Coloboma is a defect in the morphogenesis of the eye that is a consequence of failure of choroid fissure fusion. It is among the most common congenital defects in humans and can significantly impact vision. However, very little is known about the cellular mechanisms that regulate choroid fissure closure. Using high-resolution confocal imaging of the zebrafish optic cup, we find that apico-basal polarity is re-modeled in cells lining the fissure in proximal to distal and inner to outer gradients during fusion. This process is accompanied by cell proliferation, displacement of vasculature, and contact between cells lining the choroid fissure and periocular mesenchyme (POM). To investigate the role of POM cells in closure of the fissure, we transplanted optic vesicles onto the yolk, allowing them to develop in a situation where they are depleted of POM. The choroid fissure forms normally in ectopic eyes but fusion fails in this condition, despite timely apposition of the nasal and temporal lips of the retina. This study resolves some of the cell behaviors underlying choroid fissure fusion and supports a role for POM in choroid fissure fusion. In vertebrates, the eye forms through a series of coordinated interactions between tissues of different origins: the retinal neuroepithelium, non-neural surface ectoderm, and a loose array of cells arising from both neural crest and mesoderm known as periocular mesenchyme , in which apical surfaces of opposing epithelial cells meet and fuse, a process that is characterized by dynamic apical protrusions that mediate initial cell-cell contacts  signaling also contributes to ventral eye morphogenesis and choroid fissure fusion, acting both directly on the ventral optic cup, as well as regulating gene expression within the POM zf77 mw11 vu119y5 , rabbit anti-laminin , chicken anti-GFP . The secondary antibodies were: Alexa Fluor 633 anti-mouse, 488 anti-rabbit, and 488 anti-chicken . Images were collected on a Leica confocal microscope using a 40x oil immersion lens. Gain and offset were adjusted to enhance the contrast of the signal against the background.Whole-mount immunolabeling procedures were performed as previously described  for 90 s and imaged while wet without cover-slipping.To detect apoptotic cells, TUNEL labeling was carried out using the Apoptag kit .To block cell division, embryos were cultured in embryo medium containing 100 μM aphidicolin and 20 mM hydroxyurea dissolved in 2% dimethylsulphoxide from 36 to 60 hpf zf77 ct3aGT, . We have not resolved the eventual fate of the cells lining the fissure, but the retraction described above suggests some such cells may move toward the outer retina and join the RPE. However, from other movies, nuclear tracking suggests that some cells lining the fissure may actually move toward the inner retinal surface where they could incorporate into the neural retina vu119 embryos . After 3.5 h, protrusive activity is evident but without a clear directionality. However, from 6 h, the cell is more overtly polarized toward the temporal lip of the fissure, and displays protrusive activity in the vicinity of cells in the fissure with very motile nuclei that we presume belong to the POM. These protrusions could potentially be involved in the formation of new retinal cell-cell contacts during choroid fissure fusion.Figure In cell transplantation experiments, we observed that 17 out of 19 GFP positive cells targeted to the fissure divided at least once during timelapse acquisition  as in wild-types , suggesting that cell proliferation is not essential for fissure fusion (data not shown).Cell cycle progression regulates neural crest cell delamination from the neural tube vu119 and Tg(ctnna:citrine)ct3aGT embryos and embryos with mosaically GFP-labeled cells lining the fissure mw11 embryos vu119 embryos in which membrane targeted GFP is expressed by all cells . The ectopic optic vesicles had variably reduced amounts of Tgzf77 labeled neural crest cells  positive hosts; Movies Ectopic optic vesicles underwent overtly normal morphogenesis to form isolated optic cups/eyes on the yolk .We noticed that the lack of fusion is most evident superficially within the retina Figures , but in These analyses show that morphogenesis of the optic cup can occur normally in transplanted vesicles in the presence of reduced amounts of POM cells, and that the lips of the choroid fissure come into apposition in a timely manner. Nonetheless, fissure fusion fails to occur by 5 dpf, leaving the transplant eyes displaying a classic coloboma phenotype.In this study, we provide both a detailed description of many of the cellular events that accompany choroid fissure closure in zebrafish and evidence consistent with a role for POM in mediating fusion subsequent to apposition of the lips of the fissure. We show that although cells lining the fissure approach each other via their basal surfaces, at the point of fusion, it appears that there is transient apical to apical cell contact. Many fissure cells undergo cell division but few, if any, die during fusion and neither proliferation nor cell death appears necessary for fusion to occur. We show that optic vesicles depleted of POM develop into normally shaped optic cups with well-formed choroid fissures. However, choroid fissures in ectopic eyes fail to fuse indicating that the local environment in which the eye forms impacts choroid fissure fusion. This would be consistent with a normal complement of POM being required for choroid fissure fusion.Most epithelial fusion events are initiated by apical to apical contacts between the cells undergoing fusion  whereas they express rx2 after fusion , on the other hand, they remain proliferative while most other RPE cells have exited the cell cycle (Hu and Easter, As they approach each other, we find that cells lining the nasal and temporal lips of the choroid fissure have cuboidal morphology distinct from both the RPE and neural retina. Indeed several lines of evidence suggest that fissure cells are a distinct population. Not only does their morphology and behavior differ from their neighboring retinal cells, they also have a distinct gene expression profile; for example The mechanisms by which cells lining the fissure break down their initial apico-basal polarity remain to be determined but it is likely that the loss of basal lamina is a key trigger for this event. In other situations loss of basal lamina can lead to epithelial cells reorienting their apico-basal polarity. For instance, in the early evaginating optic vesicle in zebrafish, disrupting Laminin can lead to complete inversion of cell polarity such that apical proteins appear at the basal surface of the optic vesicle and cells adopt a more mesenchymal morphology (Ivanovitch et al., Young optic vesicles depleted of POM and transplanted heterotopically onto the yolk developed into optic cups/eyes almost indistinguishable from wild-type eyes. Notably, optic cup morphogenesis appeared normal, a choroid fissure formed and the nasal and temporal margins of the fissure became properly apposed. Consequently many aspects of eye formation can occur in retinal cells isolated from their normal environment. This is demonstrated perhaps most dramatically in the ability of ES cell organoids to form optic cups that contain differentiated RPE cells and retinal neurons (Eiraku et al., The inductive events leading to choroid fissure formation are poorly understood although there appears to be a requirement for BMP7 signaling and in retinal organoids, BMP signaling enhances DV polarization of optic cups (and consequently fissure formation; Morcillo et al., A striking observation was that although morphogenesis proceeded normally in isolated optic cups, the apposed lips of the choroid fissure always failed to fuse. Experiments in which eye primordia were transplanted ectopically in chick showed similar results Gayer, . This sucloche (Stainier et al., There is considerably more evidence, albeit mostly circumstantial, that POM influences fissure fusion. Indeed, in this study we find that highly motile POM cells elaborate processes that extend and contact cells on both sides of the fissure right up to the point of fusion. The POM is composed of both mesodermal and neural crest components and either or both cell populations could regulate fissure fusion. A notable absence from the ectopic eyes is the hyaloid vasculature, which normally enters the optic cup though the choroid fissure. The vasculature forms just prior to fissure fusion, with contribution from both components of the POM (Isogai et al., Foxc1 in mouse, does not appear to lead to coloboma and Foxc1 mutations in humans are only rarely associated with coloboma (Kidson et al., There is considerable evidence that disrupting neural-crest derived POM can lead to optic vesicle morphogenesis defects and coloboma (Bazin-Lopez et al., in vivo in zebrafish, mice, and humans in which genes that impact POM development are disrupted and eyes exhibit very severe morphogenesis defects (Gage et al., We show that optic cups form relatively normally when depleted of POM comparable to the situation when optic cups form in ES cell organoids (Eiraku et al., in vivo and when eyes are grown ectopically or in vitro. First, genes required for POM development frequently have additional roles in other tissues and these other roles may contribute to the severity of eye phenotypes when gene function is disrupted. Second, we suggest that eye formation in vivo is both promoted and constrained by the environment in which optic cup morphogenesis normally occurs. In the vicinity of the forming eyes there are many contemporaneous and precisely coordinated developmental events occurring and the developing eye may have limited capability to cope with environmental disruption. For instance, the presence of mis-positioned, dying or abnormal POM may have much more severe consequences on eye morphogenesis than depletion/absence of such cells.Several factors likely contribute to differences in severity of phenotypes when POM is disrupted In summary, our study shows that ectopic optic vesicles show remarkably normally morphogenesis but fail to fuse the choroid fissure. This observation is consistent with the possibility that POM cells, which are throughout the fissure during the closure process, may play a role in fissure fusion. This could be, for instance, in degrading the basal laminae that subsequently enables fissure cells to remodel their apico-basal polarity and directly appose during the fusion process.GG and SW conceived and planned the study; GG, CS, and NB-L conducted experiments; GG and SW wrote the paper with comments and input from other co-authors.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. The reviewer FC declared a past co-authorship with the authors GG and SW to the handling Editor.


