Being often deployed in remote or hostile environments, wireless sensor networks are vulnerable to various types of security attacks. A possible solution to reduce the security risks is to use directional antennas instead of omnidirectional ones or in conjunction with them. Due to their increased complexity, higher costs and larger sizes, directional antennas are not traditionally used in wireless sensor networks, but recent technology trends may support this method. This paper surveys existing state of the art approaches in the field, offering a broad perspective of the future use of directional antennas in mitigating security risks, together with new challenges and open research issues. Wireless sensor networks (WSNs) have emerged as a key technology for a broad spectrum of applications, ranging from weather forecasting  or completc. With the advancements of smart antenna technology, the omnidirectional antennas may either be replaced by directional ones or can work in tandem with them on the same motes. The advantages brought by directional antennas to WSN nodes can be seen not only in increased quality of transmissions, optimization of energy usage, decreased number of hops due to longer transmission range, but also from the security point of view.Usually, sensor nodes employ omnidirectional antennas for wireless communication due to a variety of reasons including their small size, low cost, ease of deployment, simplified transmission-related protocols, Directional antennas can mitigate the malicious attack risks in WSNs in two ways: (a) directly, by being immune to attacks launched from outside their narrow radiation region; or (b) indirectly based on node position verification—here a node equipped with directional antenna, using the received signal’s direction of arrival to compute the position of a sender node (in conjunction with other trusted nodes or beacons), can identify malicious nodes by checking their position against a trusted list. By using these two lines of defense against hostile attacks, the nodes equipped with directional antennas may identify, mitigate or even eliminate security risks when speaking about eavesdropping, jamming, wormhole attacks or Sybil attacks. From this perspective, this paper aims to survey the current state of the art in the field and to identify the major research challenges and perspectives.The remainder of the paper is organized as follows: Traditionally, communication inside WSNs is done using omnidirectional antennas which broadcast radio signal almost uniformly in all directions. Omnidirectional antennas are small, inexpensive and simply to deploy, but they suffer from poor spatial reuse, high collisions, reduced energy efficiency and are susceptible to security attacks ,5. A relIf the mentioned drawbacks dramatically affect the normal WSN operation and security, wireless nodes can be equipped with directional antennas either alone or in conjunction with existing omnidirectional antennas . A direcImproved energy consumption; the wireless data transmission is proved to be the most energy-intensive operation of a sensor node ,11. By fLonger transmission range; reporting information inside WSNs using fewer hops ,14 or reHigher security; derived either from their immunity to eavesdroppers  or jammeA brief comparison between the two types of antennas  is provietc.) have a fixed beam that can be oriented in the desired direction by mechanical rotation. Smart antenna [Directional antennas are generally constructed by combining simple antenna elements  into antenna arrays. Their overall radiation patterns are influenced by the type, the number and the geometrical configuration of the elements and also by the characteristics of the signal applied to each element. There are basically two types of directional antennas: traditional directed antennas and smart antennas. Traditional directed antennas   band or within the millimeter-wave spectrum ,26,27,28The directional antenna prototypes specifically designed to equip sensor nodes are briefly presented in Leang and Kalis  indicateet al. [Nilsson ,32 identet al.  presenteet al. [A 2.4 GHz four-beam patch antenna prototype meeting the size, cost and energy constraints of sensor nodes was proposed by Giorgetti et al. . This diet al. [Liang et al.  developeet al. proposed and tested two cost-effective and compact switched-beam antenna prototypes, in the ISM band. The first one employs a radiation structure made of eight microstrip antennas using rectangular two-element patch antenna arrays and a vertical half-wavelength dipole antenna [l-shaped slot antenna elements which are disposed in a symmetrical planar structure of 10 × 10 cm2.Catarinucci  antenna . The sec antenna ,36 uses et al. [Another directional antenna prototype for WSN nodes was developed and evaluated in a fully directional neighbor discovery protocol by Felemban et al. . For thiAlthough the research in developing directional antennas suitable for WSN nodes is in the early phases, the results obtained so far are encouraging. This allows us to envisage new models of wireless communications between sensor nodes, endowed with higher security.Directional antennas can mitigate or even eliminate the risks related to some categories of security attacks on WSNs due to their specific radiation pattern which can be materialized into mechanisms for localization of neighboring or malicious nodes, or can drastically reduce the areas from where an attack can be carried out. The main types of attacks that can be mitigated using directional antennas are: eavesdropping, jamming, Sybil attack and wormhole attack, but similar countermeasures can reduce the risks for traffic analysis, man-in-the-middle attack or node capturing attack.Directional antennas can reduce malicious attack risks in two ways, either directly by being immune to attacks launched from outside their narrow radiation region, or indirectly based on position verification procedures ,40 emploetc.) that later can be used in more destructive attacks. In WSNs, two categories of eavesdropping attacks have been identified [Eavesdropping is the attack in which a malicious entity intercepts private communication in an unauthorized real-time manner. The attacker, analyzing the stolen information packets can obtain contextual and targeted information  et al. proved that when using directional antennas, the eavesdropping probability is drastically decreased compared to the case of omnidirectional antennas. These studies make three important contributions: (i) they establish eavesdropping models for both omnidirectional and directional antennas in the context of wireless sensor networks; (ii) they prove that in the case of directional antennas the eavesdropping probability is diminished due to two factors: the number of hops to route a message is reduced and the exposure region from where malicious nodes may listen is smaller; and (iii) they validate the two eavesdropping models  and the corresponding values of eavesdropping probability through extensive simulation studies.In ,42, Dai et al. [Another analysis of the effects of using directional antennas upon eavesdropping probability in wireless networks, but this time from the attacker’s perspective, is presented by Li et al. . The proAn interesting approach to mitigate eavesdropping attacks in wireless networks is proposed in  and emplJamming is the deliberate act of broadcasting an inference radio signal aimed to disrupt wireless communication. This type of electromagnetic interference can be accomplished either in a simple manner when the jammer continually transmits interference signals or using more sophisticated approaches based on communication protocol vulnerabilities.Due to their particular radiation patterns, directional antennas can efficiently mitigate the effects of jamming attacks being able to safely communicate if the jammer’s location is outside antenna’s coverage sector . The sciad hoc communication network. By comparing the network connectivity index when either omnidirectional or directional antennas are used in jamming circumstances, the author proved a significant improvement in the second case. This result stands not only for randomly placed jammers but also for jammers optimally located in the network area. Moreover, the result can be extrapolated to diverse types of smart antennas able to concentrate the beam’s power in the receiver’s direction  [In , Noubir antenna) ,58.et al. [For mitigating the jamming effect in wireless sensor networks, Panyim et al.  proposedIn order to reduce unwanted interferences in randomly deployed wireless sensor networks Staniec and Debita  suggesteUsually, in a wireless sensor network each node has its own identity (ID), a one-to-one relationship between nodes and their unique IDs being a prerequisite for many network mechanisms . In a SyAn example of such an attack is presented in et al. [Newsome et al.  identifiFrom this comprehensive list, one of the most efficient methods to discover the Sybil nodes is undeniably the position verification technique. Accordingly, the Sybil nodes can be identified by comparing their exact position with the previously known locations of network nodes from which the Sybil nodes stole the identities. This type of methods usually employs two elements: (a) radio signal characteristics ; and (b) trusted nodes cooperation for node identification and authentication. Directional antennas are inherently offering the direction of captured signals. If two messages coming from two nodes having the same IDs are concurrently gathered from two different directions, then we can come to a logical decision: one of the two network nodes is undoubtedly malicious. Such a methodology can be derived from the one described in , which uet al. [A simplified Sybil attack named evil-twin, in which the malicious node is using only one stolen identity, is addressed by Bhatia et al.  using foet al.  is empload hoc networks (MANETs) or one of their subcategories (vehicle ad hoc networks—VANETs) can be simply particularized to address the Sybil attack in WSNs. Two such methods attracted our interest.Approaches for coping with Sybil attacks in wireless sensor networks based on directional antennas can also be derived from methods proposed for other types of wireless networks. For example, some methods developed for mobile Vaman and Shakhakarmi  proposedA cross-layer scheme to detect Sybil attacks in VANETs is proposed in . A trialThe wormhole attack ,64 occurThe methods developed to identify the wormhole attacks usually require that all/some nodes be equipped with extra hardware . When th(a)The directional neighbor discovery protocol. The proposed mechanism does not rely on any type of cooperation among nodes. The protocol works in three consecutive steps: (i) a node  of a just-deployed sensor network sends a HELLO-type message including its ID; (ii) all nodes that receive the HELLO message reply with an encrypted message that basically contains their node ID and the zone where the message was received. The encryption process is done using previously established keys, stored on each node together with corresponding neighbor ID; and (iii) the announcer will decrypt the message verifying the node ID and that the zone reported by the neighbor is opposite to its zone. After the neighbor discovery process is finished, the node will ignore any kind of messages coming from nodes that do not belong to the neighbor list. Even its effect on mitigating the wormhole attacks is reduced, the protocol is envisioned by the two authors to represent a strong basis for the following two.(b)The verified neighbor discovery protocol is based on sharing information between network nodes. It can stop attacks in which the malicious entity controls the two wormhole endpoints and when the targeted nodes have no direct communication link (are at least two hops distant). The mechanism is based on directional neighbor discovery protocol which is enhanced by a verification procedure done using verifiers (network nodes that are not in opposite direction from the wormhole endpoints). The role of verifier-nodes is to check the legitimacy of announcers.