics

The latitudinal biodiversity gradient remains one of the most widely recognized yet puzzling patterns in nature [1]. Presently, the high level of extinction of tropical species, referred to as the “tropical biodiversity crisis”, has the potential to erode this pattern. While the connection between species richness, extinction, and speciation has long intrigued biologists [2], [3], these interactions have experienced increased poignancy due to their relevancy to where we should concentrate our conservation efforts. Natural extinction is a phenomenon thought to have its own latitudinal gradient, with lower extinction rates in the tropics being reported in beetles, birds, mammals, and bivalves [4]–[7]. Processes that have buffered ecosystems from high extinction rates in the past may also buffer ecosystems against disturbance of anthropogenic origin. While potential parallels between historical and present-day extinction patterns have been acknowledged, they remain only superficially explored and plant extinction patterns have been particularly neglected. Studies on the disappearances of animal species have reached conflicting conclusions, with the rate of extinction appearing either higher [8] or lower [9] in species richness hotspots. Our global study of extinction risk in vascular plants finds disproportionately higher extinction risk in tropical countries, even when indicators of human pressure (GDP, population density, forest cover change) are taken into account. Our results are at odds with the notion that the tropics represent a museum of plant biodiversity (places of historically lowered extinction) and we discuss mechanisms that may reconcile this apparent contradiction.     

Status and the Brain

Social hierarchy is a fact of life for many animals. Navigating social hierarchy requires understanding one''s own status relative to others and behaving accordingly, while achieving higher status may call upon cunning and strategic thinking. The neural mechanisms mediating social status have become increasingly well understood in invertebrates and model organisms like fish and mice but until recently have remained more opaque in humans and other primates. In a new study in this issue, Noonan and colleagues explore the neural correlates of social rank in macaques. Using both structural and functional brain imaging, they found neural changes associated with individual monkeys'' social status, including alterations in the amygdala, hypothalamus, and brainstem—areas previously implicated in dominance-related behavior in other vertebrates. A separate but related network in the temporal and prefrontal cortex appears to mediate more cognitive aspects of strategic social behavior. These findings begin to delineate the neural circuits that enable us to navigate our own social worlds. A major remaining challenge is identifying how these networks contribute functionally to our social lives, which may open new avenues for developing innovative treatments for social disorders.

“Observing the habitual and almost sacred ‘pecking order’ which prevails among the hens in his poultry yard—hen A pecking hen B, but not being pecked by it, hen B pecking hen C and so forth—the politician will meditate on the Catholic hierarchy and Fascism.” —Aldous Huxley, Point Counter Point (1929)

