Self-assessment scores regarding the learning objectives related to upper limbs were significantly higher in experimental group than in control group. Additionally, experimental group received significantly higher academic scores than did control group. The students in peer-assisted learning perceived themselves as having a better understanding of course content and achieved better academic results compared with those who participated in faculty-led dissection. Peer-assisted dissection contributed to self-perception and to the ability to retain and explain anatomical knowledge.Peer-assisted learning encourages students to participate more actively in the dissection process and promotes thoughtful dissection. We implemented peer-assisted dissection in 2012 and compared its effects on students’ self-assessments of learning and their academic achievement with those of faculty-led dissection. All subjects performed dissections after a lecture about upper-limb gross anatomy. Experimental group ( Cadaver dissection is an irreplaceable part of anatomy education that enriches the student experience through a close encounter with human mortality –3. HandsAs a form of cooperative learning, peer-assisted learning promotes more active engagement by students in the learning process and prevents them from leaving the course . Peer tuSeveral reports have compared the effect of peer-assisted versus faculty-led learning, and indicated that students’ academic achievement after peer-assisted learning was as effective as that of faculty-led learning in clinical skills training and problem-based curricula in health sciences or law –11. HoweThe study subjects consisted of students who were in their first year of medical school at Chonnam National University Medical School in 2012 and 2008 and who had been trained in gross anatomy dissection during their first semester. All subjects had completed a lecture about upper-limb gross anatomy as part of the standard curriculum. The dissection course consisted of three 2-h sessions designed to independently address the shoulder joint, axilla, elbow joint, forearm, wrist joint, and hand. An intact cadaver was allocated to every subgroup during the dissection course.Experimental group consisted of 134 students who were in their first year of medical school in 2012 and who participated in peer-assisted learning in the dissection laboratory. This group was divided into 20 subgroups (average size: 6.7 students) and three subgroups took the roles of tutors in the other subgroups by rotation at every dissection course. In other words, twenty-one students in three subgroups served as tutors, and one or two tutors were assigned to each of the other 17 subgroups during the dissection course. The roles of students were then switched at the next course. Tutors dissected the intact parts of the upper limb in advance with the guidance of textbooks and video clips; they could ask questions to a faculty member if they were facing difficulties. Members of the three tutor subgroups provided a 10-minute explanation of this practice to each tutee subgroup before the start of the dissection. The explanation addressed issues such as important anatomical structures and how to perform a dissection and identified the structures that should be carefully manipulated. During the dissection course itself, one or two tutors assisted each of 17 subgroups while they performed the dissections.Control group consisted of 71 students who were in their first year of medical school in 2008 and who were randomly assigned to faculty-led learning groups involving prosections. They were divided into 10 subgroups. A student representative from each subgroup reviewed the parts being dissected with the faculty member using a prosection. After a 30-min introduction covering topics that would subsequently be addressed in the course, each representative shared this information with other students in her/his subgroup during the dissection course. All students of each subgroup participated actively in the dissection of the entire cadavers.The Chonnam National University Hospital Institutional Review Board determined that the study was exempt from human subject research regulations. Participation in the survey was voluntary, and students were informed that their answers were confidential. Written consent was obtained from all participants.All participating students were asked to complete a short questionnaire requesting demographic information and to rate their knowledge with regard to the upper-limb learning objectives. The learning objectives were addressed by 12 items based on the core anatomy syllabus  and the Immediately after completing the lectures and dissection courses, all students completed a written examination that consisted of multiple-choice, short-answer, and essay questions. The identical faculty, who delivered the lectures and guided the dissection courses in two groups, set the exam questions based on the learning objectives. The examination in 2012 was conducted in the same way as that in 2008, to compare academic results between two groups. The subjects in 2012 were blinded to the fact that their academic results would be compared with those of the students in 2008.U-test was used to compare self-perceptions regarding learning objectives and academic achievement between groups. All analyses were performed using SPSS software version 21.0 .We performed a chi-square test to assess similarities and differences in the general characteristics of the participants. A Mann–Whitney The general characteristics of all participants are presented in The self-assessment scores on all but one item of the 12-item questionnaire addressing upper-limb learning objectives were significantly higher in experimental group, which performed peer-assisted dissection, than in control group, which performed faculty-led dissection. With the exception of the ability to describe the position and function of the retinacula of the wrist and the tendon sheaths of the wrist and hand, students in experimental group perceived themselves as having a better understanding of the major structures and their courses and functions than did those in control group .The total self-assessment scores were also higher in experimental group than in control group. Additionally, experimental group achieved significantly higher academic scores than did control group .Peer-assisted learning in a dissection course enhanced medical students’ assessment in their understanding of the multidimensional structure of the human body and led to better academic results compared with faculty-led dissection. Specifically, peer tutoring experiences increase students’ understanding of course content and their retention of the information they taught to their peers .The stereotype of anatomy as endless memorization can cause students to approach dissection with a negative attitude . HoweverThis effect of peer-assisted learning was predictable insofar as this approach allows more time for the tutors to responsibly prepare, improve their teaching and communication skills, and to become more confident in self-directed learning . In contThis study compared the learning effect of peer-assisted and faculty-led dissection, but it was limited in its ability to perform direct comparisons. First, we used different subjects at different times, so it might have had additional effects on the results. Although it was not available to compare the medical college admission tests or other parameters between two groups, the groups did not significantly differ with respect to basic characteristics; all entered the same medical school with similar levels of competence. Upper-limb anatomy education was investigated as the area of this research, which was presented by the same teacher in the unchanged curriculum from 2008 to 2012 and its outcomes were available for the evaluation in the same way between different time periods. Second, the assessment as an outcome measure was restricted to knowledge-based examinations. This study did not assess student performance such as individual dissection skills and peer group interactions. Another limitation was that student tutors had different levels of competence and were not prepared for peer tutoring through extra-education. As Cloward suggested, tutors do not have to be highly trained or excellent students . Since sDespite these limitations, our results are still meaningful, as we used objective measures rather than merely students’ perceptions to compare the academic results of peer-assisted and faculty-led dissection. The learning outcome of peer-assisted dissection compared favorably with that of faculty-led dissection. Our study suggests several far-reaching benefits of peer-assisted learning. First, it can encourage students to participate more actively in the dissection process. Second, it is one of the effective learning methods to promote students’ academic achievement. Last, it should be an attractive alternative for the institution to alleviate the lack of faculty compared to the number of students as well.The students in peer-assisted learning perceived themselves as having a better understanding of course content and achieved better academic results. Students who serve as tutors are better able to acquire knowledge by teaching and tutees are encouraged to actively participate rather than act as bystanders. Institutions should offer programs that help students understand the meaning of peer-assisted learning, to learn how to become effective tutors, and to provide constructive feedback to peers.S1 Table(SAV)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Need for the study: The most important procedure for a successful endodontic treatment is the cleaning and shaping of the canal system. Understanding the internal anatomy of teeth provides valuable information to the clinician that would help him achieve higher clinical success during endodontic therapy.Aims: To evaluate by computed tomography—the internal anatomy of mandibular second primary molars with respect to the number of canals, cross-sectional shape of canals, cross-sectional area of canals and the root dentin thickness.Materials and methods: A total of 31 mandibular second primary molars were subjected to computed-tomographic evaluation in the transverse plane, after mounting them in a prefabricated template. The images, thus, obtained were analyzed using De-winter Bio-wizard® software.Results: All the samples demonstrated two canals in the mesial root, while majority of the samples (65.48%) demonstrated two canals in the distal root. The cross-sectional images of the mesial canals demonstrated a round shape, while the distal canals demonstrated an irregular shape. The root dentin thickness was highly reduced on the distal aspect of mesial and mesial aspect of distal canals.Conclusion: The mandibular second primary molars demonstrated wide variation and complexities in their internal anatomy. A thorough understanding of the complexity of the root canal system is essential for understanding the principles and problems of shaping and cleaning, determining the apical limits and dimensions of canal preparations, and for performing successful endodontic procedures.How to cite this article: Kurthukoti AJ, Sharma P, Swamy DF, Shashidara R, Swamy EB. Computed Tomographic Morphometry of the Internal Anatomy of Mandibular Second Primary Molars. Int J Clin Pediatr Dent 2015;8(3):202-207. In comparison with permanent teeth, the relatively thin layer of mineralized hard tissue between the external and internal surfaces leads to rapid involvement of the dental pulp by the advancing caries. Nonetheless, conservation of primary teeth is deemed advantageous for maintenance of arch length and harmonized temporal and facial development of permanent teeth. Pulpectomy of primary teeth is indicated when the dental pulp is irreversibly inflamed or non-vital.2The main objective of root canal therapy is thorough shaping and cleaning of all pulp spaces and its complete obturation with an inert filling material. The presence of an untreated canal may be a reason for failure. A canal may be left untreated because the dentist fails to recognize its presence. It is extremely important that clinicians use all the armamentaria at their disposal to locate and treat the entire root canal system. It is humbling to be aware of the complexity of the spaces we are expected