(c)The strict neighbor discovery protocol adds a supplementary requirement (the verifier region must be empty when two nodes are out of radio range) for verifier-nodes to cope with Worawannotai attack , too.The three approaches with different levels of wormhole attack mitigation, proposed by Hu and Evans , assume et al. [This ensemble of three protocols can countermeasure the wormhole attacks without clock synchronization among nodes or precise location information. Shi et al.  proposedAnother method that uses directional information gathered by trusted nodes to cope with wormhole attack is described in the case of MANETs , but canAlthough the use of sensor nodes equipped with directional antennas represents a promising tool in mitigating the risks of security attacks in WSNs, research in this field is still in the beginning stages. This research status should be significantly improved with the availability of new commercial-off-the-shelf sensor nodes equipped with directional antennas and relying on efficient network protocols.(i)designing small sized, reasonably priced and energetic-efficient directional antennas able to be integrated in highly resource-constrained sensor nodes;(ii)developing efficient MAC protocols to address deafness, directional Hidden Terminal (HT) problem or Head-of-Line (HoL) blocking problem in multi-hop wireless networks ,67;(iii)providing network protocols able to assure self-localization, self-configuration, self-synchronization and self-optimization in the case of randomly deployed sensor networks using aerial scattering or other similar procedures;(iv)designing effective and reliable neighbor discovery mechanisms, being known that traditional approaches either depend on omnidirectional announcers and on time synchronization or are two complex to be implemented in real large-scale sensor networks ,37;(v)adapting the in-network data and message aggregation mechanisms to the directional antenna-based topology of WSN;(vi)designing customized topology control mechanisms to increase effective network capacity and conserve energy; and(vii)etc.) and sensing-related parameters .providing appropriate QoS models incorporating both communication-related parameters –(vi)) are already reported in scientific literature, their validation in real-world WSNs applications is still pending. Despite all these difficulties, the use of directional antennas in wireless sensor networks has already proved several advantages: it improves the transmission reliability, increases the spatial reuse, extends the transmission range or decreases the overall network power consumption. Moreover, directional antennas offer sensor nodes additional control over signal strength and interference, which allows the use of optimization techniques for providing higher network throughput and transmission reliability. Last but not least, the directional antennas provide significant advantages in coping with various security threats.(a)Involving directional antennas in coping with other malicious attack type. While the configuration of their radiation pattern can inherently mitigate the effects associated to eavesdropping or jamming, the directional antennas can be involved in identifying, mitigating or even eliminating the security risks associated to other malicious attacks using angular information . For this, the key word is “localization”, so any malicious attack that can be addressed using localization-based techniques  can be a valid target for future research. Relevant examples in this context are the selective forwarding attack [g attack  or the Hg attack .(b)Using directional antenna-based localization mechanisms to detect security attacks on other localization schemes. The WSN’s localization infrastructure is susceptible to an assortment of malicious attacks [ attacks  that can(c)Eliminating the consequences of several attacks by benefiting from the longer transmission range of directional antennas. A concrete example can be the case of sensor nodes or groups of sensor nodes isolated from the rest of the network due to various malicious attacks . In this kind of situation the nodes can find alternative paths to regain the connectivity to the rest of WSN by contacting nodes that are further away.(d)Using sensor nodes with both directional and omnidirectional antennas to solve complex security issues inside WSN. Such an approach could combine the potential advantages brought by the two antenna types. In this case, strategies to switch from one type of antenna to the other have to be design in order to maximize the WSN capability to timely discover and eliminate the security risks.(e)Coordinating the mechanisms based on the use of directional antennas with other security related technique. Coping with the increased diversity of security threats that affect wireless sensor networks, demands the use of a complex ensemble of methodologies and protocols. The integration of security mechanisms based on directional antennas in an overall security system it’s not a simple task due to a series of factors including the power, communication and computational constraints, the heterogeneity of sensor nodes, the unattended or hostile nature of the WSN environment, etc.(f)Extending the research field by addressing the security problems of more complex versions of WSNs, where the sensor nodes are endorsed with mobility  or whernetworks  or even networks ).(g)Fusing information received from directional antennas and from other devices  for coping with security threats. In many cases, the network nodes are able to obtain supplementary information that can be used to mitigate the security attack risks. Routing information, list of neighboring nodes together, locations and battery energy levels of neighboring nodes or successive sensor measurements are only few examples of information that can be utilized in this context to mitigate the security risks. For example, multimedia sensor nodes equipped with video and audio capture capabilities can fuse such information with the ones obtained from directional antennas to address security-related issues.Studying the factors that can boost the effectiveness of such devices when coping with security attacks, we found that narrowing the radiation region of antennas favors both of the abovementioned types of approaches (direct and indirect). By this the probability of eavesdroppers/jammers to be outside the radiation zone is increased and, moreover, the localization based on signal’s angle of arrival becomes more accurate. The endeavor to narrow the radiation region for directional antennas is not a simple task knowing that the antenna’s size increases with the increase of angular resolution . From thThe use of directional antennas for equipping WSN nodes arises from the need to optimize energy consumption, to raise the quality of transmissions or to decrease the number of hops due to longer transmission ranges. Besides this, directional antennas can be seen as a valuable resource for reducing the security risks that inherently affect WSNs’ operation. In this paper, after surveying the prototypes of directional antenna suitable for WSN nodes, we presented the state of the art in mitigating the security risks associated to eavesdropping, jamming, Sybil and wormhole attacks. Even though research in this area is still in a beginning stage, the results are encouraging, demonstrating the need for further theoretical and experimental investigation. Certainly, future studies should include new research topics including the need to cope with other types of malicious attacks, to consider the potential benefits of using both directional and omnidirectional antennas on the same sensor nodes, to combine the strategies based on the use of directional antennas with other security-related methods, or to expand the research area to other more complex varieties of WSNs.


Laser in-situ keratomileusis (LASIK) is a commonly performed surgical procedure used to correct refractive error. LASIK surgery involves cutting a corneal flap and ablating the stroma underneath, with known damage to corneal nerves. Despite this, the epidemiology of persistent pain and other long-term outcomes after LASIK surgery are not well understood. Available data suggest that approximately 20-55% of patients report persistent eye symptoms  after LASIK surgery. While it was initially believed that these symptoms were caused by ocular surface dryness, and referred to as “dry eye,” it is now increasingly understood that corneal nerve damage produced by LASIK surgery resembles the pathologic neuroplasticity associated with other forms of persistent post-operative pain. In susceptible patients, these neuropathological changes, including peripheral sensitization, central sensitization, and altered descending modulation, may underlie certain persistent dry eye symptoms after LASIK surgery. This review will focus on the known epidemiology of symptoms after LASIK and discuss mechanisms of persistent post-op pain due to nerve injury that may be relevant to these patients. Potential preventative and treatment options based on approaches used for other forms of persistent post-op pain and their application to LASIK patients are also discussed. Finally, the concept of genetic susceptibility to post-LASIK ocular surface pain is presented. Laser in-situ keratomileusis (LASIK) is a common procedure to correct refractive error, with approximately 650,000 cases performed in the US each year . LASIK iQualitatively, the symptoms described following LASIK that have traditionally been conceptualized as “dry eye” are similar to other forms of persistent pain after nerve injury, suggesting that ocular surface pain that develops after LASIK represents a pathological hypersensitivity of the ocular somatosensory nerves. Descriptors frequently endorsed by patients with chronic pain after nerve injury include burning and certain forms of evoked pain , pain due to light touch or other innocuous stimuli , increased sensitivity to noxious stimuli , and expansion of this heightened pain sensitivity beyond the area of initial injury  ,9. In thBoth acute and long–term ocular pain and discomfort following LASIK have been reported ,14,15. I1–2)  1 receptor 2 single nucleotide polymorphism [SNP] rs11674595) and 1 haplotype (IL10 haplotype A8) were associated with PPP after breast cancer surgery . InflammRecently, a large twin study demonstrated that shared genetic factors underlie a number of comorbid chronic pain conditions, including chronic widespread musculoskeletal pain, pelvic pain, irritable bowel syndrome, and dry eye, and that this latent genetic factor had an estimated heritability of 66%. This finding supports the notion of common pathways of susceptibility and an underlying genetic disposition to developing chronic pain . Our ownWith this review we have attempted to make three major points. First, that perhaps the experience of persistent dry eye symptoms following LASIK is better conceptualized as a neuropathic ocular surface pain syndrome involving mechanisms of both peripheral and central sensitization. Second, given this and the well-established evidence regarding injury to corneal nerves during the LASIK procedure, we may look to other PPP syndromes as the prototype for the management of persistent neuropathic ocular pain following LASIK procedures, and should revisit the use of existing neuromodulators for prevention and treatment in well-controlled randomized trials that are appropriately powered to assess efficacy and safety of known and novel approaches. Third, that the development of PPP in the eye or anywhere else in the body is the product of the interaction of genetic and environmental factors, and that understanding the genetics of susceptibility to these disorders will be important in realizing novel preventative and mechanism based treatments. We believe that it is important to better define the role of anti-neuropathic pain treatment in modulating ocular sensory apparatus function in patients with this symptom complex, and to integrate this tactic into a multi-modal approach including the treatment of ongoing ocular surface damage with ocular surface protection and anti-inflammatory agents. Furthermore, agents such as the gabapentinoids, may be promising in reducing the likelihood of development of central sensitization by attenuating the afferent trafficking of pain signals and excitatory synaptic transmission.


