Cancer is a complex genetic disease as a result of accumulated genomic alterations which serve as the driving force in initiating tumor development and propelling tumor progression. Recent advances in molecular genomic technology and the success of human genome project have empowered investigators with new tools in analyzing cancer genome and transcriptome in great details and have expedited the discovery of new cancer-associated genes. Decoding the genetic history present in tumor DNA, as well as identification and characterization of molecular changes involving cancer-associated genes and the pathways they controlled, has not only shed new light on the molecular etiology of cancer, but also has promises for the development of new diagnostic markers and novel therapeutics. In this special issue, the papers focus on recent findings of markers identified in female reproductive cancers including breast, ovarian, endometrial, and trophoblast tumors. The selected papers are not an exhaustive representation of the area of biomarkers in female reproductive cancers. Nonetheless, they represent the rich and many-faceted knowledge that we have the pleasure of sharing that with readers. We would like to thank the authors for their excellent contributions to this special issue and the reviewers' insightful input to ensure the quality of this special issue. Of particularly interest are the two timely review articles by A. M. Karst et al. and A. L. Gross et al. focusing on the possible precursor lesions in the development of female reproductive cancer. The tubal origin of “ovarian” cancer has recently been in the research spot-light because of the identification of potential precursor lesions of ovarian cancer, termed “serous tubal intraepithelial carcinoma” in the fallopian tubes. Both articles critically review the published studies and provide unique perspectives of the impact and significance of this new hypothesis in the field of cancer research. Review articles focusing on biomarkers including genetic , microRNA , and cytokine  summarize recent advances in biomarker discovery and the translational application. Those articles will serve as the source for the investigators who wish to pursue in evaluating new potential markers in the female reproductive cancer research. They also present the new challenges in biomarker discovery, validation, and verification. In addition to the five review articles, there are several exciting original research articles describing discovery and validation of biomarkers in female reproductive cancers. Those papers include the studies of fatty acid synthase , miR200c , gene expression , and cytogenetic markers . The findings from those studies could serve as biological foundation for future exploration of how those markers contribute to disease progression. The biomarkers identified in these studies could also serve as a road map for development of novel diagnosis tools and new targeted therapies. This issue also reports a synergistic effect of Pten loss and oncogenic Kras mutation on the endometrial cancer development  and the result provides new insight into the pathogenesis of endometrial carcinoma. Finally, K. L. Yap et al. applied molecular genetic markers to demonstrate a lack of a Y-chromosomal complement in the majority of gestational trophoblastic neoplasms, suggesting that most trophoblastic tumors are derived from previous molar gestations. Because the molecular characters in gestational trophoblastic disease are understudied, this short report should provide new insight into the pathogenesis of this rare but highly interesting gynecological neoplasm. In conclusion, this special issue is by no means an attempt to be comprehensive in covering the field of female reproductive cancer, but it provides excellent reviews and research articles that may be useful for readers in this field. We hope that this special issue will inspire investigators in launching new research endeavor in this field and are grateful for the opportunity to manage this special issue.Tian-Li WangTian-Li WangBen DavidsonBen DavidsonPhillip J. BuckhaultsPhillip J. BuckhaultsChiun-Sheng HuangChiun-Sheng HuangGudi PoulGudi Poul


Existing theory of host-parasite interactions has identified the genetic specificity of interaction as a key variable affecting the outcome of coevolution. The Matching Alleles (MA) and Gene For Gene (GFG) models have been extensively studied as the canonical examples of specific and non-specific interaction. The generality of these models has recently been challenged by uncovering real-world host-parasite systems exhibiting specificity patterns that fit neither MA nor GFG, and by the discovery of symbiotic bacteria protecting insect hosts against parasites. In the present paper we address both challenges, simulating a large number of non-canonical models of host-parasite interactions that explicitly incorporate symbiont-based host resistance. To assess the genetic specialisation in these hybrid models, we develop a quantitative index of specificity applicable to any coevolutionary model based on a fitness matrix. We find qualitative and quantitative effects of host-parasite and symbiont-parasite specificities on genotype frequency dynamics, allele survival, and mean host and parasite fitnesses. Coevolution between hosts and parasites is believed to be central to a number of biological phenomena, most notably the observed patterns of biodiversity and the origins of sexual reproduction. However, classical mathematical models of host-parasite coevolution account neither for the hosts' use of bacterial symbionts for protection from parasites, nor for the potential and observed complexity of genetic interactions between the coevolving species. In this article we address both challenges by simulating a large number of models of host-symbiont-parasite coevolution based on randomly generated genotype interaction patterns. We demonstrate that the degree of “specificity” between the genotypes of the interacting species is a major factor influencing the outcome of coevolution. We also observe that the symbionts may take over from the hosts the coevolutionary arms race against the parasites. Overall, our results make clear that the complex interaction patterns and the defensive symbionts can both play vital roles in host-parasite coevolution. An additional contribution of the article is a numerical index of specificity, applicable to a wide range of existing and future coevolutionary models. Parasitism is one of the main lifestyles in nature and a major source of evolutionary pressure. Despite its central place in evolutionary ecology, however, the details of the genetic architecture underlying resistance and infectivity are not known for most host-parasite associations. Mathematical models of host-parasite coevolution compensate for the missing data by making explicit, first-principle assumptions about the interaction of host and parasite genotypes. Two such classic assumptions, and consequently two classic families of models, are known as Matching Alleles (MA) and Gene For Gene (GFG). The MA models, inspired by vertebrate immune systems One problem with the existing theory is that there is a mounting number of natural systems for which the interactions between host and parasite genotypes have been disentangled and found to be neither of the MA nor the GFG kind Hamiltonella defensa lends to aphids We aim to build on these two studies to address a new challenge to the existing theory of host-parasite interactions: the discovery of bacterial endosymbionts that increase their hosts' resistance to parasites In this paper we build a generic model of the coevolution of hosts, symbionts and parasites. We incorporate as independent, tunable parameters the strength of the reciprocal selection acting on hosts and parasites, the fitness penalty for harbouring symbionts, the efficiency of horizontal and vertical transmission of symbionts, and the genetic interactions among all players. In a straightforward extension of the standard approach, the last factor is subsumed in a real-valued matrix, and we randomly sample many such matrices and simulate our model for each. In this way we are able to cover a range of potential host-symbiont-parasite systems and, importantly, decouple the effects of genetic specialisation patterns from the other factors. In addition, we analyse separately a collection of matrices describing protective symbionts acting within the established MA and GFG frameworks. Throughout, we do not treat genetic specialisation as a binary property; instead, we devise a numerical index of specificity. We find that specificity as defined in this paper strongly influences important coevolutionary outcomes of the models, such as the genotype frequency dynamics, maintenance of allelic diversity and mean host and parasite fitness. Our simulations show that these characteristics depend also on symbiont-related processes, especially the reliability of their maternal inheritance.associations: master matrix denoted We assume that hosts and parasites reproduce asexually and consider one haploid locus and two alleles in each of the three protagonists: host, symbiont and parasite; we also model hosts without symbionts. This gives rise to two parasite genotypes: We use three additional parameters in our coevolutionary setup: the maximum strength of selection that the parasites can exert on hosts see also :(1)(2) on , and then select the two matrix collections from this sample. Unfortunately, i.u.d. sampling yields no high-specificity matrices in reasonable time, because their entries have extreme values and are highly dependent on each other see . To overWe considered three values of each of the three supplementary parameters, and  see . For eacsee also : assume For each master matrix we simulated and analysed 108 models, because there are 108 auxiliary parameter combinations . Each moWe begin by considering two simple models incorporating symbiotic protection into the Gene For Gene and Matching Alleles frameworks. We assume that the hosts' innate resistance to the parasites follows one of these two classic principles, but the symbionts may confer a partial degree of resistance We found that despite the presence of symbionts, the dynamical behaviour of the models is similar to that of their classic symbiont-free counterparts. All MA models exhibit strong oscillations of host and parasite allele frequencies, while in the GFG models the allele frequencies stabilise. The balance of cost and protection quality determines the fate of symbionts in both models. In the MA model, the symbionts become fixed in the population if We now give an intuitive specificity-based interpretation of these results. The host-parasite specificity is high in the MA models: −3. We found that while both the host-parasite and symbionts-parasite specificities broadly promote cycling and diversity —the idea that host-parasite coevolution underlies the evolution of sex and recombination Figure S1Host-symbiont specificity. The influence of host-symbiont specificity on the diversity of host-symbiont associations, likelihood of cycling, allelic diversity, and mean host and parasite fitnesses.(TIFF)Click here for additional data file.Text S1Specificity of arbitrary matrices. This appendix shows how to generalise the definition of specificity introduced in the main text to operate on matrices of arbitrary size and shape.(PDF)Click here for additional data file.Text S2Matrices of high specificity. This appendix discusses the properties of high-specificity matrices.(PDF)Click here for additional data file.