to access, shape, clean and fill. We can take comfort in knowing that even under the most difficult circumstances, our current methods of root canal therapy result in an exceptionally high rate of success.1Primary teeth may often show bizarre internal geometry of the pulp cavity, with features not commonly observed in permanent teeth, such as variable shapes in cross-section, accessory canals in furcation area and horizontal anastomoses; rendering them difficult subjects for endodontic therapy. Even in contemporary dental practice, the prevailing notion that bizarre and tortuous root canals of primary teeth may not be adequately negotiated, cleaned, shaped or filled, has brought about needless sacrifice of carious primary teeth.3The evolution of the dentistry has been paralleled by that of the systems to study the internal anatomy of teeth , with different levels of complexity and of sample precision. Novel technologies, such as computed tomography have been directed to analyze the internal geometry of root canals; employing reconstruction and visualization methods that allow the canals to be observed three-dimensionally for treatment without destroying the specimen under study.There is paucity of currently available literature on the internal anatomic variations of mandibular second primary molar teeth using contemporary noninvasive imaging technology. Hence, this study was undertaken to: Evaluate the number and shape of canals in mandibu-lar second primary molars. Evaluate the root canal cross-sectional area at three pre-determined levels, and the root dentin thickness in mandibular second primary molars.4A total of 31 mandibular primary second molar teeth extracted for therapeutic reasons, were washed under running water and all the soft tissue was scraped from their crown and root surfaces using a periodontal scaler. The teeth were then autoclaved and stored in distilled water in air-tight containers, according to ISO guidelines.The root surfaces of the selected samples were coated with a thin layer of separating media and the teeth were embedded in the unset cold cure acrylic, such that each individual sample could be separated from their respective block and again repositioned, if needed . The teeThe computed tomographic (CT) slices were then taken at three pre-determined levels. Initially, the length of each root of each of the specimen was determined using the CT slices beginning from the furcation till the root tip. This length was divided into three equal parts. The coronal two-thirds thus obtained was again divided into two subsequent equal parts. These three levels determined served as observational zones for evaluation of root canal configuration. Approaching from the furcation, the designated levels were : The appearance of furcation was designated as X1. The mid-point of the coronal two-third root length measurement designated as X2. The apical most point of the coronal two-third root length measurement designated as level X3.® Software. The number of canals present in each root was counted. The root canal cross-sectional shapes at X1, X2 and X3 were recorded as  Irregular with sharp angles or slot, including fins and perforations.To assess the root dentin thickness, the shortest distance from the canal outline to the closest adjacent root surface was measured at each predetermined level using the imaging software.To analyze interobserver agreement, 10% of the measurements were repeated at random and verified with an intraclass correlation coefficient.The mean area of the individual root canals at all the three levels was calculated in sq. mm. 5 Internal complexities of the root canal are genetically determined and have definitive importance in anthropology.6 These factors necessitate the identification of a method that accurately determines the root canal morphology.Successful endodontic therapy stems from thorough canal debridement and effective filling of the root canal system, for which knowledge of morphology of the root canals is a critical prerequisite.3There are numerous reports on the root canal morphologies of different populations, which is extremely important for an endodontist as well as general dental practitioners. Also of interest to us are the methods that have been used in these studies.3The methods most commonly used in analyzing the root canal morphology are canal staining and tooth clearing, conventional radiographs, digital and contrast medium-enhanced radiographic techniques, radiographic assessment enhanced with contrast media, and more recently computed tomographic techniques.7 A CT scan uses a fan-shaped beam and multiple exposures around an object to reveal the internal architecture of this object, thereby helping the clinician to view morphologic features as well as pathology from different three-dimensional (3D) perspectives. The distinct advantage of a CT scan is that it allows for 3D reconstruction of root canal systems. Computed tomography scanning has been suggested as the preferential imaging modality in difficult situations demanding localization and description of root canal systems because of its ability to render 3D information.89The application of CT scans in endodontics was first reported by Tachibana and Matsumoto in 1990.In our study, the number of root canals visualized in the mesial roots of mandibular second primary molars was two, i.e. mesiobuccal and mesiolingual. However, wide variations were observed when it came to the evaluation of the number of canals in the distal root. While 35.48% of the distal roots presented with a single canal, 64.52% of the roots presented two canals, i.e. distobuccal and distolingual.10 However, a more recent study reports incidence rates as high as 53%.11 Aminabadi et al have reported 100% incidence of two canals in Iranian population.1 There are several explanations for this disagreement, one of which may be ethnicity of the subjects and another may be the difference in techniques used for assessment of internal morphology.1The incidence of two canals in the distal root of man-dibular second primary molars is determined by genetics and anthropometry. Previous researchers have reported 21 to 28% incidence of two canals in the distal roots of mandibular second primary molars.12Together with diagnosis and treatment planning, knowledge of the canal morphology and its frequent variations is a basic requirement for endodontic success. Stressing the significance of canal anatomy, Peters et al reported that variations in canal geometry before shaping and cleaning procedures had more influence on the changes that occur during preparation than the instrumentation techniques themselves.The predominantly observed cross-sectional morphology in the mesial canals at all three pre-determined levels was round. However, the cross-sectional canal morphology in the distal canals was more irregular in the coronal and middle third, and predominantly round in the apical third. This highlights the need for greater emphasis on a standardized funnel shaped preparation along the entire length of the canal as there is wide variation in the cross-sectional morphologies at all three levels.11The second most common cross-sectional shape observed was the irregular shape, the least observed shapes being oblong and oval. These observations highlight the need for a biomechanical preparation procedure that results in a standardized shape to ensure satisfactory fill. Previous studies have found oval or triangular shape to be the predominant cross-sectional shape in mandibular primary molars.11The cross-sectional area of the distal canals was greater than the mesial canals at all the three pre-deter-mined levels. This could be attributed to the ribbon/ irregular shape of distal canals. The root canal area assessment revealed that the canals showed a decrease in cross-sectional area, in accordance with the root canal external morphology and it’s taper from the coronal to apical third. Similar observations were made previously by other researchers as well.For successful endodontic therapy, satisfactory preparation of the coronal and middle third portion of the root canal is mandatory; as this allows the removal of interferences permitting better access to the apical third of the root canal. However, root perforation is a possible consequence of flaring that results in treatment failure.13 Thus, the root dentin thickness (RDT) is important because the amount of root dentin remaining enables the endodontically treated teeth to resist fracture; so, more the removal of dentin during instrumentation, more is the potential for perforation or fracture.Also, flaring the canal excessively to allow instrumentation with larger files decreases the root dentin thickness, thus increasing the possibility of vertical root fracture. Recent research permits the use of rotary endodontic file systems in primary teeth as well.14 In addition, thin dentinal walls increase permeability and fracture rate of teeth.The mesial canals of mandibular permanent first molars are not located in the center of the root and the areas between the canals, and also, between the canals and the furcation area have thin dentin walls; and are therefore called ‘danger zones’. Danger zones have less dentin in the ramification areas in comparison with the peripheral safe root areas. Therefore, over-preparing the cervical and middle thirds of the root canal might result in thinning of the dentinal walls and sometimes cause strip perforations in the furcation area.15 Currently, available literature limits the concept of danger zone to the mandibular first permanent molar teeth. However, our study emphasizes that the apical third of the root canals of deciduous mandibular second primary molars are highly fragile as well. The greater curvature of the roots and the reduced root dentin thickness toward the furcation make primary teeth vulnerable to root perforations and resultant complications. Hence, it would be prudent to brand these areas as ‘danger zones’ of the mandibular second primary molars.Observations of the remaining dentin thickness point to the fact that RDT reduces as we move coronoapically and also that this thickness is minimal on the root side toward furcation as compared to the side away. Reports suggest that the thickness of the distal wall of mesial roots in the permanent mandibular molars was merely 1.2 to 1.3 mm, just 1.5 mm apical to the furcation.These observations highlight the need for exercising caution while preparing the apical portion of the root canals of these mandibular second primary molars. These findings also raise serious questions regarding the use of some of the aggressive rotary file systems in primary molars, especially in the apical thirds of the root canals.Within the limitations of the study, the following conclusions can be drawn: The mesial roots of the mandibular second primary molars commonly demonstrate two root canals, while the distal root of these teeth demonstrated two canals in 64.52% of the samples. The cross-sectional shapes of both mesial and distal canals of the mandibular second primary molars demonstrated wide variations at the pre-determined coronal, middle and apical levels. While the mesial canals predominantly demonstrated a round cross-sectional shape at all three levels, the distal canals demonstrated irregular or round cross-sectional shapes in the coronal and middle third, and round shape in the apical third of the teeth. The cross-sectional area of both mesial and distal canals demonstrated a gradual reduction from coronal third to the apical third. However, the cross-sectional area of the distal canals was considerably larger than the combined area of both the mesiobuccal and mesiolingual canals. The root dentin thickness on the distal wall of the mesial root and the mesial wall of the distal roots were reduced highlighting an existing ‘danger zone’, that is more prone for fractures and perforations during biomechanical preparation. This dentin thickness was minimal in the apical third of these walls in both the roots.This study was a modest attempt to gain insight into the internal anatomy of mandibular second primary molar teeth that would help the clinicians achieve better success in endodontic practice. This study however, did not consider the inter-racial differences commonly seen in root morphology among different populations. We recommend future studies considering these criteria.