From the schoolyard to the boardroom, we are all, sometimes painfully, familiar with the pecking order. First documented by the Norwegian zoologist Thorleif Schjelderup-Ebbe in his PhD thesis on social status in chickens in the 1920s, a pecking order is a hierarchical social system in which each individual is ranked in order of dominance [1]. In chickens, the top hen can peck all lower birds, the second-ranking bird can peck all birds ranked below her, and so on. Since it was first coined, the term has become widely applied to any such hierarchical system, from business, to government, to the playground, to the military.Social hierarchy is a fact of life not only for humans and chickens but also for most highly social, group-living animals. Navigating social hierarchies and achieving dominance often appear to require cunning, intelligence, and strategic social planning. Indeed, the Renaissance Italian politician and writer Niccolo Machiavelli argued in his best-known book “The Prince” that the traits most useful for attaining and holding on to power include manipulation and deception [2]. Since then, the term “Machiavellian” has come to signify a person who deceives and manipulates others for personal advantage and power. 350 years later, Frans de Waal applied the term Machiavellian to social maneuvering by chimpanzees in his book Chimpanzee Politics [3]. De Waal argued that chimpanzees, like Renaissance Italian politicians, apply guile, manipulation, strategic alliance formation, and deception to enhance their social status—in this case, not to win fortune and influence but to increase their reproductive success (which is presumably the evolutionary origin of status-seeking in Renaissance Italian politicians as well).The observation that navigating large, complex social groups in chimpanzees and many other primates seems to require sophisticated cognitive abilities spurred the development of the social brain hypothesis, originally proposed to explain why primates have larger brains for their body size than do other animals [4],[5]. Since its first proposal, the social brain hypothesis has accrued ample evidence endorsing the connections between increased social network complexity, enhanced social cognition, and larger brains. For example, among primates, neorcortex size, adjusted for the size of the brain or body, varies with group size [6],[7], frequency of social play [8], and social learning [9].Of course, all neuroscientists know that when it comes to brains, size isn''t everything [10]. Presumably social cognitive functions required for strategic social behavior are mediated by specific neural circuits. Here, we summarize and discuss several recent discoveries, focusing on an article by Noonan and colleagues in the current issue, which together begin to delineate the specific neural circuits that mediate our ability to navigate our social worlds.Using structural magnetic resonance imaging (MRI), Bickart and colleagues showed that the size of the amygdala—a brain nucleus important for emotion, vigilance, and rapid behavioral responses—is correlated with social network size in humans [11]. Subsequent studies showed similar relationships for other brain regions implicated in social function, including the orbitofrontal cortex [12] and ventromedial prefrontal cortex [13]. Indeed, one study even found an association between grey matter density in the superior temporal sulcus (STS) and temporal gyrus and an individual''s number of Facebook friends [14]. Collectively, these studies suggest that the number and possibly the complexity of relationships one maintains varies with the structural organization of a specific network of brain regions, which are recruited when people perform tests of social cognition such as recognizing faces or inferring others'' mental states [15],[16]. These studies, however, do not reveal whether social complexity actively changes these brain areas through plasticity or whether individual differences in the structure of these networks ultimately determines social abilities.To address this question, Sallet and colleagues experimentally assigned rhesus macaques to social groups of different sizes and then scanned their brains with MRI [17]. The authors found significant positive associations between social network size and morphology in mid-STS, rostral STS, inferior temporal (IT) gyrus, rostral prefrontal cortex (rPFC), temporal pole, and amygdala. The authors also found a different region in rPFC that scaled positively with social rank; as grey matter in this region increased, so did the monkey''s rank in the hierarchy. As in the human studies described previously, many of these regions are implicated in various aspects of social cognition and perception [18]. These findings endorse the idea that neural plasticity is engaged in specifically social brain areas in response to the demands of the social environment, changing these areas structurally according to an individual''s experiences with others.Sallet and colleagues also examined spontaneous coactivation among these regions using functional MRI (fMRI). Measures of coactivation are thought to reflect coupling between regions [19],[20]; these measures are observable in many species [21],[22] and vary according to behavior [23],[24], genetics [25], and sex [26], suggesting that coactivation may underlie basic neural function and interaction between brain regions. The authors found that coactivation between the STS and rPFC increased with social network size and that coactivation between IT and rPFC increased with social rank. These findings show that not only do structural changes occur in these regions to meet the demands of the social environment but these structural changes mediate changes in function as well.One important question raised by the study by Sallet and colleagues is whether changes in the structure and function of social brain areas are specific outcomes of social network size or of dealing with social hierarchy. After all, larger groups offer more opportunity for a larger, more despotic pecking order. In the current volume, Noonan and colleagues address this question directly by examining the structural and functional correlates of social status in macaques independently of social group size [27]. The authors collected MRI scans from rhesus macaques and measured changes in grey matter associated with social dominance. By scanning monkeys of different ranks living in groups of different sizes, the authors were able to cleave the effects of social rank from those of social network size (Figure 1).Open in a separate windowFigure 1Brain regions in rhesus macaques related to social environment.Primary colors indicate brain regions in which morphometry tracks social network size. Pastel colors indicate brain regions in which morphometry tracks social status in the hierarchy. Regions of interest adapted from [48], overlaid on Montreal Neurological Institute (MNI) macaque template [49].The authors found a network of regions in which grey matter measures varied with social rank; these regions included the bilateral central amygdala, bilateral brainstem (between the medulla and midbrain, including parts of the raphe nuclei), and hypothalamus, which varied positively with dominance, and regions in the basal ganglia, which varied negatively with social rank. These regions have been implicated in social rank functions across a number of species [28]–[32]. Importantly, these relationships were unique to social status. There was no relationship between grey matter in these subcortical areas and social network size, endorsing a specific role in social dominance-related behavior. Nevertheless, grey matter in bilateral mid-STS and rPFC varied with both social rank and social network size, as reported previously. These findings demonstrate that specific brain areas uniquely mediate functions related to social hierarchy, whereas others may subserve more general social cognitive processes.Noonan and colleagues next probed spontaneous coactivation using fMRI to examine whether functional coupling between any of these regions varied with social status. They found that the more subordinate an animal, the stronger the functional coupling between multiple regions related to dominance. These results suggest that individual differences in social status are functionally observable in the brain even while the animal is at rest and not engaged in social behavior. These findings suggest that structural changes associated with individual differences in social status alter baseline brain function, consistent with the idea that the default mode of the brain is social [33] and that the sense of self and perhaps even awareness emerge from inwardly directed social reasoning [34].These findings resonate with previous work on the neural basis of social dominance in other vertebrates. In humans, for example, activity in the amygdala tracks knowledge of social hierarchy [28],[35] and, further, shows activity patterns that uniquely encode social rank and predict relevant behaviors [28]. Moreover, recent research has identified a specific region in the mouse hypothalamus, aptly named the “hypothalamic attack area” [36],[37]. Stimulating neurons in this area immediately triggers attacks on other mice and even an inflated rubber glove, while inactivating these neurons suppresses aggression [38]. In the African cichlid fish Haplochromis burtoni, a change in the social status of an individual male induces a reversible change in the abundance of specialized neurons in the hypothalamus that communicate hormonally with the pituitary and gonads [39]. Injections of this hormone in male birds after an aggressive territorial encounter amplifies the normal subsequent rise in testosterone [40]. Serotonin neurons in the raphe area of the brainstem also contribute to dominance-related behaviors in fish [29],[31] and aggression in monkeys [41].Despite these advances, there are still gaps in our understanding of how these circuits mediate status-related behaviors. Though regions in the amygdala, brainstem, and hypothalamus vary structurally and functionally with social rank, it remains unknown precisely how they contribute to or respond to social status. For example, though amygdala function and structure correlates with social status in both humans and nonhuman primates [27],[28],[35],[42], it remains unknown which aspects of dominance this region contributes to or underlies. One model suggests that the amygdala contributes to learning or representing one''s own status within a social hierarchy [28],[35]. Alternatively, the amygdala could contribute to behaviors that support social hierarchy, including gaze following [43] and theory of mind [44]. Lastly, the amygdala could contribute to social rank via interpersonal behaviors or personality traits, such as aggression [45], grooming [45], or fear responses [46],[47]. Future work will be critical to determine how signals in these regions relate to social status; direct manipulation of these regions, possibly via microstimulation, larger-scale brain stimulation (e.g., transcranial magnetic stimulation and transcranial direct current stimulation), or temporary lesions, will be critical to better understand these relationships.The work by Noonan and colleagues suggests new avenues for exploring how the brain both responds to and makes possible social hierarchy in nonhuman primates and humans. The fact that the neural circuits mediating dominance and social networking behavior can be identified and measured from structural and functional brain scans even at rest suggests the possibility that similar measures can be made in humans. Although social status is much more complex in people than it is in monkeys or fish, it is just as critical for us and most likely depends on shared neural circuits. Understanding how these circuits work, how they develop, and how they respond to the local social environment may help us to understand and ultimately treat disorders, like autism, social anxiety, or psychopathy, that are characterized by impaired social behavior and cognition.     

Structural Ensemble Modulation upon Small-Molecule Binding to Disordered Proteins

Over the past decade, there has been a growing interest in investigating whether disordered proteins can be targeted for clinical purposes using small molecules [1], [2], [3], [4], [5], [6], [7], [8]. While small-molecule binding to disordered proteins can be seen as unorthodox, examples of this phenomenon have been reported. In order to rationalize these observations, a variety of models are emerging, sometimes in apparent contradiction. Here, we offer a “structural ensemble modulation” view as an attempt to clarify the language, organize concepts, and facilitate the comparison of different studies. In doing so, we hope to promote the understanding of the general principles underlying this phenomenon toward the development of novel therapeutic compounds targeting disordered proteins, which are prevalent in a wide range of human diseases [1], [2], [3], [4], [5], [6], [7], [8].     

