In the past, lives and wealth have been lost due to corrosion in almost all engineering fields. Not only this, the cost of reviving damaged equipments in the industry due to corrosion contributed a lot to the gross domestic product of a nation. Thus, all hands must be on desk to combat this harzadous act via time to time research on its final resolution. However, current research works have revealed effective and reliable corrosion inhibitors from pharmaceutical drugs, plant extracts and ionic liquids as organic green corrosion inhibitors (OGCIs) with accommodative attributes such as being environmentally friendly, readily available, biodegradable, non-harmful, relatively cheap and many others to mention a few. This paper opens readers mind into the detailed classifications, mechanisms and active functional groups of these eco-friendly OGCIs. Not only the corrosion efficiency calculation ways but also influencing factors on efficiency were presented. Plant extracts, pharmaceutical drugs, ionic liquids and synthetic inhibitors, as among major sources of OGCIs, used in preventing material corrosion in corrosive media were separately and comprehensively examined. The significance of values obtained from simulating presented mathematical models governing OGCIs kinetics, adsorption isotherm and adsorption thermodynamics was also included. In conclusion, beneficial recommendations for both current and prospective researchers in the field of Corrosion Engineering were presented. Scavengers reduce medium corrosivity via aggressive substances scavenging while interface inhibitors inhibit corrosion through film formation at the environment/metal interface. The scavengers work in alkaline and almost neutral solutions by cathodic-oxygen reduction reaction simply written as However, the most prominent are the liquid-phase inhibitors which are further sub-divided into cathodic, anodic or mixed OGCIs based on the reaction type inhibit which could be any of cathodic, anodic or both electrochemical reactions. In anodic OGCIs, hydroxides, oxides or salts are produced to enhance passivating films formation which inhibits anodic metal dissolution reaction. Their mechanism is best explained by an active-passive metal polarization diagram. In cathodic OGCIs, corrosion is controlled either by cathodic poisoning or cathodic precipitation. In the former, sulfides and selenides which act as cathodic poisons are adsorbed on the surface of the metal to form protective films that reduce rate of cathodic reaction through oxygen diffusion minimization on metal surface. In the latter, insoluble compounds such as carbonates of calcium and magnesium are precipitated on metal surface in order to increase alkalinity at cathodic sites. Generally, hydrogen ions reduction to hydrogen atoms to form molecules of hydrogen as written in Eqs. Lastly, approximately 80% of OGCIs are categorised as mixed inhibitors which protect metal from corrosion by chemisorption, physisorption and film formation. Physisorption is facilitated by electrostatic attraction of negatively charged (anionic) OGCI with positively charged metal surface. Chemisorption process is slower than physisorption such that inhibition rate and adsorption increase as temperature increases 1.3p-electron and functional electronegative groups with conjugated double or triple bonds have been shown to be most effective 3 electron releasing substituents proved to have better efficiency than parent molecule having no substituents The OGCIs active ingredients consist of phytochemical constituents known to be functional groups with N, O, S, P or Se hetero atoms via which they are attached onto the metal surface , 45. ComD (-) pentose sugar (ribose) which consists of a phosphate monosodium salt and three antisymmetric carbons. The bark of Rhizophora Racemosa stem investigated to be very rich in tannins has been stated as the most effective OGCI for mild steel. Its basic structure contains residues of garlic acid attached to glucose through bonds of glycosidic Chamaerops humilis plant extract which is also rich in tannins is effective in inhibiting corrosion of mild steel in 0.5M sulfuric acid solution with 5% ethanol additive ∘C. Some prominent compounds such as benzoic acid 1.4OGCIs' efficiency in inhibiting corrosion is a function of their adsorption characteristics on the metal surface. Factors that have been considered by previous studies affecting OGCIs inhibition efficiency depend majorly on their structure, concentration, temperature and exposure time. Increase in OGCIs concentration results in simultaneous decrease in corrosion rate with increase in inhibition efficiency which approaches optimum level at certain concentration value. This resulted from formation of additional inhibitor molecules being adsorbed on the surface of metal which makes it complex for further corrosive attack to occur by the electrolyte solution. The dissolution of metal increases with corrosion exposure period in the presence of OGCIs. This is linked to previously adsorbed inhibitor molecules from metal surface resulting from partial desorption. Corrosion rate increases linearly as temperature increases such that equilibrium exists between adsorption and OGCIs molecules desorption at the surface of metal at a particular temperature. Increase in temperature as a result of higher desorption rate makes the equilibrium to shift until its' re-establishment at various equilibrium constant values. Thus, OGCIs inhibitive protectiveness decreases with increasing temperature. As earlier said, OGCIs' structural behaviour has a great influence on their efficiencies in corrosive media. Presence of heteroatom in OGCIs molecule enhanced their adsorption onto metal surface through the formation of adsorptive bond by Lewis acid-base reaction in which OGCIs and metal act as electron donor and acceptor respectively. The strength of adsorption bond is a function of electron density and polarizability of reaction centre. Conclusively, studies have shown surface active OGCIs adsorption to increase with increasing molecular weight and dipole moment.1.5The first step required in measuring efficiency of OGCIs is the preparation of metal sample to be examined for corrosion. The selection of metal coupons for checking OGCIs efficiency is vital as small changes in metal composition or available impurities during fabrication reflect in the results obtained 1.5.1θ2), Many researchers used test solutions prepared from actual field solution for corrosion testing while some used synthetic solutions prepared from analytical grade reagents in the absence of the former for WLM. Before the OGCIs efficiency is determined using WLM, metal samples are cleaned by polishing with abrasive paper of different grades and washed thoroughly using solvents  after which they are dried at room temperature. Vernier caliper is used to measure the dimensions of the metal specimen. The prepared metal coupons are then weighed prior to immersion using high-accuracy digital balance. The thoroughly rinsed corroded metal coupons are re-weighed after specified period of exposure time to check the weight loss. The corrosion rate θA and θB = respective surface area coverage by compound A and B when acting separately; and θAB = surface area coverage obtained for mixture of A and B. When the value of S approaches 1, interaction between A and B is negligible. If S is greater than 1, it reveals existence of synergism while a value of S being less than 1 signifies opposite effect between A and B However, there are cases where the inhibition efficiency of OGCI is enhanced as a result of combination with another OGCI such that the inhibition efficiency is increased by an appreciable value. This is called synergism effect which can be quantified using 1.5.2(V) of the system is measured and controlled by the reference electrode while the current (I) is measured by the counter electrode. As the electrochemical reactions progress, open circuit potential (Eocp) of the metal fluntuates. At equilibrium, a stable value is then measured afterwhich potentiodynamic polarization scan is performed. Application of a potential from a value below the initially measured Eocp to higher potential (between -0.25 to +0.25 V) then gives the Tafel plot. The corrosion current (icorr) and corrosion potential (Ecorr) can then be measured from the plots. icorr in the presence and absence of OGCIs using CR = corrosion rate (mm/yr), K1 = conversion factor = 3.27 × 10−3 mm g/μA cm yr, EW = equivalent weight (grams), ρ = density (grams/cm3), A = sample area (cm2), 2) in the absence and presence of OGCIs molecules respectively.Potentiodynamic polarization is another means of measuring OGCIs efficiency, corrosion rate and corrosion mechanism protection through electrochemical-based measurements. In most cases, the basic lab set up involves using three electrodes in the electrochemical cell which are working, counter and reference electrodes for the measurement immersed in the test solution of known volume and concentration. Platinum electrode 1.5.3in situ electrochemical changes with critical understanding of physical processes occuring at the metal/electrolyte interface 2SO4 at 30 °C by means of new schiff base extract with different concentrations as OGCI is shown in Rs (electrolyte solution resistance), in series with parallel arrangement of constant phase element (CPE) and Rct (charge transfer resistance) EIS is an essential method of monitoring Rp) obtained as real impedance difference at reduced and higher frequencies to replace the usual charge transfer resistance (Rct) Rp) was noticed to include Rct, accumulation resistance (Ra) resulting from species accumulated at metal/electrolyte interface, diffusion layer resistance (Rd) and inhibitor film resistance (Rf) at metal surface. Anode-cathode charge transfer causes metal oxidation which is usually obstructed by the presence of solvent molecules in aqueous acid solution. The resistance by the electrolyte solution is called the solution resistance (Rs). The charge transfer resistance (Rct) represents protective film capacity of adsorbed organic molecules on metallic surface to impede charges transfer the metal/solution interface. Impedance parameters which include Rp, n, Cdl and η% could then be obtained from Nyquist plot by the equivalent circuit.However, a study had used polarization resistance (C) A = CPE constant, j = imaginary number (i.e i2 = −1), n = phase shift exponent which is a measure of surface irregularity/inhomogeneity. The significance of n is that a lower surface roughness is obtained at higher value of n and vice versa. Also, n value determines the nature of constant phase element and states what A in −1), Rct(i) and Rct(o) represent charge transfer resistance in the presence and absence of OGCI various concentrations respectively.For better explanation of a frequency independent phase shift existing between an applied alternating potential and its current response, a constant phase element (CPE) being represented mathematically as In general, 2With reference to 2.1Industrial applications of corrosion inhibitors from greeners can be found in petroleum production, steel pipelines making industry, refrigerating industry, automobile, paint industry, acid producing companies and so on. 2.22.2.12+ ion boundary condition at anode (2) Anodic corrosion current density accounts for Fe2+ ion generation via electrochemical reactions at the surface of metal as the source term (3) Zero concentration of Fe2+ ion is applied at cathode due to scale formation (4) Fe2+ ion concentration in the shielded solution is the same as bulk solution in chemical equilibrium and (5) H+  anodic corrosion current density was used for Fe2), 2), The anodic corrosion current density is calculated using 2), ΔH = change in enthalpy (kJ/mol), R = gas constant (J/mol·K), T = solution temperature (/K) and Tref = reference temperature (/K).The iron oxidation exchange current density (2+ at anode (2+ at anode (mol/m2·s), 2), F = Faraday's constant (C/mole) and 2+ (moles).The mass flux of Fe2.2.2The derivation of equations