Once widespread in the Eastern United States, early 20th century predator-control programs reduced red wolves to a remnant population by the 1970s. The U.S. Fish and Wildlife Service, through the Red Wolf Recovery Program, restored red wolves to northeastern North Carolina in 1987. After 25 years of restoration efforts, issues of hybridization with coyotes, inbreeding, and human-caused mortality continue to hamper red wolf recovery. To understand how these issues influence recovery efforts, we examine the history of red wolf restoration and its challenges. We then formulate areas of research that are of direct relevance to the restoration of red wolves.By the 1970s, government-supported eradication campaigns reduced red wolves to a remnant population of less than 100 individuals on the southern border of Texas and Louisiana. Restoration efforts in the region were deemed unpromising because of predator-control programs and hybridization with coyotes. The U.S. Fish and Wildlife Service (USFWS) removed the last remaining red wolves from the wild and placed them in a captive-breeding program. In 1980, the USFWS declared red wolves extinct in the wild. During 1987, the USFWS, through the Red Wolf Recovery Program, reintroduced red wolves into northeastern North Carolina. Although restoration efforts have established a population of approximately 70–80 red wolves in the wild, issues of hybridization with coyotes, inbreeding, and human-caused mortality continue to hamper red wolf recovery. We explore these three challenges and, within each challenge, we illustrate how research can be used to resolve problems associated with red wolf-coyote interactions, effects of inbreeding, and demographic responses to human-caused mortality. We hope this illustrates the utility of research to advance restoration of red wolves. Canis lupus) and red wolves (Canis rufus) existed as declining remnant populations in the contiguous United States. Although gray wolf populations in Alaska and Canada were stable and the species was not threatened with extinction, red wolves were afforded no refuge. Red wolves were likely the first New World wolf species to come in contact with Europeans and, consequently, the first to be persecuted. Prior to European colonization, red wolves were common in the Eastern United States and they inhabited an area from the Atlantic coast west to central Texas, with the Ohio River Valley, Northern Pennsylvania, and Southern New York being its northernmost range and their distribution extending south to the Gulf of Mexico (Canis latrans) population, red wolves were incapable of maintaining self-sustaining populations. They began hybridizing with coyotes when they were unable to find conspecific mates and canid populations in the region gradually became genetically admixed [Perceived threats to human enterprise have historically motivated efforts to exterminate large carnivores such as wolves, bears, and lions. In particular, wolves have been extirpated from much of their historical ranges in North America by government-supported eradication campaigns protecting agricultural and livestock interests. However, changes in American societal beliefs have resulted in profound changes to how wolves are perceived. The passage of the Endangered Species Act of 1973 (ESA) paved the way for restoration of wolf populations that were severely reduced or extirpated during the 19th and early 20th centuries. When the ESA was legislated, gray wolves  established the Red Wolf Recovery Program (Recovery Program) with the task of locating and preserving populations of red wolves in southeast Texas and southwest Louisiana . However® (SSP) that was accepted by the Association of Zoos & Aquariums (AZA) [The captive-breeding program safeguarded the last remaining red wolves and served as the last repository of the red wolf genome. The primary objectives of the captive-breeding program were to certify the genetic purity of wild-caught red wolves, increase the number of red wolves in captivity, and maintain a captive red wolf population for re-establishment of the species in the wild . Red wolms (AZA) . This enDuring 1984, Alligator River National Wildlife Refuge (ARNWR) was established on the Albemarle Peninsula of northeastern North Carolina (NENC) when the Prudential Insurance Company donated approximately 480 km²  of land to the federal government . This arThe USFWS initiated a second reintroduction in the Great Smoky Mountains National Park (GSMNP) of the southern Appalachians . During Although nearly 25 years have elapsed since red wolves were reintroduced into the wild, more than half of the red wolf population still exists in captivity. The captive-breeding program safeguards approximately 200 red wolves in more than 40 captive facilities around the United States while the reintroduced red wolf population has expanded throughout the Albemarle Peninsula to about 70–80 animals in approximately 15 packs . Since 1In the progress of overcoming these challenges to restoring red wolves to the wild, there is a need to consolidate knowledge and contemplate those experiences as recovery efforts move forward. Therefore, our objective is to provide a synopsis of the challenges to restoration of the red wolf and suggest future research needed to pursue full recovery of the species.Canis species in Eastern North America [Currently, scientists find themselves in a contentious debate regarding the taxonomy of New World wolves and its implications on the evolution, ecology, and conservation of  America . The ori America ,21,22,23 America . DespiteC. rufus. By the 1960s, federal and state agencies generally assumed that viable populations of red wolves existed in the Southeast despite a great deal of confusion about the species status. McCarley’s [Canis specimens from the Southeast, Paradiso [The unique presence of a southeastern wolf was noted during the 18th century ,25,26 anCarley’s  taxonomiParadiso ,34, and Paradiso  confirmeCanis species of Eastern North America using discriminant function analysis to evaluate the characteristics of modern and paleontological Canis skulls [Canis familiaris) into separate groups and postulated that red wolves evolved from a transitional form  between a wolf-like coyote ancestor and the gray wolf. Nowak [C. rufus that persisted in the Eastern United States 10,000 years ago.Nowak ,21 invesAlthough it had been suggested that red wolves were the result of coyote and gray wolf hybridization , the hypet al. [Canis lycaon) have mtDNA control sequences more closely related with coyotes, while exhibiting unique haplotypes not found in gray wolves and coyotes. Although these mtDNA sequences don't occur in western coyotes, they cluster among western coyote populations and Wilson et al. [et al. [C. lycaon. The disagreement among these genetic studies stems in part from differing assumptions about the nature of the coyote-like mtDNA found in eastern and red wolves. Those that support a hybrid origin interpret the coyote-like mtDNA as being from coyotes, whereas those that support the hypothesis that red wolves, eastern wolves, and coyotes share a common ancestry interpret the coyote-like mtDNA as being eastern wolf in origin and a result of incomplete lineage sorting.Examining the origin and taxonomy of wolves in eastern Canada, Wilson et al. ,46 reporn et al.  attributn et al. ,45,49 ann et al. ,31. Alth [et al.  proposedCanis species in the East makes it difficult to sort out the evolutionary history of red wolves. Recent and developing studies demonstrate that the taxonomy of red wolves is complex and morphological and molecular studies of fossilized wolves from the southeast are essential to settling the debate over red wolf origin. Significant gaps in the historic and geographic genetic data and recent hybridization makes it difficult to sort out the evolutionary history of red wolves. As a result, the taxonomy of North American wolves is complex and not without debate. Prior to and during European colonization of the Southeast, there appears to have been a small wolf species present and its modern equivalent may be the red wolf. It is also possible that red wolves are morphologically and genetically similar to coyotes because they fall within the species limits of the coyote clade . AlthougRed wolves and coyotes exist as a panmictic population in NENC and hybridization provides an exceptionally tough set of problems for red wolf recovery. Understanding how red wolves interact with coyotes is an important issue, which could dictate the success of the reintroduction project. During 1999, the USFWS re-evaluated the red wolf recovery effort by organizing a Population and Habitat Viability Assessment workshop (PHVA) . IntrogrAs history has proven, coyote populations are too resilient to state and federal eradication programs and clearing the Albemarle Peninsula of coyotes poses an overwhelming challenge. Two management techniques were developed during the PHVA to prevent hybridization. Coyotes and hybrids captured by USFWS personnel within the Recovery Area are reproductively sterilized (hereafter sterilized) and used as space holders until red wolves move in and occupy those areas. Coyotes and hybrids are taken to a local veterinary clinic in which females and males are sterilized by tubal ligation and vasectomy, respectively. This process keeps the hormonal system intact and avoids disrupting breeding and territorial behavior. Sterilized animals are fitted with mortality-sensitive radio-collars, released, and monitored for the duration of their life. This allows the Recovery Program to collect relevant information on coyote space use, habitat selection, and interaction with red wolves while suppressing coyote reproduction. In the event that a red wolf pairs with a sterilized coyote, the pair cannot produce hybrid litters. Additionally, sterilized coyotes that maintain territories keep those spaces occupied and prevent fertile coyotes from establishing breeding pairs on the landscape.Canis breeding pairs within the Recovery Area are red wolves. To implement this, Recovery Program biologists began eradicating coyotes and hybrids from Zone 1 while selectively using sterilized coyotes as space holders in Zone 2. When objectives in Zone 1 were completed, management efforts shifted west to Zone 2 in which sterilized space holders were removed to create space for red wolves. Once coyotes and hybrids were removed from Zone 1 and 2, management efforts would be undertaken in Zone 3. Implementing management goals in order of priority allowed the Recovery Program to minimize hybridization by monitoring red wolf and coyote packs throughout the Recovery Area and replacing coyotes and hybrids with red wolves when opportunities arose.As recommended during the PHVA, the Recovery Area was divided into three management zones in which management efforts varied in intensity to minimize hybridization on the landscape . The ultPrior to the PHVA, the Recovery Program assumed all canids captured within the Recovery Area were wolves unless animals were unusually small and coyote-like in appearance . Once hyScientific research is essential to understanding hybridization and the interplay between research and management offers an interesting opportunity to examine this process over the long-term. Initial scientific inquiries after the PHVA were to establish studies to measure, monitor, and manage hybridization in the Recovery Area. In doing so, a complete reconstruction of a red wolf pedigree has been established and this most likely represents the most complete database for any wild population. It's now understood that hybridization between red wolves and coyotes is not directional in terms of the wolves’ sex and hybrids backcross with both species ,55,56. Fi.e., geographic barriers) exist between red wolves and coyotes because coyotes are ubiquitous throughout the red wolf's historic range. Hybridization occurs between the two species when a red wolf and a coyote form a breeding pair that will defend a territory together until the death or displacement of a mate. Consequently, the red wolf-coyote pair will produce hybrid offspring and maintain pack dynamics similar to gray wolves [Canis [i.e., behavior) that would prevent pair formation between red wolves and coyotes. Understanding the ecology of red wolf-coyote interactions is crucial to define species traits that serve as isolating