Sexual selection and sexual conflict theories that explain evolution of sexual behaviour are often an integral part of animal studies investigating diverse research questions, e.g. in behaviour, ecology, physiology and immunology. In plants, however, relatively few studies investigate sexual selection and sexual conflict. In this review we discuss how taking these theories into account can be useful not only for our understanding of plant reproductive strategies but also in related research areas, including i) mechanisms of pollen-pistil interactions, ii) mating-system evolution in hermaphrodites and iii) plant immune responses to pests and pathogens. Today it is accepted that the theories of sexual selection and sexual conflict are general and can be applied to both animals and plants. However, potentially due to a controversial history, plant studies investigating sexual selection and sexual conflict are relatively rare. Moreover, these theories and concepts are seldom implemented in research fields investigating related aspects of plant ecology and evolution. Even though these theories are complex, and can be difficult to study, we suggest that several fields in plant biology would benefit from incorporating and testing the impact of selection pressures generated by sexual selection and sexual conflict. Here we give examples of three fields where we believe such incorporation would be particularly fruitful, including (i) mechanisms of pollen–pistil interactions, (ii) mating-system evolution in hermaphrodites and (iii) plant immune responses to pests and pathogens. Theories of ecology and evolution are often based on general principles and can be applied across taxa. However, due to research tradition we can find that some research questions are considered more important in some taxa than in others. One such example is theories of sexual selection and sexual conflict, which is a large and active field in animals while treated as relatively insignificant in plants.Application of sexual selection theory to plants in the late 1970s and early 1980s  generateIt is also noteworthy that development of sexual selection theory into the theory of sexual conflict . In Web Moreover, the theories of sexual selection and sexual conflict do not appear to be integrated in studies with the aim to learn about plant ecology and evolution in related research areas, i.e. to improve our understanding of plant fitness in general. This is in contrast to studies in animals, where these theories often are integrated in other fields, such as ecology, behaviour, physiology and immunology (e.g. Collinsia heterophylla (Plantaginaceae).Here, our aim is to discuss how taking the theories of sexual selection and sexual conflict into account have the potential to improve our general understanding of plant ecology and evolution. We will give a short introduction to sexual selection and sexual conflict in plants. We will then point out three research fields in which we think it may be particularly useful to consider these theories, including (i) mechanisms of pollen–pistil interactions, (ii) mating-system evolution in hermaphrodites and (iii) plant immune responses to pests and pathogens. Rather than providing a complete review of the literature we will use specific examples and mainly focus on pollen competition in the pistil, including results from one of the author's own studies on Arabidopsis showed that female-determined mate choice was defined by four quantitative trait loci (QTLs)  competition  Box 1)..Box 1). Box 1.Darwin first suggested the theory of sexual selection in order to solve a problem regarding his theory of natural selection ; he founAlready Darwin proposed that many traits can be influenced by both natural (viability or fecundity) and sexual selection, but stressed that distinguishing between them can be difficult. More modern definition of the relation between natural and sexual selection also suggest that traits can be influenced by either selective force or by a combination of both, and that discriminating between these forces has a value for our conceptual understanding and for separating (as a group) from neutral processes, i.e. genetic drift . For exaPistil traits that enhance pollen competition could evolve as a female choice mechanism  both before and after pollination in several species, evidence for female choice, e.g. indirect sexual selection of pollen traits during pollen competition is scare and only tested in a few studies  reduced inbreeding depression [i.e. impaired offspring fitness due to homozygous deleterious alleles, according to the partial dominance model . Thus, there is opportunity for the conflict to be of significance also under natural conditions. A piece of evidence for a history of antagonistic evolution between pollen and pistils within populations of C. heterophylla comes from a study involving four populations originating from two regions in California  only a few pollen tubes enter the open mouth of the stylar canal (Brasenia and Cobomba (Cabombaceae) pollen tubes do not enter through the open mouth but instead reach the canal by growing directly through the carpel wall allowing more pollen tubes to enter  resulted in some seeds sired from the second pollen load, despite that it takes only between 2 and 4 h for pollen to grow down the style  receptivity (as previously outlined), a floral trait that is linked to the mating system.In order to discuss how consideration of sexual conflict may influence our understanding of evolution of mating-system and correlated traits, we will use the example of mixed-mating Collinsia (ca. 22 species) variation in mating system and outcrossing rate is large, constituting one group of mainly selfing species and one group of more outcrossing, mixed-mating species  after flower opening, was recently shown to be the only trait that was significantly correlated with outcrossing rate , preventing self-pollination in the presence of pollinators . Howeverrophylla . Enhancirophylla .C. heterophylla . We also found that pollen tube growth rate was uncorrelated with outcrossing rate, contrary to expectation  barriers and inducible responses. While innate plant immune responses, causing inducible responses, are non-adaptive, recent molecular tools have revealed multiple recognition mechanisms and signalling pathways involved in pathogen infectivity and plant defence interactions and coevolution (Cucumis sativus, siring success following two-donor crosses did not differ between pollen produced by plants that were either resistant or susceptible to yellow mosaic virus, but in infected plants, susceptible pollen was more successful (C. sativus showed that soil nutrients were positively correlated with nutrients in the pistil, which influenced relative pollen competitive ability of pollen donors grown under similar nutrient conditions (The fact that plants are sessile and mate by proxy constitute increased risks of attracting antagonistic organisms. Floral traits that increase pollinator visits can also attract antagonistic insects and vectors for pathogens . Thus, sccessful . This reSilene, but also transmission of pathogens by wind dispersal of spores can occur during the flowering phase (N. attenuata plants with defective ethylene signalling allowed growth of colonizing root microbes (Insect vectors can transfer pathogens directly to flowers, e.g. smut-fungus in ng phase . Pathogeng phase , the latmicrobes , suggestmicrobes . Interesmicrobes .It could be hypothesized that effectiveness of plant defence is influenced by differences in pistil environment, either due to compatibility reactions or pistil traits with the capacity to favour some pollen over others or mitigate conflict costs caused by antagonistic pollen. Interestingly, a recent study on the Solanaceae family found that species with self-incompatibility systems had relatively more constitutive defence to herbivores while self-compatible species had relatively more induced defence . It was In many animal taxa post-mating activation of the immune system can occur independent of damage or pathogen presence following mating. Hypotheses explaining this activation include preparation for embryo implantation and responses to increased probability of pathogen infection or physical harm, but also the more recent idea that male reproductive proteins can activate the female immune system, as suggested in mammals and fruit flies (reviewed by Here, we have discussed that even though some researchers are applying the theories of sexual selection and sexual conflict to plant studies today, these theories are not considered as important and useful as in animals where these theories were first developed . We thinWe believe that much can be gained from fully acknowledging that sexual selection and conflicting male and female interests are important beyond species where we can easily observe mating behaviour. Considering sexual selection and sexual conflict theories also in a wider range of plant studies on ecology and evolution can be useful, for example in the three research areas we have discussed here. Increased knowledge about mechanisms of pollen–pistil interactions in relation to male–male competition and female choice is of particular interest as we expect direct interactions between competing pollen and pistil tissue. By studying sexual selection on haploid pollen, we have also the opportunities to learn about differences in adaptation in relation to haploid vs. diploid selection (cf. masking hypothesis, Swedish Research Council supported this work.The Å.L. wrote the first draft. The text was improved by both authors.None declared.