governing kinetics of corrosion at the cathode is based on the assumption that oxygen and water reduction in the system is negligible such that the two cathodic reactions are stated as Eqs. + reduction partial cathodic corrosion current densities and H2CO3 reduction is stated as 2), 2), A general form used in the calculation of H+ and H2CO3 reduction at cathode are determined using The exchange current densities of HFor electro-neutrality condition in the solution, Thus, 2.2.32 stated as ia = anodic current density (A/m2), Mw,Fe = atomic mass of iron (kg/mol), 3), n = number of moles of electrons involved in iron oxidation (2 mole/mol) and F = Faraday's constant.Assuming corrosion rate is governed only by electrochemical reaction, total anodic reaction current density is used in determining corrosion rate of COThe current density for iron dissolution is obtained by using The Tafel slope of Iron oxidation 2), 2), Ecorr = corrosion potential (V), Erev,Fe = reversible potential of iron oxidation (V), Thus, 2), 2) and 2).The current density of any cathodic reaction is calculated thus 2), The charge transfer current density of cathodic reactions , 3) and F = Faraday constant The limiting current is determined from the mass transfer limitation for the case of H2 hydration reaction rate being very slow, limiting current density (3), 2CO3 in water (m2/s), 2 hydration reaction and 2 hydration reaction (s−1).Suppose there is restriction of carbonic acid reduction due to COf for A theoretical flow multiplier 2.32.3.12+ ion mass flux leaving the metal surface, can be determined using 2+ ion from the metal surface by diffusion and electro-migration, and (2) Fe2+ ion distribution in the solution is governed by Fick's second law. Thus, Fe2+ flux can be solved using Nernst-Planck equation 2·s), D = diffusivity (m2/s), C = concentration (mol/L), F = Faraday's constant (C/mole), z = valence (mole/mol), ℜ = ideal gas constant (J/mol·K), T = absolute temperature (K) and The risk of pitting corrosion could be increased under stagnant conditions in which corrosive microenvironments are established on the surface. The accumulation of stagnant electrolyte at the bottom of pipes, tubes and tanks could be prevented by both drying and ventilation. The buildup of local highly corrosive conditions could also be prevented through agitation 2+ ion and electrical field in the solution such that change in concentration with time The pitting corrosion rate is determined after computing the distributions of Fe2.3.2Stress corrosion cracking in metallic materials had been discussed previously Active path dissolution and film rupture model: For this, crack growth rate or crack velocity , The pure oxidation dissolution Faradic equation is stated as The anodic current density at time t is given as Substituting If Integrating Assuming Hydrogen embrittlement: The related stress corrosion cracking model is based on the assumption that crack advance occurred due to hydrogen assisted creep fracture (HACF) of hydrogen embrittled grain boundaries. Thus, stress corrosion crack growth rate is expressed as The critical fracture strain , 2S in bulk solution (mol/m3), 2S at steel surface (mol/m3), 2S at different mackinawite layer (mol/m2·s), 2S in liquid boundary layer (m/s), 2S in water (m2/s), The corrosion rate caused by H2.4θ) and OGCI concentration together describe the most suitable adsorption isotherm for the process. Prominent OGCIs adsorption isotherms have been stated to be Langmuir, Temkin, Frumkin, Freudlich, Virial Parson and Bockris-Swinkels isotherms R2) value that is very close to unity or equal to 1.Adsorption isotherms play key role in giving detailed information about existing interaction between molecules of OGCIs and metal surface 2.5Ea) of the process. The value of adsorption equilibrium constant (Kads) obtained from the best fitted isotherm tabulated in CR = Corrosion rate (mm/yr), N = Avogadro's number (6.02 × 1023 mol−1), h = Plank's constant (6.63 × 10−34 m2 kg·s−1), R = Gas constant (8314 J mol−1 K−1), T = Absolute temperature (°K), Ea = Activation energy (kJ mol−1) and A = Pre-exponential factor.The consideration of thermodynamics studies in the OGCIs adsorption reveals the significance of Gibbs free energy of adsorption 33.11.Many of the greeners considered for extraction of OGCIs are edible and are very useful for human need in many areas such as medicinal, pharmaceutical, food consumption and so on thus, making them to be very competitive in terms of functionality.2.It has been established that many silicates also possess corrosion inhibitory attributes due to their capability to block corrosion active sites on metals in acidic medium.3.None of the examined studies presented chain reaction mechanisms and reaction pathways showing present of intermediates in the course of corrosion reaction inhibitory effects of used OGCIs and examined metals.4.as the case may be) in pure form and the spent OGCIs.Also, there is complexity of adequate separation techniques to be employed in obtaining tested acidic or alkaline solution  as a result of corrosion over period of time.12.ginkgo biloba leave Diospyros Kaki L.f husk Some corrosion inhibitors extracted from 13.2SO4 were the most widely used because of their high corrosive nature to most metals and alloys even at low concentrations 3 and H3PO4 were explored in isolated cases Acid solutions have found industrial application in mill scales removal from metallic surfaces, acid descaling, acid pickling, industrial acid cleaning and oil well acidizing. An important stimulation technique for enhancing oil production is petroleum oil well acidization. However, studies of metal corrosion using organic acid solutions were rare as compared to similar studies with mineral acids as corrosive media 14.Previous researchers have been using days and months to critically figure out adsorption corrosion process of examined materials, media  and inhibitors from greeners to truly determine percent inhibition efficiency of inhibitors. Though very few researchers have been working on methodologies and technologies to employ to reduce corrosion examination period as presented in 15.uniquely bridged the gap among the stated sources of OGCIs by providing useful information for prospective researchers in the field of corrosion engineering. Also, many researchers rarely present various isotherm and thermodynamic models used in validating their corrosion experimental data. Only results are usually presented and discussed. All applicable isotherm and thermodynamics equations that any researcher in this field could think of are well-presented for future usage. Nevertheless, various kinetics modelling corrosion equations for various corrosion types also make this review paper to be superb. Prospective researchers are implored to apply them in forcasting corrosion types and corrosion rate of materials in order to cut wasteful corrosion costs.Recently, researchers have written separate comprehensive review papers on plant extracts The following few points were concluded after comprehensive scrutiny of literatures on this subject matter:3.21.There is need for future research works to focus more on wastes from greeners constituting environmental nuisance causing harmful effects on both human and aquatic natures. Recently, Al-Zubaidi et al. 2.Researchers should focus on synthesizing composite OGCIs from greeners extracts and silicates (such as rice husk waste) to improve their efficiency. This phenomenon is refered to as synergism effect which can be quantified by applying 3.Prospective researchers should show how intermediates produced during corrosion reaction inhibitory effects of OGCIs and metals contribute or affect the inhibition via adsorption of OGCIs on metals. It is an established fact that when extracts from greeners react, intermediates are formed in the course.4.The reusability of spent OGCIs and decrease in their efficiency with time is a great challenge for prospective researchers in the field of Corrosion Engineering.5.2SO4 solutions for some period of time. In support of these, existing optimization tools such as response surface methodology and central composite design of design expert coupled with predictive tools such artificial neural network based Monte Carlo simulation, sum of square errors and so on will be of help Cost effective modern techniques that will maximize OGCIs extraction in pure form from their sources are required. Detailed financial implication from pilot scale to full industrial plant is needed to expose general public to see this as a key means of internal revenue generation. Previous studies focussed majorly on refluxing greeners in HCl, ethanol and H6.Flavonoid is a good candidate to explain the corrosion inhibition effects observed in greeners. Its detailed chemistry must be studied to enhance further works on contribution of flavonoid in OGCIs on corrosion inhibition of metals.7.Phytochemical analysis provides information on active ingredients present in plants extracts acting as corrosion inhibition of OGCIs. Therefore, it is most likely that a mixture of constituents are acting as corrosion inhibitors 8.Additional advanced characterization techniques coupled with fundamental studies are required to further differentiate OGCIs mechanism and investigate relationship binding their structure with experienced corrosion inhibition. This knowledge will help tailoring OGCIs structure in obtaining necessary corrosion inhibition properties 9.Further research works are required on using additives such as iron control agents, water wetting agents, anti-sludge agents, non-emulsifiers, stabilizers and viscoelastic surfactants with the green corrosion inhibitors. They do not have corrosion inhibition potential but can enhance corrosion inhibition performance of OGCIs and significantly reduce corrosion rate.10.Deep studies are required on kinetics of corrosion inhibition of materials by OGCIs via earlier presented modelling equations. Also, more studies are needed in the aspect of developing mathematical models with reduced assumptions involving kinetics and mechanistic studies in predicting corrosion inhibitory effects of OGCIs from plant extracts. Computer software that will enhance quick prediction and other applications pertaining to corrosion inhibitory exhibits of these OGCIs can be developed.11.Novel research works on quantitative models development to bridge chemical structure to properties using existing machine learning or statistical approaches are required. Though quantitative structure-activity relationships (QSPR) and quantitative structure-property relationships (QSPR) modelling had been presented , 202, th12.There is need to work assiduously on other metallic components such as copper, alloys, aluminium, stainless steel and so on. All these work in unison and play specific roles in material selection for domestic/industrial purposes. They also corrode when subjected to certain environmental conditions. They are also major components of automobiles whose corrosion is of major concern.13.Thorough research is required on the use of OGCIs from greeners that have strong affinity for concrete cements and constituents (pastes) to increase the life span of reinforced concrete structures damaged as a result of high alkalinity and tackle rusting of these iron rods used in building construction.14.2, H2S and NaCl besides MIC type having great impact on metals used in oil and gas industries. Nevertheless, identification of corrosion type inhibited by OGCIs based on the solution examined for the materials is necessary.More researches should be carried out on other corrosive environments like COCurrent researchers whose research works center on this subject matter and prospective researchers who may develop interest after indepth understanding of this article could develop and execute novel research works in Corrosion Engineering field by considering the following stated recommendations:All authors listed have significantly contributed to the development and the writing of this article.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.