mechanisms, describe how these traits prevent hybridization, and identify what selection forces in nature favor the maintenance of red wolves and coyotes as separate species.Hybridization between red wolves and coyotes implies the obvious break down of reproductive barriers and the two species consort and breed with one another when situations favor opportunities to mate with congenerics. Currently, no extrinsic reproductive barriers  genes to examine red wolf kin recognition and mate choice. MHC genes were originally identified in inbred mice during skin graft experiments in which MHC molecules of the host recognized graft tissue as foreign antigens and attacked them . Since tIt's well established that variation in survival and reproduction are responsible for the dynamics of populations ,102,103.A primary goal of red wolf recovery is to establish and maintain a red wolf population of 220 individuals in three disjunct populations within the species’ historical range . To evalK) of 140 individuals that was reached in 2001, it was assumed that the red wolf population would continue to expand in subsequent years because red wolves occupied approximately 60% or less of the Albermarle Peninsula land area [The 2007 five-year status review (hereafter 2007 Review) of red wolves indicated the NENC population had fluctuated between 80-130 individuals per year since 1999 . With anand area . Howeverand area . The 200Odocoileus virginianus) hunting season (October 15–December 15) than during the non-hunting season [Ursus americanus) hunting seasons and increased mortality rates during this time forces red wolf breeders to quickly replace lost mates. When red wolves lost mates to gunshots during the hunting seasons they were more likely to pair with coyotes or fail to replace their mates than to pair with red wolves [et al. [Red wolves were seven times more likely to be killed during the North Carolina white-tailed deer , red foxes (Vulpes vulpes), and coyotes are hunted with hound dogs for sport in approximately 20 states [Any discussion of red wolf recovery must occur against the backdrop of current ecological and anthropogenic challenges. Although hybridization, inbreeding, and demographics were discussed separately earlier, these three issues are intrinsically related because they are influenced by the presence and management of coyotes. Therefore, these issues are complex and controversial causing management plans to promote recovery efforts to be more difficult than planned. Prior to the mid-1990s, coyotes were rare but increasing in NENC and Recovery Program biologists anticipated eventual colonization of the Peninsula by coyotes. The use of sterilized coyotes as space holders allowed the Recovery Program to saturate the Recovery Area with territories of red wolf packs and sterile coyote pairs. During the early 2000s, most coyotes captured, sterilized, and released with radio-collars failed to establish territories or pair with a space holder. In other words, the Recovery Program effectively saturated the Peninsula with canid territories and coyotes dispersing into the Recovery Area failed to find available space or mates and eventually left. However, legislation (NCGS § 113 273) passed by the NC General Assembly allowing owners of fox pens to buy live coyotes from licensed trappers and hunt them within their fox pens  may nega0 states ,119,120.0 states . Althoug0 states .Evident by the widespread persistence and range expansion of coyotes, current policies to control their populations are ineffective and have failed to significantly reduce coyote populations. Laws promoting trafficking and nighttime hunting of coyotes increase the chances that red wolves will be accidentally or purposefully killed by hunters, and attempts to remove these hunting laws are constrained by organized hunting and trapping groups. Increased killing of red wolves by predator-control programs during the early 20th century facilitated the decline of red wolves and promoted their hybridization with expanding coyotes. Recent research showed intense harvest of eastern wolves also facilitated hybridization with coyotes by disrupting the population's social structure . Today, i.e., rates of individuals growth, reproduction, and mortality), population-level processes , and community-level interactions  are known to correlate with body-size allometries regardless of taxonomic status [Increased mortality rates of red wolves and coyotes promote high turn-over rates of territories and erode the effectiveness of sterilization methods. Despite this, continued use of sterilization and efforts to increase the number of red wolves on the landscape will likely fail to prevent hybridization if reproductive barriers do not exist in the first place . Key to c status ,122,123.c status ,128,129.A major impediment to red wolf restoration is the limited knowledge about traits that facilitate behavioral and ecological differences between red wolves and coyotes. This is critical to red wolf restoration because expanding our knowledge about mechanisms that facilitate stable and reproductively isolated red wolf populations will allow us to recognize responses of red wolves to changing environments. This knowledge guides research to make accurate inferences and predictions about the future and promotes implementation of appropriate management. The reality of incomplete reproductive isolation may present challenges to red wolf restoration but evolution is ongoing and management efforts should promote conditions that allow for the gradual evolution of reproductive barriers. Although much work remains to be done, information and experiences gained from more than 25 years of restoration efforts have made crucial contributions to the future of the red wolf. They also allow us to formulate areas of investigation that are of direct relevance to the restoration of red wolves.


Electrochemotherapy (ECT) is a combined treatment in which high voltage electroporation (EP) pulses are used to facilitate the uptake of a chemotherapeutic drug into tumor cells, thus increasing antitumor effectiveness of the drug. The effect of ECT of deep-seated tumors located close to the heart on functioning of the heart has not been previously investigated. In this study, we investigate the effects of intra-abdominal ECT of colorectal liver metastases on functioning of the heart during the early post-operative care period.For ECT high voltage EP pulses with amplitudes of up to 3000 V and 30 A were delivered in synchronization with electrical activity of the heart. Holter electrocardiographic (ECG) signals were obtained from 10 patients with colorectal liver metastases treated with ECT. ECG was recorded during the periods of 24 hours before and after the surgical procedure involving ECT. Four-hour long night-time ECG segments from both periods exhibiting the highest level of signal stationarity were analyzed and compared. Changes in several ECG and heart rate variability (HRV) parameters were evaluated.No major heart rhythm changes  or pathological morphological changes  indicating myocardial ischemia were found. However, we found several minor statistically significant but clinically irrelevant changes in HRV parameters after ECT procedures: a decrease in median values of the mean NN interval, a decrease in the low-frequency and in the normalized low-frequency component, and an increase in the normalized high-frequency component.Only minor effects of intra-abdominal ECT treatment on functioning of the heart were found. They were expressed as statistically significant but clinically irrelevant changes in heart rate and long-term HRV parameters and were as such not life-threatening to the patients. The nature of these changes is such that they can be attributed to the known effects of the drugs given to the patients in the post-operative care. Further investigation is still warranted to unambiguously resolve whether ECT with high voltage EP pulses applied in immediate vicinity of the heart is responsible for the observed effects. Electroporation-based techniques, like electrochemotherapy (ECT), gene electrotransfection and non-thermal irreversible electroporation, are becoming a viable option for treatment of internal, deep-seated tumors and tissues . In such cases, surgical, percutaneous or endoscopic procedures are used to gain access to the treatment area -18. ThesDuring the study of intra-abdominal ECT of colorectal liver metastases at the Institute of Oncology Ljubljana, Slovenia ,6 tumorsDuring the clinical study on safety and feasibility of ECT of colorectal liver metastases at the Institute of Oncology Ljubljana we experienced no obvious or clinically relevant immediate or short-term effects of EP pulses on functioning of the heart during the treatment itself. Nevertheless, the observations summarized in the previous paragraph lead to the hypothesis that EP pulse delivery in close vicinity of the heart even when synchronized with ECG could induce measurable effects on functioning of the heart that could remain visible several hours after the treatment. The aim of this study was thus to explore these effects by evaluating changes in ECG during the early post-operative care of patients within 24 hours after intra-abdominal surgery and ECT of colorectal liver metastases.http://ClinicalTrials.gov: NCT01264952). The objective of this study was to examine safety and feasibility of ECT of colorectal liver metastases [The study of changes in ECG of patients during early post-operative care after intra-abdominal electrochemotherapy (ECT) procedure was part of the prospective pilot clinical study conducted at the Institute of Oncology in Ljubljana  of approximately 24-hours duration were recorded on the day before and after intra-abdominal ECT procedure. ECG signals were sampled at 200 Hz and acquired using a holter system . These ECG signals were used to investigate changes in functioning of the heart in early post-operative care. Holter recording before and after ECT was not possible for patients number 1, 2 and 8 and the signals recorded on patients number 3, 11 and 16 could not be used due to technical reasons. ECG signals from ten patients were thus included in the analysis. The main characteristics of these ten patients and their tumors are presented in Table The protocol of the clinical study was described in detail elsewhere ,6. Here ECT procedures were performed under combined general and thoracic epidural anesthesia (TEA). Epidural catheter was placed at T8-12 interspaces before anesthesia, and a test dose of lidocaine (60 mg) was injected. 