Vector-borne diseases are responsible for significant health problems in humans, as well as in companion and farm animals. Killing the vectors with ectoparasitic drugs before they have the opportunity to pass on their pathogens could be the ideal way to prevent vector borne diseases. Blocking of transmission might work when transmission is delayed during blood meal, as often happens in ticks. The recently described systemic isoxazolines have been shown to successfully prevent disease transmission under conditions of delayed pathogen transfer. However, if the pathogen is transmitted immediately at bite as it is the case with most insects, blocking transmission becomes only possible if ectoparasiticides prevent the vector from landing on or, at least, from biting the host. Chemical entities exhibiting repellent activity in addition to fast killing, like pyrethroids, could prevent pathogen transmission even in cases of immediate transfer. Successful blocking depends on effective action in the context of the extremely diverse life-cycles of vectors and vector-borne pathogens of medical and veterinary importance which are summarized in this review. This complexity leads to important parameters to consider for ectoparasiticide research and when considering the ideal drug profile for preventing disease transmission.  •Novel drugs against ectoparasites should prohibit vectors to transmit pathogens.•Timing events of most human and animal vector-borne diseases are reviewed.•Most ticks and insects show substantial differences in transmission time.•Drug profile requirements differ between ticks and insects.•Ideal new ectoparasiticides combine fast speed of kill and repellency. Soft ticks like Ornithodoros are fast blood-feeders that need only minutes to fully engorge. Adults feed many times, and females lay eggs in small batches after each blood meal. They develop through more than one nymphal stage, increasing the number of opportunities for transmitting pathogens during their life-span  or lice (Phthiraptera) can develop in either immature stages or adults. Trypanosoma cruzi (Rickettsia prowazekii (Bartonella quitana (Reduviidae, blocking transmission via killing the insect before releasing infected feces may also work. However, as Reduviidae are fast feeders, release of the feces could occur within the first minutes of a blood meal. It remains to be demonstrated if preventing access to a host and subsequent biting with a repellent drug can effectively block T. cruzi transmission.Some major pathogens of hemimetabolic insects like true bugs (ma cruzi , Rickettowazekii , or Bart quitana  are tranPathogens of most holometabolic insects develop and multiply in an adult individual that has a life expectancy on the order of days or weeks. Their development can start immediately after ingestion and needs to reach the infective stage within the life-span of the insect vector. In these cases the pathogen strategy appears to be different and infectious stages are transmitted often within seconds to the mammalian host. Blocking transmission is therefore more challenging, and avoiding insect bite via a repellent drug could be the best option.4Babesia sp (Borrelia sp. (Ehrlichia canis transmission from Rhipicephalus sanguineus ticks by orally administered isoxazolines compared against topically applied products containing synthetic pyrethroids, the tested systemic isoxazoline ectoparasiticides gave insufficient protection of dogs from pathogen transfer (Leishmania sp. in dogs by repelling and killing sandflies (The principal feature of an ectoparasitic drug aiming to block transmission should certainly be a very fast onset of action. This requirement is generally understood by the animal health industry, and most products marketed recently have been tested and compared for the speed of their onset of action . Recent besia sp  or Borreelia sp. . Such a elia sp. . Based otransfer . Despitetransfer . It appeandflies . They arandflies . They haandflies . Howeverandflies  disquali5Meeting the requirements for new ectoparasiticides, including prevention of transmission of pathogens, is challenging if possible at all. Transmission in companion animals of some major tick-borne pathogens can be now controlled with compounds of the isoxazoline class because of their fast onset of action. Extending the use of this class of molecules to humans and farm animals may help to control some tick-borne zoonotic diseases. For other pathogens, mainly those transferred to the host by insects immediately at bite and by soft ticks, the speed of kill by isoxazolines is insufficient to effectively prevent pathogen transmission. Most insect vectors have little time for feeding before being chased away or being killed by the host, and therefore, in most cases, blood feeding and associated pathogen transmission begins immediately upon landing. In this situation, drugs having repellent or deterrent activity that hinders the vector from biting or landing on the host would be more successful at preventing disease transmission. Solutions could, therefore, be different depending on the vector, the associated pathogens and the speed of transmission. In an ideal situation, a drug or a combination of chemical entities should prevent the vector from access, or at least from biting the host. If the vector eventually succeeds in reaching the host, killing by the drug should happen very rapidly. Repellent efficacy combined with parasiticidal activity seems to be the ideal drug profile for successfully preventing vector-borne diseases in humans, pets and livestock. This easy statement unfortunately hides major difficulties especially if the repellent effect has to be long-lasting for weeks or months. Due to those substantial difficulties, the search for new vaccines or drugs targeting the pathogen should not be left aside. Novel alternative approaches, for example ones based on regulators of the immune system like the Toll pathway of the vector  should a


This review summarizes the main animal models of vulnerable plaques, with an emphasis on genetically altered mice, and the state-of-the-art preclinical molecular imaging strategies.Atherosclerosis is characterized by intimal plaques of the arterial vessels that develop slowly and, in some cases, may undergo spontaneous rupture with subsequent heart attack or stroke. Currently, noninvasive diagnostic tools are inadequate to screen atherosclerotic lesions at high risk of acute complications. Therefore, the attention of the scientific community has been focused on the use of molecular imaging for identifying vulnerable plaques. Genetically engineered murine models such as ApoE Atherosclerosis is the primary cause of myocardial ischemia, stroke and lower limb amputation. It is a chronic disease that occurs predominantly at sites of disturbed laminar flow , which are characterized by the focal subendothelial accumulation of apolipoprotein B–containing lipoproteins and inflammatory cells  on injured intima, leading to the thickening of the arterial wall and formation of atherosclerotic plaques. The majority of atherosclerotic plaques remain clinically silent , but, inThese lesions are prone to rupture and may cause the above clinical complications . The mecThe present clinical and diagnostic tools are inadequate for the early identification of atherosclerosis and lesions that have a high probability to determine an acute event. Using a predictive model of risk assessment such as the Framingham risk score, it is impossible to accurately identify patients who will develop events and who would require immediate and aggressive therapeutic intervention. Therefore, the attention of the scientific community has focused on the development of new tools that provide higher sensitivity and specificity for the detection of vulnerable plaques. Molecular imaging can reveal specific biological pathways or cellular processes for a better understanding of the molecular events responsible for plaque destabilization. Moreover, the identification of novel imaging biomarkers may aid in risk stratification, with the potential to optimize preventive interventions that would reduce disease progression and cardiovascular events.In vivo studies with experimental systems are essential to gain detailed insight into the molecular mechanisms of atherosclerotic plaque vulnerability and to develop better diagnostic and therapeutic tools. Animal models may enable us to study noninvasive imaging strategies to improve the ability to predict future risks of plaque rupture and to reduce the incidence of acute events. Although a variety of small and large animal models have been used for research on atherosclerosis, no model is currently considered ideal, and each has its own advantages and limitations with respect to the study of atherogenesis and modeling of the vulnerable plaque .Until recently, useful large animal models have included nonhuman primates, pigs, and rabbits. Lesions in primates ,9,10,11 Rabbits have also been extensively used in atherosclerosis research, and some strains that exhibit familial hypercholesterolemia ,19, suchMus musculus) is currently the most favored species to better understand the biology and pathology of atherosclerosis. The main advantages of this model include a well-known genetic background, a proportion of approximately 80% of genes in homology with humans iodo-DPA-713, which targets macrophages, to selectively detect macrophage infiltrates along the descending aorta and within the myocardium of the ApoE−/− mice. Radiotracers targeting VCAM-1 have been validated in murine experimental systems as attractive tools for imaging vulnerable plaques. Atherosclerotic lesions in the aortic arches of ApoE−/− mice have been successfully identified by Nahrendorf et al. [Foss et al.  developef et al.  using thf et al.  through 99mTc both in vitro on murine and human endothelial cells and in vivo at the level of the aortic arch in ApoE−/− mice. Dimastromatteo et al. [Similarly, Broisat et al.  tested ao et al.  demonstr−/− murine model, include other biomarkers of inflammation such as the following: P-selectin [vβ3 receptor with an RGD-galacto peptide [99mTc-annexin V [99mTc-AnxF568 and 124I-Hypericin [64Cu-labeled GPVI fragment crystallized [99mTc-antibody [−/− mice and, more recently, gold particles of 15 nm with a polyethylene glycol coating called Aurovist in the ApoE−/−C1039G+/− model [Other relevant targets of plaque vulnerability, investigated through nuclear medicine imaging in the ApoEselectin ,248; αvβ peptide  or the n peptide , both lannexin V  or 99mTcypericin  for apoptallized  or fibroantibody . Finallyantibody  or PEGylantibody  in ApoE−/− model .Currently, fluorescence imaging is emerging in preclinical research because it offers several advantages: high sensitivity, the possibility of simultaneous multi-spectral imaging, high-throughput capabilities, cost-effectiveness, and the absence of ionizing radiation. This technique can be applied both ex vivo for immunofluorescence microscopy and for in vivo applications such as intravital microscopy or tomographic imaging. In particular, FMT in the NIR window is gaining great relevance in cardiovascular research for longitudinal studies in mouse models of atherosclerosis. Atherosclerosis research using FMT is still sparse, but the previously mentioned peculiarities make it an attractive in vivo alternative to nuclear imaging. FMT is based on the collection of photons propagated in deep tissues from different points of view to obtain the tomographic distribution of fluorochromes.A number of fluorescent targeted probes are available, with the possibility of obtaining in vivo quantitative three-dimensional data of fluorescence signal distribution. These probes are excitable with a laser at the appropriate wavelength, and, after excitation, they emit light at a higher wavelength, detectable with appropriate charge-coupled device cameras. FMT offers an improved penetration depth of light in the near-infrared spectrum and limited autofluorescence but has limited spatial resolution and is currently restricted to preclinical field or ex vivo human samples . Moreove−/− mouse aortas both in vivo and ex vivo with fluorescent imaging [−/− mice. Moreover, the same authors showed that FMT-CT may be useful for monitoring the efficacy of atorvastatin treatment in ApoE−/− mice. In agreement, Larmann et al. [−/− mice through in vivo tracking of the recruitment of near-infrared fluorescent-labeled macrophages  conjugated with the near-infrared dye Cy5.5 both in vivo and in excised carotid arteries. cRGDyK-Cy5.5 co-localization with MAC-3 expression was confirmed using fluorescence confocal microscopy.More recently, Lin et al.  evaluateo et al.  have shoPhotoacoustic imaging (PAI), also called optoacoustic tomography (OAT), is a hybrid imaging modality that uses nonionizing optical radiation and ultrasonic detection. Recent advances in laser technology and detection strategies have led to significant improvements in the capabilities of optoacoustic systems. PAI is showing real promise as a convenient alternative to other imaging modalities. PAI combines the advantages of both optical and ultrasonic imaging methods, offers higher spatial resolution and allows imaging of deeper tissues compared to optical imaging techniques ,266. PAIApplications in the field of molecular imaging provide functional information regarding cellular events by using endogenous chromophores  and a great variety of exogenous contrast agents ,268,269.