Recycling waste tire rubber by incorporating it into concrete has become the preferred solution to dispose of waste tires. In this study, the effect of the volume content of crumb rubber and pretreatment methods on the performances of concrete was evaluated. Firstly, the fine aggregate and mixture were partly replaced by crumb rubber to produce crumb rubber concrete. Secondly, the mechanical and durability properties of crumb rubber concrete with different replacement forms and volume contents had been investigated. Finally, the crumb rubber after pretreatment by six modifiers was introduced into the concrete mixture. Corresponding tests were conducted to verify the effectiveness of pretreatment methods as compared to the concrete containing untreated crumb rubber. It was observed that the mechanical strength of crumb rubber concrete was reduced, while durability was improved with the increasing of crumb rubber content. 20% replacement of fine aggregate and 5% replacement of the total mixture exhibited acceptable properties for practical applications. In addition, the results indicated that the modifiers had a positive impact on the mechanical and durability properties of crumb rubber concrete. It avoided the disadvantage of crumb rubber concrete having lower strength and provides a reference for the production of modified crumb rubber concrete. The number of waste tires is continually increasing, as a result of the growing use of transport vehicles . Almost Several methods of recycling waste tires have been proposed, including use as a fuel in cement kilns and to produce carbon black ,6. TheseConcerning the reuse of waste rubber in concrete, extensive studies had been conducted ,12,13. TThe properties of crumb rubber concrete were significantly affected by rubber content. Ghaly and Cahill  studied et al. [In order to minimize the loss in strength caused by introducing crumb rubber into concrete, prior surface treatment of rubber particles by modifiers was utilized . Mohammaet al.  pretreatet al.  adopted et al.  mentioneThis paper investigates the mechanical and durability properties of concrete containing waste crumb rubber. The rubberized concrete was produced by replacing the fine aggregate and mixture with crumb rubber at different volume ratios. Compressive strength, splitting tensile strength, axial compressive strength, the modulus of elasticity, freezing-thawing resistance and sulfate resistance were evaluated for concretes with different contents of crumb rubber. In addition, the crumb rubber was pretreated in six modifiers. Additionally, the effects of modifiers on the properties of crumb rubber concrete were comparatively analyzed and discussed.Composite Portland cement of Grade 32.5  with a specific gravity of 3.12, conforming to GB 175-2007 , was useCrumb rubber with the particle size (2–4 mm) specially used to pave a rubber runway was selected as the replacement material, as shown in Modifiers for the pretreatment of crumb rubber, including emulsion, ethoxyline resin, synthetic resin, amino-acrylate (contact glue), chloroprene adhesive and unsaturated resins (marble glue), were commonly used in decoration and construction engineering. They had satisfactory performance in improving adhesion and enhancing strength. The crumb rubbers after pretreatment by modifiers are shown in This study consisted of one plain concrete as the control and 9 crumb rubber concretes. All of the concretes were designed at a constant water-cement ratio of 0.42. Crumb rubber was used as the replacement for an equal part of fine aggregate and mixture. Considering the different specific gravities of crumb rubber and mineral materials, the replacement with crumb rubber was conducted based on the volume other than weight ,30. The Concrete specimens were produced as per the JTG E30-2005 standard . Mixing Compressive strength, splitting tensile strength, axial compressive strength and the modulus of elasticity of hardened concrete were measured as per GB/T 50081-2002 . Comprescnf is the compressive strength of concrete experiencing twenty-five cycles of freezing and thawing. Additionally, cf0 is the compressive strength of the control concrete.Concrete specimens of 150 mm in size cured for 90 days were used to test the durability (freezing-thawing resistance and sulfate resistance) according to GB/T 50082-2009 . The resfK is the anti-corrosion coefficient; cnf is the compressive strength of the tested concrete after fifteen cycles of drying and soaking; cf0 is the compressive strength of the control concrete.In the case of sulfate resistance, the specimens were immersed in sulfate solution. Then, the waterish specimens were dried at a constant temperature of 80 ± 5 °C. The anti-corrosion coefficient of concrete after fifteen cycles of drying and soaking was calculated as Equation (2).The mechanical properties and durability of concrete were significantly affected by introducing crumb rubber into concrete. In this paper, the variation of properties in concrete with the changing of rubber content was investigated. Additionally, the effect of modifiers was evaluated.The mechanical properties of crumb rubber concrete were tested and are listed in The splitting tensile strength of crumb rubber concrete cured at 28 days was measured, as shown in The reduction in axial compressive strength was similar to the compressive strength. It decreased with the increasing of the percentage content of crumb rubber. When the mixture was replaced by crumb rubber, the axial compressive strength significantly decreased as compared to the concrete with fine aggregate replacement. There was a strong correlation between the axial compressive strength and the replacement volume of crumb rubber.The modulus of elasticity represented the deformation capacity of concrete. The test photograph is shown in There are many reasons accounting for the lower strength of crumb rubber concrete . FirstlyFreezing-thawing resistance and sulfate resistance are the important aspects of the durability of concrete. In this paper, the freezing-thawing resistance and sulfate resistance of crumb rubber concrete were investigated. Additionally, the effect of crumb rubber content on freezing-thawing resistance and sulfate resistance was analyzed and discussed.Crumb rubber concrete cured for 90 days was set in the refrigerator for freezing, as shown in The crumb rubber concrete after sulfate corrosion is illustrated in The crumb rubber particles in the rubberized concrete can prevent the formation of cracks and material separation. This may be the reason for the good performance to resisting freezing-thawing and sulfate attack for crumb rubber concrete ,35.As can be seen from the above study about the mechanical properties and durability of crumb rubber concrete, 5% replacement of the total volume of mixture resulted in a high decrease of strength. From a practical viewpoint, the rubber content should not exceed 5% of the total volume of the mixture due to the negative effect on strength. Therefore, crumb rubber concrete with 5% replacement of total mixture was selected to investigate the effect of modifiers on the properties of rubberized concrete.Modified crumb rubber concrete was produced by introducing crumb rubber after pretreatment using modifiers. Compressive strength, splitting tensile strength and axial compressive strength were tested. Compared to the measured strength of unmodified crumb rubber concrete, the percentage was calculated, and corresponding results are listed in The effect of pretreatment methods on freezing-thawing resistance and sulfate resistance was investigated. Additionally, the testing results are listed in Crumb rubber concrete prepared with modified rubber had an obvious improvement in mechanical properties and durability compared to untreated rubber, which could be the result of better bonds between rubber particles and cement paste. Additionally, the treated rubber by modifiers could block the early initiation of internal cracks, which enhanced the freezing-thawing resistance and sulfate resistance . ComprehIn this paper, crumb rubber concretes with different replacement forms and replacement levels were produced. The effect of the volume content of crumb rubber and pretreatment methods on the performances of concrete was investigated. The following conclusions have been obtained.(1) Adding crumb rubber into concrete resulted in a significant decrease of the mechanical properties, but increased the durability. The effect caused by replacing the mixture with crumb rubber was higher than that caused by fine aggregate replacement.(2) Compressive strength, splitting tensile strength, axial compressive strength and the modulus of elasticity were reduced with the increasing percentage content of crumb rubber, while freezing-thawing resistance and sulfate resistance were improved. A 20% replacement of fine aggregate and a 5% replacement of the total mixture with crumb rubber met the safety strength requirements of concrete and had excellent durability.(3) The negative effect of crumb rubber on mechanical strength could be minimized and avoided by pretreatment of the crumb rubber using modifiers. These pretreatments enhanced the adherence between the rubber and cement paste and achieved the uniform distribution of rubber particles in mixture.(4) Synthetic resin significantly improved the mechanical and durability properties of crumb rubber concrete as compared to the other modifiers. The compressive strength, splitting tensile strength and axial compressive strength of crumb rubber concrete prepared with pretreated rubber using synthetic resin increased 12%, 40% and 5%, respectively. Additionally, the strength loss ratio after 25 cycles of freezing-thawing was reduced to 1.2%.