To explore the perspectives of a diverse group of stakeholders engaged in medicines decision making around what constitutes an “essential” medicine, and how the Essential Medicines List (EML) concept functions in a high income country context.In-depth qualitative semi-structured interviews were conducted with 32 Australian stakeholders, recognised as decision makers, leaders or advisors in the area of medicines reimbursement or supply chain management. Participants were recruited from government, pharmaceutical industry, pharmaceutical wholesale/distribution companies, medicines non-profit organisations, academic health disciplines, hospitals, and consumer groups. Perspectives on the definition and application of the EML concept in a high income country context were thematically analysed using grounded theory approach.Stakeholders found it challenging to describe the EML concept in the Australian context because many perceived it was generally used in resource scarce settings. Stakeholders were unable to distinguish whether nationally reimbursed medicines were essential medicines in Australia. Despite frequent generic drug shortages and high prices paid by consumers, many struggled to describe how the EML concept applied to Australia. Instead, broad inclusion of consumer needs, such as rare and high cost medicines, and consumer involvement in the decision making process, has led to expansive lists of nationally subsidised medicines. Therefore, improved communication and coordination is needed around shared interests between stakeholders regarding how medicines are prioritised and guaranteed in the supply chain.This study showed that decision-making in Australia around reimbursement of medicines has strayed from the fundamental utilitarian concept of essential medicines. Many stakeholders involved in medicine reimbursement decisions and management of the supply chain did not consider the EML concept in their approach. The wide range of views of what stakeholders considered were essential medicines, challenges whether the EML concept is out-dated or underutilised in high income countries. The concept of “essential medicines” dates back to military tradition, in which therapeutic supplies (such as penicillin) were essential to be carried by soldiers, field medics, and camp infirmaries, into combat zones. This was also applied to the rationalising of therapeutic restrictions necessary during wartime economy . Ensurin“Essential medicines are those that satisfy the priority health care needs of the population. They are selected with due regard to disease prevalence, evidence on efficacy and safety, and comparative cost effectiveness. Essential medicines are intended to be available within the context of a functioning health systems at all times, in adequate amounts, in the appropriate dosage forms, with assured quality, and at a price the individual and the community can afford.”Nearly forty years since the introduction of the WHO EML, few studies have investigated the impact of EML policies on access to medicines ,8. AlthoA study by Cameron et al  showed tIn line with the EML concept, national reimbursement schemes aim to enable access to medicines for a population within a functioning health system . They arDespite efforts to evaluate the usefulness of having EML policy ,8, uncerEthics approval was obtained from the Human Research Ethics Committee (HREC) at the University of Sydney. All study participants were provided the study information statement, consent form and brief interview guide prior to interviews. As required by HREC, written consent was obtained. Participants were guaranteed confidentiality and anonymity.A qualitative semi-structured interview protocol was developed based on existing literature and research objectives  24]. Th. Th24]. Between October 2012 and January 2015, 32 Australian stakeholders were recruited for in-depth interviews through purposive and snowball sampling approaches ,31. Fortth WHO EML, published in May 2015 . However, most participants noted that in principle, the notion of an “essential” medicine in a HIC like Australia has evolved in terms of the definition and intended application of an EML [Q2]. Some participants considered the reimbursement of medicines through the Australian PBS was akin to a functioning EML [Q3]. However, many participants argued they could not distinguish between reimbursed medicines and essential medicines [Q4] [Q5]. While some believed an EML approach could support stakeholders in the supply chain to sustain fundamental health services [Q6], one participant highlighted that the consideration of a medicines’ cost and cost-effectiveness were contentious issues that undermined the notion of essential [Q7].Some participants reflected the selection of medicines for reimbursement has been increasingly driven by consumer needs [Q8]. Furthermore, some participants explained reimbursement decisions were driven by cost effectiveness and additional benefit of a medicine for some individuals rather than the essential need of a medicine by the population [Q9]. Many participants found it challenging to apply the concept of EML to the Australian context because EMLs were perceived to be useful for resource scarce settings [Q10]. However, some participants regarded essential medicines as part of a complex pharmaceutical supply chain. Even in HICs, access cannot always be guaranteed [Q11]. One participant described the characteristics of medicines that are categorised as “essential” by the pharmaceutical industry within the supply chain [Q12].The selection and supply of reimbursed medicines were complex issues. One participant reflected that the notion of essential medicines was highly confounded [Q13]. The influence of cost containment was interpreted differently amongst stakeholders. Some participants thought decisions about whether a medicine was listed on the PBS included consideration of cost containment by lowering prices of generic medicines in order to expand the PBS to list new medicines [Q14]. While some participants felt that despite high prices set by pharmaceutical companies, the PBS has helped keep medicine costs affordable for Australians [Q15]. Many participants encouraged more transparency around the pharmaceutical industry’s role and influence in the decision making process [Q16].This study found that stakeholders had a broad range of views surrounding the application and/or relevance of the EML concept within the Australian context. The findings illustrated a diversity of perspectives amongst stakeholders, often reflecting their position within the health care system. Views held by consumers and health professionals with respect to what was “essential” and what medicines should therefore be accessible and reimbursed, were sometimes in contrast to the views of supply chain managers or policy makers. This demonstrated the tussle between perceptions of an EML based on a utilitarian approach in the health system for a population versus an EML established to meet individuals’ needs.Stakeholders’opinions of what constituted an ‘essential medicine’ differed depending on whether they were being tasked with making decisions for the population or they were considering a medicine to be ‘essential’ for an individual. From a consumer’s perspective, a medicine is considered essential at the point of care. Education can empower consumers to request access to medicines they deem essential. Individuals want access to the right drug, at an affordable price, of safe quality and available at all times within close proximity . On the th WHO EML includes a range of high cost medicines that address priority diseases in a variety of settings [While participants in this study supported the EML concept and were able to explain its general function, most found it difficult to determine what constituted an essential medicine due to the confounding nature of individual or utilitarian needs, as described above. Historically, an EML was once a basic formulary of survival and emergency medicines that adhered to utilitarian principles . Today, settings . For exasettings ,5. ThereShared self-interests provide clear conditions for a health system to identify and manage national priority medicines. For example, the utilitarian-type public health program around influenza pandemic planning manages and ensures continued supply of vaccines and medicines to all Australians. Meanwhile, participants’ discussed how differing interests contributed to the wide breadth of reimbursement under the PBS, which can include multiple options within the same drug class. Lastly, tensions between stakeholders may result from individuals having opposing interests. Participants described these tensions in the deliberations involved in the selection of high cost medicines for reimbursement, and negotiations to lower prices of generic medicines. For example, cost containment may prevent few individuals from accessing rare high cost medicines through public subsidised funding. At the same time, pharmaceutical companies may have opposing interests to governments and consumers regarding pricing of their medicines, and may be pressured to lower the cost of products in order to have them listed and utilised. This demonstrates how individuals’ opposing and differing interests contribute to the expansion and costs of reimbursement lists in HICs, in contrast to shared (utilitarian) interests around priority medicines.Study participants described the PBS accommodated broader individual needs than EMLs. Some highlighted that consumer engagement in the decision-making process has contributed to the wide breadth of the PBS to include sometimes rare and costly medicines to meet individual needs and expand access. The tussle between policy makers and consumer driven decisions may be explained by the Comprehensive Theory of Collaboration which describes three types of self-interest: shared, differing, and opposing . Shared It is often assumed that an EML ensures access to prioritised medicines. Therefore, stakeholders perceived that applying the EML concept provided access to affordable medicines to individuals and the health system. Yet, even in a HIC like Australia, access to medicines can be hindered. Two examples of vulnerabilities highlighted by stakeholders include unforseen drug shortages and high costs of medicines. Despite reimbursement, essential medicines are part of a complex global supply network, in which supply cannot always be guaranteed during unpredictable drug shortages –47. ThisSecondly, participants described the affordability of medicines for consumers and health systems was a growing concern as pharmaceutical expenditures continue to rise . Health Furthermore, consumers’ inability to afford out-of-pocket expenses can hinder access to medicines, sometimes leading to “catastrophic drug costs” ,57. AlthAll participants acknowledged the PBS as a national reimbursement scheme generally effective at meeting Australians’ health needs. However, the issues raised above, led them to explore strategies from the EML concept to prioritise medicines in the supply chain. In line with Hogerzeil  and WoodThe comprehensive theory of collaboration was applied as the underpinning theory to explain the variability in the notion of what is meant by an essential medicine. “Self-interests” in collaboration surrounding EMLs influence stakeholders’ views of what they deemed essential. Hence, multiple factors may influence these self interests which guide individuals to choose medicines, as represented in Fig illustrates the disparity between health systems that focus reimbursement schemes on utilitarian population needs versus meeting the expanding needs of individuals. This is most apparent between LMIC and HIC approaches to reimbursing medicines. When consumers absorb the difference in out-of-pocket medicine costs outside of government reimbursement programs, it can sometimes become exorbitantly unaffordable [A health system is unable to meet every need of all individuals within a population. Thus, fordable . TherefoThe results in this study were not meant to be generalisable. Instead, the use of qualitative methodology allowed researchers the opportunity to explore issues facing decision makers when creating national medicines lists. The strength of this study lies in offering an in-depth exploration of a broad range of key stakeholder views through rich qualitative data. This study gathered the perspectives of leaders and senior management throughout the continuum of a complex supply chain involved in decision-making surrounding medicines management in HICs. The study however, did not explore perspectives of primary health care workers, which should be pursued in future studies. It showed there were a variety of views as to what the term EML really means and how it relates to policy in a HIC context. While not included in the scope of this study, future studies could examine how this can be applied in LMICs. Additionally, future studies could examine further each individuals work environment and its effect on their views.The EML concept is simple, idealistic, and has been widely received. However, this study showed that the notion of “essential” is not