Neuroendocrine carcinoma of the urinary bladder is a rare entity, accounting less then 1% of urinary bladder malignancies. The vast majority of the neuroendocrine carcinoma of the urinary bladder is represented by small cell neuroendocrine carcinoma while just few cases of large cell neuroendocrine carcinoma (LCNEC) have been reported. In this cases report we describe a rare case of primary bladder LCNEC.http://www.diagnosticpathology.diagnomx.eu/vs/2474700528951562.The virtual slide(s) for this article can be found here:  Neuroendocrine carcinoma of the urinary bladder is a rare entity, accounting less than 1% of urinary bladder malignancies .LCNEC was first described by Travis  in the lIn this cases report we describe a rare cases of primary bladder LCNEC.A 53 years old woman presented with asymptomatic hematuria in September 2011. Ultrasound studies revealed a 4 cm mass in the posterior wall of the urinary bladder. TC scan confirmed the presence of the lesion and demonstrated that both ureteral opening were involved by the tumor. Mesenteric lymph nodes were evident. The tumor was partially resected transurethrally. Microscopically, the tumor was composed of large pleomorphic cells with moderate amount of cytoplasm and coarse nuclear chromatin, organized in trabecular and rosette-like patterns and showed high mitotic rate  and metastasis were present in 5 right hypogastric lymph nodes (pN2) and in 2 common iliac lymph nodes (M1).Following surgery, patient underwent a 5th cycle of chemotherapy with etoposide and cisplatin and she died on March 2012, seven months after the diagnosis.Neuroendocrine tumors of the urinary bladder are relatively rare and include carcinoids, large cell neuroendocrine carcinomas and small cell neuroendocrine carcinomas, the latter being by far the most common. LCNEC was first described in the lung by Travis , as wellOnly few cases of LCNEC are present in the literature and most of them showing mixed histology, including the present case. In our case, areas of LCNEC and urothelial carcinoma were evident while in other reported cases were described areas of LCNEC mixed with a squamous cell or adenocarcinoma component. This is in line with the hypothesis that neuroendocrine cells originate from stem cells suggesting a common clonal origin of the neuroendocrine, urothelial, squamous or adenocarcinoma component .Mixed histology is also seen in other rare primary bladder tumors such as adult rhabdomyosarcoma  or giantInterestingly, around 30% of cases of small cell carcinoma or the urinary bladder are positive for TTF-1  while noIn addition, to our knowledge, this is the first case in the literature of primary LCNEC of the bladder with known status of EGFR.In our case, as well as in most of the described cases, tumor showed a very rapid local growth in few months, and this might be one of the important clinical manifestations of this rare tumor.Recognition of this rare entity could be significant considering its poor outcome and differential diagnosis considerations. The differential diagnosis of primary LCNEC includes metastatic LCNEC, extension from poorly differentiated prostatic carcinomas, high grade urothelial carcinoma, small cell neuroendocrine carcinoma and lymphoma.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no conflict of interest.CC conceived of the study, participated in its design and drafted the manuscript. PP participated in the design of the study. DG participated in the design of the study. EA carried out the IHC study. RC carried out the molecular studies. DM carried out the molecular studies. LM participated in the design of the study and drafted the manuscript. All authors read and approved the final manuscript.


Bone grafting allows reconstruction of the atrophied or destroyed alveolar process. In orthopaedics and traumatology allogeneic grafting has been used to restore defects of osseous tissue for over 60 years. In order to improve safety of the graft recipient, sterilized allogeneic grafts have been use. The aim of the study was to assess the direct and long-term outcomes following augmentation of atrophied alveolar processes with the use of radiation-sterilized allogeneic bone grafts. Sixty-eight patients were surgically treated between 2004 and 2011: 29 underwent open sinus floor elevation, post-extraction alveoli augmentation was performed in 16 subjects and 23 underwent reconstruction of the atrophied alveolar process. Augmentation of bone defects used bone granulate in 63 patients and bone blocks stabilized with titanium screws in 5 patients. PRF membranes collected from the patient’s blood were also used in all the procedures. In each of the cases optimal dimensions of the alveolar process were obtained allowing embedment of BIOMET 3I dental implant/-s. In all the patients the defects were successfully restored with implant-supported prostheses. Radiation-sterilized allogeneic bone grafts proved to be safe and effective for the patients and manageable for the surgeon constituting a good alternative to autogeneic material. Lack of adequate mass of a patient’s own osseous tissue constitutes a clinical problem, still valid in dental implantology, which precludes intraosseous implant embedment. Bone grafting at the site of atrophied alveolar process is a first-line preparatory measure for implant-prosthetic treatment  scan: 4 mm, atrophy of the alveolar process (reduced height) from the side of the maxillary sinus and normal aeration of the maxillary sinus.The procedure was performed under local anesthesia. The mucous membrane was incised at the top of the alveolar process and then the incision was extended to the vault of the oral vestibule. The mucoperiosteal flap was detached showing the anterior wall of the maxillary sinus above the site of planned implantation. With a diamond drill the bone lamella was removed creating an oval bone lid leaving the mucous membrane, which lines the sinus, untouched. The mucous membrane was subsequently dissected free and strengthened with PRF membrane, which significantly decreased the risk of perforation during augmentation . The inclusion criteria were: minimal height of the alveolar process observed in the computed tomography (CT) scan: 8 mm (the length of the shortest implant), the width of the alveolar process below 5 mm and no signs of acute inflammation in the intraoral examination and additional tests  and negative results of serologic testing were obtained. Frozen cortico-spongious bone blocks were prepared from the iliac ala. After being defatted in alcohol solution and rinsed, bone blocks were subsequently radiation-sterilized with a dose of 35 kGy. Whereas, the frozen demineralized bone matrix was prepared from the compact bone of diaphysis. After grounding and defatting, the bone grafts were decalcified in 0.6 M HCl and rinsed. The dose of 25 kGy was used for radiation-sterilization. For both graft types the radiation sterilization was performed with the accelerated electron beam in the Institute of Nuclear Chemistry and Technology in Warsaw  the number of complications related to augmentation with the use of allogeneic bone was reduced considerably. The most common ones associated with self-tissue harvest included transient mandibular nerve paralysis, problems related to wound healing and/or wound breakdown. Due to considerably poorer patient’s hygiene inflammation of the mucous membrane within the operational site was commonly observed, which hindered and extended the recovery time  membrane, which contains large number of growth factors, the process of graft union with recipient’s own bone was accelerated. Due to elasticity and viscosity these membranes adhere to the bone surface acting as mechanical barrier against the penetration of the endothelium (Simon et al. Presented results strongly suggest that radiation-sterilized bone allografts can be successfully used for reconstruction in oral surgery procedures. Allogeneic bone material is a good bone replacement formulation in case of maxillary and mandibular alveolar process reconstruction and may be used in out-patient healthcare. The use of allogeneic grafting reduces the number of operational sites and minimizes the physical burden for the patient. The use of PRF membranes allows fixation of bone allografts as well as faster union with the patient’s self-bone. Orthodontic treatment facilitates scheduling and performance of augmentation and allows ultimate implant-prosthetic restoration of the dental defect improving the final aesthetic outcome.The ease of application and availability of the material allows grafting within a considerable area of the facial skeleton. Long-term clinical outcomes confirm the safety of this surgical procedure allowing permanent restoration of dental defects.Our data confirm that surgical procedure with radiation-sterilized bone allografts constitutes a very effective and promising treatment modality in patients with alveolar atrophy.