An in-situ filtration sampler has been developed for use in measuring trace metals in precipitation. This sampler is a modification of the Aerochem Metrics Model 301 wet/dry precipitation sampler now in use in the National Atmospheric Deposition Program/National Trends Network (NADP/NTN). The sample is captured in a funnel and filtered directly into a precleaned and preweighed collection bottle. Upon collection, the bottle can be weighed to determine sample volume and immediately preserved with the appropriate amount of acid. Samples collected in this manner were compared to those collected in high density polyethylene (HDPE) buckets according the standard NADP/NTN protocols for major ion sampling. These bucket-collected samples were filtered in the laboratory at different times throughout a week to approximate time lapses normally present in a large scale monitoring study. The metals investigated were aluminum, cadmium, copper, iron, lead, manganese, and zinc. Graphite furnace atomic absorption spectrophotometry was the method of analysis.Filtration of aqueous samples followed by acidification has been shown to be necessary to preserve the natural distribution of metals since the particles present in the sample may adsorb or desorb metal ions rapidly . Immediaμm polycarbonate membrane filter is housed in a tetrafluoroethylene (TFE) filter holder. The samples are collected directly into a 2-liter polyethylene bottle. After collection, the bottle is weighed and the sample is preserved with the appropriate amount of ultrapure nitric acid.The in-situ filtration system consists of a funnel and bottle assembly with an in-line filter. The top opening of the funnel is the same diameter as the standard buckets (30 cm), so that the catch area is the same. The HDPE funnel is connected to the fluorinated ethylene propylene (FEP) tubing via a polypropylene cone. The 0.4 The counterweight bar was cut and the middle portion removed, so that tubing could be connected from the collection funnel to the receiving bottle.The bucket was shortened by 2.5 cm (off the top) to allow the funnel to nest at the top of the bucket. This was necessary to allow full closure of the sampler lid during dry periods.A hole was cut in the bottom of the bucket for passage of the polypropylene cone and FEP tubing.The Aerochem Metrics Model 301 wet/dry precipitation sampler was modified as follows:μg/L. The maximum concentrations found in the in-situ filtration system blank leachates were much lower for iron and zinc.Direct comparisons between this system and the bucket collection system involved blank leachates and natural wet deposition samples. Blank leachate results for the two systems are shown in To check for adsorption of metals onto the collector and/or filter surfaces, spike recoveries were determined for the in-situ filtration system. Results are presented in The gravity filtration system evaluated in this study has some limitations. Precipitation with a high particulate load may require a pumping system, and below freezing temperatures necessitate the use of a heating system and/or insulation. Further research into these areas is planned.The advantages of in-situ filtration are: preservation of the natural distribution of metals, decreased sample handling, and the inexpensive modification of available equipment.


Vascular cognitive impairment/vascular dementia have been the subject of a largenumber of studies, due to their high prevalence and broad preventive andcompensatory therapeutic potential. The knowledge of the cerebral anatomycorrelated to the vascular territories of irrigation enables understanding ofclinical manifestations, as well as classification into the several types ofsyndromic presentations. The central cholinergic system exercises importantneuromodulatory functions on cerebral circuits related to cognitive andbehavioral integration, as well as on vasomotor control related to cerebralblood flow adjustments. The acquisition of data on the anatomy of thecholinergic pathways, including the localization of the nuclei of the basalprosencephalon and the routes of their projections, established an importantmilestone. The knowledge of the vascular distribution and of the trajectories ofthe cholinergic pathways allows identification of the strategic points where avascular lesion can cause interruption. The ensuing denervation leads tocholinergic hypofunction in the involved territories. This information provesimportant to better evaluate the sites of vascular lesions, emphasizing theirstrategic localizations in relation to the cholinergic pathways, and offeringmore robust foundations for treatment aiming at enhancing cholinergicactivity. This knowledge underpins the widely known cholinomimetictreatment strategy, with the efficacious use of cholinesterase inhibitors5.Lesions of the BP and/or of the cholinergic pathways can be found at varied points oftheir course in VaD. The same can be seen in mixed presentations, the most commonlydescribed being AD+CVD and MD (AD+VaD)The knowledge of the cholinergic system, both in normal and pathological states, isimportant to fully understand how the cholinergic treatment strategy works in VaD andwhat benefits it offers.7.The central cholinergic system is made up of several clusters of neurons distributedacross different levels of the brain. The BP lies in the basal part and comprisesfour clusters or groups of cholinergic neurons, the large nucleus basalis of Meynert(nbM) being among them. The others include the medial nucleus of the septum (nmS)and the nuclei of the diagonal band of Broca, along with the vertical (ndbBvl) andthe horizontal (ndbBhl) limbs8.The groups of cholinergic neurons in these nuclei are named according to the Chnomenclature, and are found in nmS (Ch1), in ndbBvl (Ch2), in ndbBhl (Ch3), and innbM (Ch4)10. The nbM has about 200 000 neurons in eachhemisphere, subdivided into sectors related with particular cortical areas, inapproximately a mediolateral and anteroposterior topography15.The nmS plus ndbBvl comprise about 20 000 neurons, with 3 200 cholinergic neurons, ineach hemisphere6.All cholinergic neurons express acetylcholinesterase (AChE) and cholineacetyltransferase (ChAT). The Ch1- Ch4 clusters differ by the presence of neurons(about 90%) containing the nerve growth factor receptor (NGFr), tirosine kinase(TRKa) and the neurotrophine receptor (p75NTR), not found in cholinergic neurons atother levels20 and to vasomotor control.The central cholinergic system exercises important functions includingneuromodulation of brain circuits related to cognitive and behavioralintegrationVasomotor control is related to modulation of brain blood flow, exerted through twomechanisms:21andcircumscribed enhancement of perfusion related to increased neuralactivity in a given area caused by cholinergic stimulation,corresponding to ‘functional hyperemia’ resulting from neurovascularmetabolic coupling25.vasodilator action on arteries of varied caliber, mainly on terminalramifications  accomplished through muscarinicreceptors localized close to astrocytic terminations (gliovascularcomplexes) with liberation of nitric oxide to the smooth muscular fibersand pericytes29. An increase of perfusion was also seen in normal subjectsand patients with AD or VaD with PET and SPECT imaging related to cholinergicintervention (use of cholinesterase inhibitors)34.Vasomotor control has been studied in animal models, where vasodilatation was shownby cholinergic stimulationThus, this double activity, tissular and vascular, makes the cholinergic systemimportant in normal functional condition. On the other hand, its hypofunctionbecomes an important target for interventions aiming to enhance its modulatoryactivity.39.The projections from the BP cholinergic groups are directed toward severalsubcortical and cortical brain regionsThe projections to the hippocampal formation and entorhinal cortex originate mainlyfrom Ch1-Ch2 and have a route that accompanies the fornix. The terminals reachmainly the CA2-CA4 sectors of the hippocampus and the dentate gyrus, with a lesserdensity to sector CA1 and subiculum.The Ch3 group is directed to olfactory areas, reached through the medialprosencephalic fascicle.The projections to other regions of the cortex originate in the Ch4 group andconstitute two bundles, the medial and the lateral. Fibers detach from these bundlesand supply subcortical regions and cerebral cortex.medial cholinergic pathway originates from the nbM, passesthrough the white matter of the straight and medial orbital gyri, around the rostrumof the corpus callosum and accompanies the cingulum bundle until the splenium, whereit continues to the retrosplenial white matter. This pathway supplies ramificationsto the medial orbitofrontal, subcallosal, cingulate, pericingulate, andretrosplenial cortical regions.The lateral cholinergic pathway arises from the nbM and forms acompact bundle that subdivides in the capsular and perisylvian divisions that runthrough the external capsule and the claustrum, ramify widely in the centrumsemiovale and subcortical white matter, and distribute fibers to the inferiorfrontal, frontoparietal operculum, temporal, insular, and para-hippocampalneocortex. The amygdala also receives fibers from the lateral pathway.The 18 . There is a significantdifference in the global density of the cholinergic axons among the severalcytoarchitetonic regions. The highest fiber density is observed in the centrallimbic structures, such as the hippocampal formation and amygdala, followed by thecortical paralimbic areas, entorhinal and cingulate cortex; the cholinergicinnervation of the unimodal and heteromodal associative areas is of intermediarydensity, while that of the primary sensory areas is the lowest18  and 2. T38.The cholinergic projections of the septo-hippocampal path present a fairly compactconstitution, running through the fornix to reach the hippocampal formation. TheMeynert-cortical projections present a relatively compact origin, but once outsidethe basal ganglia territory, at the level of the centrum semiovale, the lateralpathway presents a fanlike distribution to reach their destination areas, while themedial pathway runs mainly through the cingulum and distributes ramifications alongits route51 , middle cerebral artery(MCA), anterior communicating artery (ACoA), posterior communicating artery (PCoA),anterior choroidal artery (AChA) – provide irrigation of the territories where thecholinergic projections