Emerging viral diseases, most of which are zoonotic, pose a significant threat to global health. There is a critical need to identify potential new viral pathogens and the challenge is to identify the reservoirs from which these viruses might emerge. Deep sequencing of invertebrate transcriptomes has revealed a plethora of viruses, many of which represent novel lineages representing both plant and animal viruses and little is known about the potential threat that these viruses pose.Vigna unguiculata (cowpea) plants. Providence virus particles purified from these cowpea plants were used to infect two mammalian cell lines.Providence virus, an insect virus, was used to establish a productive infection in Here, we present evidence that Providence virus, a non-enveloped insect RNA virus, isolated from a lepidopteran midgut cell line can establish a productive infection in plants as well as in animal cells. The observation that Providence virus can readily infect both plants and mammalian cell culture lines demonstrates the ability of an insect RNA virus to establish productive infections across two kingdoms, in plants and invertebrate and vertebrate animal cell lines.The study highlights the potential of phytophagous insects as reservoirs for viral re-assortment and that plants should be considered as reservoirs for emerging viruses that may be potentially pathogenic to humans. The threats posed by human viral diseases, many of which are caused by zoonotic viruses , are incHelicoverpa zea (H. zea), midgut cell line [Helicoverpa and Spodoptera [Extatosoma tiaratum, the giant prickly stick insect (NCBI nucleotide sequence GAWG01012535) and that cDNA-derived PrV transcripts are capable of launching PrV replication in insect, mammalian as well as plant cell-free extracts [Providence virus (PrV) is a small, non-enveloped insect RNA virus that was originally identified as a persistent infection in the corn earworm, ell line . Like otodoptera ,4 but moodoptera . It is iTombusviridae family [in vitro in plant cell-free extracts [All tetraviruses have monopartite or bipartite (+) ssRNA genomes, encoding a viral replicase (REP) and a single, capsid protein precursor (CP), 240 copies of which assemble into a procapsid that is autoproteolytically cleaved during particle maturation [e family . PrV ande family ,7. This extracts  raised t2.MG8 cells were grown to confluence in complete ExCell 420 medium in T75 flasks. HeLa (ATCC CCL-2) and MCF7 (ATCC HTB-22) were maintained in DMEM supplemented with 10% FBS at 37°C in an atmosphere that contained 5% COCells were treated with 0.1% Triton X-100 for 15 mins. The supernatant was clarified at 5 000 rpm for 15 mins at 4°C. Virus particles were purified as described previously  by ultra5 particles mL-1 was used to gently stroke the leaves. After 30 mins, the leaves were rinsed with water until no carborundum powder was visible on the leaves. The plants were grown in a 28°C constant environment room and watered as required. Watering regularly washed the leaves to ensure the removal of any PrV or carborundum on the leaf surface. After harvesting, plant material was stored at -80°C until analysis.Carborundum was sprinkled lightly over the two leaves of the cowpea plants. A gloved finger, wet in a buffer solution (mock infected plants) and PrV solution (infected plants) where PrV was diluted to approximately 103 particles were added to the cells and the flasks incubated until confluence. These cells were analysed by fluorescence microscopy as described below.HeLa and MCF7 cells were grown to 50% confluence in a T25 flask. A total of 10CGA GGT TAC CAC AAC CTG C or JRS77F- GTG GTA CCT CTA CAA ATG TGG TAT GGC TG) or the positive sense RNA (JRS80R- GAT GCC CTC GGC AAC C or JRS 78R- CTT GGC TCG TCT AGC GC). The cDNA was subsequently PCR-amplified for 30 cycles (with the primers JRS79-JRS80) or 53 cycles (with the primers JRS77-JRS78) using the KAPA Taq PCR Kit  as described previously [RNA was extracted with the RNA Shield Purification Kit . A total of 10 ug RNA was used in reverse transcription reactions with the QuantiTect Reverse Transcription Kit  with random primers (supplied with the kit), with primers specific for the negative sense RNA (JRS79F- Protein extracts were separated on 10% SDS-PAGE gels and transferred to Immobilon-P membrane (Millipore) for western analysis with anti-CP anti-serum  , anti-mouse AF546  and anti-rabbit AF633  in permeabilization buffer for 30 min. Cells were washed thrice, for 15 mins. In the second wash, 1 μg mL-1 DAPI was added to stain the nuclei of the cells. Cells were mounted using fluorescent mounting medium . Cells were imaged using a Zeiss LSM780 laser scanning confocal microscope using the x63, 0.75 NA objective. All images were acquired using the same exposure and detector settings for each spectral channel. The Zen 2011 Blue software was used to acquire images from the Zeiss LSM780 microscope and to perform image overlays.Cells settled overnight on glass coverslips were washed three times in PBS (pH 7.0) for 5 min after which the cells were fixed in 4% paraformaldehyde for 15 min followed by three washes in PBS as above. The cells were permeabilized in permeabilization buffer  for 15 min. The cells were incubated with anti-p40  (biotin-H. zea MG8 cells in which the virus had first been identified [PrV particles were isolated from persistently-infected entified . In thesVigna unguiculata (cowpea) seedlings, with two primary leaves, were mechanically infected with PrV particles purified from insect (MG8) cells. After two weeks, plants infected with PrV showed some lesions as well as yellowing of the leaves, which were not observed on the leaves of mock-infected plants  in leaves collected from infected plants but not in those of mock-infected plants  and all In order to be a true trans-kingdom virus, PrV must not only replicate in plants but must still retain its ability to infect and replicate in mammalian cells after being passaged through plant cells. To this end, virus particles prepared from cowpea plants were used to infect human cervical (HeLa) and breast (MCF7) cancer cells as described previously  and immuH. zea MG8 cell line bind, enter, and successfully establish productive infections in mammalian cells while retaining the ability to replicate in plants. It would be interesting to analyse the genomes of PrV isolated from MG8 insect cells, cowpea plants as well as mammalian cell lines. There is growing evidence that the same changes are observed in the sequence of the viral genome when the virus infects a specific host [In this study, we demonstrate the ability of an insect RNA virus to establish productive infections across two kingdoms, in plants as well as invertebrate and vertebrate animal model systems. Host shift, which is the ability of a virus to move from one host into a novel species, is often associated with changes in the sequence of the viral genome . These cfic host . PrV wouH. zea larva infected with a tetra-like virus. This scenario is not unreasonable, given that the ability of heliothine midgut cells to absorb nucleic acids is well known [In studies requiring the mechanical infection of plants with viruses, the conventional method involves the use of carborundum. In this study, we also used carborundum to inoculate cowpea plants with PrV particles. This method mimics the transfer of virus particles from biting insects to plant hosts and inversely this could represent the mechanism by which viruses are transferred from the plant to biting insects. Given the phylogenetic relationship between the PrV and tombusvirus replicases, it is probable that PrV evolved because of re-assortment between an ancestral tombus-like virus ingested by an ll known  and is bll known .Asfarviridae, Iridoviridae, Poxviridae), ssDNA viruses (Parvoviridae), dsRNA viruses belonging to the Birnaviridae family and numerous ssRNA viruses (the (-)-sense Nyamiviridae, Orthomyxoviridae and (+)-sense Flaviviridae and Togaviridae, (Tymoviridae and Tenuivirus are able to infect invertebrates and plants [Reoviridae (dsRNA) family includes viruses that infect vertebrates, vertebrates and invertebrates, or plants and invertebrates. The Rhabdoviridae (ssRNA) includes viruses that replicate in invertebrates and vertebrates or in invertebrates and plants. The (+/-)-ssRNA Bunyaviridae includes viruses that replicate in invertebrates, in vertebrates, in invertebrates and vertebrates, and in plants (Tospovirus). However, for all these virus families, no viruses have been reported to replicate in invertebrates, vertebrates as well as plants to date [There are several families of viruses that infect invertebrates and vertebrates, including dsDNA viruses  in Hungary [Reports in the literature do exist that suggest the possibility that some tobamoviruses may be infectious to humans. For instance, detection of the pepper mild mottle virus (PMMoV) in human faecal samples was linked with clinical symptoms of viral infection, including fever and abdominal pain , but wit Hungary . This ba Hungary  and so iS1 FigDetection of viral RNA in cDNA generated from mock infected (lanes 1 to 3) and PrV-infected (lanes 4 to 9) leaves from cowpea plants using the primers JRS79-JRS80. For the control reactions, virus preparations from mock infected plants and infected plants (lanes 10 and 11 respectively) were used. 1 μg pFLM (cDNA) was used as a positive control.(TIF)Click here for additional data file.S2 FigRNA isolated from purified particles was converted to cDNA with random primers and then probed for the presence of a 5’ region of the PrV genome (i) or the CP sequence (ii) with the primers JRS79-JRS80 or JRS77-JRS78 respectively. For the control reaction, 1 μg pFLM was used.(TIF)Click here for additional data file.S3 FigHuman cervical (HeLa) and breast (MCF7) cancer cells that had been infected with PrV isolated from cowpea plants were cultured for three months and subsequently analysed by immunofluorescence microscopy. Cells were probed with mouse monoclonal anti-dsRNA and anti-mouse AF546 . Viral r(TIF)Click here for additional data file.