30 minutes before surgical incision, all patients received the prophylactic antibiotics cefazolin (2 g) and metronidazole (500 mg) as a rapid intravenous infusion. The patients received 25-50mg of levobupivacaine epidurally during the operation, according to analgesic needs. General anesthesia was induced with sufentanyl, propofol, and vecuronium, and after tracheal intubation, maintained with sevoflurane in 40% oxygen-nitrous oxide balance. A bolus of local anesthetic levobupivacaine (between 10 to 15 mg) was administered epidurally about five minutes before surgical incision. Additional epidural levobupivacaine and intravenous vecuronium and sufentanyl were administered during the operation as appropriate. Epidural analgesia  with rate 3-6 mL/hour and with patient controlled analgesia (PCA) demand dose of 4 ml (lock out time 30 minutes) was continued in all patients postoperatively. During post-operative care, patients also received intravenously opioid analgesic piritramide, non-opioid analgesic metamizole and antiemetic metoclopramide as required. In the case of hypotension, an infusion of vasopressor norepinephrine in low dose (range from 0.01 - 0.2 µg/kg/min) was administered in order to maintain optimal blood pressure. At night, patients received another dose of the analgesic metamizole and the antipsychotic haloperidol.http://www.visifield.com.ECT was performed during open surgery that was primarily performed to treat larger, unresectable or difficult-to-treat liver metastases . To reac2. Delivery of EP pulses was started eight minutes after bleomycin administration. Eight EP pulses of 100 μs duration were delivered for each pair of the electrodes by the electric pulse generator Cliniporator Vitae .ECT procedure was performed as follows. First, needle electrodes were inserted into the tumor and the immediate surrounding tissue according to the treatment plan. The chemotherapeutic drug bleomycin was then administered intravenously in bolus with a dose of 15 mg/mThe delivery of EP pulses was synchronized with ECG via AccuSync 42, an external R wave triggering device . The ECG signal was acquired by the AccuSync 42 independently of the regular ECG monitoring by the anesthesiologist. The AccuSync 42 detects the R wave of each individual heartbeat early on the ascending slope of the R wave and provides a trigger to Cliniporator Vitae. Cliniporator Vitae delivers pulses 50 ms after a valid trigger pulse, thus avoiding the so-called vulnerable period of the ventricles (the T-wave). Validation of trigger pulses is performed by a built-in synchronization algorithm.The exact protocol of EP pulse delivery depended on the type of electrodes , noise-free 5 min long subsegments with a small number of abnormal heartbeats are required ,29. The The whole procedure thus resulted in twelve selected 5-min segments for each ECG record that were appropriate for HRV analysis. They were additionally manually inspected for correctness of R peak detection, location and heartbeat classification and, if necessary, corrected to assure correct identification and classification of every single heartbeat. We developed software  with graphical user interface for easy and rapid screening and checking for correctness of the located feature points and classification of heartbeats . After sQT interval is usually defined as the interval between the start of a Q wave and the end of a T wave within an individual normal heartbeat. However, we used an interval between the Q peak and the T peak , because Q and T peaks are much easier to detect reliably than the start of a Q wave and the end of a T wave. The corrected ppQT intervals  were determined for each heartbeat as the ppQT interval divided by the square root of the corresponding RR interval, following the common definition of the corrected QT interval.Heart rate variability (HRV) analysis deals with fluctuations in RR interval time series between normal adjacent QRS complexes. Normal QRS complexes are those resulting from the sinoatrial node depolarization, and the associated RR intervals are frequently referred to as NN intervals . The RR After manual editing of RR intervals from each selected 5-min subsegment of the ECG signals in the previous step, 1% or less abnormal heartbeats in each 5-min subsegment were required in order to minimize the error in subsequent calculation of HRV parameters. If this requirement was not met initially for a given subsegment, it was replaced with a new one, i.e., with the first next 5-min subsegment from the same 20 minutes long segment according to the requirements for selection and extraction of ECG subsegments as described above.In the next step the 5-min subsegments were edited using cubic spline interpolation method to replace abnormal RR intervals with the interpolated RR intervals. This approach is considered superior to simple deletion of abnormal RR intervals . An NN iFollowing the recommendations in the literature for short-term ECG recordings , severalFrequency-domain HRV measures were initially calculated from power density spectra estimated with non-parametric FFT-based Welch's periodogram method using Hamming window and with parametric autoregressive model-based method (AR model of the order 16 with coefficients determined based on Burg method) ,29. SincThe time course of voltage and current during EP pulse delivery was recorded and stored for further analysis. These records allowed us to determine the number of EP pulses delivered to each tumor and to calculate the average values of voltage and current and the total energy applied to individual tumor and patient. The median value of voltage and current delivered within each train of eight EP pulses was first calculated. Then, the median of these median values for all trains of EP pulses delivered to individual tumor was used as a measure of the average voltage and current delivered to the tumor. Where more than one tumor was treated in a patient, the largest average voltage and current values were taken for statistical analysis. The energy applied to individual tumors was determined by integrating the power of all EP pulses (i.e. product of current and voltage) delivered to the tumor. The total energy applied to a patient was also determined as the sum of energy delivered to all tumors in the patient. Values of these electrical parameters were intended for assessment of their association with the significant changes in ECG or HRV parameters (if found).Several statistical comparisons were performed. The median values of number of abnormal heartbeats, number of deviated ST segments, ppQTc intervals and HRV measures obtained from 4-hour ECG signals recorded before and after ECT procedure on individual patients were compared using paired Wilcoxon Signed Rank test. Linear regression method was used to determine the degree of association between the parameters of EP pulses and the ECG and HRV measures for which statistically significant changes were found. The statistical analysis was performed using SigmaPlot 11.0 package  and the p value of less than 0.05 was considered as the indication of statistically significant differences.No clinically significant adverse effects such as induction of extrasystoles, ventricular tachycardia or fibrillation) on functioning of the heart in patients due to ECT procedure were observed in the post-operative care. The analysis of ECG signals recorded before and after ECT procedure showed no increase in appearance of abnormal heartbeats in ECG signals recorded after ECT even in cases where abnormal heartbeats were present in ECG signal before ECT Table . No statECG signals recorded in patients number 6, 9 and 10 contained large amounts of high-frequency noise either before or after ECT procedure, which rendered a reliable determination of ppQTc intervals impossible Table . On the ECG signals from all 10 patients were included in HRV analysis. The Wilcoxon Signed Rank test showed statistically significant decrease in median values of parameters mean NN interval, LF and nLF, and statistically significant increase in median nHF after intra-abdominal ECT Table . StatistThe results of our study indicate that intra-abdominal surgery involving ECT of colorectal liver metastases was associated with only minor and clinically irrelevant changes in functioning of the heart detected in early post-operative care and as such presented no life threat for the patients. No major changes in functioning of the heart such as rhythm changes  or pathological morphological changes  that would suggest myocardial ischemia were found in ECG signals.According to the literature, surgery and anesthesia produce a stress response characterized by increased sympathetic and hormonal activity which may predispose the patient to arrhythmias . Post-opEP pulses delivered in immediate vicinity of the heart could potentially cause structural changes in myocardium and consequently induce myocardial ischemia. The myocardial ischemia can be detected by analysis of changes in ST segment level, by blood test of values of biomarkers of myocardial necrosis  and by cardiac imaging . Minor oHeart rate variability (HRV) analysis is a method for studying the physiological mechanisms responsible for the control of heart rate, in which the autonomic nervous system appears to play the primary role. HRV measures strongly depend on conditions under which ECG signals are recorded and are affected by numerous physiological factors modulating the normal rhythm of the heart  ,29,46. WThere are many factors that can directly or indirectly affect the autonomic nervous system. In addition, any factors that change properties of the heart itself can also have an effect on the heart rate . Even thIn our study, ECG signals recorded in the same night-time window when patients were resting (from 0:00 to 4:00 a.m.) were chosen for evaluation of HRV before and after ECT procedure in order to exclude the influence of circadian variation and patient's activity on HRV. Because HRV measures were compared for each patient before and after ECT procedure (self-control), age and gender dependence of HRV was minimized. None of the patients had clinically significant pre-existing cardiac conditions Table  - more d2O are known to cause a decrease in both the LF and HF components of the HRV power spectra [Induction and maintenance of general anesthesia with sevoflurane and N spectra -54. The  spectra . The rec spectra ,53,55. TDelivery of EP pulses could also contribute to an increased heart rate but such effects are reported to be only transient and were thus not expected to be present during the night-time after ECT procedure . InteresEpidural anesthesia decreases blood pressure and may be accompanied by a decrease in heart rate. The principal mode of action of epidural blockade is prevention of sympathetic outflow . High thTo avoid hypotension we used vassopresor norepinephrine at low infusion rates (0.01 - 0.2 µg/kg/min) in postoperative care. Norepinephrine vascular effects in the doses normally used clinically result from the simultaneous stimulation of alpha and beta adrenergic receptors in the heart and vascular system. This results in an increase in the force  of myocardial contraction. Intravenous administration of norepinephrine results in intense vasoconstriction and results in increase arterial pressure. The increase in blood pressure may cause a reflex decrease in the heart rate ,64. SchäAccording to the literature, analgesics given in post-operative care, especially opioids, depress the LF component more than the HF component of the HRV spectrum, and therefore reduce the LF/HF ratio, suggesting an increased parasympathetic dominance in the HRV activity 529 . TherefoSurgical pain and post-operative pain is known to trigger autonomic nervous and endocrine responses, which can be manifested also as accelerated heart rate ,69. ConsIntra-abdominal surgery involving ECT of colorectal liver metastases with synchronized delivery of EP pulses does not present any life threat for the patient. Namely, no major changes in functioning of the heart such as rhythm changes  or pathological morphological changes  due to ECT procedure were found in ECG signals recorded during early post-operative care. The probability for complications could increase when using EP pulses of longer durations or larger number of pulses of increased pulse repetition frequency and/or in the treatment of tumors in the immediate vicinity of the heart. Statistically significant although clinically irrelevant differences in some HRV measures after ECT procedure were identified but they can be explained by the known effects of post-operative drugs. In order to distinguish the effects of ECT itself from those of post-operative drugs, the ECG recordings before and after a similar surgical procedure but without ECT would be required, preferably obtained from the same patients on which the ECT has been performed during a previous surgical procedure. However, cases where a surgical procedure involving intra-abdominal ECT is followed by a similar surgical procedure without ECT are rare and were not available for this study. In addition, for reliable investigation of influence of different parameters of EP pulse delivery , a detailed data about distance between location of EP pulse delivery and the heart would also be needed.ECT: electrochemotherapyEP: electroporationECG: electrocardiogramHRV: heart rate variabilityTEA: thoracic epidural anesthesiaPCA : patient controlled analgesiaD. Miklavcic holds patents of which some have been licensed to IGEA SpA, Italy, the producer of a clinical device for electrochemotherapy used in this study. He also consults for IGEA SpA. Other authors declare no conflict of interest.BM collected and analyzed the ECG data and drafted the manuscript. VG was cardiology consultant. IE and EB were the main surgeons performing ECT in the study. MC helped to draft the paper. GS coordinated clinical parts of the study. BS was anesthesiology consultant. DM conceived the study and helped to draft and finalize the manuscript. TJ supervised and helped in collection and analysis of ECG signals and manuscript drafting and finalized the manuscript. All authors read and approved the final version of the manuscript.