travel51 .56. It ispossible to localize the points where lesions can interrupt these pathways byconsidering the routes of the cholinergic pathways38 and the vascular territories  due to lesions in theterritories of the ACA, ACoA and MCA, and its wide and fanlike course can beaffected in the white matter of the centrum semiovale, irrigated mainly by the ACA,MCA and PCA .Two neuropathologic studies indicate a relationship between CVD and the interruptionof these pathways, besides the anatomic relations between the cholinergic pathwaysand the vascular territories.57.One of these studies was conducted using brain tissue from a patient with CADASIL, a disease that can be considered a model for pure CVD (VaD).The material was stained with a histochemical technique to show AChE revealingcholinergic denervation in several cortical areas, except for the hippocampalformation and entorhinal cortex. Even in the more affected areas a number of AChEpositive fibers were seen. The cholinergic neurons of the nbM were undamaged, asverified by techniques for NGFr and AChEThe other study was performed using brain tissue of patients with VaD of theBinswanger subtype. The material was stained with histochemical andimunnohistochemical techniques to show AChE and ChAT. This material revealed severereduction of AChE and ChAT positive fibers in the external capsule and claustrum, incomparison to controls. The nbM had large neurons preserved, but showed somechromatolytic changes and numerical reduction.58. Therefore,underpinned by these two paradigmatic studies, we can state that CVD may causeinterruption of segments of the cholinergic pathways, leading to denervation andconsequent cholinergic hypofunction of the affected territories.A neuroimage-neuropathological correlation was possible for some of the patients. MRIshowed hyperintensities in the frontal periventricular white matter, extending tothe subinsular white matter . The brains of thesame patients at autopsy showed loss of myelin in the correspondingregions59. These functional data gave riseto the proposal of a ‘cholinergic neurovascular hypothesis22.Cholinergic hypofunction, variable according to the lesioned segment of thecholinergic pathways, causes integrative dysfunction of the target brain structuresand disturbances of vasomotor control with consequent reduction in brain blood flowof the affected areas60. The other, more detailed rating,proposes an evaluation on 4 slices , separated into 10 regions. The severity ofwhite matter lesions was visually rated on a 3-point scale (0-3) for each region,and weighted (1-4) to account for the decreasing concentration of cholinergic fibersas they project and fan out in the white matter61. The results of these studies suggest that the localizationof the hyperintensities in the white matter holds special importance, consideringthat some of these may occur at strategic points and may be related to measurableclinical manifestations61.Recently, two studies were dedicated to the relationship between the cholinergicpathways and the white matter hyperintensities, correlated to the clinicalmanifestations of VCI/VaD, with the aim of staging scales. These proposals relatethe white matter lesions with their localization in relation to the cholinergicpathways. The staging was graduated according to the visually evaluated extension,and number of lesions localized, along the anatomical known routes of thecholinergic pathways. One of these rating scales classified the lesions in thecholinergic pathways as minimal , moderate  and severe Thus, the knowledge of the anatomy of the cholinergic pathways and their relation tothose vascular territories where an interruption can occur, allied to the consequentclinical manifestations, enable better evaluation of CVD clinical expression. It mayalso be able to lend a more solid basis for treatment strategies, such as thecholinergic approach.CVD can cause clinical symptoms defining VCI/VaD according to its extension andlocalization. Two mechanisms play a role: one corresponding to tissue lesions ofcortical areas and subcortical regions, including white matter responsible fordisconnection related manifestations, while the other is related to the interruptionof the cholinergic pathways at various localizations along their routes, producingmanifestations consequent to cholinergic denervation which result in ahypocholinergic state of the affected territories.Knowledge of cognitive-behavioral and vasomotor functions of the cholinergic system,allied to that of the anatomical localization of the course of its pathways, isimportant to better assess the sites of vascular lesion. Such knowledge permitsstrategic points of the cholinergic pathways to be highlighted and provides moresolid bases for use of cholinergic therapeutic strategies.


The review tried to provide cutting edge knowledge in ZIKV research with future insights required for the development of novel therapeutic remedies to curtail ZIKV infection.One of the potential threats to public health microbiology in 21st century is the increased mortality rate caused by Zika virus (ZIKV), a mosquito-borne flavivirus. The severity of ZIKV infection urged World Health Organization (WHO) to declare this virus as a global concern. The limited knowledge on the structure, virulent factors, and replication mechanism of the virus posed as hindrance for vaccine development. Several vector and non-vector-borne mode of transmission are observed for spreading the disease. The similarities of the virus with other flaviviruses such as dengue and West Nile virus are worrisome; hence, there is high scope to undertake ZIKV research that probably provide insight for novel therapeutic intervention. Thus, this review focuses on the recent aspect of ZIKV research which includes the outbreak, genome structure, multiplication and propagation of the virus, current animal models, clinical manifestations, available treatment options (probable vaccines and therapeutics), and the recent advancements in computational drug discovery pipelines, challenges and limitation to undertake ZIKV research. The review suggests that the infection due to ZIKV became one of the universal concerns and an interdisciplinary environment of  Aedes africanus although there was no indication to cause human disease (Table 1.Zika virus (ZIKV) is a mosquito-borne flavivirus (family Flaviviridae) pertinent to West Nile virus, dengue virus (DENV), and yellow fever virus. ZIKV carry a positive RNA which is single stranded . ZIKV ha disease . The ZIK disease . An outb disease . An outb disease . The areThe ZIKV infection accreted severe global health issues due to the rise in number of GBS correlated reports and exuberant microcephaly cases in infants and fetus in Brazil by the end of 2015 . The serMore than 5600 microcephaly cases in neonates have been well-documented which delineate greater than 20-fold with respect to historical average of past 5 years by 2016. The mortality rate due to microcephaly associated with ZIKV was registered to be approximately 120 by Brazilian health authorities .According to the recent report, there were active circulations of ZIKV nearly all Caribbean and Latin American countries . Even thFigure 1A  in their protein envelope  involved in the binding of cell receptors form the envelope . E proteogenesis .The two known ZIKV lineages belong to African lineage (divided into two clades signifying two different introductions) and Asian lineage are differentiated by complete genetic analysis of RNA sequences . A stronThe region of glycoprotein E which is contiguous to Asn 154 glycan depicts the largest structural deviation from other flavivirus. Novel anti-ZIKV vaccines and drugs can be developed based on the knowledge of the function of glycans and proteins .Figure 2. Most of the arboviruses are known to replicate within the skin dendrites at the primary inoculation site, later, it spreads to the regional lymph nodes followed by the blood stream , assembly of viral particle in ER and release of the virions . The repmentally . The virmentally .in vitro in cultured monkey cell lines such as LLC-MK2 or Vero . Autophagy developed as vital ancient immune response during evolution of lysosome-mediated catabolic process . Some viAedes aegypti (other than A. africanus) from a group of mosquitoes garnered in Malaysia (1966), it furnished the indication of the first transmission cycle of ZIKV in urban areas  by virtue of the subcutaneous route leading to the recognition of clinical features. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to determine the viral load in the tissues, protein levels of cytokine, anti-ZIKV neutralizing antibody, and viremia followed by plaque reduction neutralization test (PRNT) and multiplex immunoassay. The symptoms observed were lethargy, hunched back, fever, reduced mobility, and rumpled fur. PRNT presented the anti-ZIKV neutralizing antibody in the infected guinea pigs and the infection led to rise in multiple cytokines level and increased level of growth factors in the serum. The virus was observed to be replicated in the brain and spleen , γ (type II), and aaa (type III), interferon (IFN) and copious IFN-stimulated genes were stimulated by ZIKV that prevented the infection . The mectargeted .The infection caused by Asian-lineage of ZIKV is affiliated with fetal abnormalities. However, poor knowledge of the mechanism has led to the study of rhesus macaque animal model. The study exhibited the injection of vRNA in the plasma approximately 10 days. The infection evoked an immune response such as neutralizing Ab that enable protection in the case of further infection and T-cell response specific for the virus. Nonetheless, the vRNA was detected in saliva and urine after being cleared from blood and further, the presence in CSF indicated that probable existence of ZIKV in some tissues at low levels .A number of clinical studies have examined malformations in the eye and pathology of newborns to mothers infected with ZIKV during pregnancy. Eye diseases in neonates with ZIKV included optic neuritis, colobomas, bilateral iris, chorioretinal atrophy, intraretinal hemorrhages, blindness, and lens subluxation. Uveitis is a viral infection due to the inflammation of uveal tissues  which can resulted in the permanent loss of vision, if untreated . The ant+ IFNARfl/fl C57BL/6 via vaginal route post-hormonal treatment. AG129 mice failed to persist infection in di-estrus-like phase while, infection