−/− mice to test the in vivo potential of molecular PA contrast agents to identify stable versus vulnerable plaques such as a VCAM-1–targeting gold nanoshell probe, which has highlighted a higher PA signal in the aortic arches of ApoE−/− mice fed a WTD compared to controls [−/− mice compared to the signal produced after administration of free ICG [Very recently, some pilot preclinical studies have been conducted in ApoEcontrols  and indofree ICG  (as showAtherosclerosis can start in the late years of childhood and can remain silent for many years. It becomes symptomatic when it interferes with the circulation and blood supply, causing heart attack, stroke, or ischemic symptoms. However, this mechanism is responsible for acute events in only 30%–40% of patients. A significant number of people with myocardial ischemia do not have plaques that significantly narrow the arteries. A total of 60%–70% of patients with acute coronary syndrome or sudden cardiac death have thrombosis associated with rupture-prone plaques that have been shown in post-mortem evaluation to have specific characteristics such as increased macrophage activity and a peculiar molecular pattern. These features appear in an atherosclerotic plaque prior to rupture.As outlined in the “response-to-injury” hypothesis by Ross et al. , the endMany of the currently available clinical diagnostic methods still provide minimal information about the biological characteristics of plaques and/or are invasive. The imaging methods currently used provide only partial quantitative and morphological data on the atherosclerotic plaque. The cellular and molecular processes that characterize the atherosclerotic plaques require more advanced methods that can best represent the peculiarities of the pathological events with respect to size. The development of molecular imaging using probes for specific biological functions could be of great value to study the evolution and the chemical outcome of the atherosclerotic plaque and for the prediction of vulnerability. The study of atherosclerosis in animal models is of great relevance for the advancement of knowledge, but some precautions are required when relevant data are extrapolated from animals for new diagnostic and therapeutic tools. Proper design and generation of animal models are essential to make preclinical data more reproducible and translatable to the clinic. Atherosclerosis is a complex disease. The animal models are a simple representation of this complex system. Therefore, a single animal model may not be able to reproduce all aspects and all stages of the atherosclerotic process in humans in all of its complexities but rather may represent a specific aspect of the disease. In atherosclerosis research, mice are nowadays the most commonly used animals. Generally, the lipid metabolism in wild-type mice is significantly different from humans. The major fraction of plasmatic lipoproteins in wild-type mice is HDL, which is different from humans where LDL or VLDL lipoproteins are mainly represented, and therefore mice are well protected from atherosclerosis due to a beneficial cholesterol spectrum in the blood. Nevertheless, the C57BL6 strain of inbred mice is relatively susceptible to atherosclerosis, and by feeding them with an atherogenic diet, development of atherosclerosis can be augmented to such an extent that the resulting advanced lesions are useful to study disease mechanisms. Overcoming the limits of this latter model, the advent of the genetically modified mice has revolutionized the study of the pathogenetic mechanisms of atherosclerosis.−/− mice are currently the most well-characterized mouse model of atherosclerosis used by research groups worldwide. However, the ApoE−/− mouse is a model of a rare disease in humans, named homozygous familial hypercholesterolemia, while the atherosclerosis induced by LDL accumulation is the most common pathological mechanism in humans. Additionally, several systemic factors could modify the course of the disease in the animal models, such as plasmatic levels of HDL, insulin resistance, and hemodynamic factors, which could make the process more or less different from that of humans. Moreover, considering that atherosclerosis development is largely dependent on local inflammation, it is important to consider that mechanisms of inflammation in atherosclerosis could differ between mice and humans [In particular, ApoEd humans . Further−/−Fbn1C1039G+/− mouse model has been developed. These mice exhibit vulnerable atherosclerotic plaque phenotypes, with a greater incidence of plaque rupture in comparison to the ApoE−/− or LDL−/− mice, which exhibit mainly stable plaques.Very recently, the ApoEThe new generation of imaging modalities comprises promising tools for the prediction of vulnerable atherosclerotic plaques. Although novel molecular imaging approaches for atherosclerosis have been tested in the previously mentioned experimental systems, accurate assessment of plaque characteristics and vulnerability is still in its infancy. Each imaging technique has its own strengths and drawbacks. At the moment, CEUS and molecular MRI appear to be the most suitable and promising because they have been applied to research the analysis of the compositional features of vulnerable plaques, they show high resolution and sensitivity, they use nonionizing radiation and they have potential clinical utility. The main concerns to overcome are: improvement of the sensitivity and specificity of the molecular probes; complex extrapolation of results from animal models; and scientific as well as financial challenges for translating new molecular imaging ligands from preclinical research to clinical practice. Further investigations of the mechanisms of plaque destabilization and rupture and technological advances of imaging equipment are required to reduce both mortality and morbidity worldwide. Successful translation of targets and ligands to clinical molecular imaging of the vulnerable plaque may also offer novel therapeutic perspectives for vulnerable plaques such as targeted drug delivery.


Rhizobium, Pseudomonas, Azospirillum and Bacillus, have been found to have positive impacts on crops by enhancing both above and belowground biomass and could therefore play positive roles in achieving sustainable agriculture outcomes. Thus, it is necessary to study this rhizosphere microbiome with more sophisticated culture-independent techniques such as next generation sequencing (NGS) with the prospect of discovering novel bacteria with plant growth promoting traits. This review is therefore aimed at discussing factors that can modulate rhizosphere microbiome with focus on the contributions of nitrogen fixing bacteria towards sustainable agricultural development and the techniques that can be used for their study.Rhizosphere microbiome which has been shown to enhance plant growth and yield are modulated or influenced by a few environmental factors such as soil type, plant cultivar, climate change and anthropogenic activities. In particular, anthropogenic activity, such as the use of nitrogen-based chemical fertilizers, is associated with environmental destruction and this calls for a more ecofriendly strategy to increase nitrogen levels in agricultural land. This feat is attainable by harnessing nitrogen-fixing endophytic and free-living rhizobacteria.  The rhizosphere is the zone of the soil environment that is endlessly regulated by exudates, border cells and mucilages released by plant roots ,2,3. “RhImbalance in nitrogen cycling, soil physicochemical properties, changing climatic conditions and abiotic stresses are the interwoven factors threatening sustainable agriculture. Sustainable agriculture depends on fertile soil, but land degradation and rapid desertification cause estimated loss of 24 billion tons of fertile arable land globally . Recent Moreover, factors such as soil type determine the composition of both endophytic and free-living rhizosphere microorganisms ,16,17,18Other extrinsic factors like climate change and anthropogenic activities can also modulate the microbial communities in a specific plant host . In partRhizobium spp., non-nodulating strain of Endobacter medicaginis, Micromonospora spp., Microbacterium trichothecenolyticumn and Brevibacillus choshinensis [Researchers have consequently been giving attention to endophytic bacteria because of their role in increasing N input to the soil . These ehinensis ,27. Taxohinensis  neverthehinensis .Hence, the fundamental questions to better comprehend plants and their associated microbiomes are “who is there?”, “how do they respond to variation in environmental conditions?” and “do they impact plant health and growth?” Proffering solutions to the above questions will result in better understanding of how environmental changes modulate plant microbial composition; a prerequisite to detect if and how such microorganisms might be utilized in the future to enhance plant growth and crop yield especially in arable lands faced with environmental perturbations. Genomic studies of the plant associated microbiome are of utmost importance to provide answers to the abovementioned posers. DNA based analyses of each microbial species or meta-genomic profile of entire microbial communities reveal the composition and functional potential of plant rhizosphere microbiome . Among tThis review is therefore aimed at discussing factors that can modulate rhizosphere microbiome with focus on the contributions of N fixing bacteria towards sustainable agricultural development and the techniques that can be used for their study ,33,34,35Various factors such as biotic and abiotic parameters modulate or influence the microbial diversity and composition in the region surrounding the root . The levArabidopsis thaliana cultivars revealed that soil type greatly affects rhizosphere microbial diversity [Several studies have revealed that soil has a great effect on the structure of mycorrhizal fungal communities in the rhizosphere ,39,40. Tiversity ,17. The iversity  and secoPseudomonas species in flax and tomato rhizosphere. This suggests that soil properties and soil type can determine the types of microorganisms that colonize