implicit. Although beneficial in theory, Australian stakeholders struggled to identify how the EML concept functioned in practice. In Australia, decision making around reimbursement of medicines has strayed from the fundamental utilitarian concept of essential medicines. Instead, focus is on cost-effectiveness of new technologies and meeting unmet individual needs through expansive reimbursement lists. Interestingly, many of those involved in medicine reimbursement decisions and management of the pharmaceutical supply chain did not consider the EML concept in their approach. Moreover, the results of this study challenge for whom we consider essential medicines for, and if the term essential is currently appropriate. Therefore, this challenged whether the EML concept was out-dated or underutilised in HICs such as Australia. As medicine expenditures continue to rise worldwide and global drug shortages remain frequent and problematic, the EML concept can potentially play a role in managing health resources. Therefore, further investigation is required to address innovative ways to apply EML concepts in HICs to support population wide access to prioritised medicines, while strengthening collaborations between pharmaceutical supply chain stakeholders. Transitioning the EML concept from policy to practice continues to be a work in progress.S1 FigA core list of medicines reflects shared stakeholder interests to support the fundamental basic needs of a country’s health system. Bearing in mind that all medicines on the WHO EML are not assumed to be included on a country’s EML, since they are adapted to meet each health system’s needs. In contrast, broader reimbursement schemes have wider inclusion of differing and opposing interests between stakeholders. The extent of out-of-pocket expenses individuals may incur beyond the shared priorities supported by the health system are illustrated by the difference in area from the outer layer. This model does not take into consideration that a health system with broader reimbursement may also be at risk of reimbursing inappropriate medicines. Also, countries without a national EML (ie. the US) are not illustrated in this model despite high out-of-pocket expenses, due to the high variability of private insurance schemes. And while some health systems do not have EMLs, people can still access medicines if they are willing and able to pay-out-of pocket or have alternative funding assistance available such as private insurance.(TIF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.


Finding new therapies for Parkinson’s disease (PD) is a slow process. We assembled an international committee of experts to examine drugs potentially suitable for repurposing to modify PD progression. This committee evaluated multiple drugs currently used, or being developed, in other therapeutic areas, as well as considering several natural, non-pharmaceutical compounds. The committee prioritized which of these putative treatments were most suited to move immediately into pilot clinical trials. Aspects considered included known modes of action, safety, blood-brain-barrier penetration, preclinical data in animal models of PD and the possibility to monitor target engagement in the brain. Of the 26 potential interventions, 10 were considered worth moving forward into small, parallel ‘learning’ clinical trials in PD patients. These trials could be funded in a multitude of ways through support from industry, research grants and directed philanthropic donations. The committee-based approach to select the candidate compounds might help rapidly identify new potential PD treatment strategies for use in clinical trials. Multiple new therapies for Parkinson’s disease (PD) have emerged following the initial development of levodopa therapy, including dopamine receptor agonists, MAO-B inhibitors, COMT inhibitors and deep brain stimulation. These treatments predominantly still involve an approach of dopamine-replacement to reduce symptoms acutely, without achieving slowing of disease progression. Levodopa-based therapy is complicated by the emergence of motor fluctuations and dyskinesias, and has limited effects on a range of non-motor PD symptoms  that are the source of significant morbidity. It may be possible to accelerate the process of identifying new PD treatments by repurposing medicines (drug repositioning) that are approved for other indications because of evidence that they may also have beneficial actions on PD progression.In order to try and facilitate identification of such compounds for repositioning we have set up a Linked Clinical Trials initiative (LCT), a structured approach for accelerating new treatments for PD. This initiative arose for 4 reasons; 1) basic research breakthroughs regarding PD pathophysiology; 2) previous failure in developing treatments for PD progression; 3) the realisation that many agents in clinical use for other medical conditions may have off-target effects relevant to PD; and 4) the lengthy procedures needed to take a drug from the laboratory to the clinic.Drug repositioning is highly attractive in that it partly focuses on drugs with regulatory-approved clinical safety data, and there are thousands of regulatory-approved biologically-active drugs already available. If these approved drugs include agents with efficacy in the treatment of PD progression, repositioning may represent a more efficient process –8 than oClinical trials in PD using five different repositioned drugs have been undertaken, Exenatide , a GLP-1The recently-published Exenatide pilot clinical trial  apart frThe notion that small screening studies for putative new treatments are applied to PD is not new. A series of futility studies were conducted some years ago with a similar aim; to determine cost-effectively whether a full Phase 3 clinical trial was merited for each agent tested. Notably, Coenzyme Q10, the neuroimmunophilin-ligand GPI-1485, creatine and minocycline were tested in 2 futility studies, that provided information suggesting that it was warranted to proceed with creatine –28. AddiWe therefore sought to build on this initiative and felt that the best way to do this was to set up a formal international committee tasked to prioritize which putative treatments should move quickly into clinical trials.We undertook an extensive review process of marketed drugs and drugs under development in many different therapeutic areas, as well as several natural non-pharmaceutical compounds. Essentially, this process involved rigorous and continual scrutiny of the results of an evolving wide range of pharmaceutical approaches used by basic and clinical researchers trying to influence cellular function and cellular protection in other key fields of medical research, most especially in ophthalmology, cardiology, metabolic diseases, and oncology. The aim was to identify agents that may modify the underlying pathogenesis in PD, and also to establish a process for technology transfer of new biochemical/pharmaceutical approaches where breakthroughs in other therapeutic areas could be rapidly applied to PD where there was a rationale to do so.This process initially identified 72 potential new candidate therapeutic approaches for PD that addressed specific biochemical targets of interest. On closer scrutiny, these 72 candidates were reduced to 26, mainly on grounds of safety, blood-brain-barrier penetration, or commercial/patent issues. Succinct supporting dossiers (5–7 pages) were written describing the reasons why each of the 26 proposed interventions should be considered for entry into learning (pilot) clinical trials in PD patients.These 26 dossiers were then assessed by an international committee of experts see  with theThis initial triage process removed several of the proposed therapies in terms of insufficient support for their use in PD. In view of the multiple funding sources required to facilitate a considerable number of parallel learning trials in PD patients, representatives of key funding bodies were also invited to attend including NIH/NINDS, Van Andel Institute, The Michael J Fox Foundation for Parkinson’s Research, The Kinetics Foundation, The Parkinson’s Disease Foundation, Parkinson’s UK, and The Cure Parkinson’s Trust, as well as several patients with PD. A number of other major funding bodies , who were not able to attend the committee meeting were given in-person, detailed debriefings of the outcomes of the LCT committee meeting.The 26 candidate agents presented to the committee are shown in The average pre-prioritization scores allocated by the LCT committee members (before their face-to-face committee meeting) are presented in The candidate treatments above the cut-off point on In addition, 5 other candidates  were considered potentially interesting by the committee, but they were placed on a waiting list, subject to further information becoming available on them. The awaited information included impending results of a clinical trial using the same intervention in another therapeutic area, questions about physical characteristics of the interventions (such as blood-brain-barrier penetration) or further unpublished information  that might impact on the selection of the intervention, or subsequent trial design.The 7 therapeutic candidates initially selected/prioritized are now being moved into learning (pilot) clinical trials involving a worldwide clinical trial network currently being established as part of the LCT initiative.With an increasing number of biochemical targets emerging as potentially relevant to PD progression comes a need to develop new approaches for identifying therapies that will engage those targets. Drug repositioning from existing therapeutics, or from interventions under development in other disease areas, represents an effective interim way forward whilst more specifically designed drugs can be identified, synthesized and brought through from pre-clinical studies. Since safety and toxicology data in humans is already known for these repositioned drugs, it means that success rates of such approaches can reach 30%, which is a vast improvement on traditional drug development methods , 12.How can one best identify drugs suitable for repositioning for PD? We focused on current understanding of PD-relevant biochemical and pharmaceutical characteristics involving research that straddled recent advances in many other therapeutic areas. The LCT initiative aims to use methods of candidate identification as propounded by O’Connor and Roth .As the success rates of Phase 2 clinical trials continue to fall , often tIndeed, Sherer et al.  commenteIn fact, since most drugs for use in neurology have been discovered empirically, it has been suggested that CNS diseases are far less suited to target-based approaches than other therapeutic areas . The unpWe prepared study dossiers for 26 potential interventions . These aOf the 26 interventions selected for committee evaluation, some were already known to influence multiple biochemical targets. Furthermore, some of these targets were known to be influenced by more than one of the drug candidates being considered. For example, in terms of drugs targeting G protein coupled receptors (GPCR), which include cyclic AMP receptors, there were 6 drugs considered by the committee .Indeed in the past, NINDS undertook a similar process and created a series of excellent dossiers describing putative approaches for testing in PD . Three oSome, but not all, of the candidates in the LCT initiative are owned by pharmaceutical companies which means that the future funding of any prioritized candidate might be influenced by ownership. So whether or not an agent is owned by a pharmaceutical agent, funding might well need to be secured from non-commercial sources see  if the cAlso attending the LCT Committee meeting was an executive group comprising the main funding bodies that support basic and clinical research in PD, several of whom had already been funding pre-clinical laboratory studies involving candidates now being considered for pilot clinical studies. It is hoped that by involving them at this stage, that the funding of trials related to selected targets can happen quickly.Across the treatments that were finally prioritized by the committee, it was recognised that here is an opportunity to harmonise the trial designs including control groups and outcome measures. An initiative like this also has the capacity to set up co-ordinated multi arm trials, as has been done in some cancer studies .In conclusion, the intelligent use of drug repositioning is likely to represent a powerful bridging strategy for testing drugs active on multiple targets, some of which are directly relevant to the treatment of PD. The LCT initiative seeks to formalise this process and use a comprehensive approach that annually screens the literature for new interventions. The committee then prioritizes the agents for clinical trial evaluation through an on-going, practical head-to-head screening of important new approaches. By so doing, it is hoped that the identification of safe and effective new agents can be undertaken with greater speed, as we attempt to slow down or even halt the progression of this disorder.