during estrus-like phase demonstrated resistance. However, LysMCre+ IFNARfl/fl mice in di-estrus-like phase showed short-term infection. The viral replication occurred in brain and spleen along with viremia in vaginal washes 10 days of post-infection. Thus, the study indicated that the occurrence of transgenital transmission accompanied by variation in hormones of female reproductive tract with prolonged prevalence of viral replication  in comparison with the first and second samples . However, the newborn showed delay in neuropsychomotor development, with spastic hemiplegia and global hypertonia, with severe damage to the right dominant side by the age of 6 months  experienced symptoms and gave birth to a microcephaly infected male child in 2016 in Sao Paulo, Brazil. The weight, length, and head circumference of the infant was observed to be 3095 g, 48 cm, and 32.5 cm, respectively. A reduced brain parenchyma, preferentially in the frontal and parietal lobes, foci of calcification in the sub-cortical area and compensatory dilatation of the infratentorial supraventricular system was observed by magnetic resonance imaging technique. The saliva and urine were tested for the virus on the 54th day by qRT-PCR. The studies depicted that all the three assays were positive for ZIKV RNA with 1.4 × 106 months .The symptoms of ZIKV infection are non-specific and can be confound with symptoms of other diseases such as chikungunya and dengue caused by arbovirus, the differential laboratory diagnosis plays vital role in the regions where the virus co-circulates and hence, ZIKV infection can be misdiagnosed . AsymptoLaboratory methods analyze ZIKV infection by the detection of viral nucleic acid and viral antibody or antigen . During In the earlier stages, mainly during primary acute infections, techniques such as solid phase immunosorbent assay and hemagglutination were used to diagnose flaviviral infection . The detRT-PCR analysis of serum samples attained inside the first week of clinical illness and MAC-ELISA testing of samples that were not tested or found negative by RT-PCR was expected to have the highest diagnostic yield. The uses of RT-PCR were found in detecting ZIKV in amniotic fluid, breast milk, semen, saliva, and blood products. The utility of urine samples for diagnosis of ZIKV infection demonstrated that ZIKV RNA detectable at an elevated load longer period than in serum . The posIn accordance with the recent CDC guidelines for testing pregnant women likely to be exposed to ZIKV, there was a strong explanation for testing asymptomatic pregnant women as long as sufficient laboratory facilities are available . The mosThe following are the major demerits to be considered during the diagnosis of ZIKV infections :• As a result of identical clinical features with other infections of arbovirus such as chikungunya and dengue, the diagnosis of ZIKV is challenging.• Due to the failure in differential diagnosis, most often, ZIKV is diagnosed as DENV at the incipient stage.• The scarcity of sensitive and specific laboratory test makes it complicated to detect the virus in the samples collected from the patients.• Antibodies of ZIKV infection are often cross-reactive with other flavivirus such as yellow fever, dengue, West Nile Virus, Murray valley encephalitis, Kunjin virus and that which frequently co-circulate with ZIKV, confines the use of serology and hinders the regular performance of the viral culture. The lacks of antigenic detection contribute one of the major challenges in the diagnosis.• Even if PCR-based testing of saliva, blood, and urine is highly recommended, accessibility of the preferred amount of nucleic acids in various samples possess significant encumbrance.• The short viremic duration demonstrated an impediment in the detection of significant antibodies.• To run molecular tests in an outburst situation, the laboratories require acquaintance and experience of quality control and validation, ability to access rapid reagents and other necessities from supply chains, and to augment the high throughput testing approaches.Aedes mosquito enables ZIKV to invade newer areas and impose worldwide risk as there are no preventive approaches or vaccines for ZIKV. The control measures are depending on the eradication of mosquito vector breeding foci  where four phase 1 trials have been initiated. A joint collaboration between National Institute of Allergy and Infectious Diseases (NIAID) and University of Pennsylvania, Moderna/Valera and GlaxoSmithKline (GSK) investigated several mRNA vaccines that are identical to DNA vaccines. The candidate developed by Moderna/Valera has been assessed in phase 1 clinical trial .AGS-v is a vaccine considered to be acted against several mosquito-borne infections. It is found to set off an immune response to the proteins present in mosquito saliva instead of specific virus. Four synthetic proteins found in mosquito salivary glands are utilized to formulate the test vaccine. The proteins are intended to trigger modified allergic responses that lead to the prevention of infection on being bitten by the mosquito. This vaccine currently evaluated for phase 1 clinical trial at NIH Clinical Center in Bethesda, MD, United States .An accurate understanding of ZIKV biology is essential for the development of anti-ZIKV therapeutics, vaccines, and improved diagnostics . The treTable 2.An integrated advancement in traditional Chinese medicine and western medicine were used to cure ZIKV infection. Xiyanping was used for the treatment of latest case of ZIKV due to the antiviral properties of this drug . IntraveCamellia sinensis (green tea) provides intense antiviral activities against the viruses such as herpes simplex virus (HSV), influenza virus (FLU), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) , a polyphenol (catechin) found in us (HIV) . Throughus (HIV) . Over thFigure 5.A quick development of therapeutic requires the swift analysis of mechanism underlying the pathogenesis of the ZIKV . The prMRecently, small number of FDA-approved drugs such as IFN (34.3 IU/ml), Ribavirin (143 μg/ml), 6-Azauridine (1.5 μg/ml), and Glycyrrhizin (384 μg/ml) were tested against ZIKV ,b. The pTo control more than 60% of human protein-coding genes, small non-coding RNAs (sncRNAs) which are termed as MicroRNAs (miRNA) that regulate post-transcriptional gene expression by translational repression were examined . The infFurthermore, there is high scope using alternative approaches other than molecular modeling-based methods. For example, techniques such as advanced machine learning methods  are appropriate to capture complex statistical patterns between thousands of descriptors extracted from drug compounds. Better predictions can be made using Deep Neural Networks (DNN) in comparison with standard machine learning tools based on Kaggle competition data sets. A deep machine learning network was developed to identify the site for epoxidation and to differentiate epoxidized and non-epoxidized molecules .in vivo congenital alterations. The in utero infection on pathology has not been reported in mice, and it is difficult to model this pathology in wild-type pregnant mice. The peripherally inoculation of ZIKV to AG129 pregnant mice causes fetus infection and congenital variations, because flaviviral infection leads to the development of viremia in mice when compared to the ones infected in humans (The most challenging aspect of ZIKV research is the demonstration of ZIKV-induced n humans .The nucleoside analogs such as 7DMA and T-705 Favipiravir are proficient for the treatment of several viral infections. However, the application of nucleoside analogs for the treatment of ZIKV can be unsafe in pregnant women. Currently, there are protocols established to assess the safety of Ribavirin treatment during pregnancy. The gestational use of nucleoside analogs is suggested when benefits exceed in comparison to the probable fetal harm. The safety of most nucleoside analogs should be studied for ZIKV-induced congenital malformations to examine whether these drugs exhibited competence in infected adults .The preservation of an intact immune response in mice models is essential; however, there are issues with inoculation of the virus to brain. The murine models that exist with sustained viremia are supreme to the compounds such as neutralizing antibodies and antiviral agents, which target ZIKV for preclinical testing. To elucidate the scientific challenges, various mice models of infection are required in ZIKV research .Wolbachia into the A. aegypti population. Thereby, the replication of ZIKV curtails within the mosquito and delays the distribution of virus to the salivary glands of mosquitoes. The existing limitation for the application of vector control strategies in ZIKV affected areas are the probability of environmental damage and public distrust of genetically modified organisms (An alternative vector strategy to abate the transmission of ZIKV by mosquitoes was demonstrated by the introduction of the bacterium rganisms .in vitro cellular assays and computational biology approaches probably contribute insights for the screening of molecular targets in drug design. An improvement in the existing strategies of vector control, awareness programs among people and opening novel outlook in ZIKV research to study the current challenges and limitations are the vital component in the disease management.ZIKV infections are spreading across the world and possessed great threat to the public health. The infection often associated with neurological disorders and severe birth defects. Several vector and non-vector-borne forms of transmission are reported which aid in spreading the disease. The similarity of ZIKV with other flavivirus such as DENV and West Nile virus is worrisome. Further, more severe complications such as pathogenesis and cellular mechanism are unknown. Hence, there is a high demand to undertake ZIKV research as thrust areas and develop strategies to ascertain therapeutic remediates to curtail the infection. There are several researchers and industries are working toward the development of effective anti ZIKV vaccines. The development of vaccine requires clear understanding of the pathogenesis and genome structure of the virus. An interdisciplinary environment of AS and AP collected the data and prepared the complete manuscript, SS reviewed, edited, and revised the manuscript.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.