the rhizosphere, and that different soil types can contain different microbial species , bringing about differences in disease suppressiveness [Rhizosphere bacterial analysis using PhyloChip technique of three different cultivars of potato grown at two separate sites revealed 2432 operational taxonomic units (OTUs) and 40% of the OTU abundance was site-specific . Howeversiveness ,57. Addisiveness . Certainsiveness .Bacillus cereus were detected. Furthermore, research on microbial diversity in the rhizosphere of maize showed that host genetic factor influences the composition of the rhizosphere microbiome [It has been suggested that the modern way of plant breeding has probably not taken into consideration traits that are important for plants to serve as hosts to mutualistic microorganisms . In an acrobiome ,14.In addition, microorganisms in the soil are a possible factor that influences the structure of the rhizosphere microbiome . SeveralMicroorganisms play important functions in all environments and so it is significant to understand how they respond to anthropogenic activities and climate change ,61. ClimAn unraveled problem now is how microbial communities of the rhizosphere will respond to different facets of climate change. Rhizosphere microorganisms may have a greater ability to evolve than their host plants. In addition, as a result of their vast biodiversity, rhizosphere microorganisms may comprise of taxa that are acclimatized to warmer environmental conditions. The dispersal of soil microorganisms may also permit the immigration of microbial species from warmer environments into relatively cooler soil environments. Whether rhizosphere microbial composition under a particular climate change is dependent on spread of microorganisms or on their genetic acclimatization is an important question that needs to be addressed so as to know whether the microbial communities of the rhizosphere can cope with changes caused by global climate change.Rhizobium species can survive in dry environments, but their diversities are significantly lower in dry soil environments. Moreover, drought does not only affect the ability of Rhizobium species to fix N but also the growth and development of plant legumes. At times, it is difficult to separate the consequences of drought stress from heat stress as they both occur at the same time in semi-arid and arid regions of the world [In particular, climate change can cause abiotic stresses and some of the stresses associated with it are drought, cold and heat. It has been found that he world ,69. Suithe world  and therFurthermore, since the inception of the industrial revolution, anthropogenic activities in natural ecosystem have escalated as a result of the rising human population, pollution and ecosystem degradation and these have greatly affected soil microbial community structures. For instance, anthropogenic transformation of forest to farm lands resulted in the homogenization of the indigenous soil bacterial structure . Thus, dHerbicides are chemical compounds commonly used to control weeds in agriculture and many of them cause environmental contamination cum human health problems due to their toxicity and persistence in the soil . Like chThe rhizobacteria community can be affected by the presence of herbicides such as glyphosate, frequently used where ‘genetically modified herbicide-tolerant’ crops are grown. The impact of glyphosate on rhizosphere microorganisms from agricultural crops like soybean and maize has been reviewed . CertainRhizobium species and soybean roots and there is indication that the presence of sub-lethal amounts of glyphosate in growth media results in the accumulation of shikimate and decrease in Bradyrhizobium japonicum growth [B. japonicum strains exhibit different sensitivities to the herbicide [Rhizobiaceae might breakdown glyphosate and utilize it as the main source of phosphorus in the presence of aromatic amino acids [Rhizobium spp. in soybean have been studied in field and greenhouse experiments [Bradyrhizobium, Methylosinus and Burkholderia were higher in the rhizosphere microbiome from the field of the low level N fertilizer than standard level N fertilizer. On the contrary, the relative abundance of methanogenic archaea was higher in the field amended with standard level of fertilizer (SLF) than low level N fertilizer (LNF) field [pmo/mmo and acdS responsible for methane oxidation and plant interaction respectively were more abundant in rice rhizosphere microbiome grown in LNF field. Similarly, functional genes for the metabolism of sulphur (S), iron (Fe), aromatic compounds and N were significantly higher in the LNF rhizosphere microbiome [13C-labeled methane experiment and quantitative polymerase chain reaction (qPCR) analyses for mcrA and pmoA genes coding for methyl coenzyme-M reductase and methane monooxygenase respectively indicated that methane oxidation was more active in the rice roots cultivated in LNF field than in those grown in SLF field [Herbicides do not only affect microbial diversity, they also affect microbial functions  in the soil. N fixation in legumes such as soybean can be triggered by symbiotic interaction between m growth . But, dierbicide ,84. Someno acids . The imperiments ,87 even eriments  observederiments  reportederiments  and theseriments . Furthereriments . SequencF) field . The gencrobiome , but, 13LF field . These oGluconacetobacter diazotrophicus which lacks the capacity to fix N is not able to enhance its host plant growth as much as the wild type. The most studied endophyte that has the ability to fix N is Pseudomonas stutzeri A1501, which was first isolated in China [nif genes which are needed by free living organisms is repressed in the presence of ammonia. “Interestingly, P. stutzeri can switch between denitrification, nitrification, and N fixation under anaerobic, aerobic, and micro-aerobic conditions, respectively [pnfA. PnfA is controlled by similar sigma factors as nifHDK (which code for nitrogenase). The expression of PnfA genes is not directly affected by mutation; however the mutant strain exhibits reduced nitrogenase activity particularly in micro-aerobic environment [Nodule formation, which is a very efficient process of N uptake, has been reviewed extensively ,96. Howein China . The bacectively ”. Transcironment .Azoarcus species are N-fixing plant growth promoting rhizobacteria (PGPR) and the wild type of Azoarcus sp. BH72 which inhabits kallar grass roots was able to enhance the dry weight of the grass cultivated in an environment deficient in N when compared with “nifK mutant strain of BH72”. Surprisingly, the bacterium can transform irreversibly from free living to endophytic forms and vice versa and as such it is not always feasible to re-isolate Azoarcus sp. BH72 endophytic colonies from roots [om roots .Rhizobium sp. IRBG74 as well as Azorhizobium caulinodans which colonize rice roots have been isolated respectively from Sesbania aculeata and Sesbania rostrata. Rhizobium sp. IRBG74 has similarly been recovered from Sesbania cannabina, but being an endophyte, it does not have the potential to fix N since it lacks certain nif genes like nifV. Rhizobium sp. IRBG74 has now been re-categorized from the genus Agrobacterium to Rhizobium since it does not possess Ti plasmid, fusA, rpoB and 16S rRNA gene sequences. This bacterium possesses a sym-plasmid having nifH together with nodA genes [Sesbania plants. Similarly, A. caulinodans ORS57 is able to colonize rice and fix N in endophytic form, however this bacterium should be tested with other plants species for its endophytic infection and N fixing abilities so as to know if this potential is unique to this plant sp. or is a common characteristic. “In order to determine whether it is the plant that initiates the N2-fixation in its bacterial symbiont (as regards nodulation), a common SYM pathway rice mutant should be tested for its ability to form endophytic symbiosis with ORS571 [Azorhizobium responsible for plant infection, tolerance to stress and nodulation in order to ascertain those involved in Azorhizobium-Sesbania mutualistic interaction and rhizobial-legume associations.dA genes  and it ch ORS571 ,104”. ItHerbaspirillum seropedicae is an additional endophyte of plant roots that have been studied extensively and it infects the roots of rice, sugarcane and sorghum. It enhances the growth of its host plant by fixing N even in soil deficient in nitrogen and oxygen content [H. seropedicae perhaps acquired its N fixing capability via “horizontal gene transfer” like other non-rhizobial strains. Like other disease-causing microorganisms of the genus Herbaspirillum, it is fascinating to know that H. seropedicae which is non-pathogenic has the entire genetic make-up for type I, II, III, V, VI and IV pili which it uses to facilitate communication with its host plant [Bradyrhizobium elkani and Rhizobium sp. NGR234 with their hosts [ content . H. serost plant . The typir hosts .Scientists’ attention on endophytes and their contributions to plant health and productivity should not only be theoretical and thus, these microorganisms can probably be utilized to improve nutrient absorption and plant diversity.Rhizobiome (which can be regarded as rhizobacteria) is a group of bacteria found in the rhizosphere that help to enhance the growth of their host plants. The rhizosphere has abundant microbial diversity and nutrients such as carbon substrates than the bulk soil. Microorganisms in the rhizosphere can be manipulated by wide range of complicated and highly monitored cell to cell interaction and by exploring signaling molecules to monitor their habitat and modify their activities. Rhizobacteria can affect nutrients absorption by plant roots directly or indirectly through nutrient mobilization/immobilization or alteration in root structure/physiology respectively. They can also enhance host plant nutrient status by increasing bioavailability of rhizospheric nutrients and improving other symbiotic interactions of the host . Many miRhizobacteria are a major component of PGPR, a terminology that was coined over 30 years ago . PGPR arBurkholderia cepacia can destroy the pathogen Fusarium spp. and also promote maize growth under iron-deficient environments through siderophore production [It should be noted that some PGPR cannot be considered as biofertilizers. PGPR that enhance plant growth by eradicating pathogens are bio-pesticides and not biofertilizers. Amazingly, some PGPR seem to promote the growth of plants by functioning as biofertilizer and bio-pesticides. For instance, oduction . Moreoveoduction .However, some microorganisms such as arbuscular mycorrhizal fungi (AMF) present in the rhizosphere have been reported to have pesticidal traits ,114; a pIn particular, studies have shown how several rhizobiome influenced both above and below-ground biomass  115,116116,117. Bradyrhizobium spp.) that have the potential to improve crop growth while providing protection from biotic stresses (such as plants pathogens and pests) and abiotic stresses .The global world requires novel ideas for farming so as to be able to generate farm produce that can cater for world population of 6.9 billion. Actualizing food security which is the process of producing sufficient food and enhancing its quality to sustain the ever increasing population without undermining environmental protection is termed global green revolution . SustainSeveral studies on isolation, identification and application of microorganisms as an alternative method for chemical fertilizer utilization have been reported ,128. EnhRhizobium spp.) as briefly discussed below.Hence, accomplishing food security is dependent on the enhancement of plant growth and diversity including seed yield right from the field and one of the ways this feat can be attained is through inoculation of agricultural crops (such as soybean) with rhizospheric rhizobacteria  inoculated with Rhizobium significantly increased the crop growth and yield constituents such as number of branches bearing pod per plant, total number of pod per plant and seed number per pod. Amendment of Vicia sativa L. (vetch) with Azospirillum in greenhouse and field experiments increased significantly N fixation activity, percentage N and N content. Rhizobium leguminosarum introduced into pea and lentil seeds was able to enhance pea nodulation, shoot/root diversity and yield of pea seed. Also, seedling height, nodule and shoot biomass of lentil were enhanced. There was also enhancement on nodulation of peanut treated with Rhizobium species while chicken pea treated with same species in greenhouse and field experiments resulted in significant increase in plant growth, root dry weight and number of nodules. Ravikumar [Vigna mungo and Vigna radiate inoculated with Rhizobium when compared to control experiments  ability, nutrient uptake and crop yield [Rhizobium with Bacillus spp. [Azospirillum and Bacillus spp. were inoculated with Rhizobium in pigeon pea [Streptomyces lydius WYEC108 interaction with Rhizobium was noticed to promote pea growth including nodule number, perhaps by Streptomyces colonization of root and nodule. Furthermore, Enterobacter species are PGP bacteria that enhanced the yield of nodules in plant upon co-inoculation with a species of Bradyrhizobium. There was increase in infection sites area and the number of root hair formed when Medicago truncatula was amended with Pseudomonas fluorescens WSM3457 and Sinorhizobium [P. aeruginosa and Mesorhizobium sp. [Rhizoctonia solani and Fusarium oxysporum have been noticed on chickpea amended with a bacterial consortium of Azotobacter chroococcum, Trichoderma harzianum, Mesorhizobium and P. aeruginosa [Rhizobium introduction in leguminous crops is known for growth stimulation and is used as a substitute to the expensive conventional chemical fertilizers [op yield ,141,142 lus spp. . Improvegeon pea ,144. Strhizobium . Also, sbium sp. . Similarruginosa . Althougruginosa . Rhizobitilizers . The useThe availability and absorption of mineral nutrients like nitrogen (N), phosphorus (P), magnesium (Mg), sulphur (S), calcium (Ca) and potassium (K) are very essential for the growth of plants especially in Africa where various cropping systems involving leguminous crops such as soybeans are practiced.Rhizobium spp. may be a major source of N in most cropping systems with about 80% of the needed N emanating from biological N fixation [Phaseolus vulgaris indicated that N fixation from rhizobial inoculation contributed N equivalence of 20–60 kg of N per hectare in Brazil while Clayton and Rice [Rhizobium increased plant N from 19–42 mg per plant.In particular, symbiotic interactions between legumes and fixation . Indeed,fixation  reportedand Rice  found thRhizobium species and essential mineral nutrients into the rhizospheric soil since Rhizobium and even other bacterial species such as Bacillus, Burkholderia, Azospirillum, Pseudomonas and Erwinia have been reported to have phosphate solubilizing potential [Also, P absorption is important for the development and proper functioning of plants and there is a large reserve of P in the soil environments, mainly in non-soluble forms that cannot be absorbed by plants and thus limiting plants’ growth. PGPR can solubilize the non-soluble forms of P through chelation, enzymatic activities and acidification ,152. Theotential ,154.Rhizobium inoculation into Pigeon pea (Cajanus cajan L. Millsp) and increase in the amount of nutrient in the environment increased the chance of plants uptake. Study carried out by Makoi and Bambara [Rhizobium strains on mineral nutrient absorption by Phaseolus vulgaris showed significant increase in the uptake of P, K, Mg, Ca and S in the entire plant parts. It was reported by the author that even though the concentration of P and K skyrocketed in the root region due to Rhizobium introduction, the increase was only significant in the greenhouse experiment and not in the field condition. Rhizobium inoculation enhancement of micronutrient  uptake in the shoots, roots, pods and the entire plant with the exception of Mo intake in the roots has been reported. Rhizobium has also been shown to cause significant increase in Ca and sodium (Na) content and the pH of the soil [B. japonicum and P under greenhouse and field conditions [B. japonicum caused significant increase in the intake of Zn, Fe, Cu and Mn in soybean (G. max L.) under greenhouse condition while uptake of Fe, Cu and Mn increased significantly and that of Zn decreased under field condition [Besides deficiencies in major nutrients, micronutrients such as zinc (Zn), boron (B), molybdenum (Mo) and iron (Fe) are also limiting nutrients that work against legumes productivity. It was reviewed by Mfilinge, Mtei , that th Bambara  on the ithe soil . The uptnditions . B. japoondition .Rhizobium, P and K interaction have not been studied in detail and it is therefore important to study the potential role of Rhizobium, P and K on the bioavailability of other mineral nutrients in leguminous crops such as soybean grown in each ecological zone in Africa.There is however limited information on the roles of different rhizobial species of legumes on the bioavailability of other mineral nutrients in bean cultivars especially in Africa. Similarly, the impacts of The crucial regulating element for plant growth is N due to its limited availability and also beans require this element more than other mineral nutrients. N is a component of most organic compounds such as proteins, growth regulators and chlorophylls. N deficiency can lead to stunted growth, yellowing of leaves and reduced branching in beans. N is one of the monomeric units of proteins and is greatly required for the entire enzymatic process in the cells of plants. It is also present in many vitamins and chlorophyll and plays a role in photosynthetic process . In a stB. japonicum showed increase in chlorophyll content and growth indices such as height of plant, leaves number on a plant, stem width, day’s number to 50% flower and pod development as against the control counterparts [B. japonicum on cowpea (Vigna unguiculata L.) growth, it was discovered that the chlorophyll content of the leaf of cowpea significantly skyrocketed for treatment inoculated with B. japonicum in the field. There is however little information on the impacts of K, P and Rhizobium inoculation and their interactions on formation of chlorophyll in Phaseolus vulgaris in Tanzania which is a gap that needs to be covered research wise [It has similarly been reported that soybean amended with terparts . Upon strch wise .3+) is more available in the soil, plants usually prefer to absorb Fe in the form of reduced ferrous ion (Fe2+) [3+ and helps to retain it in soil solution. The chelators release the Fe3+ to the surface of the root where it is converted to Fe2+ and absorbed immediately while phyto-siderophores secreted by grasses are absorbed with Fe3+ across plasma membrane [Besides inorganic P and N, Fe is an additional mineral nutrient that plants can obtain through symbiotic interactions with soil rhizobacteria. Although, ferric ion (Fen (Fe2+) . Plant rmembrane .3+-siderophore complexes [3+-siderophore absorption to plants’ iron nutrition is controversial. Some researchers reported that the contribution of Fe from this source to plants’ overall Fe requirement is small [On the other hand, some PGPR are able to sequestrate the insoluble form of Fe from the soil with the aid of siderophores making it available for the host plant  and theromplexes  for theiis small  while otis small ,162. Baris small , who preis small , reporteThe sequestration or acquisition of Fe through PGPR siderophores decreases the bioavailable Fe in the rhizosphere and as such affect the growth of fungi that might be pathogenic to the plant ,165. In Enterobacter, Staphylococcus, Herbaspirillum, Pseudomonas, Ralstonia, Stenotrophomonas, Burkholderia and Ochrobactrum have rhizosphere-associated species that can interact with both human and plant hosts. The mechanisms by which these bacteria colonize plants’ rhizosphere and control plants’ pathogens are similar to those responsible for colonization of human cells. Multiple antibiotic resistances are not only common with clinical isolates of these bacterial groups but also with isolates from the rhizosphere. These antibiotic resistances are caused by presence of different antibiotics, high competition and horizontal gene transfer in the rhizosphere. Several PGPR are phylogenetically related to some of the human opportunistic microorganisms and their potential to cause disease can easily be assessed by their ability to survive at 37 °C [Stenotrophomonas maltophilia and S. rhizophila DSM14405T (PGPR) are genomically similar to S. maltophilia K279a (a human pathogen) but the former possess genes involved in the breakdown of cell walls of bacteria and plants, Fe sequestration, tolerance to salinity and spermidine synthase production [Stenotrophomonas species produce indole-3-acetic acid but environmental species seem to produce more of the phytohormones [There are some rhizospheric microorganisms that have the potential to cause opportunistic infections in humans. Berg and Eberl  suggesteat 37 °C . The disoduction . Irrespehormones . Moreovehormones .Klebsiella pneumoniae Kp342 is an endophytic N-fixer of some agricultural crops and has human pathogenic close relative (strain MGH78578). Strain Kp342 is different from strain MGH78578 since it is able to fix N and lack the genes that encode “global secondary messenger cdi-GMP” known for control of virulent components and biofilm formation. “In total, 4205 proteins  were shared between these two strains, and 1107 proteins were unique to the plant associated Kp342”. Surprisingly, none of the projected coding sequence of Kp342 was similar to that of the already sequenced Azoarcus sp. BH72 [Many other N fixing endophytes are also closely related to some human pathogens. In particular, sp. BH72 .Caenorhabditis elegans and the slime mould fungus Dictyostelium discoideum have been used as models to study the pathogenesis of Pseudomonas, Burkholderia Stenotrophomonas infection [Azoarcus sp. BH72 responsible for adhesion on fungal and plant cells have amino acid sequence that is similar to the human pathogens P. aeruginosa and Neisseria gonorrhoeae indicating that the plant-associated bacterium may be able to cause opportunistic infections in humans.In addition, detection of virulence factors in most of these rhizosphere related opportunistic human pathogens is difficult because of the absence of animal models. However, alternative models  have been developed for some of these bacteria. For instance, the nematode nfection . Studiesnfection . Dörr etnfection  suggesteStenotrophomonas infections. There is even a projection that in future, opportunistic infections will increase because of the increasing numbers of at-risk patients [The category of humans that are susceptible to opportunistic pathogens are the immuno-suppressed patients including AIDS patients, the aged with chronic diseases and those that have been subjected to long-term antibiotic therapy. According to Denton and Kerr , long-tepatients . TherefoBradyrhizobium species and Beta-proteobacteria are capable of forming nodules on the roots of leguminous crops where they transform atmospheric N to ammonia for plant use and gain carbon substrate from the plant in exchange [Frankia also form nodules in interaction with plants like Casuarina and Alder. Several bacteria are free living in the environment or may exist as endophytes in plant roots, and similarly have the capacity to fix N [R. leguminosarum are still able to infect plant roots [Some members of the Rhizobiales such as exchange ,174. Witexchange . Actinobto fix N . Nodule to fix N  long aftto fix N . Althougnt roots  suggestiChanges in plants’ rhizosphere microbiome caused by the aforementioned rhizosphere modulators can be detected by various techniques. Therefore, researchers can gain insight into the composition and abundance of microorganisms present in the rhizosphere of plants in natural and agricultural ecosystem by using either what is broadly classified as traditional  or molec6–107 cells/cm2; this count was obtained by culturing soil samples in nutrient media under different growth conditions  in soil ecosystems in the USA [Recently, researchers have studied the rhizosphere microbial community of soybean ( the USA . Yet, mu the USA ,99,187.  