Here the rate-yield tradeoff also has important implications. I believe the seemingly contradictory points of view expressed in the literature can be reconciled by considering the growth conditions, ecological strategies and level of organization treated by these various studies. This opinion article is not intended as a comprehensive review. Excellent reviews and analyses of various aspects of this literature exist . Pirt  defined G, as the yield in the absence of maintenance costs. In this paper I deal mainly with Y, as this is what most studies measure). This results in a linear relationship in the double reciprocal plot (1/Y vs. 1/μ), which produces the hyperbolic relationship shown on the left side of Figure 2 stress responses. He points out that costs such as storage and extracellular losses should not be strictly considered part of physiological maintenance (or endogenous metabolism), and that cell death can also contribute to Y. Wang and Post . Discounting other fates of consumed substrate, namely extracellular products, BGE is analogous to Y. Low BGE are observed under oligotrophic conditions (Del Giorgio and Cole, There is considerable theoretical and empirical support for a negative relationship between growth rate and yield in microbial metabolism. A tradeoff between rate and efficiency is intuitive in the mechanical analogy: hot rods and monster trucks get lower gas mileage than slower, less powerful, fuel-efficient models. However, the physiological basis for this tradeoff in microbes is less obvious. Some authors (Westerhoff et al., E. coli, though not among different populations. The classic work of Monod (E. coli, with yield decreasing and rate increasing with increasing temperature (see SI of Beardmore et al., Cobetia marina (Figure 1 in Yumoto et al., Arthrobacter species from Arctic soil (Figure 4A in Panikov and Sizova, There are several examples of microbes shifting between high yield/low rate and the converse state depending on growth conditions (see references in Kreft, of Monod  shows a Negative relationships between growth rate and yield are sometimes found among species and within communities. In experiments on soils incubated with substrate, a rate-yield tradeoff was observed across seasonal gradients and among samples that varied in fungal: bacterial ratio (Lipson et al., The form of rate-yield tradeoffs is sometimes observed to be linear (Lipson et al., Sorangium cellulosum, grows slowly and has a very low apparent yield, partly owing to its large investment in lipids and secondary signaling compounds (Bolten and Muller, Despite the wide variety of contexts in which the preceding theories and data were developed, there is no reason they cannot be reconciled under a broader perspective. Positive relationships between growth rate and yield are observed in oligotropic aquatic environments and in chemostats at low dilution rates. Rate-yield tradeoffs are observed in richer growth media, soils and other environments varying in resource availability, and between planktonic and spatially structured environments. I propose that all of these observations are part of the same continuum Figure . The oveThere is a growing effort to incorporate microbial growth kinetics into larger scale ecosystem studies (Schimel and Weintraub, The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of “intact” intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections. Genome expression studies based on high-density tiling arrays and RNA deep sequencing (RNA-seq) show that a relatively high percentage of the bacterial genome is transcribed as non-coding RNA molecules  the identification of those sRNAs that are actually relevant for infection; (ii) the infection phase(s) at which their regulatory activity takes place; (iii) the mode by which the pathogen activates and de-activates these regulatory circuits; and (iv) the impact of sRNA activity in the infection process.The role of bacterial non-coding RNAs in virulence has received much attention in the last few years. Relevant data have been obtained in both cellular and animal models  and/or mild phenotypes linked to their absence  and a dormant small cell variant (SCV), identified 15 sRNAs in bacteria isolated from eukaryotic cells and axenic cultures  isolated from fibroblasts  in non-growing intracellular bacteria , which contribute to build up the transcriptome at a precise time. An ectopically-expressed sRNA will be just able to bind mRNAs that are already “there.” In order to identify a specific sRNA target relevant for infection, the sRNA and the target should be both co-expressed in time and space, which might not be the case for bacteria growing in laboratory media. The ectopic induction of the sRNA might also lead to “off-target” effects on mRNAs that would never be naturally co-expressed with the sRNA in the infection scenario. These observations indicate that the most rational way to get functionally relevant information from regulators is to determine both global gene expression and proteome dynamics along the infection. Proteomic analyses performed in bacteria isolated from cultured eukaryotic cells are known for  Bumann, . AlthougIn this section we refer to sRNAs studied directly in intracellular bacterial pathogens obtained from infected eukaryotic cells. Table L. pneumophila expresses the non-coding 6S RNA at much higher levels inside macrophages than in laboratory media  5′-UTR of the lmo0514 gene, which encodes a surface protein of unknown function that is abundant in the cell wall of intracellular bacteria  to elementary bodies . Two chlamydial histone H1 homologs; Hc1, encoded by the gene hctA, and Hc2, bind to and compact the bacterial chromosome making the EBs transcriptionally and translationally inactive. IhtA (for inhibitor of hctA translation) is only expressed in RBs where it inhibits hctA translation without affecting hctA mRNA transcription or stability. Conversely, Hc1 is only present in purified EBs where it densely compacts the chromosome, rendering Chlamydia in a metabolically inert stage  assay that uses a common oligonucleotide probe to the yfp sequence. Signals are recorded on a microfluidic chamber in which each strain is grown in independent cells  the limitation that the genetic procedure has for analyzing non-essential genes or essential genes whose function is not perturbed by the tag; and, (ii) the possibility that tagging might affect function in protein(s) contributing to the infection. Nonetheless, these factors influence any type of screening and they should not impact negatively the benefits that these massive analyses might have for studying RNA regulation in intracellular bacterial pathogens.Infection of cultured eukaryotic cells has been widely used to characterize major virulence factors in intracellular bacteria, their cellular targets and the underlying signaling pathways , a ratio often used disparately by different labs even for the same cell line and pathogen. An optimal adjustment of this parameter prevents over-infection and lysis of heavily-infected cells due to massive bacterial load  in individual bacteria. However, to date most of those studies have been performed in bacteria grown in axenic cultures and it is not trivial to extend such advances to an Salmonella-macrophage infection model whereas 0.4% SDS is needed to lyse efficiently fibroblasts infected with this pathogen  were infected with S. Typhimurium to obtain sufficient RNA for qRT-PCR assays  with L. monocytogenes to obtain RNA for northern blotting assays  acidic phenol, and 19% (v/v) ethanol . This technique was first developed in the context of an infection model by two pioneer studies aimed to characterize genes expressed by + mRNA), which is followed by selective capture of bacterial rRNA. A positive aspect of this procedure, as in the SCOTS method, is that no physical separation of intact intracellular bacteria is required since the procedure is performed on “total” (host-pathogen) RNA. Moreover, it guarantees that bacterial sRNAs remain in the sample. To date, no study has reported yet the usage of these kits for gene expression profiling in pathogens located inside eukaryotic cells, although they should be evaluated given its potential benefits. A comparative study examining the efficacy of protocols relying on bacteria isolated from infected host cells relative to those methods based on “total RNA” will be, undoubtedly, of extraordinary value. This type of studies might provide useful guidelines for future research involving global gene expression in intracellular bacteria.Taking into account these facts, investigators face two important questions when their objective is to determine gene expression profiling in intracellular bacteria. The first decision is whether or not to physically separate intracellular bacteria from host cell debris. These intact intracellular bacteria are precious material for any type of subsequent expression analyses, including tiling arrays, RNA-seq and individualized gene expression assays . The second decision is whether to directly obtain gene profiling data directly from the infected sample avoiding separation of bacterial and eukaryotic materials. In this latter case, the investigator could use SCOTS if pathogen information is the ultimate goal. There are also commercial kits that allow recovery of bacterial RNA from samples of infected eukaryotic cells containing host-bacterial RNA mixtures. These kits are based on a prior purification step that removes eukaryotic RNA (28 S, 18S rRNAs, and polyAin silico analysis allows to identify eukaryotic and prokaryotic sequences and to assign them to both the host cell and the pathogen, respectively. Advantages of this method are that it does not require physical separation of the intracellular bacteria and that it avoids the usage of microarrays. The information relative to gene expression in the eukaryotic cells derives however from both infected and non-infected cells. This factor could be a major caveat in infection models in which the infection rate is relatively low, even when using a high MOI. Moreover, most infection models show that