From Dinosaurs to Modern Bird Diversity: Extending the Time Scale of Adaptive Radiation

What explains why some groups of organisms, like birds, are so species rich? And what explains their extraordinary ecological diversity, ranging from large, flightless birds to small migratory species that fly thousand of kilometers every year? These and similar questions have spurred great interest in adaptive radiation, the diversification of ecological traits in a rapidly speciating group of organisms. Although the initial formulation of modern concepts of adaptive radiation arose from consideration of the fossil record, rigorous attempts to identify adaptive radiation in the fossil record are still uncommon. Moreover, most studies of adaptive radiation concern groups that are less than 50 million years old. Thus, it is unclear how important adaptive radiation is over temporal scales that span much larger portions of the history of life. In this issue, Benson et al. test the idea of a “deep-time” adaptive radiation in dinosaurs, compiling and using one of the most comprehensive phylogenetic and body-size datasets for fossils. Using recent phylogenetic statistical methods, they find that in most clades of dinosaurs there is a strong signal of an “early burst” in body-size evolution, a predicted pattern of adaptive radiation in which rapid trait evolution happens early in a group''s history and then slows down. They also find that body-size evolution did not slow down in the lineage leading to birds, hinting at why birds survived to the present day and diversified. This paper represents one of the most convincing attempts at understanding deep-time adaptive radiations.

“It is strikingly noticeable from the fossil record and from its results in the world around us that some time after a rather distinctive new adaptive type has developed it often becomes highly diversified.” – G. G. Simpson ([1], pp. 222–223)

George Gaylord Simpson was the father of modern concepts of adaptive radiation—the diversification of ecological traits in a rapidly speciating group of organisms (Figure 1; [2]). He considered adaptive radiation to be the source of much of the diversity of living organisms on planet earth, in terms of species number, ecology, and body form [1]–[3]. Yet more than 60 years after Simpson''s seminal work, the exact role of adaptive radiation in generating life''s extraordinary diversity is still an open and fundamental question in evolutionary biology [3],[4].Open in a separate windowFigure 1An example of adaptive radiation and early bursts in rates of speciation and phenotypic evolution.(a) The adaptive radiation of the modern bird clade Vanginae, which shows early rapid speciation, morphological diversity, and diversity in foraging behavior and diet [15],[32]. (b) Hypothetical curve of speciation rates through time that would be expected in adaptive radiation. The exponential decline in speciation rates shows that there was an “early burst” of speciation at the beginning of the clade''s history. (c) Hypothetical curve of rates of phenotypic evolution through time that would be expected in adaptive radiation, also showing an early burst of evolution with high initial rates. Part (a) is reproduced from [32] with permission (under CC-BY) from the Royal Society and the original authors.To address this question, researchers have looked for signatures of past adaptive radiation in the patterns of diversity in nature. In particular, it has been suggested that groups that have undergone adaptive radiation should show an “early-burst” signal in both rates of lineage diversification and phenotypic evolution through time—a pattern in which rates of speciation and phenotypic evolution are fast early in the history of groups and then decelerate over time (Figure 1; [3]–[5]). These predictions arise from the idea that clades should multiply and diversify rapidly in species number, ecology, and phenotype in an adaptive radiation and that rates of this diversification should decrease later as niches are successively occupied [2].Early bursts have been sought in both fossils and phylogenies. Few fossil studies have discussed their results in the context of adaptive radiation (but see [6]), but they often have found rapid rises in both taxonomic and morphological diversity early in the history of various groups [7], ranging from marine invertebrates [8] to terrestrial mammals [9]. However, fossils often lack the phylogeny needed to model how evolution has proceeded [7]. On the other hand, studies that test for early bursts in currently existing (extant) species typically use phylogenies, which allow us to model past evolution in groups with few or no fossils [5]. Phylogenies have most often been used to test early bursts in speciation (see, e.g., [10]). However, such tests may be misled by past extinction, which will decay the statistical signal of rapid, early diversification [11]. Furthermore, diverse evolutionary scenarios beyond adaptive radiation can give rise to early bursts in speciation [12]. By contrast, studies of phenotypic diversification may be more robust to extinction [13] and they test the distinguishing feature that separates adaptive from nonadaptive radiation [2],[12].Thus, studies of adaptive radiation in extant organisms increasingly have focused on phylogenetic tests of the early-burst model of phenotypic evolution. Some studies show strong support for this prediction in both birds [14],[15] and lizards [5],[16]. However, the most extensive study to date showed almost no support for the early-burst model. In this study, Harmon et al. [17] examined body size in 49 (and shape in 39) diverse groups of animals, including invertebrates, fishes, amphibians, reptiles, birds, and mammals. They found strong support for the early-burst model in only two of these 88 total datasets.This result raises an important question: if adaptive radiation explains most of life''s diversity [1], how is it possible that there is so little phylogenetic evidence for early bursts of phenotypic evolution? One possibility is that early bursts are hard to detect. This can be due to low statistical power in the most commonly employed tests [18]. It may also be due to a lack of precision in the way “early burst” is defined (and thus tested), as the ecological theory of adaptive radiation suggests that the rate of phenotypic evolution will decrease as species diversity increases in a group, not just over time [14],[16]. Indeed, recent studies [14],[16] detected a decline in rates with species diversity in clades that were also in the Harmon et al. [17] study, yet for which no decline over time was detected.A second possible reason for why early-burst patterns are uncommon is more fundamental: the patterns of phenotypic diversity that result from adaptive radiation may be different at large time scales. Many of the best examples of adaptive radiation are in groups that are relatively young, including Darwin''s finches (2.3 million years old [myr]; [19]) and Lake Malawi and Victoria cichlids (2.3 myr; [20]), whereas most groups that are examined for early bursts in phenotypic evolution are much older (e.g., 47 of 49 in Harmon et al. [17]; mean ± sd = 23.8±29.2 myr). So there may be an inherent difference