the USA .Nowadays, virtually all investigations involve molecular analyses which are necessary for detailed characterization of species and investigation of microbial interactions with host plants in the rhizosphere. “Though there are many technical innovations in HTS that lead to insightful and better understanding of the microbiome phylotypes and functions; Dini-Andreote and van Elsas  have empAzospirillum. PGPR strains development is hampered mainly by the fact that these organisms are sometimes unable to survive harsh environmental conditions, including high concentrations of environmental contaminants, salts, extremities of pH and temperature. Genetic engineering can be used to develop PGPR strains that are effective at low inoculum doses and under a variety of environmental conditions. It is urgent to develop more effective PGPR strains with longer shelf lives to achieve sustainable crop production in dry land production. Recent advances in the fields of microbiology, biotechnology, molecular biology and bioinformatics have opened up the way to identify novel genes involved in drought tolerance. Concepts of micro biotechnology application in agriculture should be employed to isolate indigenous PGPR from the stress affected soils, and screening on the basis of their stress may be useful in rapid selection of efficient strains that could be used as bio-inoculants for crops grown in dry lands [“The future trend needs to be in developing genetically modified PGPR over transgenic plants for boosting plant performance, as it is simpler to modify a bacterium than complex higher organisms. Moreover, instead of engineering individual crops, a single, engineered inoculant can be employed for several crops, especially when using a nonspecific genus like ry lands ”.There are myriads of microorganisms including rhizobacteria found in the ecosystem of the rhizosphere and these bacterial interactions with the plants root have been declared beneficial to sustainable agricultural development. This group of bacteria, among other merits can enhance plant development and diminish the occurrence of plant disease. New as well as uncultured microbial candidates in the rhizosphere can better be captured and studied using culture independent techniques which have the potential to analyze broad spectrum of microbial species unlike the culture based techniques. The mechanism by which these microorganisms achieve the mutual benefits on their hosts is not completely comprehended; however, it has been observed that virtually all of their traits enable them to accomplish these benefits. To add to this, rhizospheric microorganisms must be competent in the sense that they should be able to strive within rhizospheric soil that is being influenced by several factors including soil type, plant cultivar and agricultural practices. It is advisable to properly match suitable PGPR with compatible host plant cum environmental condition so as to accomplish better benefits on the plant. This feat will help to reduce the usage of conventional chemical fertilizers as well as pesticides most especially if these microbial inoculants are delivered effectively to the target plant and environment.


Wind farm development can combat climate change but may also threaten bird populations’ persistence through collision with wind turbine blades if such development is improperly planned strategically and cumulatively. Such improper planning may often occur. Numerous wind farms are planned in a region hosting the only cinereous vulture population in south-eastern Europe. We combined range use modelling and a Collision Risk Model (CRM) to predict the cumulative collision mortality for cinereous vulture under all operating and proposed wind farms. Four different vulture avoidance rates were considered in the CRM. Cumulative collision mortality was expected to be eight to ten times greater in the future (proposed and operating wind farms) than currently (operating wind farms), equivalent to 44% of the current population  if all proposals are authorized (2744 MW). Even under the most optimistic scenario whereby authorized proposals will not collectively exceed the national target for wind harnessing in the study area (960 MW), cumulative collision mortality would still be high (17% of current population) and likely lead to population extinction. Under any wind farm proposal scenario, over 92% of expected deaths would occur in the core area of the population, further implying inadequate spatial planning and implementation of relevant European legislation with scant regard for governmental obligations to protect key species. On the basis of a sensitivity map we derive a spatially explicit solution that could meet the national target of wind harnessing with a minimum conservation cost of less than 1% population loss providing that the population mortality (5.2%) caused by the operating wind farms in the core area would be totally mitigated. Under other scenarios, the vulture population would probably be at serious risk of extinction. Our ‘win-win’ approach is appropriate to other potential conflicts where wind farms may cumulatively threaten wildlife populations. Harnessing the kinetic energy of moving air is widely recognized as an environmentally friendly strategy to combat climate change, and an ambitious global target for humanity to satisfy 20% of its energy demands through wind power by 2050 has been recommended , requiriOne such impact of concern is mortality for species prone to collision with turbine blades, such as birds –11. Old The massive expansion of wind farm developments is a modern example of the well-known issue of the “tyranny of small decisions” , implyinAegypius monachus) in south-eastern Europe . Thi. Thi49].Given the expected sharp increase of wind farm developments in the study area in the near future, the nationally-derived Greek target within our study area (960 MW) will be exceeded by three fold, if all proposed projects were authorized. Of course, not all wind farm proposals placed into planning systems gain authorization, for various reasons. What is of serious concern, nevertheless (see later), is that the guiding instrument for wind farm developers in Greece, the WPA , paid scA real challenge therefore lies in influencing government policy through a balanced and objective approach to manage and spatially guide developers’ interest in a way that a positive cost-benefit ratio is gained in favour of both economic developments that hamper further global warming, and biodiversity conservation, within all relevant legislative frameworks . AddressIn this study we combined spatial modelling  and a CoGyps fulvus) was found buried under rocks in the base of a wind turbine evincing a likely underestimation error, even from independent carcass surveys [pers. comm.). We suggest that a post-construction carcass survey in the study area and in other parts of the world would further contribute to model validation and calibration, when predicting mortalities with our method.Our model was validated  on the b surveys ,53,54  which dOur approach can be criticised in that it was by definition static. We had to assume that future wind farms would operate simultaneously, as their real operation time remains unknown. Non-simultaneous operation, however, would only protract the collective impact, not dilute the overall impact; as we have documented, this impact is likely potentially severe.We also assumed that the cinereous vulture maintains the same current standing population (relevant demographic data were unavailable) as well as having the same space use in future years. We accounted for the former by primarily expressing mortality proportionately, rather than absolutely . The latter assumption, besides that recently was found valid for other vultures and raptor species ,57,58, tSex and age class are known not to affect the space use of cinereous vulture in our study area , but it Our findings clearly indicated poor implementation of the SEA Directive  relevantNeophron percnopterus) in Greece [Aquila chrysaetos nests (Sidiropoulos L. pers. comm.). Besides the high number of raptors colliding with wind turbines (17 carcasses), wind turbines have a clear negative impact of bats as well [Spatial planning of the WPA has apparently been performed in the absence of any biological criteria, which is further supported for other species of conservation concern, since the WPA is located in an area that includes all of the few remaining and small colonies of griffon vultures (two in Bulgaria and three in Greece) of Eastern Rhodope, more than half of the few remaining breeding pairs of globally endangered Egyptian vultures . Furthermore, we could consider approximately 10% reduction of predicted annual cumulative mortality for this scenario, given the overestimation produced at the coarse conservation zone level . This more realistic scenario would still account for a large part of annual population loss (39% of the standing population). Even under the most optimistic but improbable scenario that no further proposals are authorized once the national target is met for the study area, the cumulative collision mortality would still be high (17% of the standing population). Although a population viability analysis, integrating the predicted mortalities within baseline species demographics, would allow a more accurate population trajectory , our resOur findings emphasized the need of officially designating the population core area as a wind farm exclusion zone, since it is the most vital area for the population survival , and it We further provided a solution to the spatial planning problem, suggesting the authorization of the proposed wind farms first in the periphery and second in the outer zone of the non-core area, while excluding the other zones. This win-win solution allows harnessing 1095 MW of wind power, readily meeting and even exceeding the national target for the study area, while minimizing the collision mortality effect to the vulture population to only one death per year from prospective future developments. To achieve this minimal 1% population loss per year, collision mortality should be eliminated in the population core area, either by the displacement ,64 of alOur research has revealed the magnitude of a potential conflict between large-scale industrial wind-farm developments as an environmentally friendly strategy to combat climate change on the one hand, but on the other hand its detrimental impact on a protected species of conservation concern, when relevant protective legislation is poorly implemented. We have shown that if all wind farms  are to be authorized in south-eastern Europe, the cinereous vulture population is at great risk of extinction in the near future. On prior evidence, proper environmental assessments of wind farms are not expected to hamper authorization of many future harmful developments. Our results provide strong evidence for the official designation of the cinereous vulture population core area as a wind farm exclusion zone and suggest new spatial planning for future