These observations open a fundamentally new paradigm for clinical approaches that include in vivo stone dissolution and identify high-frequency layering of organic matter and minerals as a template for biomineralization in natural and engineered settings.More than 10% of the global human population is now afflicted with kidney stones, which are commonly associated with other significant health problems including diabetes, hypertension and obesity. Nearly 70% of these stones are primarily composed of calcium oxalate, a mineral previously assumed to be effectively insoluble within the kidney. This has limited currently available treatment options to painful passage and/or invasive surgical procedures. We analyze kidney stone thin sections with a combination of optical techniques, which include bright field, polarization, confocal and super-resolution nanometer-scale auto-fluorescence microscopy. Here we demonstrate using interdisciplinary geology and biology ( In 1868, Beale3 reported that urine contains suspended crystal sediments of calcium oxalate  that are “difficult” to dissolve. Research then focused on mineralogy, chemistry and protein incorporation as primary controls on stone growth, with little evaluation of urine chemistry6. These minerals originally form as tetragonal bipyramid crystals of CaOx dihydrate  that “transform during the partial loss of water” into monoclinic lozenge-shaped crystals of CaOx monohydrate 9. A third common but minor mineral phase in CaOx stones is orthorhombic crystalline uric acid 10. During the 1960s, stone research transitioned to focus on the role of 24-hour urine solution chemistry and calculated supersaturation as an index of stone formation risk11. It is now known that a complex variety of factors other than simple urine chemistry influence stone formation, including protein degradation products, cellular responses, and even the likely presence of a kidney microbiome14. Moreover, a host of complex human health conditions including dehydration, pregnancy, diet, diabetes, hypertension, obesity, genetics, and climate15 also affects kidney stone susceptibility. Thus, current clinical strategies to employ hydration, diet and/or drugs to “correct” urine chemistry are at best only partially effective in preventing stone growth.Kidney stones have been formally studied by western medicine since 1802geobiology) techniques are applied within the chronological and spatial framework of crystal growth and stratigraphic layering patterns  that comprise each stone fragment. Bulk mineralogical analyses with infrared (IR) spectroscopy confirm that all samples are primarily composed of CaOx (see Methods). We analyze ~20 μm-thick stone fragment sections polished on both sides using a wide range of optical microscopy (250 nm-resolution), which include brightfield, phase contrast, polarization, single- and two-photon spectral confocal, and fluorescence lifetime imaging. In addition, we use Airyscan super-resolution microscopy (~140 nm-resolution) to bring the optical resolution beyond the diffraction limit of light18. To determine mineralogy and chemistry in thin section, we use scanning electron microscopy and energy-dispersive x-ray spectroscopy. Only stone fragments from patient MP2 contain all events of crystalline architecture collectively identified in all six patient specimens. Therefore, we present MP2 as an exemplar of CaOx kidney stone growth.In the present study, more than 50 CaOx kidney stone fragments, collected from six Mayo Clinic patients during standard percutaneous nephrolithotomy (PCNL) procedures, are analyzed. Integrated geology and biology  is revealed by auto-fluorescence 17 generated by changes in organic matter composition 20.Distinct stratigraphic layering on the scale of 10’s to 100’s of nanometers  COD and COM with internal concentric zonations consistent with free-floating growth in the renal calyx and/or pelvis .The earliest stage of kidney stone growth begins with the precipitation of 5–250 μm-diameter perfectly geometrically formed  of COMC takes place, in which the original fine laminations of the cortex are completely to partially preserved   then grow on outer surfaces of the stone complex  that COMC encrusts  called Randall’s plaques, or in the ducts of Bellini called Randall’s plugs31. Randall’s plaque commonly erupt into the renal calyx and pelvis, or are released through the nephron, and begin to accrete CODFF and eventually COMC (HSE 4) while bathed in urine12. Instead of the initial stages of apatite nidus growth32, CaOx kidney stones in the present study have a nidus composed of free-floating CODFF and COMFF 33. In addition, although the entombment of organic matter has been previously documented15, the clinical importance of these biomass-rich nano-layers remains undetermined28. Our results indicate that kidney stones repeatedly dissolve in vivo  and highlight the intrinsic relationship between organic-rich and mineral-rich nano-layering.Results from the current study modify the long-held working assumption that COM is strongly insoluble diagenesis)24. The sector zoning in COMCE indicates that these crystals may be more soluble and thus more susceptible to diagenesis than expected with respect to calculated urine supersaturation states25, as controlled by the differential entrapment of organic matter on specific COM crystal faces35. The biomolecules present within the human kidney may also play a major role in driving the multiple events of dissolution as recorded in the HSE 13. However, the composition and potential effects of these biomolecules are currently unknown37.The repeated dissolution, crystallization and resultant remodeling of crystalline architecture that takes place during CaOx stone growth is analogous to the commonly observed post-depositional physical, chemical and biological alteration observed in natural mineral deposits  frequency analyses to compare the nano-layering within individual COM C Figs.  and 2 aners Fig.  and 12. ers Fig. . While S nm Fig. , previouC couplets  promote and maintain CODFF growth, continue CODFF surface dissolution, then completely dissolve CODFF prior to COMC encrustation; 3) periodically induce CODFF growth during COMC formation to create layers that can be dissolved; 4) enhance any of the later-stage HSE dissolution events; and 5) disrupt crystal, urine and biomolecule components that combine to control the switching between organic matter-rich and crystal-rich COMC nano-layers. All of these therapeutic targets would involve the introduction of macromolecules that either inhibit or promote crystal growth and dissolution, and lead to less crystal aggregation. The development of tools to read the crystalline architecture of kidney stones in a clinical setting would allow rapid evaluation and determination of which of these interventions to pursue. It has been confirmed for over a decade that precursor forms of amorphous CaOx exist as they do in calcium carbonate and calcium phosphate biomineralization57. Through a multi-step process, these amorphous CaOx phases fully transform into CaOx crystals. Although they leave behind no obvious record of their existence, clinical interventions targeting these early upstream events of biomineralization should also be considered for the treatment of kidney stones to target these transient states to destabilize the forming crystals. Taken together, these geobiology approaches will also have a transformative impact on the study of gallstones, atherosclerosis, osteoporosis and a variety of other human biomineralization conditions that involve multiple events of crystal growth and dissolution.Human beings almost constantly create urine supersaturated with respect to CaOx, yet the question remains as to why only 1 in 11 people actually grow symptomatic kidney stonesHSE Fig.  is a newAll methods reported in this manuscript were carried out in accordance with the basic medical research study, which was reviewed and approved by the Mayo Clinic Institutional Review Board (IRB 09-002083), and the outcomes of this study will positively affect the future management of kidney stone formers. Written informed consent was obtained from all six Mayo Clinic patient participants.Kidney stones were collected and analyzed from all six patients by a single urologist using standard percutaneous nephrolithotomy (PCNL) procedures under sterile operating room conditions at the Mayo Clinic in Rochester, Minnesota. A line of section for thin sectioning for each stone was carefully selected under a Zeiss Axio Zoom.V16 Microscope , along the orientation that would exhibit a complete cross-section of earliest-to-latest crystalline stone growth. Photographs and descriptions were sent with the six stones to Wagner Petrographic Ltd. in Linden, Utah. Here they prepared standard-sized (24 mm × 46 mm), uncovered (no cover slip), doubly polished thin sections. Stones were first vacuum impregnated and then mounted on borate silicate glass slides with clear low-viscosity cathodoluminescence-resistant epoxy impregnation to prepare double-sided polished, 20 µm-thick petrographic thin sections.Thin sections were imaged on a wide variety of optical modalities  using an FEI Quanta FEG 450 FESEM .2+) and oxalate (C2O42−) concentrations of 1 mM each (pH of 7.2) was prepared and injected into the micromodel at a rate of 100 µL/h to promote crystal formation in the micromodel. CaOx crystal growth was tracked with a Nikon Eclipse Ti-E epi-fluorescent inverted microscope with an Andor Zyla color camera attachment. Mineralogy of the COM and COD crystals that precipitated within in the micromodel was confirmed with Raman Spectroscopy  collected between 0–1600 cm−1 with a 532 nm DPSS laser and confirmed with the RRUFF online database.A pilot study of initial kidney stone nidus crystal growth HSE 1, Fig. , Fig. 1 Supplementary MaterialsSupplementary Video 1Supplementary Video 2


P. falciparum malaria has been recognized as one of the prominent evolutionary selective forces of human genome that led to the emergence of multiple host protective alleles. A comprehensive understanding of the genetic bases of severe malaria susceptibility and resistance can potentially pave ways to the development of new therapeutics and vaccines. Genome-wide association studies (GWASs) have recently been implemented in malaria endemic areas and identified a number of novel association genetic variants. However, there are several open questions around heritability, epistatic interactions, genetic correlations and associated molecular pathways among others. Here, we assess the progress and pitfalls of severe malaria susceptibility GWASs and discuss the biology of the novel variants.HbS and ABO blood group were replicated across endemic populations, only few novel variants were convincingly identified and their biological functions remain to be understood. We estimated SNP-heritability of severe malaria at 20.1% in Gambian populations and showed how advanced statistical genetic analytic methods can potentially be implemented in malaria susceptibility studies to provide useful functional insights.We obtained all severe malaria susceptibility GWASs published thus far and accessed GWAS dataset of Gambian populations from European Phenome Genome Archive (EGA) through the MalariaGen consortium standard data access protocols. We noticed that, while some of the well-known variants including The ultimate goal of malaria susceptibility study is to discover a novel causal biological pathway that provide protections against severe malaria; a fundamental step towards translational medicine such as development of vaccine and new therapeutics. Beyond singe locus analysis, the future direction