between what unfolds over the relatively short time scales emphasized by Schluter [2] and what one sees at macroevolutionary time scales (see [21] for an in-depth discussion of this idea as it relates to speciation).The time scale over which adaptive radiations unfold has been little explored. As a result, the link between extant diversity and major extinct radiations remains unclear. Simpson [1] believed that adaptive radiation played out at the population level, but that it should manifest itself at larger scales as well—up to phyla (e.g., chordates, arthropods). He suggested that we should see signals of adaptive radiations in large, old clades because they are effectively small-scale adaptive radiation writ large [1]. Under this view, we should see the signal of adaptive radiation even in groups that diversified over vast time scales, particularly if adaptive radiation is as important for explaining life''s diversity as Simpson [1] thought it was.Part of the reason why potential adaptive radiations at deep time scales remain poorly understood is that studies either focus on fossils or phylogenies, but rarely both. In this issue, Benson et al. [22] combine these two types of data to address whether dinosaurs show signs that they adaptively radiated. Unlike most other studies, the temporal scale of the current study is very large—in this case, over 170 million years throughout the Mesozoic era, starting at 240 million years ago in the Triassic period. This characteristic allowed Benson et al. to shed light on deep-time adaptive radiation.The authors estimated body mass from fossils by using measurements of the circumference of the stylopodium shaft (the largest bone of the arm or leg, such as the femur), which shows a consistent scaling relationship with body mass in extant reptiles and mammals [23]. They then combined published phylogenies to obtain a composite phylogeny for the species in their body-size dataset. The authors finally conducted two types of tests of the rate of body-size evolution—tests of early bursts in phenotypic evolution that are the same as those of Harmon et al. [17], as well as an additional less commonly used test that estimates whether differences between estimated body size at adjacent phylogenetic nodes decreases over time.Benson et al. [22] found two striking results. First, in both of their analyses, the early-burst model was strongly supported for most clades of dinosaurs. This early burst began in the Triassic period, indicating that diversification in body size in dinosaurs began before the Triassic-Jurassic mass extinction event would have opened competition-free ecological space (as commonly hypothesized; [24],[25]). Rather, the authors [22] suggest that a key innovation led to this rise in dinosaurs, though it is not clear what this innovation was [26]. In general, though, the finding of an early burst in body-size evolution in most dinosaurs—if a consequence of adaptive evolution—suggests that adaptive radiation may play out over large evolutionary time scales, not just on the short time scales typical of the most well-studied cases of extant groups.Second, one clade—Maniraptora, which is the clade in which modern-day birds are nested—was the only part of the dinosaur phylogeny that did not show such a strong early burst in body-size evolution. Instead, this clade fit a model to a single adaptive peak—an optimum body size, if you will—but also maintained high rates of undirected body-size evolution throughout their history. Benson et al. [22] suggest that this last result connects deep-time adaptive radiation in the dinosaurs, which quickly exhausted the possibility of phenotypic space, with the current radiation in extant birds, which survived to the present day because their constant, high rate of evolution meant that they were constantly undergoing ecological innovation. This gives a glimpse into why modern birds have so many species (an order of magnitude higher than the nonavian dinosaurs) and so much ecological diversity.The use of fossils allowed Benson et al. [22] to address deep-time radiation in dinosaurs and its consequence on present-day bird diversity. Nevertheless, the promise of using fossils to understand adaptive radiation has its limits. The paleontological dataset presented here is exceptional, yet still insufficient to explore major components of adaptive radiations like actual ecological diversification. As in many paleontological studies, Benson et al. used body-size data to represent ecology because body size is one of the few variables that is available for most species. But it is unclear how important body size really is for ecological diversification and niche filling, because body size is important for nearly every aspect of organismal function. Consequently, evolutionary change in body size can result not only from the competition that drives adaptive radiation, but also from predation pressure, reproductive character displacement, and physiological advantages of particular body sizes in a given environment, among other reasons [27].Despite the broad coverage of extinct species presented in Benson et al. [22], the data were insufficient to study another major part of adaptive radiation: early bursts of lineage diversification. While new approaches are becoming available to study diversification with phylogenies containing extinct species [28],[29] or with incomplete fossil data [30], these approaches are limited when many taxa are known from only single occurrences. This is the case in the Benson et al. dataset, and more generally in most fossil datasets.Given that few fossils exist for many extant groups, a major goal for future studies will be the incorporation of incomplete fossil information into analyses primarily focused on traits and clades for which mostly neontological data are available. For example, Slater et al. [31] developed an approach to include fossil information in analyses of phenotypic evolution. They showed that adding just a few fossils (12 fossils in a study of a 135-species clade) drastically increased the power and accuracy of their analyses of extant taxa. Thus, the combination of fossil data and those based on currently living species is important for future studies, as are new approaches that allow analyzing early bursts of lineage diversification along with phenotypic evolution in fossils.So what answers do Benson et al. [22] bring to Simpson''s original question of the importance of adaptive radiation for explaining diversity on earth? The authors present an intriguing and unconventional link between adaptive radiation and the diversity of modern-day birds. They argue that bird diversification was possible because the dinosaur lineage leading to birds did not exhaust niche space, potentially thanks to small body sizes; in contrast, other dinosaur groups adaptively radiated, filled niche space, and thus could not produce the ecological innovation that may have been necessary to survive the Cretaceous-Paleogene mass extinction. This intriguing hypothesis suggests an important role for the relative starting points of successive adaptive radiations in explaining current diversity, giving a new spin to the pivotal question raised by Simpson more than 60 years ago.     