For the latter, we attached glass fibers of different stiffness to the stereocilia and observed the induced changes in the spontaneous bundle movement. When driven with sinusoidal deflections, hair bundles displayed phase-locked response indicative of an Arnold Tongue, with the frequency selectivity highest at low amplitudes and decreasing under stronger stimulation. A striking broadening of the mode-locked response was seen with increasing stiffness of the load, until approximate impedance matching, where the phase-locked response remained flat over the physiological range of frequencies. When the otolithic membrane was left intact atop the preparation, the natural loading of the bundles likewise decreased their frequency selectivity with respect to that observed in freely oscillating bundles. To probe for signatures of the active process under natural loading and coupling conditions, we applied transient mechanical stimuli to the otolithic membrane. Following the pulses, the underlying bundles displayed active movement in the opposite direction, analogous to the twitches observed in individual cells. Tracking features in the otolithic membrane indicated that it moved in phase with the bundles. Hence, synchronous active motility evoked in the system of coupled hair bundles by external input is sufficient to displace large overlying structures.Active hair bundle motility has been proposed to underlie the amplification mechanism in the auditory endorgans of non-mammals and in the vestibular systems of all vertebrates, and to constitute a crucial component of cochlear amplification in mammals. We used semi-intact  Experiments performed Apart from spontaneous oscillation, the active process in decoupled hair bundles manifests itself in rapid active movements or “twitches” in response to transient step stimuli The complementary metal oxide semiconductor (CMOS) imaging and motion-tracking software described in the To fully characterize the effects of loading on the response function of a hair bundle, we measured the motility evoked by mechanical stimuli, applied over the physiological range of the sacculus. Glass fibers were attached to the tallest row of stereocilia and used to deliver lateral sinusoidal displacements at increasing amplitudes and in discrete frequency increments. All of the hair bundles recorded exhibited spontaneous oscillations prior to the attachment of the stimulus probes.Upon loading with fibers that were elastic with respect to the hair bundles  were anesthetized with 200 to 600 Prior to performing experiments, all animal-handling protocols were approved by the UCLA Chancellor's Animal Research Committee (Protocol Number ARC 2006-043-13C) in accordance with federal and state guidelines. Adult bullfrogs  using a 20Borosilicate glass capillaries were pulled with a micropipette puller (Sutter Instruments Co. Flaming/Brown P-97). The tapers were cut with surgical scissors, then pulled again at For measurements of individual hair bundles, the probe tips were dipped in 2 mg/mL concanavilin A (Sigma-Aldrich) to improve adhesion to the stereocilia. Probes were mounted on a piezoelectric stack actuator (PiezoJenna PA 4/12), the position of which was controlled with an electric micromanipulator. Probes were positioned above the preparation and brought into contact with either the tallest row of stereocillia or the otolithic membrane. The piezoelectric stimulator was controlled by a function generator (Tektronix AFG 3022), which simultaneously triggered the CMOS camera recording. For experiments on tuning in the coupled system, stimulus frequencies were varied across the physiological range (up to 200 Hz) in 5 or 10 Hz increments, at fixed stimulus amplitudes, either 250 or 500 nm. The frequency selectivity of the evoked motion in the underlying hair bundles was characterized by the dimensionless response function Pulse trains  were delivered to the overlying otolithic membrane, and hair bundle motion was recorded at 1000–5000 fps. To improve the signal-to-noise ratio for the transient response, each trace was averaged over presentations of the pulse train. At both frame rates, we were able to measure the magnitude of the active response in the averaged trace. The phase-locked amplitude of each bundle was measured by fitting the first 5 ms of the trace to a sine wave. For the bundles recorded at 5000 fps, we measured the relaxation time following the twitch from the remainder of the trace. For 30/39 bundles recorded the recovery was well described by a single exponential decay  fit to tA linear stimulus isolator (World Precision Instruments A395) was used to pass constant electric currents though the saccular epithelium. Freshly chlorided silver wires were placed in both the apical and basolateral compartments of the chamber, and the current values were controlled with a function generator. To avoid a large transient at the onset and end of each step stimulus, the command signals were low-pass filtered at 10 Hz. Positive currents are defined as passing from the apical to basal sides of the epithelium. Mechanical pulse trains were delivered the the otolithic membrane as described above, with the transepithelial current clamped at 0, +10, and −10


Cbfa1 gene, also called Runx2, located on the short arm of chromosome 6. It primarily affects bones which undergo intramembranous ossification. This condition is of clinical significance to dentistry due to the involvement of the facial bones, altered eruption patterns and multiple supernumerary teeth.Cleidocranial dysplasia is a rare congenital defect of autosomal dominant inheritance caused by mutations in the Our patient, a 15-year-old Indian girl, presented with the typical features of prolonged retention of deciduous dentition and delayed eruption of permanent teeth, that is, mandibular prognathism along with other skeletal abnormalities like shrugged shoulder and the absence of clavicles. A multidisciplinary approach was followed, comprising orthodontic, surgical and pedodontic teams for management.Successful treatment of such a case lies in a holistic approach that takes care of all aspects, including the primary pathology, the deformity itself and even the psychological angle. Cleidocranial dysplasia is a rare congenital defect of autosomal dominance inheritance -3 that pThe pathology relating to this condition is due to an early developmental disorder of mesenchyme or connective tissue. This causes retarded ossification of bone precursors, especially at junctions, which can lead to defective ossification, or even failure of ossification, of portions of the skeletal structure.Cleidocranial dysplasia presents with skeletal defects, partial or complete absence of the clavicles, late closure of fontanelles, presence of open skull sutures and multiple wormian bones ,8. The mA 15-year-old Indian girl was referred to our Department of Pedodontics and Preventive Dentistry with the chief complaint of unerupted teeth. Her medical history revealed delayed closure of the anterior fontanelle, a fracture of her right humerus at three years of age and delayed puberty. Our patient was poorly built, short statured, moderately-nourished with a concave facial profile. She had shrugged shoulders with more than normal mobility of the shoulder girdle. Oral findings include Class III malocclusion with anterior and posterior crossbite and retained deciduous teeth  or Multiplex Ligation-dependent Probe Amplification (MLPA), needs to be done [Runx2, molecular genetic analysis is recommended.It has been suggested that 70% of patients with cleidocranial dysplasia have a point mutation involving  be done -12. ThouDental findings in cleidocranial dysplasia are characterized by a decreased eruptive force of both primary and permanent dentition, prolonged retention of primary teeth  and an iThe treatment objective in our case was to consider both the physical and psychological aspects of our patient. Redistribution of the space in her oral cavity and guidance of the permanent teeth to erupt in a proper alignment were planned. These were achieved by extracting the retained teeth and surgical exposure followed by orthodontic traction for the eruption of the permanent teeth . SimultaStabilization of the periodontal health of her permanent teeth and necessary orthodontic treatment will be continued in further follow-up appointments .Cleidocranial dysplasia patients with compromised aesthetics are usually seen as an unexpected event in the course of observing or treating a patient. Early diagnosis allows proper orientation to the treatment and offers a better life quality. A holistic approach takes care of all the aspects, including the primary pathology and the psychological aspects.Written informed consent was obtained from our patient's parents for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.NC analyzed and interpreted the patient's data regarding the associated dental problems. BSS, SM, NHK and RY performed jointly the treatment procedures and were major contributors in writing the manuscript. All authors read and approved the final manuscript.


Ultrasound (US) is one of the most important diagnostic tools available for the detection and evaluation of carotid stenosis. The case of a 70-year-old woman with recurrent right-sided amaurosis fugax presented here highlights the way in which tissue Doppler imaging (TDI) and contrast-enhanced US (CEUS) may aid in the diagnosis of carotid plaque vulnerability. Furthermore, the novel inverse fly-through technique was used for the three-dimensional visualization of the carotid stenosis. Stroke risk in patients with carotid stenosis may vary widely. The risk of ipsilateral ischemic stroke is increased in patients with a previous stroke. Furthermore, stroke risk increases progressively with the severity of carotid stenosis. The risk of a recurrent stroke or transient ischemic attack (TIA) is the greatest immediately after the initial ischemic event and decreases over time, as does the potential benefit which may be derived from carotid endarterectomy (CEA) . The casA 70-year-old woman was admitted after transient right monocular visual loss on the day of presentation and on the previous day. The medical history revealed that, eight years before, the patient had undergone right CEA for symptomatic stenosis of the internal carotid artery (ICA). Preceding that operation, the patient had suffered two TIAs with reversible paresis of the left arm. US examination on admission revealed a 60 percent right-sided ICA stenosis . The proAn extended US examination including CEUS  demonstrated neovascularization of the carotid plaque (arrows in In summary, the most likely explanation of the recurrent transient monocular visual loss of our patient was amaurosis fugax secondary to right-sided 60 percent ICA stenosis. After weighing all risks and benefits, a recommendation for CEA was made. The procedure was performed promptly and went without complication. A good correlation was found between US findings and the microscopic examination of the CEA specimen Figures . In partThe recent advances in noninvasive carotid imaging promise the identification of new carotid plaque vulnerability markers. It is to be hoped that this will further improve the assessment of stroke risk associated with carotid stenosis.Symptomatic carotid artery disease is associated with plaque neovascularization, intraplaque hemorrhage, and invasion of inflammatory cells. CEUS increasingly provides the means to quantify the degree of neovascularization of an atherosclerotic plaque. Since neovascularization is associated with an inflammatory infiltrate, CEUS may also indicate increased invasion of inflammatory cells –4.Hypoechogenicity is already well established as a marker of high-risk carotid lesions . TDI offWhile there is already good data showing the value of CEUS in the diagnosis of carotid plaque neovascularisation, more work will be required to delineate the specific value of novel US techniques such as TDI and the three-dimensional fly-through technique.