of malaria susceptibility requires a paradigm shift from single -omics to multi-stage and multi-dimensional integrative functional studies that combines multiple data types from the human host, the parasite, the mosquitoes and the environment. The current biotechnological and statistical advances may eventually lead to the feasibility of systems biology studies and revolutionize malaria research. Plasmodium falciparum, the causative agent of severe malaria, has been infecting humans for at least 5000-10,000 years following the advent and expansions of agriculture [P. falciparum infects millions and kills hundreds of thousands of African children each year. However, this constitutes only a small proportion (1%) of the populations in endemic areas in which the infections progress to severe malaria such as profound anemia or cerebral malaria [iculture . Malariaiculture , 5. P. f malaria , 7.HbS and ABO blood group [Comprehensive understanding of the genetic basis of resistance and susceptibility to severe malaria is crucial to understand the molecular mechanisms of host-parasite interactions that can inform the development of effective therapeutics, vaccination, diagnostics and risk prediction strategies , 9. To tod group –13. Despod group –13; suggHere we review the current status of malaria susceptibility GWASs and provide guidance to future research directions. We begin by assessing the progress and pitfalls of severe malaria susceptibility GWASs and discuss the biology of the novel variants. We then provide an overview of the recent progresses in post-GWAS approaches and discuss how these methods can be implemented in severe malaria susceptibility studies to better understand the underlying biology. We conclude by discussing on research areas where further works are needed in light of the global malaria eradication efforts.P. falciparum infection is very common, the majority of children recover from malaria. However, a small proportion of infections progress to the severe form of the disease such as severe anaemia, cerebral malaria, acidosis and respiratory distress [P. falciparum parasitaemia). Severe anaemia is defined as a haematocrit of < 15% or haemoglobin < 5 g/dl in the presence of P. falciparum parasitaemia [P. falciparum malaria severity. However only small proportions (~ 2%) of heritability is explained by the well-known variants such as sickle-cell anaemia and α-thalassaemia [In malaria endemic areas where repeated distress . Cerebrasitaemia . Althougassaemia . The conassaemia  and the assaemia , 19 haveGWAS in malaria susceptibility study was motivated to address the acute limitations of the conventional approaches and provide better understandings of the underpinning genetics at genome wide scale. To this effect, a global partnership of malaria researchers, named as Malaria Genomic Epidemiology Network  was established in 2008 –13 and rOwing to the fact that Africa is the origin of modern humans, there is high level of genetic diversity and weak linkage disequilibrium (LD) in Africans compared to non-African populations –28. ThesHbS locus, a well-known variant conferring resistance to severe malaria demonstrated a weak signal  because of the weak LD between causal variants and the SNPs that were genotyped. After which authors sequenced the locus, undertook multipoint imputation, used proper reference panel and dramatically improved the signal to p-value ~1x10-14. However, coverages of the genotyping chips have been enormously improved to be able to capture the genetic diversities among global populations following the recent technological advances and availability of diverse reference data sets [This might have affected the power of previous malaria GWASs. For instance, in the first malaria GWAS , HbS locata sets , 30–32. ata sets .. [Such developments have also facilitated imputation-based studies in African populations. For instance, Band et al.  showed t. . We beli-8) is usually needed to declare evidences of genuine associations to minimize false discovery rate that can arise from multiple testing [In GWAS, a stringent p-value  and ATP2B4 that were well-replicated across malaria endemic populations. We then extend our discussion to other novel variants.Severe malaria GWASs have replicated some of the well-known variants such as se-where . Below wATP2B4 gene with severe malaria susceptibility was reported by Timmann et al. [ATP2B4 encodes a ubiquitous plasma membrane calcium-transporting protein (PMCA4b) [PMCA4b is widely expressed in different tissues and is the main transporter of Ca2+ in erythrocyte membrane [The association of variants in n et al.  in Ghanan et al. , 46. SNPn et al. . ATP2B4 (PMCA4b) . PMCA4b membrane .ATP2B4 haplotype named as ‘haplotype-1' and individuals with this haplotype exhibit a reduced PMCA4b expression level [PMCA4b expression significantly decreases the calcium extrusion in RBCs. Consistent with this, a study conducted by Lessard et al. and colleagues elegantly characterized the ATP2B4 locus using a combination of transcriptomic, epigenomic and gene-editing study approaches [ATP2B4 knocked-out mice express an elevated level of MCHC. Then, they conducted expression quantitative trait locus (eQTL) mapping studies using UK biobank dataset and showed strong associations between ATP2B4 erythroblast specific variants and RBC related traits including MCHC level, decreased RBC distribution and increased hemoglobin levels.A recent study showed that the GWAS SNPs are localized in a previously unrecognized on level . In thisproaches . The autATP2B4 and eQTL mapping showed that the GWAS SNPs are mapped to an erythroid specific enhancer element. Deletion of this enhancer from human erythroid cell line using CRISPR-Cas9 system showed a dose dependent reduction of ATP2B4 expression level. Bi-allelic deletion of the enhancer resulted in eighty three percent reduction of ATP2B4 expression level compared to the wild type while mono-allelic deletion resulted in moderate reduction of the ATP2B4 expression level [Further analysis of DNase I hypersensitivity sites (DHSs) at on level .ATP2B4 gene on calcium homeostasis, Lessard et al. measured the calcium concentration in unedited and edited (ATP2B4 enhancer deleted) HUDEP-2 cells. The edited cells demonstrated higher intracellular calcium level compared to wild cells indicating that ATP2B4 expression is essential for plasma membrane calcium pump. The disturbance of intracellular Ca2+ homeostasis might play an important role in impairing the invasion, development and reproduction of malaria parasite in RBCs [ATP2B4 region can potentially be targeted for development of vaccine and therapeutics.To determine the effects of the regulatory variants at  in RBCs , 52. The. [FREM3 gene and cluster of 3 glycophorin genes (GYPA/B/E) on Chromosome 4. A haplotype (at SNP rs184895969) within this region was reported to reduce the risk of developing severe malaria by about 40% and is common in Kenyan populations with allele frequency reaching 10% [The largest multi-center malaria susceptibility GWAS which included eleven populations was conducted by Band et al. . In thishing 10% .GYPA and GYPB genes [DUP4 was reported to reduce the risk of severe malaria by about 40% in eastern African (Kenya) populations. Further characterization showed that, this variant is composed of complex GYPB-A hybrid and encode Dantu antigen in MNS blood group system [GYPA and GYPB encode the MNS blood group system and are known to be receptors for P. falciparum during RBC invasion [GYPA and GYPB serve as an erythrocyte membrane receptor for EBA-175 and EBL-1 proteins of the parasite respectively [P.falciparum and humans [A subsequent study in the same populations identified a large number of copy number variants which are characterized by deletion, duplications and hybrid structures in PB genes . Of whicp system . The assp system . The glyinvasion . GYPA anectively . This ged humans  . Furthers6503319) is located close to SCO1 (synthesis of cytochrome c oxidase) gene on chromosome 17p13. SCO1 is a multi-functional signaling protein which plays an essential role in mitochondrial cytochrome c oxidase (COX) copper delivery pathways [COX catalyzes electron transfer from reduced cytochrome c to oxygen and is abundantly expressed in muscles, brain and liver [COX caused by mutations in SCO1 gene can lead to respiratory distress and severe metabolic acidosis [SCO1 gene are associated with the pathological pathways of cerebral malaria.Notable association signals were identified by the first malaria susceptibility GWAS conducted in Gambian population . The firpathways . COX catnd liver . Deficieacidosis  which aracidosis . FurtherDihydroxypheny-alanine decarboxylase (DDC) on Chromosome 7p12.2. A recent study in Tanzanian populations replicated the association of DDC variants with cerebral malaria [DDC gene encodes Aromatic-L-amino-acid decarboxlase enzyme which is involved in biosynthesis of neurotransmitters such as dopamine and serotonin [DDC is an essential enzyme for brain and nervous developments and its deficiency is associated with reduced cognitive functions [DDC is involved in cellular immunity and contributes in protection against parasitic disease in invertebrates [DDC gene was reported to be associated with refractoriness of Anopheles gambiae mosquito against P.falciparum parasites [The second notable association signal identified in this study was  malaria . DDC generotonin . DDC is unctions . DDC is tebrates . Furtherarasites ATP2B4, Timmann et al. [rs2334880) on chromosome 16p 22.2 which is linked to MARVELD3. However, this association has not been replicated in other studies. MARVELD3 is one of the components of tight junction proteins in several epithelial and endothelial tissues and is expressed as two alternative spliced variants [In addition to the n et al.  identifivariants . These pvariants .IL-23R and IL-12RBR2), in ketch-like proteins (KLHL3) and Human Leucocyte Antigen (HLA) regions. Interleukin-12 is formed from a hetrodimer of IL12B (ILp40 subunit) and IL-12A (ILp35 subunit) [IL-12 plays a vital role in stimulating cell-mediated immune responses against intra-cellular pathogens through binding to high affinity IL-12RB1 and IL-12RBR2 receptor complexes. It promotes the development of T-helper cells (Th1) and enhances the production of INF-γ, both of which are known to mediate the clearance of intracellular pathogens [IL-12 has been implicated in mediating the protective immunity both in experimental animals and in humans [IL-23 is an important pro-inflammatory cytokine that shares p40 subunits with IL12 [CD4 T-cells to IL-17 which plays key roles in pathogenesis of autoimmune diseases [The most recent malaria susceptibility GWAS was conducted in Tanzanian population . In thissubunit) . IL-12 pathogens . In malan humans  . IL-23 ith IL12 . It indudiseases .HLA-Bw53) and HLA class II variant (DRB1*1302-DQB1*0501) were reported to confer protections against severe malaria in Gambian populations [HLA-Bw53 [DRB1*1302-DQB1*0501 variant might possess efficient antigen presentation mechanism that can lead to rapid clearance of blood stage parasites [HLA is encoded by the Major Histocompatibility Complex (MHC), the most polymorphic genes known in human genome. The diversity of MHC is believed to be driven by selection pressure from infectious pathogens and known to be associated with the risk of several infectious diseases . HLA varulations . HLA claHLA-Bw53 . On the arasites .Variants in immune pathways are of great interest because of their potential to inform the development of effective malaria vaccines . The curPolygenic view of genetic architecture is gaining ground in genetic epidemiological