Scherffelia dubia, 137,161 bp; Tetraselmis sp. CCMP 881, 100,264 bp). Characterized by a very small single-copy (SSC) region devoid of any gene and an unusually large inverted repeat (IR), the quadripartite structures of the Scherffelia and Tetraselmis genomes are unique among all core chlorophytes examined thus far. The lack of genes in the SSC region is offset by the rich and atypical gene complement of the IR, which includes genes from the SSC and large single-copy regions of prasinophyte and streptophyte chloroplast genomes having retained an ancestral quadripartite structure. Remarkably, seven of the atypical IR-encoded genes have also been observed in the IRs of pedinophycean and trebouxiophycean chloroplast genomes, suggesting that they were already present in the IR of the common ancestor of all core chlorophytes. Considering that the relationships among the main lineages of the core Chlorophyta are still unresolved, we evaluated the impact of including the Chlorodendrophyceae in chloroplast phylogenomic analyses. The trees we inferred using data sets of 79 and 108 genes from 71 chlorophytes indicate that the Chlorodendrophyceae is a deep-diverging lineage of the core Chlorophyta, although the placement of this class relative to the Pedinophyceae remains ambiguous. Interestingly, some of our phylogenomic trees together with our comparative analysis of gene order data support the monophyly of the Trebouxiophyceae, thus offering further evidence that the previously observed affiliation between the Chlorellales and Pedinophyceae is the result of systematic errors in phylogenetic reconstruction.The Chlorodendrophyceae is a small class of green algae belonging to the core Chlorophyta, an assemblage that also comprises the Pedinophyceae, Trebouxiophyceae, Ulvophyceae and Chlorophyceae. Here we describe for the first time the chloroplast genomes of chlorodendrophycean algae ( Tetraselmis and Scherffelia  and 100,264 bp [GenBank:KU167097], respectively  with an average length of 798 bp and that of the Tetraselmis genome a total of 651 reads  with an average length of 855 bp. The general features of both chlorodendrophycean genomes are compared with those previously reported for selected core chlorophytes in Choricystis minor [Floydiella terrestris [Schizomeris leibleinii [Scherffelia cpDNA relative to its Tetraselmis homolog is attributable to an enlarged IR; the remaining fraction is accounted for by longer intergenic regions (i.e. a lower gene density), the presence of five extra genes, and the occurrence of seven introns , 27 tRNAs (trn genes) that can read all codons present in the genome, and the RNA subunit of RNase P (rnpB). The latter RNA species shares 36.6% sequence identity with its homolog in the prasinophyte Nephroselmis olivacea and displays the typical secondary structural elements reported for RNase P RNA subunits  as well as trnR(ccg) and rnpB. These five genes are absent from the chloroplast genomes of other core chlorophytes and a number of prasinophytes [chl genes most probably completely vanished from Tetraselmis, because Blastp searches of the transcriptome shotgun assembly protein database of NCBI (tsa_nr) using the Scherffelia chlB, chlL and chlN sequences as queries revealed no significant similarity with the transcriptome of the halophilic microalga Tetraselmis sp. GSL018 which is included in this database. Both Scherffelia and Tetraselmis are missing six protein-coding genes that are present in other core chlorophytes , JAC66565  and JAC64732 . JAC64732 was confirmed to be the genuine PsbM  in BlastP searches of the nr database and consistent with this result, a subcellular localization analysis using TargetP [accD and cysA gene products and TargetP predicted the presence of an N-terminal mitochondria-targeting signal in each protein. Hence, although it remains to be confirmed that psbM is lacking in the chloroplast genome of Tetraselmis sp. GSL018, our results support the notion that this gene migrated to the nucleus before the emergence of the Chlorodendrophyceae. In prasinophytes, psbM disappeared from the chloroplast on three independent occasions [Similarities of the ed genes , which vsubunits . Relativnophytes , 18. Therophytes , suggest TargetP  stronglyccasions  and was ccasions .Tetraselmis chloroplast genome is lacking introns, seven are found in the Scherffelia genome  with its chlorodendrophycean homologs, it has retained the ccsA gene [Chromera velia, which adopts a linear conformation with terminal inverted repeats [Chromera SSC region would have occurred concomitantly with the linearization of the genome. However, the situation differs in the Chlorodendrophyceae, as both the Tetraselmis and Scherffelia genomes adopt a circular conformation. There is no doubt that these two green algal genomes are circular-mapping molecules considering that we obtained several plasmid clones and individual sequence reads extending over both IR/SSC junctions of Tetraselmis and that we recovered independent PCR fragments and several sequence reads spanning both IR/SSC junctions of Scherffelia.Unlike all IR-containing chlorophyte genomes that have been examined so far, the C region . At 3,38e cpDNAs . Prior tst sizes , 20. To csA gene . Concept repeats , could bScherffelia and Tetraselmis cpDNAs is compensated by the rich gene complement of their IRs. Among all completely sequenced IR-containing green algal cpDNAs, the Scherffelia and Tetraselmis IRs are the most rich in conserved genes and as will be discussed below, this situation is partly due to the acquisition, through multiple IR expansions, of genes typically found in the LSC and SSC regions. In addition to the rRNA operon, the 32,310-bp IR of Scherffelia contains 14 protein-coding genes and nine tRNA genes, whereas the 21,342-bp pair IR of Tetraselmis contains 15 protein-coding genes and 14 tRNA genes , are found within the IRs of Scherffelia and Tetraselmis. Besides supporting the affinities of the Chlorodendrophyceae with the Pedinophyceae and Trebouxiophyceae, these observations indicate that the IR of the common ancestor of the core chlorophytes had already expanded by acquiring a set of seven genes from the LSC region. However, the exact gene organization of this ancestral IR cannot be inferred on the basis of the available data because of the great variability of this cpDNA region in the Pedinophyceae, Chlorodendrophyceae and Trebouxiophyceae.Despite their differences in gene content and gene organization, the tructure . All sevScherffelia and Tetraselmis chloroplast gene organizations with those of other core chlorophytes, we analyzed all possible gene pairs found in the core chlorophyte genomes listed in To compare the The chloroplast phylogenomic and structural analyses reported in this study support the notion that the Chlorodendrophyceae is an early lineage of the core Chlorophyta, although its precise placement relative to other chlorophyte lineages could not be resolved. Despite these ambiguities, our results provide a better understanding of the relationships within the core Chlorophyta by shedding light on the monophyletic/paraphyletic status of the Trebouxiophyceae. Indeed, our finding of synapomorphic gene pairs uniting the Chlorellales and core trebouxiophyceans together with the recovery of the Trebouxiophyceae as a monophyletic group in the trees inferred from the 108-gene data sets offer further evidence that the previously observed affiliation between the Pedinophyceae and Chlorellales is incorrect. As pointed out by Lemieux et al. , the affScherffelia and Tetraselmis chloroplast genomes is unique among the core Chlorophyta. This unusual structure appears to have evolved by remodeling, through multiple expansions of the IR, of an ancestral core chlorophyte genome that was likely partitioned in the same fashion as extant pedinophycean and trebouxiophycean cpDNAs. These gene rearrangements occurred concomitantly with the transfer of psbM to the nucleus and the losses of five other protein-coding genes  from the chloroplast genome. Following the divergence of the Scherffelia and Tetraselmis lineages, the IR underwent further expansions/contractions and gene shuffling, highlighting the dynamic evolution of this cpDNA region in the Chlorodendrophyceae.Characterized by a gene-rich IR and a SSC region devoid of any gene, the quadripartite architecture of the S1 FigE. coli RNase P RNA, and helical regions are numbered accordingly [Nephroselmis olivacea RNase P RNA [The model is based on the secondary structure of the ordingly . The resse P RNA .(PDF)Click here for additional data file.S2 FigScherffelia atpA and cemA introns. (B) Consensus secondary structure of the Scherffelia petA and petB introns. Intron modeling was according to the nomenclature proposed for group II introns [atpA and cemA introns in panel A, petA and petB introns in panel B). Nucleotides in boldcase letters in panel A are conserved in the group II intron identified in Euglena myxocylindracea psbA [(A) Consensus secondary structure of the  introns . Exon secea psbA .(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


As communities’ fears of Ebola virus disease (EVD) in West Africa exacerbate and their trust in healthcare providers diminishes, EVD has the potential to reverse the recent progress made in promoting facility-based delivery. Using retrospective data from a study focused on maternal and newborn health, this analysis examined the influence of EVD on the use of facility-based maternity care in Bong Country, Liberia, which shares a boarder with Sierra Leone - near the epicenter of the outbreak.Using a case series design, retrospective data from logbooks were collected at 12 study sites in one county. These data were then analyzed to determine women’s use of facility-based maternity care between January 2012 and October 2014. The primary outcome was the number of facility-based deliveries over time. The first suspected case of EVD in Bong County was reported on June 30, 2014. Heat maps were generated and the number of deliveries was normalized to the average number of deliveries during the full 12 months before the EVD outbreak (March 2013 – February 2014).Prior to the EVD outbreak, facility-based deliveries steadily increased in Bong County reaching an all-time high of over 500 per month at study sites in the first half of 2014 – indicating Liberia was making inroads in normalizing institutional maternal healthcare. However, as reports of EVD escalated, facility-based deliveries decreased to a low of 113 in August 2014.Ebola virus disease has negatively impacted the use of facility-based maternity services, placing childbearing women at increased risk for morbidity and death. As we pass the one-year anniversary in early 2015 of the first reported death in Guinea from Ebola virus disease (EVD) the statistics are staggering. The World Health Organization reports the total number of cases attributed to EVD in West Africa has now surpassed 20,000 with the death toll