Understanding how signals propagate through signaling pathways and networks is a central goal in systems biology. Quantitative dynamic models help to achieve this understanding, but are difficult to construct and validate because of the scarcity of known mechanistic details and kinetic parameters. Structural and qualitative analysis is emerging as a feasible and useful alternative for interpreting signal transduction.In this work, we present an integrative computational method for evaluating the essentiality of components in signaling networks. This approach expands an existing signaling network to a richer representation that incorporates the positive or negative nature of interactions and the synergistic behaviors among multiple components. Our method simulates both knockout and constitutive activation of components as node disruptions, and takes into account the possible cascading effects of a node's disruption. We introduce the concept of elementary signaling mode (ESM), as the minimal set of nodes that can perform signal transduction independently. Our method ranks the importance of signaling components by the effects of their perturbation on the ESMs of the network. Validation on several signaling networks describing the immune response of mammals to bacteria, guard cell abscisic acid signaling in plants, and T cell receptor signaling shows that this method can effectively uncover the essentiality of components mediating a signal transduction process and results in strong agreement with the results of Boolean  dynamic models and experimental observations.http://www.phys.psu.edu/~ralbert/ESM.This integrative method is an efficient procedure for exploratory analysis of large signaling and regulatory networks where dynamic modeling or experimental tests are impractical. Its results serve as testable predictions, provide insights into signal transduction and regulatory mechanisms and can guide targeted computational or experimental follow-up studies. The source codes for the algorithms developed in this study can be found at  The normal functioning of biological organisms relies on the coordinated action of a multitude of components. The interactions between genes, proteins, metabolites and small molecules form networks that govern gene regulation, determine metabolic rates, and transduce signals ,2. InterGiven the topology (i.e. the nodes and edges) of a network, it is natural to wonder just how important  each node is to the network's connectivity and functionality. Not surprisingly the issue of node centrality and its correlation with node influence has attracted the attention of many researchers. A large number of graph measures have been developed for evaluating node centrality in complex networks, from degree centrality , closenep, of which one, the edge from A to Cp, is negative . Moreover, combinatorial regulation is ubiquitous in biological networks; this means that multiple interactions that regulate a component may act in a synergistic  fashion , with 1 indicating a node whose loss causes the disruption of all ESMs between the input and output node(s). As a comparison we also define the analogous measure using simple paths instead of ESMs (Methods).Our method ranks the importance of signaling components by the effects of their perturbation on the ESMs of the network. We characterize each node EESM) and the simple path (SP) centrality measure (denoted by ESP). We compare these measures to a traditional centrality measure, node betweenness centrality (denoted by BC) , with 1 indicating a node whose loss disrupts all ESMs between the input and output node(s).where We also consider a more traditional graph measure for the essentiality of signaling components based on the number of all simple paths (SPs) from inputs to outputs:NSP(G) and NSP(ΔvG) denote the total number of simple paths from the input(s) to the output(s) in the original expanded network G and the damaged network Δv Gafter deleting v, respectively. This SP measure also takes values in the interval , with 1 indicating a node whose loss causes the disruption of all paths between the input and output node(s). There are polynomial algorithms for computing the paths between any pair of nodes in a graph [where  a graph . Here weterval 0,, with 1 bintprog in the Matlab Optimization Toolbox which uses a branch-and-bound algorithm to solve binary integer linear programming problems. Programs are available at http://www.phys.psu.edu/~ralbert/ESM.All the algorithms in this study were coded and implemented in Matlab 7.6 . The depth-first algorithms are the Matlab implementation of the pseudo code given in the Additional file RSW and RA conceived and designed the study. RSW performed the study. RSW and RA analyzed the results. RSW and RA wrote, read, and approved the final manuscript.Algorithms developed and used in this study. This file contains the algorithms developed and used in this study, including the depth-first-search algorithm for enumerating all simple paths from the input node(s) to the output node(s) of a signaling network, the iterative integer linear programming algorithm for enumerating elementary signaling modes, the depth-first-search algorithm for estimating the number of elementary signaling modes, as well as the dynamic programming algorithm for finding shortest elementary signaling modes.Click here for fileEssentiality of the components from dynamic models of the three signaling networks. This file describes the essentiality of the signaling components obtained by dynamic simulation of Boolean models for the host immune response network and the guard cell ABA signaling network, and logical steady state analysis of the T cell receptor signaling network (Tables S1-S4).Click here for fileEssentiality of the guard cell ABA signaling components from our method. This file contains the importance values of the guard cell ABA signaling components obtained by single-node deletions  and two-node deletions , and literature support of the uncovered essential components.Click here for fileThe expanded T cell receptor signaling network. This file contains the expanded T cell receptor signaling network  and the importance values of the T cell receptor signaling components found by our method with AP as the input node .Click here for file


Hz to occur.Cortical oscillatory signals of single and double tremor frequencies act together to cause tremor in the peripheral limbs of patients with Parkinson's disease (PD). But the corticospinal pathway that transmits the tremor signals has not been clarified, and how alternating bursts of antagonistic muscle activations are generated from the cortical oscillatory signals is not well understood. This paper investigates the plausible role of propriospinal neurons (PN) in C3–C4 in transmitting the cortical oscillatory signals to peripheral muscles. Kinematics data and surface electromyogram (EMG) of tremor in forearm were collected from PD patients. A PN network model was constructed based on known neurophysiological connections of PN. The cortical efferent signal of double tremor frequencies were integrated at the PN network, whose outputs drove the muscles of a virtual arm (VA) model to simulate tremor behaviors. The cortical efferent signal of single tremor frequency actuated muscle spindles. By comparing tremor data of PD patients and the results of model simulation, we examined two hypotheses regarding the corticospinal transmission of oscillatory signals in Parkinsonian tremor. Hypothesis I stated that the oscillatory cortical signals were transmitted via the mono-synaptic corticospinal pathways bypassing the PN network. The alternative hypothesis II stated that they were transmitted by way of PN multi-synaptic corticospinal pathway. Simulations indicated that without the PN network, the alternating burst patterns of antagonistic muscle EMGs could not be reliably generated, rejecting the first hypothesis. However, with the PN network, the alternating burst patterns of antagonist EMGs were naturally reproduced under all conditions of cortical oscillations. The results suggest that cortical commands of single and double tremor frequencies are further processed at PN to compute the alternating burst patterns in flexor and extensor muscles, and the neuromuscular dynamics demonstrated a frequency dependent damping on tremor, which may prevent tremor above 8  Hz is originated from oscillatory neuronal activities in subcortical and cortical networks Resting tremor in Parkinson's disease (PD) is elicited by the reciprocal and alternating activities of antagonistic muscles There are two major descending pathways from primary motor cortex to spinal motor neurons, a mono-synaptic cortico-motoneuronal (CM) pathway, and a multi-synaptic cortico-motoneuronal pathway via interneurons Observations of cortical signals and peripheral behaviors of PD tremor may provide the input-output information that allows us to investigate the corticospinal mechanism of tremor transmission We also designed experiments to record resting tremor and EMGs signals from PD patients with their arm performing postural tasks in the horizontal plane with gravity-support cast on a frictionless glass surface In this study, a model was developed to simulate tremor behaviors driven by cortical oscillatory inputs  with idiopathic tremor-dominant Parkinson's disease were recruited for this study. All subjects had no other neurogenic disease. The study was approved by the Ethics Committee of Animal and Human Subject Studies of Shanghai Jiao Tong University. All subjects signed the informed consent form, and were instructed by the attending physician to take their prescribed medications as usual while participating the experiment session.seconds in each trial. The task was repeated 10 times at each posture for each subject. The movement was captured using the MotionMonitor II system with a 120 frame per second resolution. Surface EMG signals were amplified with a gain of 5000, then band-pass filtered with 1 Hz high-pass and 1000 Hz low-pass filter, and then sampled at 2410 Hz. The spectrum of sampled EMG and motion data were calculated using FFT algorithm in MATLAB . The temporal patterns of tremor kinematics and RMS EMG were used later for comparison with simulated tremor behaviors.An experimental method was designed to record resting tremor in PD patients without the effect of gravity Ia and Ib afferents of muscle spindles and Golgi Tendon Organ (GTO), as well as recurrent inhibition of Renshaw cells. The outputs of The corticospinal-virtual arm (CS-VA) model consisted of a propriospinal neuron (PN) network At the bottom of the system, the generic VA model included components of virtual muscle (VM), biomechanical model of musculoskeletal dynamics, proprioceptor models (muscle spindle and GTO), and spinal reflex circuitry. The VA model was validated to capture the realistic neurophysiological and neuromechanical characteristics of the sensorimotor system of human upper extremity Ia, Ib and recurrent feedback information. It was shown that the values of reflex gains must be kept relatively low in order to maintain the VA in a stable state, except for Ib afferent The spinal reflex circuitry regulated the outputs of The C3-C4 propriospinal neuronal system is unique since much of its organization and function is well known Ia afferent to PN; Ia afferent discharge frequencies of spindles arising from flexor and extensor muscles. Subscripts f and e denote variables pertaining to flexor and extensor respectively. Note that Based on these neurophysiological connections of PN The central commands originated from primary motor cortex (M1) may activate the  pathway . The mulBased on the findings of Taylor et al. Hz, and that of the motor command of Hz. The range of Hz, which appeared to be the median tremor frequency we observed in PD patients In simulation experiments, we chose sinusoidal signals of single and double tremor frequencies to approximate the  0.5 see . This biSimulation experiments were performed with the protocol established in previous simulation studies ture see ; and a sIa and Ib origins, as well as recurrent inhibition The first set of simulation experiments was performed using the CS-VA model without the PN network. In this case, postural commands,  and joint movements were analyzed off-line in MATLAB. Joint kinematic data were filtered by low-pass Butterworth filter with zero-phase shift, and cut off frequency of 20 Time delay and phase shift between the bursts of antagonist muscle inputs (U) were evaluated. A publically available MATLAB code  multi-synaptic corticospinal pathway. In this scenario, the PN network is involved in processing the descending cortical oscillatory signals.Test of hypotheses was performed by comparing the simulated behaviors of the CS-VA model with and without PN network to those observed in PD patients. Decision of accepting or rejecting a hypothesis was based on the ability of the model to reproduce the signature features of kinematic tremor and EMG signals of PD patients qualitatively and/or quantitatively. The test criteria included (1) the occurrence of single tremor frequency peak in the spectra of simulated muscle inputs and joint movements, and (2) the occurrence of alternating activation pattern of antagonistic muscles, i.e. there was a significant phase shift between the simulated inputs to antagonistic muscles.Hz with an average of 4.6 HzTremor and/or alternating burst EMGs in flexor and extensor muscles were observed in all seven PD patients during posture maintenance in the horizontal plane. The frequencies of tremor and EMGs of these PD patients ranged from 3.8 to 5.4 0 and elbow joint at about 850. In Hz in both the shoulder and elbow. Showing in msec and 146.21±18.25 deg, from FDS to ED was 121.98±15.48 msec and 180.35±22.89 deg  showed an in-phase co-contracting bursts, but not the alternating pattern as seen in PD patients  . This agHz, but there were also declining harmonic components at 10 Hz and 15 Hz in the muscle inputs, which were similarly observed in EMGs of PD patients. The virtual arm displayed tremor like behavior of 5 Hz primarily at the elbow joint accompanied by a smaller oscillation of 5 Hz at the shoulder joint. The higher frequency components in muscle inputs (U) were filtered out by the low-pass filter effect of neuromuscular dynamics (see section 4 in the following). The simulated joint oscillations captured the general characteristics of pathologic Parkinsonian resting tremor and the signature feature of PD patients presented in The results from second set of simulations were presented in The simulated tremor behaviors within the full range of cortical commands of sec on the average, and the phase shift was about 180 deg. Thus, the PN network was able to create a phase shift between flexor and extensor activations that were favorable to produce oscillatory movements.The calculated time delay between neural inputs of flexor and extensor muscles  was abouHz was analyzed with constant amplitude of cortical oscillations. In Hz, the tremor amplitude diminished to almost