Dopamine-Induced Conformational Changes in Alpha-Synuclein

Background

Oligomerization and aggregation of α-synuclein molecules play a major role in neuronal dysfunction and loss in Parkinson''s disease [1]. However, α-synuclein oligomerization and aggregation have mostly been detected indirectly in cells using detergent extraction methods [2], [3], [4]. A number of in vitro studies showed that dopamine can modulate the aggregation of α-synuclein by inhibiting the formation of or by disaggregating amyloid fibrils [5], [6], [7].

Methodology/Principal Findings

Here, we show that α-synuclein adopts a variety of conformations in primary neuronal cultures using fluorescence lifetime imaging microscopy (FLIM). Importantly, we found that dopamine, but not dopamine agonists, induced conformational changes in α-synuclein which could be prevented by blocking dopamine transport into the cell. Dopamine also induced conformational changes in α-synuclein expressed in neuronal cell lines, and these changes were also associated with alterations in oligomeric/aggregated species.

Conclusion/Significance

Our results show, for the first time, a direct effect of dopamine on the conformation of α-synuclein in neurons, which may help explain the increased vulnerability of dopaminergic neurons in Parkinson''s disease.     

The “Balance of Nature”—Evolution of a Panchreston

The earliest concept of a balance of nature in Western thought saw it as being provided by gods but requiring human aid or encouragement for its maintenance. With the rise of Greek natural philosophy, emphasis shifted to traits gods endowed species with at the outset, rather than human actions, as key to maintaining the balance. The dominance of a constantly intervening God in the Middle Ages lessened interest in the inherent features of nature that would contribute to balance, but the Reformation led to renewed focus on such features, particularly traits of species that would maintain all of them but permit none to dominate nature. Darwin conceived of nature in balance, and his emphasis on competition and frequent tales of felicitous species interactions supported the idea of a balance of nature. But Darwin radically changed its underlying basis, from God to natural selection. Wallace was perhaps the first to challenge the very notion of a balance of nature as an undefined entity whose accuracy could not be tested. His skepticism was taken up again in the 20th century, culminating in a widespread rejection of the idea of a balance of nature by academic ecologists, who focus rather on a dynamic, often chaotic nature buffeted by constant disturbances. The balance-of-nature metaphor, however, lives on in large segments of the public, representing a fragile aspect of nature and biodiversity that it is our duty to protect.The notion of a “balance of nature” stretches back to early Greeks, who believed gods maintained it with the aid of human prayers, sacrifices, and rituals [1]. As Greek philosophers developed the idea of natural laws, human assistance in maintaining the balance did not disappear but was de-emphasized. Herodotus, for instance, the earliest known scholar to seek biological evidence for a balance of nature, asked how the different animal species each maintained their numbers, even though some species ate other species. Amassing facts and factoids, he saw divinely created predators'' reproductive rates lower than those of prey, buttressing the idea of a providentially determined balance with a tale of a mutualism between Nile crocodiles beset with leeches and a plover species that feeds on them [1]. Two myths in Plato''s Dialogues supported the idea of a balance of nature: the Timaeus myth, in which different elements of the universe, including living entities, are parts of a highly integrated “superorganism,” and the Protagoras myth, in which gods created each animal species with characteristics that would allow it to thrive and, having run out of biological traits, had to give man fire and superior intelligence [1]. Among Romans, Cicero followed Herodotus and Plato in advancing a balance of nature generated by different reproductive rates and traits among species, as well as interactions among species [1].The Middle Ages saw less interest in such pre-set devices as differential reproductive rates to keep nature in balance, perhaps because people believed in a God who would maintain the balance by frequent direct intervention [1]. The Reformation, however, fostered further development of the concept of a providential balance of nature set in motion at creation. Thomas Browne [2] added differential mortality rates to factors maintaining the balance, and Matthew Hale [3] proposed that lower rates of mortality for humans than for other animals maintain human dominance within a balanced nature and added vicissitudes of heat from the sun to the factors keeping any one species from getting out of hand.The discovery of fossils that could not be ascribed to known living species severely challenged the idea of a God-given balance of nature, as they contradicted the idea of species divinely created with the necessary features for survival [4]. John Ray [5] suggested that the living representatives of such fossils would be found in unexplored parts of the earth, a solution that was viable until the great scientific explorations of the late 18th and early 19th centuries [4]. Ray also argued that what would now be termed different Grinnellian ecological niches demonstrated God''s provision of each species with a space of its own in nature.According to Egerton [1], the earliest use of the term “balance” to refer specifically to ecology was probably by Ray''s disciple, William Derham [6], who asserted in 1714 that:

“The Balance of the Animal World is, throughout all Ages, kept even, and by a curious Harmony and just Proportion between the increase of all Animals, and the length of their Lives, the World is through all Ages well, but not over-stored.”

Derham recognized that human populations seemed to be endlessly increasing but saw this fact as a provision by God for future disasters. This explanation contrasts with that of Linnaeus [7], who saw human and other populations endlessly increasing but believed the size of the earth was also increasing to accommodate them. Derham grappled with the issue of theodicy but failed to reconcile plagues of noxious animals with the balance of nature, seeing them rather as “Rods and Scourges to chastise us, as means to excite our Wisdom, Care, and Industry” [1].Derham''s contemporary Richard Bradley [8],[9] focused more on biological facts and less on Providence in sketching a more comprehensive account of an ecological balance of nature, taking account of the rapidly expanding knowledge of biodiversity, noting that each plant had its phytophagous insects, each insect its parasitic wasps or flies and predatory birds, concluding that “all Bodies have some Dependence upon one another; and that every distinct Part of Nature''s Works is necessary for the Support of the rest; and that if any one was wanting, all the rest must consequently be out of Order.” Thus, he saw the balance as fragile rather than robust, in spite of a constantly intervening God. Linnaeus [10] similarly marshaled observations of species interactions to explain why no species increases to crowd out all others, adding competition to the predation, parasitism, and herbivory adduced by Bradley and also emphasizing the different roles (we might now say “niches”) of different species as allowing them all to coexist in a sort of superorganismic, balanced whole.Unlike Derham, Georges-Louis Leclerc, Comte de Buffon [11] managed to reconcile animal plagues with a balanced nature. He perceived the balance of nature as dynamic, with all species fluctuating between relative rarity and abundance, so that whenever a species became overabundant, weather, predation, and competition for food would bring it back into balance. Buffon''s successor as director of the Jardin des Plantes in Paris, Jacques-Henri Bernardin de Saint-Pierre [12], was probably the first to associate ecological damage caused by biological invasions with a disruption of the balance of nature. Observing damage to introduced trees from insects accidentally introduced with them, he argued that failure to introduce the birds that would eat the insects led to the damage. William Paley [13], perhaps the inspiration for today''s advocates of “intelligent design,” analogized nature to a watch. One would assume a smoothly running watch was designed with purpose, and so too nature was designed by God with balance and a purpose.In the 19th century, evolution burst on the scene, greatly influencing and ultimately modifying conceptions of a balance of nature. Fossils that seemed unrelated to any living species, as noted above, conflicted with the balance of nature, because they implied extinction, a manifestly unbalanced event that furthermore could be seen to imply that God had made a mistake. Whereas Ray had been able to argue that living exemplars of fossil species would be found in unexplored parts of the earth, by the 19th century, this explanation could be rejected. Jean-Baptiste Lamarck [14] resolved the conflict in a different way, arguing that species continually change, so the balance remains the same. The fossils thus represent ancestors of living species, not extinct lineages. Robert Chambers [15], another early evolutionist, similarly saw fossils not as a paradox in a balanced nature but as a consequence of the fact that, as the physical environment changed, species either evolved or went extinct.Alfred Russel Wallace was perhaps the first to question the very existence of a balance of nature, in a remarkable notebook entry, ca. 1855:

“Some species exclude all others in particular tracts. Where is the balance? When the locust devastates vast regions and causes the death of animals and man, what is the meaning of saying the balance is preserved… To human apprehension there is no balance but a struggle in which one often exterminates another” [16].