there is heterogeneity regarding the number of intracellular bacteria per host cell as the infection progresses. There is also evidence that intracellular bacteria can behave in a heterogeneous manner within the infected cell. For example, for S. Typhimurium it has been shown that actively growing and persistent bacteria co-exist in the same macrophage (Helaine et al., S. Typhimurium intracellular populations with distinct intracellular locations, cytosol vs. intravacuolar, have also been observed in epithelial cells (Knodler et al., Lastly, it should be noted the recent efforts aimed to analyze simultaneously gene expression in both host cells and intracellular bacteria. Technologies as “dual RNA-seq” allows to reach such goal (Westermann et al., in vivo that might not be possible to model using regular genetic approaches in vitro (Diard et al., Infection is a multidimensional complex event and several reasons provide a high degree of uncertainty when investigators try to model it. Thus, infection proceeds through distinct stages with different type of associated stresses. The pathogen also interacts during this journey with varied and complex systems, like the resident microbiota, the immune response, and the intracellular eukaryotic cell milieu. In addition, bacterial populations are not phenotypically homogeneous and display complex responses For example, the study of intracellular bacterial pathogens needs a homogenization in the infection experimental set-ups. Infection protocols differ substantially from lab to lab even for the same pathogen: the way to prepare bacteria for infections, the MOI, the host cell type or animal model, and the protocols for sample preparation and gene expression analysis. Most times, there is not a scientific reason behind but it is just the “the way we do it here.” It would be interesting to reduce the experimental variability at this point since this is critical for investigations dealing with intracellular bacterial pathogens that have inherent technical difficulties.S. Typhimurium that showed varied expression of many sRNA along the infection (Ortega et al., sRNA-regulon” once we know which sRNAs are regulating which transcripts or proteins.Another important aspect highlighted in this review is the dynamics of the intracellular infection. Although a glance was obtained in a study with non-growing intracellular in vitro infection model are also urgently needed.The valuable “dual” gene expression analyses existing to date (Westermann et al., E. coli cells used to feed Caenorhabditis elegans was shown to interfere expression of worm genes related to chemosensory behavior and lipid metabolism (Liu et al., Agrobacterium tumefaciens (Wilms et al., Bartonella spp, Rickettsia spp., Brucella spp., and Helicobacter pylori, also encode T4SS and evidence for DNA transfer to the eukaryotic cell has been found for some of them (Llosa et al., L. monocytogenes and S. Typhimurium engineered to undergo lysis upon entry into the host cell (Kuo et al., Lastly, recent original studies have suggested a probable action of bacterial sRNAs in eukaryotic cells. Thus, the production of the sRNAs OxyS and DrsA in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This database is associated with user-friendly tools to facilitate searching, retrieving and downloading of disease-related information.There is an urgent need for a unified resource that integrates trans-disciplinary annotations of emerging and reemerging animal infectious and zoonotic diseases. Such data integration will provide wonderful opportunity for epidemiologists, researchers and health policy makers to make data-driven decisions designed to improve animal health. Integrating emerging and reemerging animal infectious and zoonotic disease data from a large variety of sources into a unified open-access resource provides more plausible arguments to achieve better understanding of infectious and zoonotic diseases. We have developed a model for interlinking annotations of these diseases. These diseases are of particular interest because of the threats they pose to animal health, human health and global health security. We demonstrated the application of this model using brucellosis, an infectious and zoonotic disease. Preliminary annotations were deposited into VetBioBase database (http://vetbiobase.igbb.msstate.eduDatabase URL:  AnoAt the molecular level, various infectious agents have different genetic makeups that may cause similar or different signs and symptoms. The molecular diagnosis of infectious diseases using nucleic acid-based technologies such as the polymerase chain reaction, ligase chain reaction, transcription-mediated amplification and nucleic acid sequence-based amplification have become tools in understanding key molecular factors related to ERAIZD causal agents. These techniques provide highly accurate diagnosis of infections caused by bacteria, viruses, fungi, parasites and others . For exaEmerging and reemerging diseases are caused by diverse range of biological agents that exit their reservoirs, enter susceptible hosts through different routes and cause tissue damage. Provision of a resource that links findings from epidemiological and basic research investigations could help answer fundamental questions regarding factors responsible for disease development and so forth. We have developed a model for annotating multidimensional diverse ERAIZD data from a large variety of sources and integrating it into an open-access unified resource we call ERAIZDA . The model is referred to as ‘ERAIZDA model’ throughout the article. We envision that availability and utility of an ERAIZDA resource will (i) promote global data sharing beyond usual boundaries which could lead to improved interactions in multi- and interdisciplinary research teams; (ii) furnish the animal/human health and veterinary research communities with integrated disease information for developing effective joint policies and guidelines for controlling animal infectious and zoonotic diseases; (iii) lead to better planning of coordinated research strategies including setting up research priorities, developing testable data-driven hypotheses and identification of effective and efficient integrative interventions and (iv) lead to establishing database and data sharing standards used by multi- and interdisciplinary communities for collaborative data sharing, solving much of the current problems in data curation and database application programming interface and web services among related data sources. We strategically selected brucellosis to demonstrate implementation of the ERAIZDA model. Globally, brucellosis is one of the most significant zoonotic diseases  with pubThe ERAIZDA model adopts an annotation approach that takes into consideration all sources of information for an infectious disease that occurs at the animal–human–pathogen–ecosystem interface. Identification of information sources that collectively provide animal infectious, and zoonotic diseases is a first step when implementing this model. Five main sources of disease information were identified and designated as PADER , where PComprehensive annotation of animal infectious and zoonotic diseases using the PADER approach is a complex process that involves coordinated and dedicated effort of diverse expertise and skills to integrate multidisciplinary disease information into a unified resource. The complete ERAIZDA model herein described  demonstrSupplementary File S1). We acknowledge that debate may exist around the description of these parameters which could lead to better and standardized disease annotation terms.The ERAIZDA model is designed for comprehensive annotation of animal infectious and zoonotic diseases from diverse sources (PADER) to generate multidisciplinary disease-related information. We have defined parameters to be used as reference to guide the annotation process in each PADER source. These parameters are grouped into three broad categories representing (i) diseases, (ii) pathogens  and (iii) molecular information, specifically disease biomarkers. The model adopts different terminologies for describing infection, frequency and typology of an infectious or zoonotic disease . A detaiBrucellosis was chosen as a classical example of a significant endemic zoonotic disease to demonstrate the implementation of the ERAIZDA model. The annotations of brucellosis described herein are not by any means exhaustive but serve as model for annotating animal infectious and zoonotic diseases from diverse sources.Supplementary File S1) for guiding comprehensive annotation of animal infectious and zoonotic diseases using this model. In order to provide structured annotations the parameters were organized in an Excel spreadsheet where rows represented diseases and columns represented parameters.Preliminary annotation of selected PADER sources enabled us to establish important parameters (Supplementary File S2).The process of annotation started by searching important keywords [brucellosis OR (brucellosis) OR (brucellosis zoonotic disease) OR  OR (brucellosis in human)] in Google as First-Pass Analysis to get a clear picture about the expected sources of information and initial knowledge of brucellosis. The search returned 180 000 google hits. Clustering of the top 100 hits using PADER codes identified the most important sources of brucellosis information (Supplementary File S1). In the process of annotating brucellosis we found that some parameters may not apply or be relevant to some diseases and data may not be available for some parameters. In such cases, the corresponding fields are represented with triple hyphen (—) until such data become available in the subsequent updates.From the First-Pass Analysis, we were able to generate preliminary brucellosis information that enabled us establish custom terms  for describing disease parameters (Supplementary File S3). In the past 15 years, the number of brucellosis articles has been increasing progressively. A dedicated effort to annotate these articles can yield a very significant information that may provide insight of new ways for managing the