Irritable bowel syndrome (IBS) is one of the most common gastrointestinal diseases in humans. It is characterized by visceral pain and/or discomfort, hypersensitivity and abnormal motor responses along with change in gut habits. Although the etio-pathogenesis of IBS is only partially understood, a main role has been attributed to psychosocial stress of different origin. Animal models such as neonatal maternal separation, water avoidance stress and wrap restraint stress have been developed as psychosocial stressors in the attempt to reproduce the IBS symptomatology and identify the cellular mechanisms responsible for the disease. The study of these models has led to the production of drugs potentially useful for IBS treatment. This review intends to give an overview on the results obtained with the animal models; to emphasize the role of the enteric nervous system in IBS appearance and evolution and as a possible target of drug therapies. Irritable bowel syndrome (IBS) is a highly prevalent (12–20% in western countries) and multifactorial gastrointestinal disorder ,4 and eoThe etiology of IBS is still poorly known; however, psychosocial stress is the most widely acknowledged risk factor for the development and/or relapse of IBS ,10. NotaIn summary, it is possible that hyperalgesia and psychosocial stressors are able to aliment each other in a vicious circuit responsible for the chronic progression of the disease. The possibility to interrupt this circuit is a challenge for researchers and clinicians.Psychosocial stress belongs to the exteroceptive stressors ,13 and aPresently we will review three of the most used psychosocial stress models: the wrap restrain stress (WRS), an acute stress and two models of chronic stress: the maternal separation (MS) and the water avoidance stress (WAS). In parallel we will focus our attention on those data, obtained by us and other researchers, that highlight the role played by the enteric nervous system in the genesis of IBS symptomatology and the available experimental or clinical pharmacological therapies affecting, directly or indirectly, the enteric neurotransmission .The WRS was introduced in the scientific literature more than 30 years ago as the model for human IBS . Since tBeyond the chronic course, accurate anamneses of IBS patients have attested a significant association between IBS and childhood traumas of different origin or the presence of repeated stress situations in adulthood . Thus, tThe stress consists in removing the puppies from the mother for 3 h per day during the first two weeks of life. Since maternal care affects the hypothalamic-pituitary-adrenal (HPA) axis and the cognitive and emotional functions , the MS Interestingly, the experimental data demonstrated a greater sensitivity to MS of females compared to males . NotablySeveral reports agree that daily chronic stress predicts the severity of the IBS symptoms ,35,36. ILesser known and still a matter of investigation is the possibility that the two models of chronic stress presently reviewed possess also the predictive validity i.e., how well the models are responsive to treatment showing some efficacy in alleviating visceral pain to predict triggered treatment. Noteworthy, WAS shows some peculiarities, i.e., the absence of a tolerance after repeated applications and the maintenance of hyperalgesia and hypermotility after the stress is removed, that make it particularly suitable to investigate the ability of drugs to prevent or attenuate the appearance and evolution of the colonic alterations induced by stress, and also to evaluate long lasting beneficial effects of the drug after its withdrawal.It has been suggested that pain signals originate in intrinsic primary afferent neurons and are transmitted by extrinsic primary afferent neurons . AfferenPsychological stress at any point of the lifetime can bring permanent alterations of several systems such as the descending pain modulatory system, the immune system, and the gut microbiota. Preclinical evidence has accumulated over the years suggesting that stress-related alterations of colonic motor and visceral functions are primarily mediated by the activation of the brain CRFr/CRF1 signaling pathway, while CRF2 receptor activation may exert a counteracting influence ,40,41. CAlthough CRF has been the subject of 33 years of effort in research by academia and the pharmaceutical industry that resulted in several thousand papers and patents, yet little progress has been made to identify and market diagnostic or therapeutic CRF peptides and small molecule ligands, and no modulators of this peptide has reached the pharmaceutical market for IBS so far .https://www.alosetronrems.com/AlosetronUI/rems/pdf/letterforhealthcareproviders.pdf; https://www.fda.gov/Drugs/DrugSafety/ucm103223.htm). Newer 5-HT4 agonists such as prucalopride, naronapride, velusetrag, and YKP10811 have been found active only in chronic idiopathic constipation. Further studies are needed in order to develop an optimal treatment in the field of serotonin.Several lines of evidence indicate that altered 5-HT signaling in the central nervous system and in the gut contributes to the pathophysiology in IBS . FurtherNK2r antagonists have been proven effective in animal models of visceral hyperalgesia induced by inflammation ,58 or WAAlso, the NK3r  could play a role in visceral hyperalgesia, due to the reported effectiveness of either intraperitoneal  or intraIn spite of these promising experimental results, none of these NKr antagonists have reached the market due to limitation of their effect in humans and the presence of side effects found in the development clinical phases.It has been reported that T-type calcium channels encoded by the Ca(V)3.2 isoform are expressed in colonic nociceptive primary afferent neurons and that they contribute to the exaggerated pain perception in a butyrate-mediated rodent model of IBS . These pSince irritation, immune challenge and inflammation releases both tachykinin and calcitonin gene-related peptide (CGRP) from extrinsic afferents and from intrinsic neurons inside the gut, these peptides may either increase the peripheral sensory gain of extrinsic afferents or contribute to the afferent transmission of algetic signals within the central nervous system. As gut extrinsic afferents do not possess receptors for CGRP, it is probable that peptide-induced sensitization or excitation of sensory neurons is an indirect consequence of peptide action on gastrointestinal effectors . In expeThe increase in the vanilloid receptor TRPV1 significantly correlated with the decrease in heat and distension sensory threshold confirming that both afferent and intrinsic fibers take part in the change of signaling pathways of visceral hypersensitivity. Further, WAS could induce the upregulation of TRPV1 and TRPA1 in the colonic afferent DRG indicating these receptors as candidate molecules in stress-induced hyperalgesia in rats . An antiIt should be noted that while modulators of vanilloid receptors are under preclinical development, activators of guanylate cyclase (GC) receptor has produced a drug successfully introduced in the market for IBS with predominant constipation (IBS-C) such as linactotide. Linaclotide has shown potent anti-nociceptive effects in several mechanistically different rodent models of visceral hypersensitivity  is a five-domain transmembrane protein that is highly expressed in steroid-producing tissues, including the glial cells within the brain. ONO-2952 is a novel and selective inhibitor of TSPO that reduces stress-induced defecation and visceral hyperalgesia in rat models .https://www.fda.gov/Drugs/DrugSafety/DrugSafetyPodcasts/ucm547907.htm), GABAergic agents are alpha2β ligands that generally bind potently to an auxiliary protein associated with voltage-gated calcium channels, reducing depolarization-induced calcium influx at nerve terminals. This reduces the release of several excitatory neurotransmitters, including glutamate, noradrenaline, substance P, and calcitonin gene-related peptide (CGRP), which are involved in pain mechanisms. Gabapentin and pregabalin did not show a good efficacy/risk ratio in clinical trials.Other classes of drugs challenged in IBS are those acting on opioid or GABA receptors. Among opioid agonists, loperamide and diphenoxylate are anti-diarrheal agents that have been used in IBS for many years but their efficacy is already discussed and not completely proven while the recent one eluxadoline has been licensed in USA for IBS-D with some limitations due to its side effects  are able to induce visceral hypersensitivity that is one of the main signs of IBS. To translate the visceral hypersensitivity measured in the animals to the abdominal human pain is not so likely. Several differences in pain threshold, central processing of nociceptive modulation, emotional and cognitive contributions between rodents and humans limit this translation. However, the three animal models have demonstrated that numerous neurotransmitters are involved in this nociception and that modulators of them are generally active in reducing experimental pain. Nevertheless, because of the complexity of this phenomenon in humans, only few of these latter molecules have been found active in clinical studies and this has hampered the development of new drugs for IBS. Up to date, the focus on targets related to specific symptoms has brought the production of drugs aimed to treat constipation or diarrhea with alternate results. This choice did not have attention on the possibility to look for drugs useful for global IBS symptoms. A more integrated view on the disease seems to be desirable for now, in order to develop efficient therapies that can counteract the chronic natural history of IBS.