In modern parlance, Wallace appears almost to be asking how “balance” could be defined in such a way that a balance of nature could be a testable hypothesis.Darwin''s theory of evolution by natural selection certainly explained the existence of fossils, and his emphasis on inevitable competition both between and within species downplayed the role of niche specialization propounded by Plato, Cicero, Linnaeus, Derham, and others [1]. Darwin nevertheless saw the ecological roles of the diversity of species as parts of an almost superorganismic nature, and his main contribution to the idea of a balance of nature was his constant emphasis on competition and other mortality factors that kept all species'' populations in check [1]. His many metaphors and examples of the interactions among species, such as the tangled bank and the spinsters-cats-mice-bumblebees-clover stories in The Origin of Species [17], contributed to a sense of a highly balanced nature, but one driven by natural selection constantly changing species, rather than by God either intervening or creating species with traits that ensure their continued existence. Unlike Wallace, Darwin did not raise the issue of whether nature was actually balanced and how we would know if it was not.As ecology developed in the late 19th and early 20th centuries, it was inevitable that Wallace''s question—how to define “balance”—would be raised again and that increasingly wide and quantitative study, especially at the population level, would be brought to bear on the matter. The work of the early dominant plant ecologist Frederic Clements and his followers, with Clements'' notion of superorganismic communities [18], provided at least tacit support for the idea of a balance of nature, but his contemporary Charles Elton [19], a founder of the field of animal ecology and a leading student of animal population cycles, forcefully reprised Wallace''s concern:

“‘The balance of nature’ does not exist, and perhaps never has existed. The numbers of wild animals are constantly varying to a greater or lesser extent, and the variations are usually irregular in period and always irregular in amplitude. Each variation in the numbers of one species causes direct and indirect repercussions on the numbers of the others, and since many of the latter are themselves independently varying in numbers, the resultant confusion is remarkable.”

Despite Elton''s explicit skepticism, his depiction of energy flow through food chains and food webs was incorporated as a superorganismic analog to the physiology of individuals (e.g., [20]). Henry Gleason, another critic of the superorganism concept, who depicted populations distributed independently, rather than in highly organized communities, was ignored at this time [21].However, beginning with three papers in Ecological Monographs in 1947, the superorganism concept was increasingly questioned and, within 25 years, Gleason was vindicated and his views largely accepted by ecologists [22]. During this same period, extensive work by population biologists again took up Elton''s focus on population trajectories and contributed greatly to a growing recognition of the dynamism of nature and the fact that much of this dynamism did not seem regular or balanced [21]. The idea of a balanced nature did not immediately disappear among ecologists. For instance, a noteworthy book by C. B. Williams [23], Patterns in the Balance of Nature, described the distribution of abundances within communities or regions as evincing statistical regularity that might be construed as a type of “balance of nature,” at least if changes in individual populations do not change certain statistical features (a hypothesis that Williams considered untested at the time). But the predominant view by ecologists of the 1960s saw the whole notion of a balance as, at best, irrelevant and, at worst, a distraction. Ehrlich and Birch [24], for example, ridiculed the idea:

“The existence of supposed balance of nature is usually argued somewhat as follows. Species X has been in existence for thousands or perhaps millions of generations, and yet its numbers have never increased to infinity or decreased to zero. The same is true of the millions of other species still extant. During the next 100 years, the numbers of all these species will fluctuate; yet none will increase indefinitely, and only a few will become extinct… Such ‘observations’ are made the basis for the statement that population size is ‘controlled’ or ‘regulated,’ and that drastic changes in size are the results of upsetting the ‘balance of nature.’”

Another line of ecological research that became popular at the end of the 20th century was to equate “balance of nature” with some sort of equilibrium of numbers, usually of population sizes [25], but sometimes of species richness. The problem remained that, with numbers that vary for whatever reason, it is still arbitrary just how much temporal variation can be accommodated within a process or phenomenon for it still to be termed equilibrial [26]. Often the decision on whether to perceive an ecological process as equilibrial seems to be based on whether there is some sort of homeostatic regulation of the numbers, such as density-dependence, which A. J. Nicholson [27] suggested as an argument against Elton''s skepticism of the existence of a balance. The classic 1949 ecology text by Allee et al. [28] explicitly equated balance with equilibrium and cited various mechanisms, such as density-dependence, in support of its universality in nature [25]. Later similar sorts of mathematical arguments equated the mathematical stability of models representing nature with a balance of nature [29], although the increasing recognition of stochastic aspects and chaotic mathematics of population fluctuations made it more difficult to perceive a balanced nature in population trajectories [21].For academic ecologists, the notion of a balance of nature has become passé, and the term is widely recognized as a panchreston [30]—a term that means so many different things to different people that it is useless as a theoretical framework or explanatory device. Much recent research has been devoted to emphasizing the dynamic aspects of nature and prominence of natural or anthropogenic disturbances, particularly as evidenced by vicissitudes of population sizes, and advances the idea that there is no such thing as a long-term equilibrium (e.g., [31],[32]). Some authors explicitly relate this research to a rejection of the concept of a balance of nature (e.g., [33]–[35]), Pickett et al. [33] going so far as to say it must be replaced by a different metaphor, the “flux of nature.”The issue is confounded by the fact that the perception of balance can be sought at different levels (populations, communities, ecosystems) and spatial scales. Much of the earlier discussion of a balance was at the population and community levels—Browne, Hale, Bradley, Linnaeus, Buffon, Bernardin de Saint-Pierre, and Darwin saw balance in the limited fluctuations of populations and the interactions of populations as one force imposing the limits. The proponents of density-dependent population regulation fall in this category as well [36],[37]. As a balance is sought at the community and ecosystem levels, the sorts of evidence brought to bear on the matter become more complicated and abstract [37],[38]. It is increasingly difficult to imagine what sorts of empirical or observational data could test the notion of a balance. For instance, Williams''s balance of nature—evidenced by a particular statistical distribution of population sizes—would not be perceived as balanced by many observers in light of the fact that entire populations can crash, explode, or even go extinct within the constraint of a statistical distribution of a given shape. Early claims of a balance at the highest level, such as the various superorganisms (Plato''s Timaeus myth, Paley''s watch metaphor, Clements''s superorganismic plant community) can hardly be seen as anything other than metaphors rather than testable hypotheses and have fallen from favor. The most expansive conception of a balance of nature—the Gaia hypothesis [39]—has been almost universally rejected by scientists [40]. The advent and growing acceptance of the metapopulation concept of nature [41] also complicates the search for balance in bounded population fluctuations. Spatially limited individual populations can arise, fluctuate wildly, and even go extinct, while suitable dynamics maintain the widespread metapopulation as a whole.Yet, the idea of a balance of nature lives on in the popular imagination, especially among conservationists and environmentalists. However, the usual use of the metaphor in an environmental context suggests that the balance, whether given by God or produced by evolution, is a fragile balance, one that needs human actions for its maintenance. Through the 18th century, the balance of nature was probably primarily a comforting construct—it would protect us; it represented some sort of benign governance in the face of occasional awful events. When Darwin replaced God as the determinant of the balance with natural selection, the comfort of a balance of nature was not so overarching, if there was any comfort at all. Today, ecologists do not even recognize a balance, and those members of the public who do, see it as something we must protect if we are ever to reap benefits from it in the future (e.g., wetlands that might help ameliorate flooding from storms and sea-level rise). This shift is clear in the writings of Bill McKibben [42],[43], who talks frequently about balance, but about balance with nature, not balance of nature, and how humankind is headed towards a catastrophic future if it does not act promptly and radically to rebalance society with nature.     