studies and widely implicated for the ‘missing’ of heritability in GWAS analysis  . The raHowever, a slight inflation of the test statistics  which cannot be corrected by GC was initially observed across the genome in a Schizophrenia GWAS . SubsequCo-evolution of host-pathogen model predicts that multiple host loci are involved in resistance/susceptibility to infectious diseases due to the complex interactions between the multi-locus parasite genotype and the corresponding defense from the host-genome , 81. IndThus, implementation of polygenic analytic methods in malaria studies may potentially shed more light to the underlying biology. For instance, heritability can be estimated and partitioned in to different cell-types and functional groups and molecular pathways which enable to localize causal variants. Furthermore, these approaches can be extended to explore the genetic correlations between susceptibility to malaria and  susceptibility to other infectious diseases. The existence of shared genetic basis between infectious diseases susceptibility/protection is well-documented . However< 1 × 10 − 3) and SNPs missing proportion of (p > 0.02)) was  applied, the estimation became stable . Neither the inclusion of more PCs  as fixed effects nor SNP phasing further brought down the estimate. Using the same stringent QC threshold, the estimation was approximately the same for Mandinka ethnic group . We couldn’t estimate for other ethnic groups because of smaller sample sizes. Furthermore, the use of PCGC model which is designed for case/control approximately showed the same estimate .To figure out how polygenic analysis can be implemented in malaria susceptibility, we accessed the Gambian malaria susceptibility GWAS dataset from European Phenome Genome Archive (EGA) through data application procedure and estimated heritability of malaria susceptibility using MLM approaches. The Gambian GWAS data is the largest MalariaGen dataset obtained from a single country comprised of 4920 samples (2429 cases and 2491controls) and 1.6 million SNPs that passed GWAS quality control (QC). We first excluded the known malaria susceptibility associated loci and performed stepwise extra QC filtering. Specifically, we focused on sample relatedness, SNP missingness proportion and SNP differential missing proportion which are well-known to affect the accuracy of heritability estimation . We thenAlthough our heritability estimation is fairly stable when stringent QC is implemented, care should be taken in interpreting these results: First, all polygenic methods perform better in less structured data obtained from homogenous populations than the MalariaGen dataset which is comprised of diverse populations spanning most of the Malaria endemic belt in Africa. Second, the methods are designed and perform well in highly polygenic traits/diseases in which effects of each variant is mostly modest. In contrast, a considerable proportion of malaria protection trait might be attributed to rare variants of large effect sizes that might not be in LD with common variants and can’t not be ‘tagged’ by SNPs chip which means that the contributions of such variants will not be accounted for. Third, subtle population structure that cannot be corrected by conventional methods such as unmatched case/control can potentially create systematic biases to the estimates.Epistasis is becoming one of the hot research topics in genetic epidemiological studies in the last few years because of the fact that none additive genetic variations are shown to have significant influence on the phenotype of complex traits/diseases than previously expected . The avaHbS) and α thalassaemia were shown to demonstrate negative epistatic interactions such that the protection against severe malaria offered by HbS is reduced when co-inherited with α+thalassaemia [Epistasis between malaria risk loci have been well documented and implicated as one of the possible reasons for lack of replication of susceptibility variants in different populations and the ‘missing’ heritability. For instance, sickle-cell trait  variants in predicting the risk of severe malaria [α+thalassaemia and Hp2–1 variant synergistically increase the protection against severe malaria by about 37%. However, the protective effect is decreased to 13% when α + thalassaemia is inherited with Hp1–1 and further diminished to neutral (zero) when inherited with Hp2–2. Similarly, in a multi-center case control study, the existence of negative epistasis interaction between HbC and ATP2B alleles was reported [A case-control study in Kenyan population also reported that  malaria . In thisreported .S12 and α+thalassaemia in which the protective effect of S12 higher in children with normal α-globin [Another recent case control study of severe malaria in Kenya reported the existence of negative epistasis between a compliment receptor called α-globin . The extThe ultimate goal of genetic susceptibility studies is to identify causal variants and understand the underlying biological pathways which can lead to translated medicine such as effective vaccines and therapeutics. However, translating GWAS signals in to biological themes remains an open problem because of the confounding effects from LD between association SNPs, limited knowledge of gene functions and localization of the majority of GWASs hits in none protein coding regulatory regions (regulatory SNPs) –93. In aBCL11A, β-globin and HBS1L-MYB), undertook dense genotyping, performed step-wise conditional analysis and revealed previously un-recognized SNPs that explain additional genetic variation. Similarly, a recent fine mapping study of HLA region identified several susceptibility loci for multiple infectious diseases [Alternatively, several fine mapping statistical tools have recently been developed following the advances in annotation data bases and improved reference panels. These include Bayesian approach, heuristic approach and penalized regression methods . The pridiseases . More sodiseases .In addition to fine mapping approaches, pathway and interaction analysis can be another avenue for exploring molecular basis of Malaria susceptibility/resistance. Instead of emphasizing on single-variant analysis, these approaches test the coordinated effects of several variants at systems level using biological information from annotation data basis . PathwayP. falciparum infected individuals from Fulani, an ethnic group that is less susceptible to malaria [Epigenetics refers to heritable phenotype changes that do not involve alterations in the DNA sequences such as methylation, post-translational histone modification, histone variation, chromatin remodelling and non-coding RNAs . Epigene malaria . The aut malaria .However, the majorities of epigenetic studies including those of malaria susceptibility and resistance have been limited to either small sample sizes or inadequate genome coverage and thus, lack adequate power to decipher the epigenetic impacts on complex diseases . In effoDUP4 which reduces the risk of severe malaria by about 33% . In addition to this, a genome-wide gene expression study was conducted in Kenyan children and reported increased expression of genes related to neutrophil activation during malaria infections [Today, there are significant advances in high throughput technologies that can generate big ‘-omic’ data from all spectrum of molecular biology . The ‘-ofections . The autP. falciparum parasites, as well as fusion of these human miRNAs with parasite mRNA transcripts to inhibit the translation of enzymes that are vital for the parasite development. Specifically, two micro-RNAs, miRNA-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes and these miRNAS along with miR-223 were shown to attenuate the growth of parasite [In another host-parasite interaction study, the importance of miRNA in inhibiting parasite growth in erythrocyte was reported . The autparasite .However, ‘-omics’ studies are limited to single data-type analysis and lack adequate power to explain the complexity of molecular processes and usually lead to identification of correlations than causations . Thus, i. [Multi-omics approaches aim to integrate big ‘-omics’ data, undertake ‘multi-step’ and ‘multidimensional’ analysis for elucidating complex biological problems  . Driven. Malaria susceptibility and resistance is influenced by several host, parasite and environmental factors as depicted in Fig. The ultimate goal of malaria susceptibility study is to discover a novel causal biological pathway that provide protections against severe malaria; a fundamental step towards translational medicine such as development of vaccine and new therapeutics that can facilitate the global malaria eradication efforts. To achieve this goal, various study approaches have been implemented at least for the last three decades and successfully identified several association variants.Recently, a number of GWASs have been implemented in malaria endemic areas to better understand the underlying biology. While some of the well-known variants were replicated, only few novel variants were convincingly identified and their biological functions remains to be understood. Several limiting factors including genetic diversity of population in malaria endemic areas, allelic heterogeneity of protective variants, small sample sizes, lack of proper reference panel and proper genotyping chips might have negatively impacted the malaria GWASs.Another challenge is that we don’t know much about the genetic architecture of malaria protective trait. There are at least two scenarios in which GWAS approach might fail; First, malaria protective trait might largely be attributed to rare variants of large effect sizes that might not be in LD with common variants and can’t be captured by the GWAS approach. Second, malaria might have left multiple genetic variants distributed across human genome; the majority of which have effects too small to be detected by the standard GWASs , 7. TheoOn the other hand, the recent advances in statistical techniques is enabling to extract useful information from the present-day GWAS sample sizes. For example, a number of statistical approaches have been developed to capture polygenicity in complex diseases. We showed how these methods can potentially be implemented in malaria susceptibility studies and provide useful insights. We believe that further studies with larger sample sizes can elucidate the polygenic effects in malaria protective trait by extending the analysis to genome partitioning, risk prediction and genetic correlations.Beyond singe locus analysis, multi-step and multi-locus analyses including pathway analysis, fine mapping and interaction analysis can potentially be implemented in malaria susceptibility GWASs to gain new insights to the underpinning biology. For instance, pathway analysis can provide important information by analyzing the coordinated effects of several variants at systems level using biological information from annotation databases. Methods that combine statistical and functional fine-mapping strategies can potentially be implemented to pinpoint the causal variants from the GWAS association signals.Most importantly, the future direction of malaria susceptibility requires a paradigm shift from single ‘-omics’ to multi-stage and multi-dimensional integrative functional studies that combines multiple data types from the human host, the parasite, the mosquitoes and the environment. The current biotechnological advances, an ever-increasing annotation data bases and availability of advanced analytical techniques, will eventually lead to feasibility of systems biology studies and revolutionize malaria research.