nearing 8000 across the three most affected countries: Guinea, Sierra Leone and Liberia [In post-conflict countries such as Sierra Leone and Liberia, strained health care systems witnessed healthcare workers leaving their clinical posts – and patients shunning healthcare facilities . In counPrior to the EVD outbreak, facility-based deliveries with a skilled birth attendant were on the rise in rural Liberia . PrelimiThis case series study explored how EVD has influenced utilization of facility-based delivery care in one rural county in Liberia. We used data collected between 2012 and 2014 from a large, USAID funded study designed to evaluate the effectiveness of maternity waiting homes (MWHs) in Bong County, Liberia, one of the areas most affected by the EVD. This USAID funded study allowed a longitudinal analysis of EVD’s effect on women’s use of facilities for childbirth in Bong County, Liberia. Indeed, the data provided an opportunity to examine the natural history of facility-based deliveries before and after the EVD and determine the effect of the EVD crisis on access to facility-based care during childbirth.This case series, examining the impact of EVD on rural facility-based delivery with a skilled birth attendant, was conducted through a retrospective record review of all deliveries reported at ten of 39 rural health care facilities and the two major referral hospitals in Bong County, Liberia. Institutional review board approval was obtained from the University of Michigan and the Liberia Ministry of Health and Social Welfare. Because this study used retrospective, de-identified data, informed consent was waived by the granting agencies.Bong County is one of 15 counties in Liberia with a population of approximately 330,000, making it one of the most populous counties in the country. It shares a common border with Guinea near the epicenter of EVD and where the same indigenous groups reside, moving freely across the two countries’ borders. The first suspected cases of EVD in Bong County were reported in June, 2014   and small clinics . All facilities are staffed by registered midwives. Both small and large clinics are equipped to provide basic emergency obstetric and newborn care. Hospitals are able to provide comprehensive obstetric emergency and newborn care.To demonstrate the temporal change in the number of facility-based deliveries and MWH stays since the onset of the EVD outbreak, heat maps were generated for cumulative EVD cases, number of facility-based deliveries, and relative number of MWH stays in Bong County, Liberia. For purposes of constructing the maps, Bong County was divided by the estimated catchment area for each facility. The cumulative number of EVD cases in Bong County during each month since the onset of the outbreak was aggregated from Liberia Situation Reports generated by the Ministry of Health and Social Welfare. The number of facility-based deliveries and MWH stays were normalized to the average number of facility-based deliveries and MWH stays during the full 12 months before the EVD outbreak (March 2013 – February 2014) within the catchment area for each facility. A linear scale was used to generate all heat maps using a red-green-blue color scale.Since 2012 and prior to the EVD outbreak, the use of facility-based deliveries steadily increased in Bong County, although seasonal fluctuations were apparent. In May 2014 the facility-based deliveries were at an all-time high at the study sites and appeared to be on an upward trajectory. In the first seven months of 2014 a combined total of 3436 facility-based deliveries were reported from study sites in Bong County, with average monthly facility-based deliveries between 400-500. However, as reports of EVD escalated, a nadir of facility-based deliveries occurred: in August there were only 113 facility-based deliveries and 160 in September. From July to August 2014, the number of EVD cases in Bong County tripled and the number of deaths began to rise  a health system unprepared to care for large numbers of highly infectious patients, and 2) the major risk to staff for exposure to the Ebola virus during childbirth without the proper personal protective gear caused healthcare workers to remain at home rather than report to work and risk infection.Fear among healthcare providers had a substantial impact on the accessibility and availability of facilities  as evideThe visible decline in the number of women seeking facility-based deliveries also highlights concerns about trust between patients and healthcare providers. Current media reports indicate a heightened distrust of health workers resulting in patients avoiding the health system –12. At pThere are several limitations to this study including a short post EVD period and the use of retrospective pre-existing regional data. Additionally, the study design used is not optimal for making inferences about causality. Without data from deliveries that occurred outside facilities, this study was unable to correlate the decrease in the use of facility-based delivery services with poor maternal and newborn outcomes. Nonetheless, our data indicate what many have speculated – that EVD placed pregnant women and their unborn babies at increased risk for morbidity and death .This case series study was exploratory and designed to provide insights into the effect of the EVD outbreak on facility-based deliveries in Bong County, Liberia. Our findings highlight the collateral effect of EVD on maternal and newborn health and suggest that more sophisticated studies are needed to determine the many ways in which EVD is impacting maternal health.


Obesity and type 2 diabetes are increasing all over the world at an alarming rate due to unhealthy lifestyles. The fight against childhood obesity represents one of the biggest social and health problems all over the world. Diabetes mellitus reduces quality of life and life expectancy and represents a serious economic burden. The correction of unhealthy lifestyles is possible through cost-effective measures and it might prevent about 80% of cases of heart disease, stroke, and type 2 diabetes and 40% of cancers. At present, there is a need to advance in the science of delivering health promotion and reproducible multidisciplinary models for the prevention and treatment of obesity and diabetes using lifestyle change. Particularly alarming is the childhood obesity which represents the most common metabolic disorder in children and adolescents. Three reasons other than the high prevalence justify the urgency of treating and preventing childhood obesity: (i) persistency of childhood obesity into adulthood; (ii) metabolic and nonmetabolic morbidity obesity associated; (iii) higher mortality in adulthood. The American Academy of Pediatrics published guidelines with the targets for prevention and treatment of childhood obesity, based on consistent scientific evidence. Nutrition and physical activity are the main tools of intervention. Recent evidence suggests that prevention should start early, from intrauterine life and infancy, when the sensitivity of the organism to metabolic long-term programming processes is high and the potential impact of the intervention may be considerable. The discouraging results of the impact of available interventions in prevention and treatment of childhood obesity stimulate new emphasis in both clinical and experimental research in the field.The present special issue has been published to enlarge the knowledge in the field of the battle against childhood and adult obesity and type 2 diabetes.Four papers address the issue of childhood obesity prevention and treatment. G. Valerio et al. report that perception of task's difficulty level may reflect an actual difficulty in obese children. These findings have practical implications for approaching physical activity in obese children. In order to design exercise programs that allow safe and successful participation it is relevant to explore both the perception of a task's difficulty level and physical performance of obese children. H. Zheng and coworkers originally looked for possible associations between the metabolome and gender, pubertal development, and physical activity in overweight adolescents, which is an important subject group to approach in the prevention of obesity and life-style related diseases. No relations between physical activity and the metabolome could be identified, whereas gender differences in the metabolome are being commenced already in childhood and might be used for identification of individuals susceptible to an early pubertal development. C. Mazzeschi et al. describe the design of the intervention programme EUROBIS (Epode Umbria Region Obesity Intervention Study), an EPODE community-based intervention combined with a clinical trial, which provides an innovative valuable model to address the childhood obesity prevention and treatment. Finally, A. Verrotti et al. explored a possible relationship between prevalence, frequency, and severity of migraine and obesity in children. Interestingly, data from literature reviewed in this paper suggest that obesity can be linked with migraine prevalence, frequency, and disability in both paediatric and adult subjects, suggesting the importance for the quality of life of children of lifestyle interventions.Regarding adult obesity, the paper by K. M. J. Azar et al. examined whether baseline BMI may influence behavioural weight-loss treatment effectiveness. The authors show that participants with baseline BMI had greater reductions in mean BMI, body weight, and waist circumference in the coach-led group intervention, compared to usual care and the self-directed individual intervention. These results indicate the importance of taking into account individual's baseline BMI when developing or recommending lifestyle focused treatment strategy. B. Strasser and D. Pesta reviewed the evidence about the role of resistance training in counteracting the metabolic dysfunction in patients with type 2 diabetes. The authors report several beneficial adaptations exerted by resistance training which include increased GLUT4 translocation in skeletal muscle, increased insulin sensitivity, increased energy expenditure, and excess postexercise oxygen consumption. This paper supports the importance of using combined aerobic and resistance exercise as a therapeutic tool for type 2 diabetic patients. A positive message about the beneficial effects of walking in obesity and type 2 diabetes comes out of the paper of C. Mazzeschi et al. who originally report the effects of prolonged exercise  on several health and psychological outcomes. The results of this study demonstrate that long-distance treks are a safe activity for trained overweight/obese people that should be recommended because they improve mood, health status, and the relationship of participants with themselves and with the regular practice of exercise with effects similar to those obtained by healthy normal weight subjects.Pierpaolo De FeoJean-Michel BorisClaudio Maffeis