Epidemiology and Genetic Diversity of Rotavirus Strains in Children with Acute Gastroenteritis in Lahore,Pakistan

Pakistan harbors high disease burden of gastro-enteric infections with majority of these caused by rotavirus. Unfortunately, lack of proper surveillance programs and laboratory facilities have resulted in scarcity of available data on rotavirus associated disease burden and epidemiological information in the country. We investigated 1306 stool samples collected over two years (2008–2009) from hospitalized children under 5 years of age for the presence of rotavirus strains and its genotypic diversity in Lahore. The prevalence rate during 2008 and 2009 was found to be 34% (n = 447 out of 1306). No significant difference was found between different age groups positive for rotavirus (p>0.05). A subset of EIA positive samples was further screened for rotavirus RNA through RT-PCR and 44 (49.43%) samples, out of total 89 EIA positive samples, were found positive. G and P type prevalence was found as follows: G1P [4] = 3(6.81%); G1P [6] = 9(20.45%); G1P [8] = 1(2.27%); G2P [4] = 21(47.72%); G2P [8] = 1(2.27%); G9P [4] = 1(2.27%); G9P [6] = 1(2.27%) and G9P [8] = 7(15.90%). Phylogenetic analysis revealed that the VP7 and VP4 sequences clustered closely with the previously detected strains in the country as well as Belgian rotaviruses. Antigenic characterization was performed by analyzing major epitopes in the immunodominant VP7 and VP4 gene segments. Although the neutralization conferring motifs were found variable between the Pakistani strains and the two recommended vaccines strains (Rotarix™ and RotaTeq™), we validate the use of rotavirus vaccine in Pakistan based on the proven and recognized vaccine efficacy across the globe. Our findings constitute the first report on rotavirus’ genotype diversity, their phylogenetic relatedness and epidemiology during the pre-vaccination era in Lahore, Pakistan and support the immediate introduction of rotavirus vaccine in the routine immunization program of the country.     