This study determined the circulating levels of TGF-β1 and its association with aortic dilation and elastic properties in congenital heart patients. Forty-six patients after tetralogy of Fallot (TOF) repair, 21 patients post arterial switch and 15 patients post atrial switch for transposition of the great arteries (TGA), 27 patients post Fontan procedure, and 36 controls were studied. Aortic dimensions and elastic properties and ventricular function were assessed by echocardiography. Serum TGF-β1, metalloproteinase (MMP)-2 and MMP-9 levels were quantified. Compared with controls, all groups of patients had significantly larger ascending aortic dimensions and worse elastic properties . Aortic stiffness correlated positively with sinus dimension  and negatively with indices of ventricular deformation . Patients with repaired TOF had significantly higher levels of TGF-β1 (p = 0.005), MMP-2 (p = 0.001) and MMP-9 (p < 0.001) than controls, while patients after atrial switch operation (p = 0.034) and Fontan procedures (p < 0.001) had higher MMP-2 levels. In patients as a group, circulating TGF-β1 levels correlated with MMP-9  and aortic sinus dimension . In conclusion, increased circulating TGF-β1, MMP-2, and MMP-9 levels were found in patients with repaired TOF, and increased circulating MMP-2 levels were also evident in patients after atrial switch operation and Fontan procedure. While alteration of haemodynamics has been hypothesized to cause aortic dilation, there is evidence of a primary aortopathy4. Histological abnormalities of the aortic root as characterized by medionecrosis, fibrosis, and fragmentation of elastic fibres have been found in different congenital heart conditions including tetralogy of Fallot (TOF) with or without pulmonary atresia, complete transposition of the great arteries (TGA), and functionally univentricular hearts5. Functional alteration of aortic elastic properties has further been demonstrated in these patients9. The interplay between functional and structural abnormalities may result in a feedback loop that leads to progressive aortic dilation. A better understanding of vascular remodeling in these congenital heart patients may shed light on the management of progressive aortic root dilation and its complications.Progressive dilation of the aortic root has become a growing concern in the adolescent and adult congenital heart population, and it is associated with aortic regurgitation, stiffening, and dissection10. In a mice model of Marfan syndrome, deficiency of normal fibrillin may lead to the release of sequestered latent form of TGF-β1, increasing its activity11. Importantly, increased circulating levels of TGF-β1 found in patients with Marfan syndrome have been shown to be associated with the magnitude of aortic root dilation, the occurrence of cardiovascular events including aortic dissection, and the need for aortic root replacement13. Interestingly, overexpression of TGF-β1 has also been reported in the ascending aorta of congenital heart disease14. Nonetheless, the clinical relevance of circulating levels of TGF-β1 in congenital heart patients who are at risk of aortic root dilation is unknown.The importance of the transforming growth factor-beta (TGF-β) superfamily of cytokine in vascular remodeling is increasingly recognized. In aortic aneurysms related to bicuspid aortic valves, the altered TGF-β1 signaling activity may increase production of matrix metalloproteinase (MMP) −2 and −9 and lead to matrix degradationGiven the increasing evidence of involvement of TGF-β1 signaling in aortic remodeling, we hypothesized that circulating TGF-β1 levels are altered in patients with repaired congenital heart disease at risk of ascending aortopathy. In this study, we aimed to determine the circulating levels of TGF-β1 and to explore their associations with aortic dilation and aortic elastic properties in adolescents and young adults after TOF repair, arterial and atrial switch operations for complete TGA, and Fontan procedure for univentricular hearts.The inclusion criteria of the study was adolescent and adult congenital heart patients who had undergone surgical repair of TOF, arterial switch operation and atrial switch operation for complete TGA, and Fontan procedure for functionally univentricular hearts. They were consecutively recruited from our congenital heart clinic. The following clinical data were retrieved from the case notes: age at operation, follow-up duration, associated cardiac lesions, and residual postoperative cardiac lesions. Healthy subjects, who are staff members of the hospital and their relatives and friends, with no known cardiovascular disease were recruited as controls. None of the controls are family members of patients. This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster, Hong Kong, and all of the methods as described were performed in accordance with the approved guidelines and regulations. All of the adult subjects and parents of minors gave written informed consent.The body weight and height of the subjects were measured and the body mass index and body surface area were calculated accordingly. All of the subjects rest for at least 15 minutes before vascular and echocardiographic assessments as described below.Vascular and echocardiographic assessments were performed using the Vivid 7 ultrasound machine . The average values of the echocardiographic indices from three cardiac cycles were obtained.The size of the aortic root was measured at diastole from the parasternal long-axis view at four levels: annulus, sinus of Valsalva, sinotubular junction, and proximal ascending aorta. Severity of aortic regurgitation was assessed semi-quantitatively by colour Doppler flow mapping.15. Systolic (AoS) and diastolic (AoD) dimensions of the proximal ascending aorta were measured, while systolic (SBP) and diastolic (DBP) blood pressures of the right upper arm were obtained using an automated oscillometric device . The following parameters of aortic elastic properties were calculated: (i) strain = (AoS-AoD)/AoD x 100%, (ii) distensibility = [2 × (AoS-AoD)])/[AoDx(SBP-DBP)], and (iii) stiffness index = ln(SBP/DBP)/[(AoS-AoD)/AoD].Elastic properties of the ascending aorta were determined using M-mode echocardiography performed from the parasternal long-axis view6. Briefly, oscillometric cuffs were wrapped around the brachium and ankles to register the pressure pulse and the right and left brachial-ankle pulse wave velocities were calculated by dividing the pulse wave traveling distance by the transit time. The traveling distance was determined automatically based on the height of the subject.For assessment of regional arterial stiffness, the brachial-ankle pulse wave velocity was determined using an automated device To assess the potential impact of altered aortic elastic properties on systemic ventricular function, echocardiographic assessment of the systemic ventricle was determined by tissue Doppler imaging and two-dimensional speckle tracking echocardiography.s) myocardial tissue velocity, peak early (e) and late (a) diastolic myocardial tissue velocities, e/a ratio, and myocardial acceleration during isovolumic contraction. For Fontan patients, the average of the right- and left-sided annular tissue Doppler velocities was used for analysis.Tissue Doppler imaging was performed with the sample volume positioned at the basal systemic ventricular free wall-valvar annular junction to obtain the following indices: peak systolic .Global longitudinal and circumferential strain and strain rate of the systemic ventricle were assessed by two-dimensional speckle tracking echocardiography as reported previouslyBlood samples were collected by venepuncture and serum samples were stored at −80 °C until assay. Total TGF-β1 level was measured by commercially available assay . Serum levels of MMP-2 and MMP-9 were also determined using enzyme-linked immunosorbent assays . These assays measured respectively the total MMP-2 levels and total pro- and active MMP-9 levels.All data are presented as mean ± SD. The aortic dimensions were measured by a single investigator (EKS) to avoid inter-observer bias. Comparisons of cardiovascular indices and circulating levels of TGF-β1, MMP-2, MMP-9 among different groups were performed using simple analysis of variance. Comparisons of cardiovascular and blood indices between controls and each of the patient cohorts were performed using unpaired Student’s t test. Relationships between TGF-β1 and MMP levels and those between TGF-β1 and aortic dimensions and elastic properties were assessed by Pearson correlation analysis. Multivariate analysis that takes also into account of the age, sex, type of congenital heart disease, aortic sinus dimension and stiffness was performed to identify significant correlates of the circulating levels of TGF-β1. A p value of <0.05 is considered statistically significant. All statistical analyses were performed using SPSS Statistics 19 .Table Compared with controls, patients as a group had similar body weight (p = 0.46), body height (p = 0.46), body mass index (p = 0.35), and body surface area (p = 0.45).Table The prevalence of aortic regurgitation was 76% and 65% in patients after arterial switch operation and repair of TOF, respectively. On the other hand, the prevalence was lower in Fontan patients and in patients after atrial switch operation at 48% and 40%, respectively Table .For the aortic elastic properties, all of the four patient groups had significantly lower aortic strain  and aortic distensibility  and greater aortic stiffness index  than controls , distensibility , and stiffness index  correlated significantly with aortic sinus dimension.s and e velocities and systemic ventricular IVA .Table Strain imaging revealed significantly lower global ventricular systolic longitudinal strain and strain rate and early and late diastolic strain rates in all four groups of patients compared with controls . On the other hand, the circumferential strain was similar between all patients and controls .s  and e  velocities.For the entire cohort, aortic stiffness correlated negatively with systemic ventricular global longitudinal systolic strain , systolic strain rate , and early diastolic strain rate   Table . PatientIn all patients, the circulating TGF-β1 levels correlated significantly with MMP-9  but not MMP-2 (p > 0.05) levels. Furthermore, the circulating levels of TGF-β1 in patients as a group were found to correlate significantly, albeit weakly, with the size of the aortic sinus   of circulating TGF-β1 levels after adjustment for other