Epilepsy is the most common serious neurological disorder worldwide, however, the specific causative factors and mechanisms underlying epilepsy remain unclear. The current study aimed to study the potential genes or pathways associated with epilepsy, based on rat miRNA expression profiles. The microarray dataset GSE49850 was downloaded and analyzed with the TimeCourse R software package, which was used to generate comparisons between the control and electrically-stimulated groups. The target genes of differentially expressed miRNAs were queried in the miRWalk database and functional enrichment was conducted using the Database for Annotation, Visualization and Integrated Discovery software tools. The interaction network of the target genes was constructed based on the Biomolecular Interaction Network Database and clustered using ClusterONE. In total, 152 differentially expressed miRNAs were identified, with rno-miR-21-5p being the most significantly differentially expressed. A total of 526 target genes of the differentially expressed miRNAs were obtained. Functional analysis indicated that these genes were predominantly involved in responses to stimuli. The interaction network showed that the GRIN and STX gene family, which are involved in synaptic signal transmission, were significant. In conclusion, the present study identified that the development of epilepsy was closely associated with neuronal calcium signaling pathways. Epilepsy is the most common serious neurological disorder worldwide . It is cMicro RNAs (miRNA) are a group of non-coding RNAs (20–30 nucleotides in length) that regulate the expression of target genes by binding to the 3′-untranslated regions of target messenger (m)RNAs ,11. The et al  software ,28. ThisGenes and proteins associated with the target genes were screened in the BIND  database2 test. The top 10 most significantly differentially expressed miRNAs are listed in The microarray data were processed with background correction and standardization to obtain the differentially expressed miRNAs. At a P-value of <0.01, a total of 152 differentially expressed miRNAs (including 144 up- and 8 downregulated) were obtained with Hotelling’s TThe expression patterns of 152 differentially expressed miRNA in 40 samples exhibited a significant distribution character . MultiplFollowing removal of the duplicate genes, 526 target genes associated with miRNA were identified. The interaction network  was consGO terms and KEGG pathway enrichment analysis indicated that there were 30 KEGG pathways and 630 GO terms in 113 types of cluster categories, including external stimuli response, gene transcription regulation and steroid response. The 3 most significantly enriched terms in each category are described in Epilepsy is the most common serious neurological disorder worldwide , and theet al  receptors are vital for healthy brain development . The NMDThe results of the interaction network for these target genes indicated that signal transduction of normal nerve cells suffers significant interference in the process of epilepsy, thus the nerve cells may not function properly, leading to the occurrence of disease. Understanding the mechanisms of the differential expression of miRNAs in epilepsy is crucial for understanding the pathophysiology of epilepsy and may constitute interesting candidate targets for therapy. However, to understand the mechanism of the different miRNAs on the function of epilepsy requires in-depth knowledge of the targets of each miRNA, and a larger scale of miRNA-target gene screening. In the current study, the targets of the miRNAs were unknown and require further investigation.In conclusion, the present study, based on miRNA expression profiles, demonstrated that the development of epilepsy is closely associated with the neuronal calcium signaling pathways involving the GRIN and STX families. The results suggested an important function for miRNAs in molecular mechanisms of epilepsy, and provided a potential direction for future research into the disease, and its treatments.


Adding, omitting or changing pre-specified outcomes can result in bias because it increases the potential for unacknowledged or post-hoc revisions of the planned analyses. Journals have adopted initiatives such as requiring the prospective registration of trials to promote the transparency of reporting in clinical trials.A review of all 3156 articles submitted to The BMJ between September 2013 and July 2014. Trial registry entries, protocols and trial reports of randomised controlled trials published by The BMJ and a random sample of those rejected were reviewed to determine the frequency and type of outcome discrepancies between pre-specified and reported outcomes. Editorial, peer reviewer comments and author responses were also examined to ascertain any reasons for discrepancies.In the study period, The BMJ received 311 trial manuscripts, 21 of which were published by the journal. In trials published by The BMJ, 22% (75/339) of the pre-specified outcomes were not reported and 8% (25/297) of reported outcomes were not pre-specified. Discrepancy rates for rejected articles were similar. The majority of reasons provided by authors for outcome discrepancies were not bias related.Mandating the prospective registration of a trial and requesting that a protocol be uploaded when submitting a trial article to a journal has the potential to promote transparency and safeguard the evidence base against outcome reporting biases as a result of outcome discrepancies. Further guidance is needed with regards to documenting reasons for outcome discrepancies and making these reasons available.


Medicago plants the bacteria undergo a host-directed multistep differentiation process culminating in the formation of elongated and branched polyploid bacteria with definitive loss of cell division ability. The plant factors are nodule-specific symbiotic peptides. About 500 of them are cysteine-rich NCR peptides produced in the infected plant cells. NCRs are targeted to the endosymbionts and the concerted action of different sets of peptides governs different stages of endosymbiont maturation. This review focuses on symbiotic plant cell development and terminal bacteroid differentiation and demonstrates the crucial roles of symbiotic peptides by showing an example of multi-target mechanism exerted by one of these symbiotic peptides.The symbiosis between rhizobia soil bacteria and legumes is facultative and initiated by nitrogen starvation of the host plant. Exchange of signal molecules between the partners leads to the formation of root nodules where bacteria are converted to nitrogen-fixing bacteroids. In this mutualistic symbiosis, the bacteria provide nitrogen sources for plant growth in return for photosynthates from the host. Depending on the host plant the symbiotic fate of bacteria can either be reversible or irreversible. In  Agrobacterium or Brucella) than to each other. Rhizobia tend to have large genomes (up to 10.5 Mbp) which in fast growing rhizobia are dispersed on multiple replicons  both of which are indispensable and carry the majority of symbiotic genes. However, many S. meliloti strains contain further auxiliary medium sized plasmids and thus, the S. meliloti genome may contain up to 9,000 genes . The symbiosis is triggered by nitrogen starvation of the host plant which has to select its Rhizobium partner from billions of bacteria in the rhizosphere. This is achieved by secretion of flavonoid signal molecules from the root which act as chemo-attractants but most importantly as inducers of the Rhizobium nodulation genes  which by constant generation of new cells provokes continuous growth and elongated nodule shape. The cells leaving the meristem do not divide anymore and enter a differentiation phase. The infection thread releases the bacteria into the submeristematic cells, which differentiate gradually along the 12–15 cell layers of the infection zone (zone II), leading to the development of nitrogen-fixing symbiotic cells in nodule zone III (M. truncatula nodules the volume of the nitrogen-fixing cells is 80-fold larger than that of the diploid meristematic cells. The growth of infected cells occurs stepwise in zone II and is the consequence of repeated endoreduplication (ER) of the genome without mitosis. In zone II the cell cycle machinery is still active but the lack of mitotic cyclins inhibits mitosis and transforms the mitotic cycles to endoreduplication cycles  sudden growth of bacteroids is visible reaching practically their final size, however, nitrogen-fixation takes place only in zone III.The bacteria released from the IT are present in the host cytoplasm as organelle-like structures, called symbiosomes. The bacteria have no direct contact with cytoplasm as they are surrounded by a peribacteroid membrane, known also as symbiosome membrane (SM). The bacteroid, the SM and the space between them comprise the symbiosome . The SM formance . TerminaM. truncatula the nodule cells produce at least 600 nodule-specific symbiotic peptides (symPEPs). The symPEP genes are only activated in the S. meliloti infected polyploid symbiotic cells . However, why does the host cell produce an arsenal of NCRs? What can be the advantage of such a diverse peptide repertoire? Is it necessary for interaction of the host with various bacteria? The symbiotic partners of M. truncatula are S. meliloti and S. medicae, however in the soil there are countless strain variants of both species. M. truncatula is also represented by many different ecotypes and accessions differing in the number, sequences, and expression profile of NCR genes and in their symbiotic interactions with different S. meliloti and S. medicae strains . AMPs with broad spectrum of microbial cell-killing activity are most frequently cationic provoking cell death by pore formation, membrane disruption and consequent lysis of microbial cells. The fact that the cell division ability is definitively lost during endosymbiont differentiation indicates that at least certain symPEPs have antimicrobial activities. Treatment of bacteria with synthetic cationic NCRs indeed provoked rapid and efficient dose-dependent elimination of various Gram-negative and Gram-positive bacteria including important human and plant pathogens . This exRhizobium-legume symbiosis. In the weevil Sitophilus, the symbiotic cells produce the antimicrobial peptide coleoptericin-A (ColA) which provokes the development of giant filamentous endosymbionts by inhibiting cell division and protects the neighboring insect tissues from bacterial invasion  which resemble the Medicago NCR peptides, however the functions of these symbiotic peptides have not been reported yet  interacts also with GroEL . This perception results in the activation of signaling cascades, and the production of antimicrobial effectors. AMP-like host peptides such as the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.