The success of the clinical use of sequencing based tests (from single gene to genomes) depends on the accuracy and consistency of variant interpretation. Aiming to improve the interpretation process through practice guidelines, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) have published standards and guidelines for the interpretation of sequence variants. However, manual application of the guidelines is tedious and prone to human error. Web-based tools and software systems may not only address this problem but also document reasoning and supporting evidence, thus enabling transparency of evidence-based reasoning and resolution of discordant interpretations.http://calculator.clinicalgenome.org.In this report, we describe the design, implementation, and initial testing of the Clinical Genome Resource (ClinGen) Pathogenicity Calculator, a configurable system and web service for the assessment of pathogenicity of Mendelian germline sequence variants. The system allows users to enter the applicable ACMG/AMP-style evidence tags for a specific allele with links to supporting data for each tag and generate guideline-based pathogenicity assessment for the allele. Through automation and comprehensive documentation of evidence codes, the system facilitates more accurate application of the ACMG/AMP guidelines, improves standardization in variant classification, and facilitates collaborative resolution of discordances. The rules of reasoning are configurable with gene-specific or disease-specific guideline variations . The software is modular, equipped with robust application program interfaces (APIs), and available under a free open source license and as a cloud-hosted web service, thus facilitating both stand-alone use and integration with existing variant curation and interpretation systems. The Pathogenicity Calculator is accessible at By enabling evidence-based reasoning about the pathogenicity of genetic variants and by documenting supporting evidence, the Calculator contributes toward the creation of a knowledge commons and more accurate interpretation of sequence variants in research and clinical care.The online version of this article (doi:10.1186/s13073-016-0391-z) contains supplementary material, which is available to authorized users. Successful clinical diagnostic application of exome and genome sequencing hinges on the ability of clinical laboratories to consistently and accurately assess pathogenicity of genetic variants. Several studies have identified inconsistencies in variant classification across laboratories, reference databases, and even within individual databases –4. InterTo improve the standardization of variant interpretation by clinical testing laboratories, the American College of Medical Genetics (ACMG) and the Association of Molecular Pathologists (AMP) have recently updated guidelines for the interpretation of sequence variants . The newThe formal structuring of evidence-based arguments for or against pathogenicity and their implementation in software provide several opportunities for enhancing the emerging knowledge of genetic variation. One such opportunity is efficient evidence tracking that facilitates critical evaluation of variant classifications. Recent studies , 4 suggeThe Calculator client-side, including the main interface, is implemented using JavaScript and ExtJS. The main Calculator interface Fig.  presentshttp://reg.clinicalgenome.org (manuscript in preparation).As illustrated in Additional file The registry enables linking and viewing of pathogenicity assessments that are independently generated by different users  for each evidence tag within the evidence grid  and aggregated evidence for the variant being pathogenic or benign (encoded as a set of evidence tags) and produces a conclusion Fig. . The reawww.redmine.org) application.GenboreeKB is implemented in Ruby as an enhancement to the Genboree server. Thus, the group-based data sharing is managed by the Genboree access control system. A web-based GenboreeKB management interface employing the ExtJS Javascript framework is available as a plugin of the Redmine Rails . GenboreeKB stores both data models and the data itself as document collections and exposes them via the APIs.Numerous tools and systems for variant curation and evaluation have been developed and used by various laboratories. Recognizing that some of those tools may benefit from the functionalities of the Calculator, all the data and functionality of the Calculator are programmatically accessible via REST APIs. The Calculator may be used as a web service (with authentication and collaborative features such as group-based access control) or by installing it on local hardware.Two adaptations of the original guidelines had to be introduced to facilitate formal algorithmic reasoning. First, several metarules were introduced to capture implicit “common sense” rules practitioners were expected to apply without explicit instruction. For example, if there are two strong and three moderate-strength evidence tags in favor of pathogenicity, then the variant can be pathogenic (≥2 strong evidence tag) or likely pathogenic (≥3 moderate-strength evidence tags). In such a case the metarules specify that the variant is pathogenic. Conversely, evidence tags that can be combined to yield both Likely Benign and Benign are scored as Benign. The Calculator distinguishes between uncertain significance that arises due to insufficient evidence from uncertain significance due to conflicting evidence.Second, the guidelines allow the practitioners to change the strength level originally assigned to certain tags based on perceived strength of supporting data for the tag . To accThe ACMG/AMP guidelines and the Calculator were tested in the context of a multi-site study  involvinGLA) and condition name and mode of inheritance . A new assessment is initiated by providing an HGVS allele identifier  can access the functionalities of the Calculator via Genboree REST-APIs. Using the services, users can upload evidence tags for several variants at a time. The uploaded data become accessible through the interface as well as through REST-APIs. In the future, the bulk upload functionality will be made accessible even for regular users without any background in computer programming.Sharing of pathogenicity assessments of pathogenicity of variants by clinical laboratories via databases such as ClinVar aids in the practice of clinical genome interpretation. However, currently, the format of information by which pathogenicity for a given variant was determined by multiple submitters to ClinVar often appears in a free text unstructured format, if it is provided at all. It would be useful if this knowledge sharing was further extended to include the reasoning and evidence codes that supported conclusions about pathogenicity in a standardized format. For example, evidential support for an early assessment that a variant is pathogenic may not have addressed more recent information about the high frequency of the variant in a specific population, thus explaining discordance with a newer independent assessment that includes this recent information. With the evidential support available for inspection, a user of the database may benefit from being able to rapidly and critically evaluate both assessments based on the evidence provided in a structured way. Toward this goal, the Calculator facilitates the sharing of the reasoning and the evidential support behind every assessment. Access to this information is provided via the interfaces Fig. , Panel 3Recent studies indicate that the initial application of the ACMG/AMP guidelines did not increase concordance in laboratory assessment ; howeverThe Calculator does not share information about pathogenicity of variant publicly without explicit user request. The information entered is protected using a login and password. It provides optional free-text fields summarizing tag assignments and links to supporting evidence. The Calculator does not require or have fields for providing identifying information about patients. Public sharing per user request will be enabled in future releases.Although ACMG/AMP guidelines provide a general framework for Mendelian sequence variant interpretation, it was always recognized that this framework would evolve and be customized to meet requirements for specific genes and disease conditions , 16–19. While each assessment refers to a specific guideline, different guidelines may exist within the same system. For example, for the purpose of assessing variants within genes causing inborn errors of metabolism, metabolite levels could be used as an evidence type in pathogenicity assessments. Because the original ACMG/AMP guidelines do not specify this type of evidence or potentially others that will be added in the future, we have designed flexibility into the Calculator to allow for addition of new evidence types (new rows in the evidence grid) and corresponding evidence tags and of new rules without programming.By automating the formal guideline-based assessment of genetic variants, the ClinGen Pathogenicity Calculator can reduce human error. The Calculator extends functionality of other currently available calculators  along seBy providing an intuitive user interface and by addressing other key computational requirements, the Calculator facilitates formal and collaborative evidence-based reasoning about the pathogenicity of genetic variants, thus paving the way toward more effective and accurate clinical genomic interpretation.