zero. This decrease in tremor amplitude was due to the low-pass filter nature of neuromuscular dynamics Hz were typically observed in PD patients In the third set of simulation experiments, how Parkinsonian tremor of different frequencies may be attenuated by neuromuscular dynamics were evaluated, and the results were shown in In this study, we address the question whether the PN network is involved in corticomuscular computation of antagonist muscle activations from cortical oscillatory commands in the generation of Parkinsonian tremor. Evidence accumulated from normal rhythmic movement studies A bottom-up modeling and computational approach is taken in this study to elucidate this question combined with experimental observation of tremor kinematic and EMG behaviors in PD patients Hz to occur. Indeed, only tremors between 3–7 Hz are clinically observed in PD patients Hz, which is well within the range of clinical PD tremor. Our model is able to make such a prediction because it contains realistic neuromuscular dynamics and accurate biomechanics of the limb Simulation results indeed demonstrates that the CS-VA model with PN network can reliably reproduce the signature feature of tremor behaviors of PD patients, i.e. the alternating bursts of flexor and extensor muscle activities in the full range of central parameters of cortical commands and frequencies . The altThe results clearly illustrate that without the PN network, the tremor behaviors cannot be reproduced with the CS-VA model. In this case, a high level of co-contraction bursts persists in the antagonist muscles, which prevents the generation of tremor at joints. This observation alone is sufficient to reject the first hypothesis, and rule out the possibility that the cortical oscillatory signals of Parkinsonian subjects are transmitted via the mono-synaptic corticospinal pathway bypassing the PN network. This outcome also suggests that the spinal reflex circuits alone are not sufficient to partition the central oscillatory signals into alternating burst activities in the antagonistic muscles in the range of stable spinal reflex gains.The results of simulation substantiate the second hypothesis that a corticomuscular computation is performed at the premotor spinal level in order to produce alternating bursting activities at the antagonist muscles. The PN network is shown to be able to perform the computation of the alternating activations of flexor and extensor muscles for a wide range of tremor behaviors. This function can be accredited to the mirror-gaiting inhibition of nditions  & 9, thuBasal ganglia circuitry has two outputs, one of which is fed back to cortex via thalamocortical pathway. This signal is relayed to spinal C3–C4 propriospinal neurons through primary motor cortex. The other output of basal ganglia is passed down to the brainstem and to the lumbar segment of spinal cord to control locomotion The effect of proprioceptive afferents on tremor in PD patients is largely unclear. However, the spinal reflex gains in PD patients did not seem to be drastically different from those of normal subjects A relevant issue regarding the organization of cortical oscillations of PD tremor can also be implicated using the CS-VA model of In this study, a dual set of cortical command signals are implemented for postural maintenance  system is applied to replicate tremor behaviors in PD patients. The agreement of simulated tremor behaviors with those observed in PD patients supports the hypothesis that cortical tremor commands are transmitted downstream to the motoneuron pools of muscles via the propriospinal neuron (PN) pathway. The PN network in the C3–C4 levels is revealed to play the pivotal role in computing the alternating burst pattern of flexor and extensor EMGs by a mirror-gaiting mechanism. This computational role is further evaluated in the full range of tremor generating conditions in comparison with behaviors observed in PD patients. The CS-VA model also demonstrates the frequency dependent damping effect on tremor activities by neuromuscular dynamics. The high frequency damping may have prevented tremor above 8


The Sertoli cells of the testes secrete anti-Müllerian hormone  and inhibin B (InhB). AMH triggers the degeneration of the uterine precursor in male embryos, whereas InhB is part of the gonadal-pituitary axis for the regulation of sperm production in adults. However, both hormones are also putative regulators of homeostasis, and age-related changes in these hormones may therefore be important to the health status of elderly men. The levels of AMH in elderly men are unknown, with limited information being available about age-related changes in InhB. We have therefore used ELISAs to measure Sertoli cell hormone levels in 3 cohorts of community-dwelling men in New Zealand. In total, 615 men were examined, 493 of which were aged 65 or older. Serum AMH and InhB levels inversely correlated with age in men older than 50 years (p<0.001) but not in the younger men. A minority of elderly men had undetectable levels of AMH and InhB. The variation in hormone levels between similarly aged men increased with the age of men. AMH and InhB partially correlated with each other as expected . However, the ratio of the two Sertoli hormones varied significantly between men, with this variation increasing with age. Elderly men selected for the absence of cardiovascular disease had AMH levels similar to those of young men whereas their InhB levels did not differ from aged-matched controls. These data suggests that Sertoli cell number and function changes with age, but with the extent and nature of the changes varying between men. The testes contain two distinct endocrine cells, the androgenic Leydig cells and the Sertoli cells, which produce AMH and InhB. In male embryos, AMH triggers the degeneration of the uterine precursor Recent studies also implicate serum AMH in human non-gonadal development. The level of AMH varies greatly between boys of similar age, with the interpersonal differences remaining stable over years InhB is part of the gonadal pituitary axis and contributes to the regulation of sperm production The classical functions of both AMH and InhB are independent of each other. However, AMH and InhB cooperate to suppress testicular cancer in mice As noted above, both Sertoli cell hormones have well characterized classical functions, but with little exploration of their wider functions. Consequently, much of the basic information relating to these hormones is absent, including their circulating profiles across the lifespan of men. Ovarian production of both AMH and InhB decline during the menopausal transition, and is absent after the menopause. Testosterone secretion declines with age in most men This paucity of basic information makes it difficult to determine whether changes in Sertoli cell hormones might contribute to the deterioration of elderly men, and/or whether sex differences in age-related diseases are partially due the loss of gonadal protein hormones. We have therefore examined the levels of AMH and InhB, and the ratio of these two hormones, to produce the first description of Sertoli cell function in elderly men. We report that, on average, both Sertoli cell hormones are lower in elderly men, with wide variation between the two hormones suggesting age-related changes in both the regulation and the number of Sertoli cells.All study participants gave written informed consent. The study of the Otago elderly subset cohort, and Vascular cohort were both approved by the Lower South Regional Ethics Committee of New Zealand, whilst the LiLACS NZ cohort study was approved by the Northern X Regional Ethics Committee of New Zealand. The younger Otago men study was approved by the University of Otago Human Ethics Committee.Serum samples were collected from 615 men aged from 19 to 93 years, with a focus on elderly men (n = 496). The participants were drawn from three distinct cohorts . A non-fThe Otago cohort was a cross-sectional study of 99 community dwelling men aged 70 or older, with 119 younger men as a comparison. The men were predominantly Caucasian and resided in Dunedin, a small university city in New Zealand. The exclusion criteria for this cohort was limited to a history of testicular tumor, testicular trauma, orchidectomy, undescended testes, self-reported infertility, chemotherapy or abdominal or pelvic radiation therapy. None of the men were on anti-androgen medication or steroid medications. The elderly men were recruited from community groups and clubs, including men receiving outpatient rehabilitation for physical or mental health (mood disorders or cognitive decline). The younger men were recruited from various facilities including staff and students at the local university, medical and non-medical hospital staff, light and heavy industrial occupation, civil service, and office-based private sector employees.The vascular cohort was a distinct Dunedin cohort comprised of 153 community dwelling men aged 54–93 years, with no history of cardiovascular disease. These men were recruited from advertisements and screened for peripheral arterial disease and carotid stenosis. Men with a carotid stenosis of greater than 50% and men with an ankle-brachial pressure index of less than 0.9 or greater than 1.3 were excluded from this cohort. Their sera were obtained from the Vascular Research Group Biobank, University of Otago.āori aged between 80–90 years old, and non-Māori aged 85 years old were recruited, with a response rate from men of 65%. Study participants had varying health and physical performance status measured, with the 257 men examined being those that had consented for their sera to be used for collaborative research. None of the participants reported a history of orchidectomy.Sera from 257 elderly men were analyzed from the LiLACS NZ Research Group . This is an ongoing epidemiological study investigating factors that lead to successful aging in elderly New Zealanders Serum AMH levels were measured in duplicate, with 575 of the 615 samples being measured with the enzyme immunoassay AMH/MIS kit . The other 40 were analyzed MIS/AMH ELISA , which became unavailable after Beckman Coulter acquired DSL. The two kits give similar results Serum InhB levels was measured in duplicate by immunoassay . The intra and interassay coefficient of variation (CV) were 6.5% and 12.6%.The levels of the two Sertoli cell hormones varied between individuals. If this variation is examined using a simple ratio of the hormones (AMH/InhB) then the resulting distribution will be non-symmetrical. Individuals with high AMH will be represented on a scale from 1 to infinity whereas those with high InhB will have values between 0 and 1. The ratio was therefore calculated using the ratio of the natural logs of the hormones (ln(AMH)-ln(InhB)) which produces a symmetrical distribution, without bias to either hormone. When this ratio is standardized using the mean levels of the hormones in the young men, then the distribution centers around zero. Any variation from a normal distribution indicates biological skewing, with the width of the bell curve being a measure of the variation in the population: as per the normal distribution of each individual hormone.The associations between AMH, InhB and age were tested by linear regression. The AMH and InhB levels in the Otago cohort were compared with the data from the other two cohorts using the nonparametric Wilcoxon-Mann-Whitney test. Skewness was calculated and its significance tested using the SKTEST. All calculations were performed in Stata/IC 12.1 .The levels of Sertoli cell hormones in a cross-section of community dwelling men  were examined, with the sole exclusion criteria being pharmacological or physical manipulation of the testes.The levels of AMH were highly variable between men, with a distribution that was positively skewed in both the younger and older men . All youThe profile of InhB was similar to that of AMH. InhB was less variable between individuals than AMH cf , 2A, andThe relative levels of AMH and InhB varied between individuals, in both the young and the elderly . HoweverThe Otago cohort was then compared to two other cohorts of elderly men, to determine the generalizability of the observations. The first comparison was between the community-dwelling Dunedin men, described above, and a second Dunedin cohort (n = 153) that had been selected for the absence of cardiovascular disease (vascular cohort). The levels of AMH in the vascular cohort were higher than the age-matched portion of the Otago cohort , p<0.001The elderly portion of the Otago cohort (n = 99) was then compared with the LiLACS NZ cohort (n = 257), which was collected from a more northern community, with a different socioeconomic and ethnic composition. The age-matched AMH and InhB levels were not different between the cohorts, although 3.5% of the LiLACS NZ cohort had atypically high levels of InhB, which could not be attributed to race .When the three cohorts were combined, the elderly men displayed larger divergence in the ratio of AMH and InhB, in both the positive and negative directions. The vascular group had a prominent AMH bias .This study shows that the hormonal output of Sertoli cells persists in the very old, but with characteristics that differ on average from younger men. The levels of both AMH and InhB were highly variable in the elderly men, with mean values approximating half those of younger men. This is consistent with histological studies of the testes showing age-related attrition of Sertoli cells There was an age-related increase in the inter-person variation in the levels of AMH and InhB, suggesting that the regulation of Sertoli cell output changes in some elderly men. A dysynchrony between LH and testosterone occurs in elderly men The vascular cohort, which was free from cardiovascular disease, had higher levels of AMH and tended to have a higher AMH to InhB ratio. The most obvious difference between the cohorts is the proven absence of cardiovascular disease in the vascular cohort. The importance of this is currently unknown, although it is consistent with recently observations linking AMH to atherosclerosis in rhesus monkeys As noted previously, the levels of InhB were lower in older men, with high statistical significance. This contrasts with a previous study by Mahmoud et al, who reported higher InhB levels in the elderly, with minimal age-related deterioration Secular changes have been observed in the androgen status of elderly men, with men born in earlier generations having higher levels of testosterone than more recent generations In summary, this study suggests that the plasma levels of Sertoli hormones decline with aging, but with high variability. There are associations between AMH, InhB and the ratio of AMH/InhB to the health status of elderly men. This identifies an area for future research, particularly into the role of Sertoli hormones in cardiovascular disease and frailty in elderly men.Figure S1(TIF)Click here for additional data file.Figure S2(TIF)Click here for additional data file.