wind farm establishment, whereby the national target for wind energy harnessing would be met, while minimising vulture deaths through collision. Our study is a typical example of problem-solving in conservation biology, when full scientific knowledge is lacking, a crisis discipline that calls for immediate conservation decisions, while minimizing the error that stems from incomplete data and/or inherent uncertainties related to the target species’ ecology and behaviour ,68. Our S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Following publication of the results of the National Lung Screening Trial in the United States, a randomized controlled trial in Italy (ITALUNG) and two simulation studies in China reported similar findings in 2017 favoring lung cancer screening with low-dose computed tomography among smokers. With such advances in lung cancer screening, worldwide interest has gradually shifted from evaluating whether refining lung cancer screening protocols is effective in preventing deaths. However, there are several practical problems to be resolved, including the balance of enrollment criteria and cost effectiveness, precise measurements to reduce false positive findings, risk-based optimization of screening frequency, challenges associated with cancer heterogeneity, strategies to combine image screening with novel biomarkers, dynamic monitoring of the natural history of cancer, accurate identification and diagnosis of cases among huge populations, and the impact of tobacco control policy and environment protection. As one in three individuals with lung cancer worldwide resides in China, these questions pose great challenges as well as research opportunities for population screening programs in China. One in five deaths from lung cancer can be averted by conducting low-dose computed tomography (LDCT) screening among smokers, as evidenced by results from the United States (US) National Lung Screening Trial (NLST) in 2011 , 4.With a micro-simulation model adapted from the US National Cancer Institute, Sheehan et al.  predicteAnother study published in July 2017 by Wang et al.  analyzedGenerally, the results from ITALUNG and the two simulation studies in China were similar and consistent with the previous NLST report. This strengthens the feasibility of LDCT screening for lung cancer. In addition, the Dutch-Belgian lung cancer screening trial (NELSON), currently the largest ongoing lung cancer screening trial, is nearing the reporting of mortality outcomes . If simiThe results of these two simulation studies are timely and meaningful, as one in three individuals with lung cancer resides in China . In factA major conclusion of the two recent studies , 4 on luAccording to Wang et al.’s work , effectiThe NELSON trial used three intervals between screens  to determine the best screening frequency. A recent report from that study observed a significantly increased rate of “missed” cancers and a decreased proportion of early-stage cancers with the longest screening interval , which hThe high heterogeneity of cancer is another issue that has important implications for lung cancer screening. According to our recent meta-analysis , indolenAlthough evidence from population-level studies is scarce, biomarkers are considered the best way to assist in lung cancer screening . They caAn inherent but usually neglected advantage of lung cancer screening studies is the ability to gain a better understanding of the natural history of the disease. A recent report from the NELSON trial revealed that the volume growth pattern of screen-detected lung cancers can be fitted to an exponential function . In a prAs incidences and mortality of cancers are relatively low, a longitudinal cancer study calls for a large number of subjects and is heavily reliant on the accuracy of collecting and confirming incident or mortal cases. This task is challenging for lung cancer screening studies, particularly when participants were not diagnosed at the hospital that conducted the screening. A project was recently initiated in Shandong Province, China, that aimed to create a database linking the cancer registry with health insurance claims , which sSmoking and other environmental factors are well-known risk factors that are targeted in primordial or primary prevention of lung cancer. As screening is a secondary prevention approach, secular variation in these risk factors may have non-linear effects on lung cancer incidence  and, thuIn summary, research on lung cancer screening is about to reach a turning point that will shift the focus from evaluating whether the refinement of such screening protocols for increased cost efficiency is effective in reducing deaths. Several challenges and major research opportunities remain to be considered in future studies, especially studies conducted in China where more than 0.5 million people may benefit from such scientific progress.


North America is currently experiencing an overdose epidemic due to a significant increase of fentanyl-adulterated opioids and related analogs. Multiple jurisdictions have declared a public health emergency given the increasing number of overdose deaths. In the province of British Columbia (BC) in Canada, people who use drugs and who are unstably housed are disproportionately affected by a rising overdose crisis, with close to 90% of overdose deaths occurring indoors. Despite this alarming number, overdose prevention and response interventions have yet to be widely implemented in a range of housing settings.There are few examples of overdose prevention interventions in housing environments. In BC, for example, there are peer-led naloxone training and distribution programs targeted at some housing environments. There are also “supervised” spaces such as overdose prevention sites (similar to supervised consumption sites (SCS)) located in some housing environments; however, their coverage remains limited and the impacts of these programs are unclear due to the lack of evaluation work undertaken to date. A small number of SCS exist globally in housing environments , but like overdose prevention sites in BC, little is known about the design or effectiveness, as they remain under-evaluated.Implementing SCS and other overdose prevention interventions across a range of housing sites provides multiple opportunities to address overdose risk and drug-related harms for marginalized people who use drugs. Given the current overdose crisis rising across North America, and the growing evidence of the relationship between housing and overdose, the continued implementation and evaluation of novel overdose prevention interventions in housing environments should be a public health priority. A failure to do so will simply perpetuate what has proven to be a devastating epidemic of preventable death. Drug overdose deaths remain among the most pressing public health challenges in North America, which is currently experiencing an overdose epidemic. The proliferation of fentanyl-adulterated opioids and related analogs have given rise to an unprecedented overdose crisis, with opioid-related overdoses now the leading cause of accidental death in North America . The ovePeople who use drugs (PWUD) and who are unstably housed have been disproportionally affected by the overdose crisis in British Columbia, as elsewhere . AccordiIn BC, a small number of overdose prevention interventions have begun to be implemented within housing environments that serve as models to inform overdose responses in other settings. These include housing-based overdose prevention sites, peer-led naloxone training and distribution, peer witness injection programs, and shared-using rooms. Drug user organizations  and other community groups have also scaled-up naloxone training and distribution within housing environments to ensure that PWUD are properly equipped and trained to administer naloxone in the event of an overdose . FurtherAdditionally, peer witness injection programs were established in two temporary winter emergency shelters in Vancouver from December 2016 to March 2017. In each shelter, a designated room was set up with a table, chairs, and harm reduction supplies  for shelter residents to inject drugs. Peer staff were hired part-time and trained to monitor drug use onsite and respond in the event of an overdose. Finally, “shared-using” rooms have been implemented in select supportive housing sites in Vancouver to expand witnessed injection programming to tenants, particularly those who are socially isolated. Shared-using rooms are spaces separate from residents’ personal rooms where they can go to inject drugs. While these rooms vary between buildings in how they are monitored  or designed , they are intended to provide a ‘supervised’ space for people to use drugs, as an alternative to using alone in their rooms where they may be at greater risk of dying from an overdose [Moreover, overdose prevention sites (OPS) have been established in communities across BC and are similar to supervised consumption sites (SCS) in that PWUD are monitored by trained staff or volunteers to intervene in the event of an overdose. Unlike SCS, which require an exemption to operate legally under current federal law, OPS operate under a provincial ministerial order given the public health emergency . As suchSCS currently exist in a variety of settings, with over 90 located in eight countries . SCS areGiven the known effectiveness of SCS in reducing overdose morbidity and mortality, it is somewhat surprising that these services have yet to be widely implemented and evaluated in housing environments, especially given the disproportionate rate of fatal overdoses occurring in private housing environments . There aDespite SCS developments, there have been few interventions for PWUD who are unstably housed. Implementing SCS and other overdose prevention interventions across housing models provides multiple opportunities to address overdose risk and drug-related harms for marginalized PWUD. For example, incorporating SCS in emergency shelters or supportive housing would provide services beyond the capacity of part-time staff and volunteers. This integration could include having medically trained staff onsite to provide harm reduction supplies and education, supervise drug use, make referrals to health and social services, and respond to overdoses and other health issues that may arise. Additionally, the rapid gentrification of urban areas highlight the need for innovative programming  that extend support to displaced and vulnerably housed PWUD. Complementing fixed overdose prevention services, including those within housing environments, with more flexible interventions may provide a more effective response to the various overdose risks faced by PWUD who are vulnerably housed.Until we can achieve more large-scale legal and policy-based harm reduction goals , the scaling up of SCS and other novel overdose prevention interventions in housing environments represents an innovative public health opportunity deserving of immediate attention. Further efforts should now be focused on the ways in which such interventions can be embedded in a range of housing and shelter environments to adequately respond to the overdose epidemic. These services should also be rigorously evaluated, including an examination of their impacts on rates of using alone and fatal overdose, impacts on residents who do not use drugs, effects on specific at-risk populations, and their cost effectiveness. Given the current North American overdose crisis, and growing evidence of the relationship between housing and overdose, the continued implementation and evaluation of novel overdose prevention interventions in housing environments should be a public health priority. A failure to do so will simply perpetuate what has proven to be a devastating epidemic of preventable death.