congenital heart disease categories, age, sex, aortic sinus dimension, and aortic stiffness index.The main findings of the present study are (1) significant elevation of circulating levels of TGF-β1, MMP-2, and MMP-9 in patients with repaired TOF, (2) a positive correlation between circulating levels of TGF-β1 and MMP-9, and (3) an association, albeit relatively weak, between circulating TGF-β1 levels and the aortic sinus dimension in the four groups of congenital heart patients studied. Furthermore, increased aortic stiffness in the four groups of patients was found to be associated with greater aortic root dilation and worse systemic ventricular systolic and diastolic function. To our knowledge, this is the first study to explore circulating TGF-β1 and MMP levels in adolescents and young adults with congenital heart disease and their relationships with aortic structure and function.17. Alteration of TGF-β1 signaling has been shown in bicuspid aortopathy and recognized as a key component in the pathogenesis of thoracic aortic aneurysms10. Single nucleotide polymorphism of genes, including TGF-β1, involved in the maintenance of the extracellular matrix of the arterial wall has been associated with the risk of development of intracranial aneurysms18. In Marfan syndrome, deficient amount of fibrillin has been proposed to increase the release of sequestered TGF-β1 and hence its tissue activity in a mouse model11, although it remains unclear whether it is the structural deficiency or functional deficiency in fibrillar protein that triggers the pathogenesis of aortic aneurysms. Increased circulating levels of TGF-β1 have been found in the mouse model and in patients with Marfan syndrome12. In patients with Marfan syndrome, the circulating TGF-β1 levels are associated with aortic root dilation13.Given the central role of TGF-β1 in vascular morphogenesis and extracellular matrix homeostasis, there is an increasing interest in the role of TGF-β1 in vascular remodeling in patientset al. described overexpression of TGF-β1 with concomitant moderate fragmentation of elastic fibres14. It is possible that leaching of the aortic tissue TGF-β1 into the circulation, as hypothesized in the mouse model of Marfan syndrome12, might have accounted for our findings in repaired TOF patients, although this remains speculative. While subgroup analysis showed mild elevation of TGF-β1 levels in patients after atrial switch operation and Fontan procedures, the increase was not statistically significant. Nonetheless, increased circulating MMP-2 levels were found to be increased in these three groups of patients, and increased MMP-9 levels was also found in repaired TOF patients 23 and angiotensin-receptor blocker (ARB)24 to slow progression of aortic root dilation through modulation of the TGF-β1 signaling pathway in Marfan syndrome are encouraging. Doxycycline, which inhibits MMPs25, has been shown to reduce the progression of abdominal aortic aneurysms in a mouse model of Marfan-associated aneurysm26. Taken together, our findings provide a basis for trials to explore the potential usefulness of ACEI, ARB, and doxycycline in patients after TOF repair, atrial switch operation, and Fontan procedure with aortopathy. Indeed, ACEI27 and ARB28 may further benefit our patients through reduction of the aortic stiffness. While we did not find associations between indices of aortic elastic properties and the circulating levels of TGF-β1 and MMPs in patients as a group, the adverse effects of arterial stiffening on aortic root dilation and ventricular dysfunction have been shown in the present and previous studies31.An evidence-based approach to the management of aortopathy in congenital heart disease is lacking32. Nonetheless, the reported strong correlations between MMP-2 and -9 levels measured in serum and in plasma33 provide support on the validity of the interpretation of our findings. Fourth, echocardiographic imaging of the anterior retrosternal aorta in patients with TGA after atrial switch operation may be difficult. Finally, we did not assessment the lifestyle, physical activity levels, and cardiovascular risk factors in patients and controls. These factors are of relevance when interpreting the levels of circulating MMPs35 and should be included in subsequent studies.Several limitations to this study require comments. First, this cross-sectional study cannot provide data on the trajectory of circulating levels of TGF-β1 and MMPs in all patients since surgery. Longitudinal studies are required to determine the prognostic value of these circulating markers and the effect of therapies that modulate the TGF-β1 signaling pathway or the activity of MMPs. Second, the relatively small number of patients in each of the patient cohorts might have limited the power to detect small differences in circulating TGF-β1 levels. However, the finding of increased MMPs in different patient groups, except for patients after arterial switch operation, supports the proposition of associations between alteration of TGF-β1 signaling and its downstream pathway and aortopathy. Nonetheless, the relatively weak correlation between TGF-β1 level and aortic sinus dimension suggests that other factors are also contributing to aortic root dilation in these congenital heart patients. A larger scale study is undoubtedly required to confirm our initial findings. Third, the origin of the circulating TGF-β1 and MMPs is unclear. Further studies are required to study the relationships between the expression of tissue TGF-β1 and MMP in the dilated aortic root of congenital heart patients and the corresponding circulating levels. Overlapping of TGF-β1 and MMP levels between all patients and controls may perhaps be related to the mild degree of aortic root dilation in our young patient cohorts. Serum MMP levels have been found to be higher than the corresponding plasma levels, related possibly to the release of MMPs from blood cellIn conclusion, increased circulating levels of TGF-β1, MMP-2, and MMP-9 are found in patients with repaired TOF, and increased circulating levels of MMP-2 are also evident in patients with complete TGA post atrial switch operation and in those with functional single ventricles after Fontan procedure. Trials of therapies that target at the TGF-β1 pathway and MMPs for the management of aortopathy in these congenital heart patients are warranted.


Recent studies of the patterns of chemokine-mediated immune cell recruitment into solid tumors have enhanced our understanding of the role played by various immune cell subsets both in amplifying and inhibiting tumor cell growth and spread. Here we discuss how the chemokine/chemokine receptor networks bring together immune cells within the microenvironment of skin tumors, particularly melanomas, including their effect on disease progression, prognosis and therapeutic options. Chemokines and their receptors are an intrinsic part of immune cell trafficking. They orchestrate cell-to-cell interactions during normal immune responses and are fundamental for directing immune cells to sites of inflammation . ChemokiChemokine receptor-ligand pairs play a key role in tumorigenesis in addition to infection and inflammation –4. WithiTumors can be broadly divided into T cell-inflamed and non-inflamed tumors . T cell-In addition to their primary role as chemoattractants, chemokines are involved in other tumor-related processes and may be exploited by tumors to promote tumor cell growth , angiogeInnate immune cells, among them DCs, macrophages, neutrophils and NK cells form an important part of the tumor immune milieu. Their role in skin cancers can be 2-fold: either they can mediate direct anti-tumor toxicity through promoting cytotoxic T cell recruitment and activation or they can contribute to inhibition of anti-tumor responses through inhibitory interactions with other cells and secretion of suppressive molecules .DCs are not only the professional APC responsible for activating naïve T cells in secondary lymphoid tissue , but canMacrophages are also frequently found in solid tumors including melanomas where they may have dual roles leading to their classification into anti-tumor M1 and inhibitory M2 subtypes . M2 macrThe macrophage chemoattractant CCL2 is expressed on melanoma cells  and its Neutrophils are the third member of the innate immune cell repertoire to play a vital role in skin cancer , 53. TheMelanoma grown in mice lacking CXCR2 display reduced inflammation, neutrophil recruitment and tumor growth . NotablyNK cells are innate lymphocytes with a crucial role in anti-viral and tumor defense –59. TheiT cells have crucial roles to play in controlling certain infections and cancers including those located in the skin. The presence of CD8+ T cells in melanomas –63 as weOne of the earliest studies to link chemokine expression in human melanoma to CD8+ T cell infiltration used gene expression arrays to identify CCL2-5 and CXCL9 as preferentially expressed in T cell rich patients' tumors , 29. TheExpression of CCR5 and CXCR3  on CD8+ T cells has emerged as another important regulator of effector T cell recruitment and prognosis in melanoma. For instance, higher expression of CCR5 and CXCR3 was associated with increased T cell infiltration of tumors, lower relapse rates  and incrThe role of other chemokines involved in T cell migration into melanoma is less well defined. On the one hand, CXCL12 has been detected in normal and neoplastic tissue  and lossCCL5 expression in a murine model of spontaneous melanoma correlated with CD3γ expression and T cell recruitment, while increased level of CCR5, the receptor for CCL5, was associated with positive outcomes in melanoma models due to T cell retention in tumors . FurtherMycobacterium bovis bacillus Calmette-Guérin injection in melanoma patients increased recruitment of γδ T cells via CXCL9-11 and CXCR3  in uncovering chemokine-mediated cellular interactions in melanoma and mechanisms of resistance to immunotherapy –93. For Immune evasion by skin cancers like melanoma represents one of the mechanisms whereby they proliferate and metastasize . ImmunosBlocking infiltration of cytotoxic effector cells into tumors is another possible mechanism of evasion and may be one of the reasons why checkpoint inhibitors are not always effective for melanoma treatment . EvidencThe clinical importance of chemokine-dependent immune cell recruitment into tumors is illustrated by the most recent advances in cancer immunotherapy, in particular checkpoint inhibitor therapy , 98, whiAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.