Simultaneous recordings of the firing activity of pairs of cortical neurons have shown that spike-count correlation coefficients (CCs) cover a wide range of values. According to recent theoretical and experimental work ,2 recurrWe examine spike-count correlations obtained from pairs of simultaneously recorded premotor cortex (PC) neurons while trained monkeys performed a vibrotactile detection task in which the stimulus was often absent or weak, and the time of its application was variable ,4. By anOur results suggest that sensory detection is partly due to a purely internal signal whereas the stimulus, if finally applied, adds a contribution to this initial processing later on . Noise cThe observation that single neurons covary with the subject's response  is usually explained by the existence of variability correlations among the cells in a neuronal population . Here weN index to quantify the amount of covariation with behavior of arbitrary linear combinations of oppositely-tuned neural pools. We found that pools of PC neurons exhibiting persistent activity become fully correlated with the subject's choice soon after stimulus onset and during the entire delay period of the task.Although the CP index is useful to study the role of single neurons in decision-making tasks, in reality, the decision is formed through the coordinated action of several pools of neurons. Hence, the relevant quantities to investigate the elaboration of the choice are population variables combining the activity of several pools. By extending the notion of CP from single neurons to neural pools, we defined the CP


Plasmodium and transmission-control was held April 5-6, 2011, in Bethesda, MD. The meeting was sponsored by the Bill & Melinda Gates Foundation and the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIH/NIAID) in response to the challenge issued at the Malaria Forum in October 2007 that the malaria community should re-engage with the objective of global eradication. The consequent rebalancing of research priorities has brought to the forefront of the research agenda the essential need to reduce parasite transmission. A key component of any transmission reduction strategy must be methods to attack the parasite as it passes from man to the mosquito (and vice versa). Such methods must be rationally based on a secure understanding of transmission from the molecular-, cellular-, population- to the evolutionary-levels. The meeting represented a first attempt to draw together scientists with expertise in these multiple layers of understanding to discuss the scientific foundations and resources that will be required to provide secure progress toward the design and successful implementation of effective interventions.A meeting to discuss the latest developments in the biology of sexual development of  It is now recognized that understanding and attacking the parasites responsible for infection of the mosquitoes is critical to the international efforts striving to eliminate malaria . FurtherThe objectives of the meeting were to:Plasmodium gametocyte biology,• Develop a comprehensive landscape of research initiatives, programmes, and funding vehicles in • Identify and prioritize key gaps in the knowledge base and research agenda  created an opportunity for international researchers and stakeholders to share research updates, identify critical experiments, and discuss the resources required to advance these objectives. The key research questions addressed by separate sessions of the meeting included:1. What are the critical factors comprising/regulating gametocyte biology?2. What are the critical factors influencing transmission dynamics?3. What are the best ways to target current and future interventions?4. What are the critical needs for the field to advance?Plasmodium, it was highlighted that in a minority of parasite species (in the subgenus Laverania) e.g. Plasmodium falciparum sexual development differs substantially from the majority (in the subgenus Plasmodium) e.g. Plasmodium vivax Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi and Plasmodium berghei. The importance of recognizng the underlying molecular differences in developmental strategies between these species-groups is critical both to experimental analysis, and to the rational design and understanding of interventions in the clinical/field setting. For example, the protracted (9-12 day) developmental period of gametocytes in P. falciparum whilst offering a near unique opportunity to dissect gametocyte maturation at the molecular level, results in a totally different relationship between morbidity (and therefore presentation to the clinic) and transmission, i.e. while cases of vivax may be more infectious to mosquitoes prior to presentation at a clinic, falciparum cases are more infectious after presentation.In recognizing the meeting would discuss sexual development of all species of P. falciparum, are expressed 24 hours into their development, Christian Doerig added that Pfnek-4 expression might also identify sexually committed gametocytes. It has been shown that all the progeny from a single blood stage schizont are already committed to become asexual parasites or either male or female gametocytes [P. falciparum which identified 11 genes up-regulated in committed parasites, three of which could be identified in 'asexual' schizonts. Christian Doerig noted that the normal excess of schizonts that are not committed to gametocytogenesis makes studying the biochemistry of the committed-few very challenging. Pietro Alano noted that recently has it been possible to purify to virtual homogeneity Stage-Ia gametocytes, thereby enabling differential proteomics [Pietro Alano discussed commitment to, and differentiation of, sexual stages at a cellular level. Pfs16 and Pfg27/25-the first molecular markers of gametocytes of the human malaria parasite etocytes . Until rP. falciparum is consistent with a constitutive 10% rate of induction. She too recognized that conversion rates are more complex in the field, where parasitaemia alone does not predict gametocyte production, and as suggested by the early studies, she surmised the onset of clinical symptoms, if they occur, is a better predictor.Following these presentations Chris Drakeley commented that, while there is laboratory work indicating that 'stress' induces gametocytogenesis, as posited by the early studies of Sinton , in natuSarah Reece offered an evolutionary ecology perspective on malaria gametocytogenesis emphasizing the need for its better analysis in order to understand malaria transmission. For example, she suggested the conventional view that malaria transmission will cease when basic reproductive rate (Ro) is reduced to less than one (see below) may not hold. Under transmission reducing measures, theory suggests parasites may adjust their gametocyte sex ratio to maximize their reproduction and thereby raise Ro again . BecauseP. falciparum do nevertheless sequester and there is a need to identify the parasite molecules involved.Pietro Alano reported that kinase knockouts (KO's) do not affect gametocyte production. Compared with merozoites, he noted that young gametocytes have a very high level of expression of export-related proteins , that erBob Sinden discussed the cellular organization of mature gametocytes. Mature gametocytes of both sexes while sensitive to inhibitors of respiration, are less sensitive to anti-metabolites and schizonticides than the immature forms and asexual stage parasites -10. MatuKim Williamson further discussed the biochemistry and gene expression of gametocytes. As indicated above, protein synthesis and degradation are essential for gametocyte maturation. The Alamar Blue viability stain confirms that gametocytes are metabolically active and can be targeted with antimetabolites e.g. epoxomicin and cycloheximide . Work isP. berghei. Questions that remain unanswered include: "What is the origin of XA in the blood meal?", and "Can one activate gametocytes in the blood stream to eliminate their transmission potential?"Oliver Billker inferred gametogenesis is optimized for speed not quality of outcome. From the moment of their induction to undergo gametogenesis, gametocytes of both sexes take around ten minutes to complete "rounding up" and emergence from the RBC, simultaneously the male undergoes "exflagellation" to release the 8 male gametes. Induction is triggered by a drop in temperature, an increase in intracellular pH, and exposure to the mosquito exflagellation factor xanthurenic acid (XA) in the mosquito blood meal . Within pfkch1) leads to loss of gametocyte infectivity in the mosquito [Nirbhay Kumar discussed cellular events and molecules in gametogenesis. To achieve this inside a mosquito blood meal, the parasite must adapt to a new pH-, temperature-, ionic/nutritional- environments, and to the components of the vertebrate and mosquito hosts immune systems to which the parasites are exposed. Potassium (K+) channels could play a role as possible drug targets. Knockout of the encoding gene  or cross-fertilize (forming heterozygotes) with equal frequency. Mating appears to be random between gametes of different genotypes ,25 the rmosquito . LinkageChlamydomonas, appears to be a useful model for fertilization in Plasmodium. It is easy to grow, easy to manipulate, has well-characterized biology, and can be manipulated by the introduction of transgenes, but current evidence suggests this rarely occurs by homologous recombination [Chlamydomonas and Plasmodium [Trypanosoma, Toxoplasma, and Eimeria. The fusion mechanism is likely to be conserved across these groups of organisms; on the other hand the initial coupling of cells is expected to be achieved by species-specific proteins.Bill Snell discussed fertilization in protists. The free-living protist, bination . Hap2 waasmodium . Hap2 isThe main objective of this session was to describe what is known about the dynamics of natural infectivity and the human reservoir of infection, and subsequently to identify some of the knowledge gaps related to the fundamental basic biology of gametocytes. It was recognized that malaria transmission is focal, i.e. it occurs within spatially limited areas - each focus of transmission is determined by the locations of vector mosquito breeding sites and the flight patterns of the resident anopheline vectors responsible for parasite dissemination. As defined by the Ross/MacDonald equation, malaria transmission is dependent upon many different, and targetable, biological events, which must be considered in concert and not in isolation. If the malaria case multiplication rate, Ro, is greater than 1, malaria transmission will be sustained within each focus. If Ro consistently falls below 1, the focus will cease to sustain transmission. As a very rough approximation, it was hypothesized any single intervention may be expected to reduce Ro by two- to four-fold when randomly applied . It was suggested targeting multiple interventions to sites of high transmission may increase their impact by a further two- to four-fold. By combining different interventions, powerful overall reductions in malaria transmission may be achieved if, and when, the reductions due to the individual interventions multiply together. Within a focus of transmission, malaria case incidence rates vary markedly across distances as short as a few tens of meters, and between foci malaria