Hemorrhage is a major cause of early mortality following a traumatic injury. The progression and consequences of significant blood loss occur quickly as death from hemorrhagic shock or exsanguination often occurs within the first few hours. The mainstay of treatment therefore involves early identification of patients at risk for hemorrhagic shock in order to provide blood products and control of the bleeding source if necessary. The intended scope of this review is to identify and assess combinations of predictors informing therapeutic decision-making for clinicians during the initial trauma assessment. The primary objective of this systematic review is to identify and critically assess any existing multivariable models predicting significant traumatic hemorrhage that requires intervention, defined as a composite outcome comprising massive transfusion, surgery for hemostasis, or angiography with embolization for the purpose of external validation or updating in other study populations. If no suitable existing multivariable models are identified, the secondary objective is to identify candidate predictors to inform the development of a new prediction rule.We will search the EMBASE and MEDLINE databases for all randomized controlled trials and prospective and retrospective cohort studies developing or validating predictors of intervention for traumatic hemorrhage in adult patients 16 years of age or older. Eligible predictors must be available to the clinician during the first hour of trauma resuscitation and may be clinical, lab-based, or imaging-based. Outcomes of interest include the need for surgical intervention, angiographic embolization, or massive transfusion within the first 24 h. Data extraction will be performed independently by two reviewers. Items for extraction will be based on the CHARMS checklist. We will evaluate any existing models for relevance, quality, and the potential for external validation and updating in other populations. Relevance will be described in terms of appropriateness of outcomes and predictors. Quality criteria will include variable selection strategies, adequacy of sample size, handling of missing data, validation techniques, and measures of model performance.This systematic review will describe the availability of multivariable prediction models and summarize evidence regarding predictors that can be used to identify the need for intervention in patients with traumatic hemorrhage.CRD42017054589PROSPERO The online version of this article (doi:10.1186/s13643-017-0480-0) contains supplementary material, which is available to authorized users. Significant hemorrhage following a traumatic injury progresses quickly within the first few hours and can result in significant morbidity or mortality . It is rOngoing bleeding that is not rapidly identified and corrected results in a state of global hypoperfusion which in turn can lead to multiple organ dysfunction or failure . The maiThe Advanced Trauma Life Support (ATLS) guidelines provide an algorithmic approach, adapted worldwide, to the initial assessment and resuscitation of the trauma patient . The guiIn recent years, several clinical prediction models for hemorrhage have been proposed. These include the Trauma Associated Severe Hemorrhage (TASH) Score  or the AWhile some patients arrive in hospital with an obvious need for early intervention, a subset of the trauma population does not manifest the classical clinical or biochemical extremes at presentation that prompt urgent action. It is this group in particular that is at risk of having their degree of hemorrhagic injury underestimated and therefore requires a systematic, evidence-based approach to early diagnosis. There is literature to suggest that elderly patients are significantly more prone to having massive bleeding missed on primary survey due to the absence of vital sign abnormalities and high incidence of non-cavitary, multi-site bleeding . UnfortuThe primary objective of this systematic review is to identify and critically assess any existing multivariable models predicting significant traumatic hemorrhage that requires intervention, defined as a composite outcome comprising massive transfusion, surgery for hemostasis, or angiography with embolization. These are prognostic models intended to predict future events and inform therapeutic decision-making. We will evaluate these models for usefulness, relevance, and the potential for external validation and updating in other study populations. The intended scope of this review is to identify and assess a specific combination of predictors informing therapeutic decision-making for clinicians during the initial trauma assessment. If no existing models meeting our pre-specified criteria are identified, we will then secondarily identify potential candidate predictors for future derivation of a new prediction model.This systematic review protocol was designed using the Preferred Reporting Items for Systematic Review and Meta-Analyses Protocol (PRISMA-P) checklist  as well We will include all studies examining adult patients, aged 16 years or older presenting to hospital with a traumatic injury. Blunt or penetrating mechanisms of injury involving the thorax, abdomen, or pelvis will be acceptable. Studies evaluating only patients with isolated head injury or isolated limb injuries will be excluded.Multivariable models will be eligible if they include any predictors typically available to the clinician during the first hour of the trauma assessment in the emergency department. This includes any pre-hospital or in-department variables. For the purposes of this review, these will include lab-based predictors such as the complete blood count, blood gases, and coagulation tests. Any concerns regarding time of availability for a laboratory test will be reviewed with a clinical expert in laboratory medicine to determine eligibility. Clinical predictors will include vital signs and point-of-care cardiopulmonary testing such as heart rate variability. Imaging-based predictors will include focussed assessment with sonography in trauma (FAST) ultrasound or computed tomography (CT) scanning. Scoring or injury classification systems that are applied retrospectively  will be excluded. Reliance on serial measurements following an admission will be excluded. There is no pre-defined categorization of these variables, and they will be extracted as defined by the study authors. The model is intended to be used at the time of initial trauma assessment and resuscitation in the ED.The outcome is defined as the need for any life-saving intervention for traumatic hemorrhage, which serves as a surrogate for clinically significant bleeding. This includes the need for hemostatic surgical intervention, angiographic embolization, or massive transfusion within the first 24 h in hospital. Studies describing surgeries without a documented indication or for non-hemorrhagic reasons, such as hollow viscus perforation, will be excluded. Angiography without embolization will similarly be excluded. There exist a variety of definitions of massive transfusion within the literature including: 10 units over the first 6, 12, and 24 h or complete circulating volume over 24 h . We willWe will include all clinical study designs evaluating predictors for life-saving intervention in traumatic hemorrhage, including randomized studies and cohort studies. There will be no date or language restrictions. Attempts will be made to translate any foreign language papers. This systematic review is primarily intended to identify and evaluate existing multivariable prediction models for our composite outcome of interest. We will include any models evaluating any of our individual outcomes of interest whether developmental, internally validated, or externally validated. Should no appropriate multivariable prediction models for our composite outcome be available, we will secondarily seek to identify potential candidate predictors for a future planned prediction rule derivation study.A comprehensive search strategy was developed in tandem with a health information specialist with expertise in systematic reviews and a clinical expert in the field of trauma. Individual search strategies were created for the EMBASE and MEDLINE databases and are available in Additional files The literature results will be captured and uploaded to Covidence, a web-based reference manager, for facilitation of screening  . The scrData extraction will be performed independently by the two reviewers using a pre-defined and piloted data collection form informed by the CHARMS checklist . AbstracGiven the anticipated paucity of literature on this topic, this systematic review is intended to be inclusive, exploratory, and descriptive in nature. If any suitable multivariable prognostic models for our composite outcome of interest are identified, the quality of each identified multivariable model will be described based on items identified in the CHARMS checklist , includiI2 statistics [Should no multivariable prediction models for our composite outcome be identified, we will secondarily seek to identify potential candidate predictors for a planned prediction rule derivation study. To inform the selection of candidate predictors for this derivation study, we will tabulate the frequency of use of each candidate predictor among the studies identified in our review, as a measure of potential importance of each predictor. We will also extract measures of predictor-outcome associations  from any bivariable or multivariable tests involving any of the three outcomes comprising our composite. For predictors used in multivariable models meeting adequate quality criteria across multiple studies, strength of association will be described using individual forest plots. Statistical heterogeneity will be examined using atistics . BecauseThere are no planned assessments of meta-bias or strength of evidence statements.The assessment of patients for significant traumatic hemorrhage can prove quite challenging and at times overwhelming. The clinician is tasked with processing inputs from a plethora of clinical, lab-based, and diagnostic imaging-based data sources and utilizing them in a manner that allows for rapid identification of patients at risk of significant bleeding. When such a scenario is rapidly and correctly identified, the patient is able to receive a much needed intervention—often in the form of transfusion, surgery, or angiography. In this systematic review, we will rigorously identify, describe, and summarize the existing literature evaluating predictors of intervention in patients with traumatic hemorrhage.Additional file 1:Appendix 1 EMBASE search strategy. (DOCX 15 kb)Additional file 2:Appendix 2 MEDLINE search strategy. (DOCX 15 kb)