Many new innovative diagnostic approaches have been made available during the last 10 years with major impact on patient care and public health surveillance. In parallel, to enhance the cost-effectiveness of the clinical microbiology laboratories (CMLs), European laboratory professionals have streamlined their organization leading to amalgamation of activities and restructuring of their professional relationships with clinicians and public health specialists. Through this consolidation process, an operational model has emerged that combines large centralized clinical laboratories performing most tests on one high-throughput analytical platform connected to several distal laboratories dealing locally with urgent analyses at near point of care. The centralization of diagnostic services over a large geographical region has given rise to the concept of regional-scale “microbiology laboratories network.” Although the volume-driven cost savings associated with such laboratory networks seem self-evident, the consequence(s) for the quality of patient care and infectious disease surveillance and control remain less obvious. In this article, we describe the range of opportunities that the changing landscape of CMLs in Europe can contribute toward improving the quality of patient care but also the early detection and enhanced surveillance of public health threats caused by infectious diseases. The success of this transformation of health services is reliant on the appropriate preparation in terms of staff, skills, and processes that would be inclusive of stakeholders. In addition, rigorous metrics are needed to set out more concrete laboratory service performance objectives and assess the expected benefits to society in terms of saving lives and preventing diseases. Infectious diseases currently contribute about 20% of the global annual death causes  and 10% In the past decade, clinical microbiology experienced revolutionary advances in terms of culture-independent molecular detection assays, laboratory automation, information systems linkage, and point-of-care testing, to name but a few. The initial optimism associated with these technological strides forward has now entered a phase of realistic discussion on the challenges and complexities posed by clinical translation of microbial genomics , 10, biomicrobiology laboratories network.” Although the volume-driven cost-savings associated with such laboratory networks seem self-evident, the consequence(s) on the quality of patient care and infectious disease surveillance remain less obvious. This article examines from the clinical to the public health perspective the challenges and opportunities of the wide-spread consolidations that the European clinical microbiology sector currently faces.At present major emphasis is placed on the operational streamlining and consolidation of CMLs. Through this consolidation, an operational model emerged that combines large centralized CMLs performing most tests on one high-throughput analytical platform connected to several distal laboratories dealing locally with urgent analyses at near point of care . The cenHow can the consolidation make the laboratory operations more advantageous both financially and clinically, given available resources?”Laboratory service consolidations have to answer the following: “From a clinical laboratory perspective, a centralized laboratory network enables addressing different levels of complexity in analytical processes, while concurrently servicing increased volumes of routine tests. The challenge is to simultaneously address the demand for detection and characterization of unusual or exotic pathogens, drug resistance, or virulence traits as appropriate, and meeting the requirement for health-care cost containment and financial efficiency. In this perspective, a number of high profile microbiology services consolidations have already taken place in the private sector over the last decade: (i) BioReference Laboratories, Inc., acquiring Edge BioServ (2013) and merging with OPKO Health (2016); (ii) Sonic Healthcare Limited acquiring CBLPath, Inc. (2010) and an additional ten such acquisitions (2010–2017) in Europe alone; (iii) LabCorp, Inc., with seven such acquisitions (2010–2017); Quest Diagnostics, Inc., with six such acquisitions (2010–2017); and others. It was expected that this trend of CML consolidation would expand to larger academic health-care providers, such as the Karolinska University Hospital merger in Stockholm (2005–2008); the Charité Hospitals mergers in Berlin (2003); or the multi-faceted mergers led by the Royal Free London and University College London Hospitals forming UCL Partners . Due to the projected cost efficiencies and anticipated health-care improvements, regional or local authorities support such consolidation initiatives often in partnership with the academic sector. The laboratory network of the Assistance Publique—Hôpitaux de Paris (AP-HP) or the recent (2015) launch of the University Laboratory of Brussels (LHUB-ULB) are representative examples.An effective approach includes the creation of task-specific teams representing multiple skills built around infectious diseases specialists and CMs. The need for an increase in professional diversity sometimes contradicts the established specialty training and certification pathways and creates complexities with regard to the required staffing structure and laboratory organization. In general, the utilization of cross-disciplinary teams forced the move in the direction of a flatter hierarchical structure within healthcare organizations –18.Even though CMs play a pivotal role in the management of infected patients ; the tec6 inhabitants  remains a clear motivating force to rationalize operations; CMLs constitute only a small percentage (about 4%) of most hospitals budgets . TherefoThe increasing positioning of health-care providers from condition-centric to patient-centric care impacts the CML organization, workflow, and specimens flow. Microbiology results, once viewed as confirmatory and often delivered after patient management decisions were made, are now integrated into the clinical workflow and decision-making algorithms. In addition, the potential value of the CML itself has been transformed by the superior diagnostic performance of innovative molecular and biophysical technologies .However, improved TTR due to new diagnostic tools, e.g., molecular diagnostics or the Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry, will only have clinical impact if used by without delay; therefore, good communication between health professionals remains fundamental. Only a few studies have evaluated the true impact of rapid microbial identification (RMI) including direct DNA identification from positive blood cultures –38. For Furthermore, the recent availability and affordability of high-throughput sequencing methods is expected to impact clinical microbiology. It is now possible to sequence bacterial genomes in less than a week at a cost similar to that of other medical diagnostic procedures. Besides outbreak investigations, genomic applications currently focus on pathogenicity and the pathogen AMR profile , 42. TheAs the consolidation of CMLs gathers pace, this change might have a positive impact on the quality of the surveillance information but also alter the ability of the public health authorities to fulfill their missions.The gradual integration of new molecular detection and typing data into the European surveillance and alert systems represents one of the most exciting and challenging developments that could revolutionize the understanding and enhance control of communicable diseases . In ordeIt is conceivable that consolidated CMLs and their regional health-care networks would pave the way to new public health information and cooperation models. Examples of such developments include regional health-care hospital networks with interactive surveillance for AMR control in EU cross-border regions . Due to In addition, the ability of networked CMLs to access multiple different partners, geographies, and clinical specialties can enhance their capabilities to provide advanced disease surveillance and early outbreak recognition. For example, in the Brussels region, the LHUB-ULB laboratory structure provided 68% of all infection notifications reported in 2016 to the Belgian sentinel laboratory network .Considering the broad range of scientific expertise and technological capabilities they host, consolidated CMLs may also act as public health reference laboratories. This is the case in Belgium, 26 of the 41 NRL were outsourced through competitive tendering to academic clinical laboratories. A similar figure is found in France where 24 of the 43 NRL are hosted in clinical laboratories .The use of high-throughput whole genome sequencing (WGS) platforms available in large CMLs forms an additional advantage. Outsourced WGS analysis of human pathogens has been recently shown to be portable across countries for supporting multi-state outbreak investigations . The avaHowever, the reduction in the number of small clinical laboratories and the aggregation of the remaining ones, may condition the ability to detect epidemiological changes. The sensitivity and representativeness of laboratory-based or confirmed national surveillance systems should be, therefore, carefully assessed using coverage measures, which indicate the proportion of the target population included within the surveillance system. In countries using the “reimbursement system,” the evaluation of the ratio of reimbursed tests performed by the laboratories reporting surveillance data to the total number of tests performed by all laboratories can be used as reliable proxy measure to assess the case ascertainment sampling fraction for these systems .The inadvertent increase in volume in clinical microbiological testing has created a substantial and continuous market-driven need for the implementation of high-throughput analytical systems. A number of high profile translational research initiatives  are meant to support the eventual technological transfer of high-throughput analytical approaches from research into the CML , 53. A cThe impact of CML consolidations has been evaluated so far mainly in the narrow financial sense. It would be particularly interesting to explore the impact of this new model on the wider quality and efficiency of health services, correlating with patient outcome metrics, such as length of hospital stay, mortality, and readmission rates. This broader health system view should include the impact on the clinical management in clinical fields that require significant laboratory input. This suggests the need for more complex studies, not just assessing assay performance for example, but rather entire process performances including the time, expertise and costs both of reporting results and of downstream clinical actions. Beyond the clinical care impact, CMLs also provide key information outputs for the protection of population health, which need to be assessed. In this perspective, the European Centre for Disease Prevention and Control (ECDC) and microbiology experts designed and implemented the EULabCap monitoring system of national public health microbiology performance indicators toward ensuring sufficient capabilities at clinical and reference laboratory levels for well-informed infectious disease prevention and control actions .The availability of increased amounts of high-resolution data at a lower cost creates an anticipation, requirement, and downstream cost(s) for the accommodation, analyses, and interpretation of these data. The inherent systemic flexibility that is necessary to receive different types of data at different speeds and from different locations—and link all that to routinely collected clinical data and report back—is not an insignificant task by itself. A number of questions are raised regarding the new pathways that might be necessary, the different regulatory approaches within Europe to handling this data under the EU personal data protection directives, and data quality issues . The ethConclusively, the fast changing landscape of CMLs in Europe gives us a range of opportunities to contribute to improving the quality of patient care but also the early detection and enhanced surveillance of public health threats caused by infectious diseases. The success of this transformation of health services is reliant on the appropriate preparation in terms of staff, skills, and processes that would be inclusive of stakeholders. In addition, rigorous metrics are needed to set out more concrete laboratory service performance objectives and assess the expected benefits to society in terms of saving lives and preventing disease.All authors contributed constructively in the conception, drafting, and final approval of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The views and opinions expressed herein are the authors’ own and do not necessarily state or reflect those of ECDC. ECDC is not responsible for the data and information collation and analysis and cannot be held liable for conclusions or opinions drawn.The authors declare that this manuscript was written in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Dr. Vandenberg reports personal fees from BD diagnostics, outside the submitted work. Dr. Schrenzel received grants from Abbott and bioMérieux.