transmission rates can vary by orders of magnitude across distances of a few hundred metres to kilometres -33, distP. falciparum have, of necessity, relied on data generated from a limited number of laboratory strains in highly controlled experimental conditions. Data from the first half of the 20th century [P. falciparum gametocyte carriage and infectivity. He suggested one of the most important recent field observations is the confirmation of prior 'malaria-therapy' data [Recent descriptions of the basic biology of  century  suggest py' data  that higpy' data . This mipy' data . They inP. vivax in SE Asia where a higher proportion of patients are infectious compared to P. falciparum (Jetsummon Sattabongkot). Similar data were observed in South America (Jo Vinetz) with a variety of parasite and host factors incriminated, including immune and metabolic mediators, of these the impact of sexual schizogony on P. vivax gametocyte infectivity has been particularly well-documented [Recognizing the numerous extraneous factors that regulate the infectivity of mature gametocytes , the relcumented . The rolP. falciparum gametocyte sequestration, and specific reagents to investigate these phenomena. The identification of biomarkers to identify persons harbouring infectious gametocytes was considered paramount. Using data obtained from a flow-based technique to examine the deformability of gametocytes at different stages of development, Pierre Buffet and Catharine Lavazec described interactions between the giRBC and the spleen - a site implicated in gametocyte sequestration.The session continued with detailed presentations on the interactions between gametocytes and their human hosts (Teun Bousema). Little is known about the ligands that mediate interactions between the gametocyte-infected RBC (giRBC) and the human host, though there are several candidates such as the STEVOR and the RIFIN families of exported proteins. Gambian children showed antibody responses to the surface of late stage gametocyte-infected erythrocytes independent of asexual parasitaemia and these responses correlated with reduced gametocyte carriage (Colin Sutherland). The field currently lacks both assays to evaluate It was noted that little has been done to look at the co-evolution of human and parasite genetics in relation to infectivity to the vector. It was muted that the human genotypes influencing the development of asexual parasites may directly or indirectly modulate transmission (David Modiano), examples of such interactions have been documented previously .The practical implications of targeting gametocytes and infectious individuals in intervention campaigns were presented. Lucy Okell proposed that mass drug administration will have most success in reducing transmission. The practicality of targeted approaches depends on the sensitivity of the diagnostic to identify parasite carriers. Recognizing that a majority of infected persons are gametocyte carriers, it was asked whether simple, more robust and sensitive methods to detect asexual parasites might provide a more cost effective approach to the identification of potentially infectious individuals. Geoff. Targett and Gillian Stresman highlighted that many of the problems in targeting interventions to the appropriate population are shared with other control approaches, notably how one identifies the individuals to be targeted in both short-term and stable foci, in hotspots within these foci, and in travellers? In the absence of a transmission-blocking vaccine (TBV), current active transmission reduction approaches rely on drugs. There remain issues as to the best anti-malarials to use, primaquine currently being the only available gametocytocidal compound. The effect of artemisinin combination therapy (ACT) on transmission may be over-estimated because despite reducing gametocyte carriage  the few and the mosquito vector, with for example drugs, vaccines, biological control  or genetic strategies to reduce mosquito populations or their susceptibility to Plasmodium.Recognizing the focus of the malaria eradication agenda has now expanded from the previous focussed objective of reducing morbidity and mortality in the human host, to protection against neo-infections, it is necessary to embrace the key target of transmission reduction. An expanded array of interventions must therefore be envisaged. These may include measures to either inactivate or eliminate parasites in both the human host Pseudomonas aeruginosa exo-protein A (EPA) [Corynebacterium diphteriae cross-reactive material 197 (CRM197) [Patrick Duffy reviewed the current interest in TBV. Rational development of TBVs requires that putative target proteins, on the surface of extracellular parasites in the mosquito vector, or components of obligate pathways and interactions, are better characterized by 'omics' approaches. To date, 24 proteins have been described as being inducers of potential transmission-blocking antibodies. Of the current lead candidates, two are expressed on the surface of gametes and in the intracellular compartment of gametocytes (P48/45 and P230), two others, P25 and P28 on the surface of the ookinetes. While producing protein antigens of appropriate immunogenicity remains challenging, it was suggested that solutions do exist e.g.  A (EPA)  and Cory(CRM197)  have beeThe question of deciding on the right target product profile for a TBV needs to be revisited carefully and should include integration of vaccines with other interventions. Identifying the target population for a (long-lived) TBV may be more straightforward than for a (short-lived) drug, Dr Duffy suggested every infected individual should be considered to be capable of transmitting the disease and therefore everyone in the area of transmission should be immunized with a TBV. This is in marked contrast to existing vaccine delivery systems in malaria endemic regions which target infants. There is now a trend toward performing Phase-1 trials in endemic settings. Considering that malaria transmission is focal, understanding micro-epidemiological transmission patterns in such settings is an essential pre-requisite to the determination of which geographic foci within the community should be preferentially targeted.P. falciparum has been much more successful than molecular target-based approaches and has generated up to 25,000 hits with potencies below 1.5 μM. This is now being complemented effectively with work on a limited number of genetically validated molecular targets with essential roles in parasite development e.g. the protein kinases PfPKG, and Pfnek2 which potentially play key roles in the formation of gametes and ookinetes respectively. Similarly PfPK7 - essential for oocyst maturation in the mosquito midgut, might be targeted by long-lasting drugs, these might prove to be challenging to develop (see below). Despite the arsenal of new generation high tech assays and readouts, no medium/high throughput solution is currently available to screen sporonticidal compounds. It is urgent that the generation of 'omics' data is completed to improve understanding of the molecular regulation of sexual development, e.g. in late gametocytes, the digestion of haemoglobin is turned off, and 'simultaneously' mitochondrial function and the tricarboxylic acid (TCA) cycle are turned on.Didier Leroy reviewed the agenda for discovery of transmission-blocking drugs (TBD). The identification and validation of new potential drug targets are two highly challenging and time consuming processes whatever disease is considered. To date, screening of asexual blood stages of In attempting to reduce Ro, gametocytes, and particularly their mature forms (stage V), may prove to be key targets for transmission-blocking agents. Clinical studies have shown primaquine efficiently eliminates gametocytes , notablyP. falciparum (where mature gametocytes can survive for up to three weeks). Targeting the oocyst with drugs (or vaccines) is even more problematic, because the deliverability and exposure of oocysts to drugs is impossible to control, potentially leading to the risk of accelerated selection of resistance.Today, better assays are needed to investigate the downstream impact that drugs, administered to the gametocyte in the human host, might have on development within the mosquito vector and transmission. From a drug development perspective such drugs are attractive because of the comparative simplicity of drug delivery. By contrast the design of drugs specifically targeting the ookinete requires that the compound has a half life that matches that of the mature gametocytes. While this is readily achieved in the majority of malaria species , it is more difficult with Considering the current level of knowledge of anti-malarials targeting the asexual parasites in human blood and the constraints of drug development, one ongoing strategy to identify transmission-blocking drugs is to screen known blood schizonticides against late stage gametocytes. Today, the possibility of pursuing a drug development programme on a molecule that would block transmission but wouldn't cure the disease in the treated host is also recognized as a valid and valuable addition to our armamentarium. This will necessitate unbiased screening of large compound libraries against gametocytes and ookinetes. A novel concept of using drugs to induce gametogenesis in the human host was suggested. This has a two-fold attraction: first, the treated parasite will be incapable of transmission and second, the formation of gametes in the human host would require the expression of known potent transmission-blocking (neo) antigens  thus enhancing the impact of natural transmission-blocking immunity. Recognizing that, naturally, > 99% of gametocytes will die within the human host and thus present antigens to the immune system, it was appreciated that responses to gametocyte antigens per se  would not be significantly enhanced.Carolina Barillas-Mury reviewed interventions targeting the parasite in the vector; she emphasized past wisdom that the only malaria control strategies that have been successful to date have included a component that controls the vector. The mosquito's immune system lacks an adaptive immune response arm and, therefore, no malaria-specific immune response awaits a subsequent infection. Recognizing the innate immune response is the only protection the mosquito can rely on to fight against the parasite, an interesting feature is the ability of the IMPer/Duox pathway to regulate the response when the midgut is invaded by parasites or bacteria. When this system is silenced/inhibited, the mosquito's midgut immune system efficiently reduces oocyst numbers. This interesting regulatory pathway needs to be further investigated and the possibility to inactivate this system specifically in mosquitoes, either directly with insecticides or indirectly with a partner drug in a combination therapy, has to be assessed. It would be interesting to know whether other similar pathways might be open to attack.Plasmodium to the vector have been neglected for ~40 yrs. It might be anticipated that the substantial body of available knowledge accrued in this period of neglect might rapidly be converted into new and effective interventions.The following synopsis divides the conclusions reached into two distinct areas: firstly outstanding questions concerning the biology of the sexual stages