Viral OTU Deubiquitinases: A Structural and Functional Comparison

Recent studies have revealed that proteases encoded by three very diverse RNA virus groups share structural similarity with enzymes of the Ovarian Tumor (OTU) superfamily of deubiquitinases (DUBs). The publication of the latest of these reports in quick succession prevented proper recognition and discussion of the shared features of these viral enzymes. Here we provide a brief structural and functional comparison of these virus-encoded OTU DUBs. Interestingly, although their shared structural features and substrate specificity tentatively place them within the same protease superfamily, they also show interesting differences that trigger speculation as to their origins.The covalent attachment of ubiquitin (Ub) to protein substrates, i.e., ubiquitination, plays a pivotal regulatory role in numerous cellular processes [1]–[5]. Ubiquitination can be reversed by deubiquitinases (DUBs) [6] and, not surprisingly, various virus groups encode such DUBs to influence ubiquitin-mediated host cell processes [7]–[21]. Some of these viral DUBs resemble proteases belonging to the Ovarian Tumor (OTU) superfamily [22]–[28]. Makarova et al. previously identified OTU proteases as a novel superfamily of cysteine proteases from different organisms [29], and their bioinformatics-based analysis included several of the viral enzymes discussed here. Recently reported structures of these viral DUBs include the OTU domains of the nairoviruses Crimean-Congo hemorrhagic fever virus (CCHFV) [22]–[24] and Dugbe virus (DUGV) [25], the papain-like protease (PLP2) domain of the arterivirus equine arteritis virus (EAV) [26], and the protease (PRO) domain of the tymovirus turnip yellow mosaic virus (TYMV) (Figure 1A–1D) [27], [28]. These viruses are strikingly diverse, considering that nairoviruses are mammalian negative-strand RNA viruses, while the mammalian arteriviruses and plant tymoviruses belong to separate orders of positive-strand RNA viruses.Open in a separate windowFigure 1Viral and eukaryotic OTU domain structures and viral protein context.Crystal structures of (A) CCHFV OTU (3PT2) [23], (B) DUGV OTU (4HXD) [25], (C) EAV PLP2 (4IUM) [26], (D) TYMV PRO (4A5U) [27], [28], (E) yeast OTU1 (3BY4) [57], and (F) human OTUD3 (4BOU) [46]. The β-hairpin motifs of CCHFV OTU and DUGV OTU are indicated in boxes in panels A and B, respectively, and the zinc-finger motif of EAV PLP2 is boxed in panel C. Active sites are indicated with arrows. The CCHFV OTU, DUGV OTU, EAV PLP2, and yeast OTU1 domains were crystallized in complex with Ub, which has been removed for clarity. Structure images were generated using PyMol [60]. (G) Schematic representation of the CCHFV large (L) protein [61], [62]. A similar organization is found in the DUGV L protein, but is not depicted. The OTU domain resides in the N-terminal region of this protein and is not involved in autoproteolytic cleavage events [48]. (H) Schematic representation of the EAV polyprotein 1ab [63]. PLP2 resides in nonstructural protein 2 (nsp2) and is responsible for the cleavage between nsp2 and nsp3 [51]. (I) Schematic representation of the TYMV ORF1 polyprotein [50]. PRO resides in the N-terminal product of this polyprotein and is responsible for two internal cleavages [49], [50]. Key replicative enzymes are indicated in G, H, and I. Colored arrowheads denote cleavage sites for the indicated protease domains. HEL, helicase; PLP, papain-like protease; RdRp, RNA-dependent RNA polymerase; SP, serine protease.Ubiquitination often involves the formation of polyubiquitin chains [1], which can target the ubiquitinated substrate to the proteasome for degradation [2] or modulate its protein–protein interactions, as in the activation of innate immune signaling pathways [3], [4]. Interestingly, several cellular OTU DUBs were found to negatively regulate innate immunity [30]–[33]. Likewise, both nairovirus OTU and arterivirus PLP2 were recently shown to inhibit innate immune responses by targeting ubiquitinated signaling factors [7]–[9], [26], [34], [35]. In contrast to eukaryotic OTU DUBs, both of these viral proteases were found to also deconjugate the Ub-like protein interferon-stimulated gene 15 (ISG15) [7], [36], which inhibits viral replication via a mechanism that is currently poorly understood [37]. Interestingly, coronaviruses (which, together with the arteriviruses, belong to the nidovirus order) also encode papain-like proteases targeting both Ub and ISG15 that were shown to inhibit innate immunity [11]–[13], [38]–[42] but belong to the ubiquitin-specific protease (USP) class of DUBs [6], [43], [44]. The presence of functionally similar, yet structurally different proteases in distantly related virus families highlights the potential benefits to the virus of harboring such enzymes.The proteasomal degradation pathway is an important cellular route to dispose of viral proteins, as exemplified by the turnover of the TYMV polymerase [45]. Moreover, the degradation of this protein is specifically counteracted by the deubiquitinase activity of TYMV PRO, which thus promotes virus replication [10]. The functional characterization of viral OTU DUBs remains incomplete and future studies will likely reveal additional roles in replication and virus–host interplay.Polyubiquitin chains can adopt a number of different configurations, depending on the type of covalent linkage present within the polymer [1]. A distal Ub molecule can be linked via its C-terminus to one of seven internal lysine residues present in a proximal Ub molecule via an isopeptide bond. Alternatively, in the case of linear chains, the C-terminus of the distal Ub is covalently linked to the N-terminal methionine residue of the proximal Ub via a peptide bond. While human OTU proteases often show a distinct preference for one or two isopeptide linkage types [46], nairovirus OTUs and TYMV PRO appear to be more promiscuous in their substrate preference [22], [25]. However, like most human OTU proteases, they seem unable to cleave linear polyubiquitin chains in vitro [22], [25], [46]. Arterivirus PLP2 has not been extensively studied in this respect.It is important to note that many positive-strand RNA viruses, including arteriviruses and tymoviruses, encode polyproteins that are post-translationally cleaved by internal protease domains [47]. Thus, while CCHFV OTU is not involved in viral protein cleavage and its activity seems dispensable for replication (Figure 1G) [48], both arterivirus PLP2 and tymovirus PRO are critically required for viral replication due to their primary role in polyprotein maturation (Figure 1H, 1I) [49]–[53]. Interestingly, while both EAV PLP2 and TYMV PRO can process peptide bonds in cis and in trans [50], [51], PRO does not cleave peptide bonds in linear polyubiquitin chains in vitro [25]. To date, activity of EAV PLP2 towards linear polyubiquitin chains has not been reported.Based on mutagenesis of putative catalytic residues, arterivirus PLP2 and tymovirus PRO were initially generally classified as papain-like cysteine proteases [51], [54], [55]. Now that crystal structures of these proteases are available, it is possible to search the DALI server [56] in order to identify structurally similar domains. Using the 3-dimensional coordinates of TYMV PRO, the most recently solved structure of a viral OTU protease, such a query identifies structural similarity with eukaryotic OTU DUBs as well as the nairovirus OTU domains and EAV PLP2 ([57] further highlights their similarities (Figure 2A–2C), and these comparisons together clearly position them within the OTU DUB superfamily. Sequence comparisons alone were insufficient to demonstrate this conclusively, as the similarity of viral OTU domains to each other and to eukaryotic OTU proteases is very limited and mostly restricted to the areas surrounding the active site residues [29].Open in a separate windowFigure 2Superpositions of the viral OTU proteases with yeast OTU1 and one another.Superpositions of yeast OTU1 (3BY4) [57] with (A) CCHFV OTU (3PT2) [23], RMSD: 1.8 Å over 112 residues, (B) EAV PLP2 (4IUM) [26], RMSD: 2.8 Å over 69 residues, and (C) TYMV PRO (4A5U) [27], [28], RMSD: 1.4 Å over 76 residues. Superpositions of the yeast OTU1-Ub complex with (D) the CCHFV OTU-Ub complex and (E) the EAV PLP2-Ub complex, highlighting the difference in the orientation of Ub between the two viral OTU domains versus the eukaryotic yeast OTU1 domain. The Ub that is complexed with yeast OTU1 is depicted in yellow, while the Ub complexed with CCHFV OTU or EAV PLP2 is depicted in orange. (F) Superposition of EAV PLP2 and TYMV PRO, RMSD: 2.5 Å over 53 residues. (G) Close-up of the active site region (boxed) of the superposition depicted in F. Side chains of the catalytic cysteine (Cys270 and Cys783 for EAV PLP2 and TYMV PRO, respectively) and histidine (His332 and His869 for EAV PLP2 and TYMV PRO, respectively) residues are shown as sticks, as well as the active site Asn263 for EAV PLP2. The backbone amide group of Asp267 likely contributes to the formation of the oxyanion hole in the active site of EAV PLP2, yet a functionally equivalent residue is absent in TYMV PRO. The Gly266 and Gly268 residues flanking Asp267 in EAV PLP2 are depicted as sticks as well, for clarity. Note the alternative orientation of the active site cysteine residue of TYMV PRO which, unlike EAV PLP2, was not determined in covalent complex with an Ub suicide substrate. All alignments were generated using the PDBeFOLD server [64], and thus the reported RMSD values differ from those reported in [60]. RMSD, root-mean-square deviation.

Table 1

Three-dimensional structural alignment of viral OTU domains against selected structures in the Protein Data Bank using the DALI server [56].

Inhibition of mTOR by Rapamycin Abolishes Cognitive Deficits and Reduces Amyloid-β Levels in a Mouse Model of Alzheimer's Disease

Background

Reduced TOR signaling has been shown to significantly increase lifespan in a variety of organisms [1], [2], [3], [4]. It was recently demonstrated that long-term treatment with rapamycin, an inhibitor of the mTOR pathway[5], or ablation of the mTOR target p70S6K[6] extends lifespan in mice, possibly by delaying aging. Whether inhibition of the mTOR pathway would delay or prevent age-associated disease such as AD remained to be determined.

Methodology/Principal Findings

We used rapamycin administration and behavioral tools in a mouse model of AD as well as standard biochemical and immunohistochemical measures in brain tissue to provide answers for this question. Here we show that long-term inhibition of mTOR by rapamycin prevented AD-like cognitive deficits and lowered levels of Aβ₄₂, a major toxic species in AD[7], in the PDAPP transgenic mouse model. These data indicate that inhibition of the mTOR pathway can reduce Aβ₄₂ levels in vivo and block or delay AD in mice. As expected from the inhibition of mTOR, autophagy was increased in neurons of rapamycin-treated transgenic, but not in non-transgenic, PDAPP mice, suggesting that the reduction in Aβ and the improvement in cognitive function are due in part to increased autophagy, possibly as a response to high levels of Aβ.

Conclusions/Significance

Our data suggest that inhibition of mTOR by rapamycin, an intervention that extends lifespan in mice, can slow or block AD progression in a transgenic mouse model of the disease. Rapamycin, already used in clinical settings, may be a potentially effective therapeutic agent for the treatment of AD.