brucellosis.The primary quantifiable sources of reliable brucellosis information, including epidemiology of the disease and molecular characterization of the causal agents, are a combination of articles published in peer-reviewed journals and controlled databases represented in the NCBI. The NCBI PubMed database is the most famous gateway for browsing information published in scientific journals. For example, searching the PubMed database (as of 5 May 2015) using brucellosis-related keywords [(brucellosis OR (brucellosis) OR (brucellosis zoonotic disease) OR  OR (brucellosis in human) OR  OR (Mediterranean fever) OR (undulant fever)] retrieved 16 845 articles, including nine articles  that werBrucella melitensis was the first brucellosis causal agent to be characterized. Sir David Bruce isolated Micrococcus melitensis (now B.melitensis) from spleen of a British soldier who died from Malta fever (brucellosis) in Malta in 1886 .Preliminary annotation of few selected articles indicated that  in 1886 . Subsequnopinata  (SupplemB.abortus is the most characterized species as evidenced by the number of subspecies represented in the NCBI Taxonomy Databases and has more open-access PubMed articles (Supplementary File S5).Currently, over 500 subspecies have been classified under these species and documented in the NCBI Taxonomy Database . B.aborBrucella species facilitated identification of species-specific molecular records of the causal agents of brucellosis (Supplementary File S6). These molecular data, especially the nucleotide (transcripts) and amino acid (protein) sequences, are very important in identifying unique features of indistinguishable causal agents. The ERAIZDA model includes a link for accessing most current molecular data for each annotated causal agents indexed in the Entrez databases.Entrez is the NCBI's primary text search and retrieval system that integrates diverse databases such as PubMed and Taxonomy databases with molecular databases such as DNA and protein sequences, genes, genomes, single-nucleotide polymorphs and gene expression data . TraditiSupplementary File S7. This data can also be retrieved from the VetBioBase database  as a reference to guide the disease annotation process from diverse sources referred (in this article) as PADER. Each parameter describes significant information related to a particular infectious and/or zoonotic disease. The model encourages data integration into an open-access unified resource for use by the animal/human and veterinary research communities to accelerate integrative translational interventions aimed at achieving better understanding of infectious and zoonotic diseases. Using brucellosis as an example, we demonstrated that multidisciplinary disease-related information from epidemiological investigations, clinical studies, case reports and basic science functional genomics investigations can be linked together and made available through a single resource. Preliminary annotations of brucellosis were used as a foundation to create a unified resource known as ERAIZDA. Our future focus is to use the ERAIZDA model to generate annotations of all animal infectious and zoonotic diseases of national and international priority, starting with the notable diseases listed by the World Organization for Animal Health (OIE).We present the ERAIZDA model, a comprehensive model for annotating animal infectious and zoonotic diseases. This model uses over 50 pre-defined parameters (Supplementary File S1) as reference is the most feasible way to ensure that every piece of available information is represented. It is most likely that a single source of disease information would not be able to provide all the parameters . This is why PADER becomes such an important approach. For example, when annotating molecular biomarkers using the ERAIZDA model, we recommend including supporting evidence. The best evidence is to link the biomarker with a reference such as PubMed articles and text that will enable users to have confidence in the annotated biomarker. For instance, sequences with amino acid replacement may signify useful molecular biomarkers for detecting mutations that could also be basis for detecting antimicrobial resistance and virulence determinants. Adding reference to the sequence information is particularly important, especially if the annotation involves cross referencing between related diseases. Here is an example of cross-species annotation from two articles; PubMed ID 21151656  and PubMed ID 17021120 : ‘Mycobacterium bovis, a bacterium that causes bovine tuberculosis is naturally resistant to pyrazinamide  that can be used by ontology developers to improve the existing ontologies such as pathogen Transmission Ontology and Symptom Ontology or designing new ontologies. The model also integrates some aspects of host-pathogen interactome features related to the pathogen and hosts at molecular level. For example, we understand that virulence of a pathogen is one of possible effects of host-microbe interaction. In that case, a microbial virulence could depend on host factors to be effective. This is why the model links possible molecular biomarkers including virulence factors with pathogen characteristics to ascertain the effects of interaction on, e.g., the microbial virulence or pathogenicity in relation to effects induced by host genes.Although ERAIZDA model intends to link disease names with Disease Ontology terms, we are aware that there are other ontologies that are relevant to the ERAIZDA model. Since data generated using the ERAIZDA model is intended to benefit not only the research community but also a wide range of animal and human health-based communities, we have been very careful not to link ontological terminologies that are not familiar to the audience. Indeed, the model is expected to provide very specific quantitative data guided by pre-defined parameters (as described in Supplementary File S1) to guide the annotation process in each PADER source. Having a standard annotation procedure will also facilitate data integration and updates.It should be emphasized that the integrated data by itself is not useful, unless it is freely accessible by the intended communities for further interventions. User-friendly tools are provided to offer such access to the interested communities. Generally, the ERAIZDA model encourages multidisciplinary collaboration of all key players involved in the disease management process to share research and non-research data without their usual boundaries. It is anticipated that the ERAIZDA data will continue to grow progressively and sustainably as more animal infectious disease annotations are added, and the research community will utilize and benefit from this data. Although curation is one of the most likely challenging aspects of maintaining a sustainable ERAIZDA database, having a uniform annotation procedure can lessen the challenge. To maintain accuracy and uniformity of annotations, curators are encouraged to use special forms containing the pre-defined parameters (Annotation of animal infections and zoonotic diseases from diverse sources is a central initiative to reveal important information for new interventions. Most importantly, integrating the disease annotations into an open-access unified resource and provision of user-friendly tools for searching, retrieving and downloading specific information could save users considerable time and effort. We expect to increase the depth and breadth of the ERAIZDA dataset by continuing to annotate additional animal infectious diseases of national and international priority. We believe that since zoonoses can infect both animals and humans, both the medical, public health, basic research and veterinary communities will definitely benefit from this unified resource. We encourage interested communities to use the ERAIZDA data to develop additional models needed to understand the mechanism and pathology of animal infectious and zoonotic diseases and translation of functional genomics of infectious diseases into biological value.Supplementary data are available at Database Online.


The modulation of sirtuin activity has been highlighted as a promising approach to reduce neurodegenerative processes. In this review, we summarize the recent discoveries regarding the potential relationship between SIRTs and depression caused by metabolic disorders (Mets). Ultimately, we suggest the possibility that SIRTs will be novel targets to alleviate neuropathogenesis induced by depression.Depression is now widely regarded as a common disabling disorder that affects negatively the social functioning all over the world. Depression is associated with diverse phenomenon in brain such as neuroinflammation, synaptic dysfunction, and cognitive deficit. Recent studies reported that depression occurs by various metabolic changes, leading to metabolic syndrome. Sirtuins (SIRTs) are NAD The prevalence of depression continues to rise all over the world and yearly prevalence rate is close to 10%  concentrations of inflammatory markers in serum showed the elevation of pro-inflammatory factors such as interleukine (IL)-6, tumor necrosis factor (TNF)-α, and C-reactive protein compared to non-depressed patients , norepinephrine and dopamine Nutt, . In patiIn addition, depression is influenced by reduced levels and activity of acetylcholine  SIRTs may control the inflammatory response in depression; (2) SIRTs might regulate the insufficient level of neurotransmitters in depression; (3) SIRTs may improve the synaptic dysfunction caused by depression; and (4) sirtuis may alleviate the cognitive decline caused by depression. As society ages, people suffer from metabolic syndrome such as diabetes, obesity, and cardiovascular disease and subsequently they suffer from depression by metabolic changes. We should investigate to search the therapeutic solution. SIRTs is the metabolism related genes and also ameliorates depression related pathogenesis (Abe et al., JS wrote the preliminary draft and revised details of the manuscript. JK revised all manuscript in detail.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.