of malarial parasites that are considered to be too fascinating and fundamentally important to our understanding of life processes to be overlooked, irrespective of whether they may have potential application; and secondly those areas where it is essential to develop a greater understanding to discover, develop and apply effectively interventions targeted to the sexual stages. It was clear to many participants that all too obvious opportunities for potent interventions against transmission of P. falciparum have identified for the first time genes essential for gametogenesis that are detected in cells that morphologically cannot be identified as gametocytes; this could be key to the identification of a switch described in the preceding sexually committed asexual schizont.The basic questions as to how the developmental decision between asexual and sexual development is induced and regulated remain at the forefront of the key unknowns. Fundamental issues as to whether the switch is constitutive and/or subject to qualitative or quantitative 'environmental regulation' remain unresolved and contested. Investigators must be prepared to ask whether this regulation differs between the parasite species. Whilst it is clear that in some species merozoites released from the pre-erythrocytic schizont can be committed to the sexual pathway (as it is in many 'primitive' haemoproteids) , it remaAlthough this field is developing rapidly the molecular basis underpinning the morphological, metabolic and functional maturation of gametocytes is poorly understood. The potential power of systematic 'omics' technologies remains compromised by the large number ~50% of the malarial genes that remain un-annotated. Data generated to date have provided a rational explanation for the induction and specificity of natural immunity to gametocyte antigens. The fascination of understanding the genetic and 'environmental' control of sexual development; the roles of genetic master-regulators' e.g. AP2, and DOZI; the roles of individual genes; of the design, assembly and functions of molecular machines and organelles was apparent to all. Nowhere is this curiosity more challenged than in the 'explosive' process of male gamete formation, where rates/mechanisms of DNA replication and axoneme assembly challenge any current understanding. From, and for, such studies improved markers of the numerous stages of sexual development must emerge.Probably the biggest gap in the understanding of gametocyte maturation is the molecular description of gametocyte metabolism. Inhibitor/drug studies suggest significant differences between the immature (trophozoite-like) and morphologically mature (arrested in Go) gametocytes (protein synthesis being down-regulated in the latter). Mitochondrial/energy production pathways throughout sexual development and early sporogonic development may provide a rich vein to be mined. Such studies will benefit from standardized culture methodologies, and would provide one route to the development of effective drugs to block transmission.P. falciparum gametocytes, and the need for appropriate markers/tools to study the phenomenon, the relevance of this topic, if any, to any parasite species outside the subgenus Laverania whilst unclear, was not discussed.There was considerable debate as to the molecular basis for the sequestration of immature Clearly despite significant recent advances, the molecular basis of gamete-gamete recognition, gamete fusion and onward zygote development remains poorly understood. The parallel influences of immunological drivers of polymorphism in gamete surface proteins and the evolution of species-specific mechanisms of fertilization/zygote survival proffer fertile ground for theorists. Whilst recognizing the 'dominant' role of the large female gamete it is appropriate to ask what roles the incredibly simple male gamete plays in development e.g. determination of the polarity and subcellular coordination of the assembly and functions of the zygote/ookinete.The complexity of malaria transmission through endemic populations was evident, key issues raised (but not fully understood) include differential transmissibility of parasite species, mixed species infections and genotypes; differential susceptibility of vector species and genotypes; the impact of human genotype; focality of transmission and intensity of transmission. Not least amongst the theoretical and practical questions raised was how these variables contribute to the definition of the reservoir of infection.The necessity of understanding the impact of spatial and temporal distributions of infectious persons in modelling malaria transmission, and tailoring the design of intervention campaigns to areas of differing endemicity is clear, but recognizing the high prevalence of gametocyte carriage in infected persons the meeting was asked whether, during the implementation of an intervention campaign it is necessary to distinguish infectious from infected persons? The method by which infections are detected (active vs. passive case detection) remains critical, because the contribution of asymptomatic persons to the infectious reservoirs needs to be understood for an appropriate plan of intervention to be designed in each locality. If the operational model in the field remains either mass drug administration (MDA), or the detection and treatment of all malaria-infected individuals, every infected person will merit treatment to prevent transmission. The combined formulation of transmission-blocking compounds with schizonticidal drugs obviously addresses this need, and has been shown to be a most effective intervention strategy .The potential of vaccines with transmission-blocking potential undoubtedly has a key role to play in any elimination/eradication agenda, whether in the treatment of focal areas of transmission or more broadly. All else being equal  the theoretical advantage of a vaccine that might be boosted by any subsequent infection is clear, but still requires experimental validation. It was recognized that there would be significant advantage if the delivery of a transmission-blocking drug could be engineered to emulate that of a vaccine (prolonged delivery from a depot). It was also recognized that despite notable individual successes the discovery and development of both transmission-blocking vaccine and drug targets have to-date lacked rational genome-based discovery and prioritization.P. vivax, and agreement on the development pathway, including standardized/harmonized assays of vaccine efficacy.A review of current progress in TBV development (Ashley Birkett) emphasized the current TPP is for a protein vaccine with a non-controversial adjuvant, which, if administered in no more than three doses within a six-month period would result in 85% blockade of mosquito infection intensity. Current focus is on Pfs48/45; Pfs25 and the mosquito antigen APN1. Recognizing that other candidates are under active investigation ,50 it woP. vivax; P. ovale); and sexual development , for which the TPP is for fast-acting, long-lived drugs. It is recognized that the very small number of parasites at these critical phases of development theoretically offers the promise of slow selection of resistance, a problem that has blighted the development of the current generation of schizonticides. Such drugs might usefully complement diverse intervention strategies.The objectives of development of new anti-malarials has been broadened, schizonticide discovery/development has been complemented by the clear recognition of the need to inhibit malaria transmission. Whilst acknowledging that early delivery of effective schizonticides will have a significant impact upon gametocyte production, and therefore transmission, two biological transmission targets have been prioritized: the hypnozoite  thinking reminded the attendees of the now-proven potential of GM vectors, and para-transgenic approaches using mosquito symbionts/pathogens to express anti-parasitic moieties. The group highlighted the recent observations as to how the mosquito midgut microflora moderates the innate immune response and malaria infections, and raised interesting questions as to the indirect impact of current/future interventions upon the balances within this particular ecosystem. Significant thought will be needed to determine how these methods might be applied effectively in field situations.Many participants highlighted the need for powerful markers of expression of all sexual-stage genes. The value of expanded repositories of research tools e.g. gene knockouts, tagged proteins, monoclonal antibodies and the wider availability of high resolution in vivo imaging technologies cannot be overemphasized. With these improved tools it will be possible for community-wide action to analyse the function/impact of sexual stage genes, their chemical biology, and undertake HTP immunological studies on parasite biology. All HTP methods of parasite protein analysis remain critically dependent upon improved genome annotations of all relevant organisms, without this the recognized need for the discovery of the best vaccine, drug and other targets cannot be undertaken rationally or effectively.P. berghei) and the parasites of man was noted. In particular it was recognized that some of the tools currently used to determine the impact of transmission-blocking strategies on the human parasites are slow, expensive and available to few groups, e.g. Cat III culture facilities and secure insectaries. Even more exclusive were facilities to work on primate hosts with the consequence that abilities to study the transmission of P. vivax, or P. knowlesi are rare, and of the remaining species  or of mixed-species infections almost non-existent. The challenge of developing new safe and widely useable (preferably at the HTP level) analytic methods must be addressed. An area of immediate need is the requirement to understand the correlation between the various assays used to report the impact of transmission-blocking vaccines.The contrast between the experimental tractability of the rodent malaria parasites (notably One proven, albeit still low throughput, approach to some of the problems above has been the production of transgenic rodent malarial parasites in which the relevant endogenous gene of interest has been replaced by the homologue from the human parasite. Where such exchanges are not complicated by fine molecular incompatibilities, these have a unique additional benefit in that the organism may not infect man thus removing major cost and security limitations to their study.Outstanding questions of transmission biology that confound the development of rational mathematical models include first, the need to understand heterogeneities at all levels of biology, from the gene through the biologies of the interacting organisms, of inter-personal infectiousness to the vector, to the spatial and environmental variations in parasite distributions in endemic communities. A second requirement is the need to recognize and define the recently recognized non-linearities in parasite dynamics ,52 as thOne outstanding question that impacts upon the design of any effective transmission-blocking intervention is how to design and conduct appropriate field trials. Whilst this has been addressed in significant depth by other meetings  it is clThe authors declare that they have no competing interests.