It is well recognized that disinfection of the complex root canal system at the apical root canal remains the most critical therapeutic measure to treat apical periodontitis.Observational and experimental data in relation to the anatomy of the apical root canal in different tooth types and the cross sectional diameters of the apical part of the most commonly used hand and rotary files are critically reviewed.Ex vivo investigations clearly indicate that a specific design of the chemo-mechanical preparation is needed at the onset of RCT, more particularly in infected teeth. Design should be based on specific anatomical parameters, and must determine the appropriate size and taper of preparation as pre-requirements for effective and safe apical irrigation.The present data analysis confirm that the challenging issue of antibacterial efficacy of modern preparation protocols in non-surgical endodontics requires more attention to apical root canal irrigation as a balance between safety and effectiveness. The optimal irrigation protocols might be designed on the basis of technical specifications of the preparations procedures, such as the penetration depth, the type of the needle, the required time for continuous irrigant flow, the concentration of NaOCl, and the activation parameters. Key words:Endodontics, root canal treatment, instrumentation, irrigation, apical root canal. Chemo-mechanical preparation is considered as the most essential procedure of RCT aiming to clean and shape the total root canal system, more especially to eliminate microorganisms and pathologic debris from this complex tooth area. A variety of instruments and techniques in combination with disinfecting irrigation solutions and intracanal medications have been proposed for the chemo-mechanical preparation of infected root canals. The cleaning and shaping efficiency of root canal instruments, aim to achieve a well-tapered root canal form, sufficient for the required irrigant flow in the whole canal and optimal 3D obturation. The objectives of chemomechanical preparation is continuously evaluated in s in RCT -3.It is well recognized that the biological prerequisite for treatment of apical periodontits is the elimination of pathogenic bacteria colonizing the apical part of the root canals at subcritical level compatible with periapical tissue healing ,5. Studiex vivo investigations in focusing on the minimal prerequisites for efficient and safe apical canal irrigation during the RCT of infected teeth. Data were systematically reviewed as follows:The aim of the study is to critically review observational data from relative i. The cross sectional diameter of the apical part of the most commonly used hand and rotary files were calculated according to their technical specifications.ii. The variation of diameters in the apical root canal in different tooth types was reviewed by bringing together data from classical and recent literature.iii. The observations from experimental studies approaching the risk for apical irrigant extrusion in relation to the distance of the apex and the size of apical enlargement were set.-Data Sources and Resources Selection This review is based on a comprehensive literature search using the Medline/Pubmed data base covering the period from 1960 to early 2016. The database search was performed using the keywords “apical root canal morphology”, “apical root canal preparation”, “apical root canal irrigation”. Eligible for inclusion in this study were scientific articles that were published in the English language, in international peer-reviewed journals with no limitations implemented by country of origin. The relevant papers included the abstracts and full text of clinical trials  that met the eligibility criteria. Unpublished research and studies that were reported only in abstract form, review articles, letters to the Editor, clinical guidelines and clinical studies and case reports were not considered for inclusion.Titles and abstracts were screened and then full texts of all potentially relevant publications were obtained and reviewed. Full paper copies of peer-reviewed papers were acquired electronically and cross references were further screened to identify relevant studies. Any disagreements on study inclusion and exclusion criteria were discussed and resolved either by consulting a third reviewer.-Data interpretation Part I. Data including the cross sectional diameter at 5 different file levels (D0-D5) of the most commonly used hand and rotary files according to their technical specifications were calculated by one reviewer. All hand files no #25 - #50 with 3 different tapers , the Protaper next and Universal systems (Dentsply Maillefer), and the iRace (FKG Dentaire), Revo, K3 , Wave –one (Dentsply Maillefer) and Reciproc systems  were comparatively evaluated.Part II. Data concerning the variation of diameter at 5 different root canal levels (0-5 mm from the apex) in different tooth types were extracted by two independent reviewers and placed in different tables for maxillary and mandibular teeth.ex vivo investigations on extrusion of irrigants in relation to the methods for apical canal instrumentation and irrigation were extracted by two independent reviewers and placed together in a table.Part III. Data from -ReviewPart I. In Part II. Five and eight studies including data for maxillary and mandibular teeth respectively were included in the final evaluation. In -Data analysisThe comparative data of two root canals (taken as indicative examples) and five file systems were analysed. Median maximum and minimum vales of the apical canal diameters in the distal root canal of mandibular molar and the mesiobuccal-1 root canal of maxillary molars were used. Data are shown in figures a. In both root canal types all file systems potentially leave unprepared root canal, when the median value of maximum diameters are considered. In these cases the 27-g needle tip easily reaches at a distance of 3 mm from the apex.b. In the case of distal root canal of mandibular molar some file systems potentially leave unprepared root canal, even when median value of minor diameters are considered. In this case only the 30-g needle tip reaches at a distance of 3 mm from the apex.c. In the case of mesiobuccal-1 root canal of maxillary molar all file systems seem to prepare adequately the root canal, when the median value of minor diameters are considered. In this case, as well as in the distal root canal of the mandibular molar, the 27-g needle tip cannot reach at a distance less than 4 mm from the apex after their preparation with files systems with reduced taper.ex vivo observations and data concerning the risk of apical irrigant extrusion as shown in table IV, clearly indicate that issues related to apical canal irrigation should be re-considered. Among these issues the need of minimal size of apical enlargement, the required file taper, the appropriate needle-tip and the safe distance from the apex seem to represent important prerequisites for effective and safe irrigation during RCT of infected teeth.These et al. (Irrigation fulfils several important chemical and microbiological functions. According to Haapasalo et al.  irrigatiet al. ,20. The The untouched dentinal walls by the files in the root canals, more particularly in the apical part of oval-shaped ones, underline the need for more detailed technical specifications for the effective control of all parameters of irrigation. Furthermore, other features of complex apical root canal anatomy, the lateral canals and isthmuses which cannot be reached by the mechanical canal preparation, emphasizes also the need for understanding of requirements of apical canal irrigation, in terms of dynamics of fluid flow. Deeper penetration of irrigant with increased wall shear stress effect can achieve better results in removal planktonic and biofilm bacteria . HoweverEx vivo investigations on the rate of apical extrusion in relation to instrumentation and irrigation parameters of RCT have showed interesting findings with a clinical significance. Altundasar et al. (et al. (ex vivo study with straight root canal prepared to size 35, 0.06 taper NaOCl with open-ended and close-ended needles. Manual dynamic, sonic and ultrasonic agitation were compared. Significantly more irrigant extruded with the open- than the closed-ended needles . Extrusion decreased as needles moved away from the apex. The effect of apical constriction diameter was not significant. More extrusion with manual dynamic agitation was found. Yost et al. (r et al.  performe (et al.  performet et al.  preparedThe present data indicate that for optimal therapeutic results, a specific design of the chemomechanical preparation is needed at the onset of RCT in infected teeth. Anatomical parameters, such as the type of the tooth, the shape of the root canal, the existing curvature, etc must determine the appropriate size and taper of preparation of the apical root canal, which is required for the optimal antibacterial efficacy of apical irrigation. The optimal irrigation protocols might be further specified by the technical specifications of the irrigation procedure, such as the penetration depth of the needle, the time of continuous irrigant flow, its concentration, activation parameters etc. in order to provide the best apical canal walls cleaning and effective and safe apical root canal irrigation ,24.In conclusion these data strongly confirm the previously stated assumptions that the apical root canal, which is particularly important for the successful outcome of RCT in infected teeth, poses a special challenge to irrigation as the balance between safety and effectiveness. Further critical analysis of observational data will lead scientists to design the directions of new protocols for effective and safe instrumentation and irrigation in the clinically important area of apical root canal.


Pain due to osteoarthritis (OA) is one of the most frequent causes of chronic pain. However, the mechanisms of OA pain are poorly understood. This review addresses the mechanisms which are thought to be involved in OA pain, derived from studies on pain mechanisms in humans and in experimental models of OA. Three areas will be considered, namely local processes in the joint associated with OA pain, neuronal mechanisms involved in OA pain, and general factors which influence OA pain. Except the cartilage all structures of the joints are innervated by nociceptors. Although the hallmark of OA is the degradation of the cartilage, OA joints show multiple structural alterations of cartilage, bone and synovial tissue. In particular synovitis and bone marrow lesions have been proposed to determine OA pain whereas the contribution of the other pathologies to pain generation has been studied less. Concerning the peripheral neuronal mechanisms of OA pain, peripheral nociceptive sensitization was shown, and neuropathic mechanisms may be involved at some stages. Structural changes of joint innervation such as local loss and/or sprouting of nerve fibers were shown. In addition, central sensitization, reduction of descending inhibition, descending excitation and cortical atrophies were observed in OA. The combination of different neuronal mechanisms may define the particular pain phenotype in an OA patient. Among mediators involved in OA pain, nerve growth factor (NGF) is in the focus because antibodies against NGF significantly reduce OA pain. Several studies show that neutralization of interleukin-1β and TNF may reduce OA pain. Many patients with OA exhibit comorbidities such as obesity, low grade systemic inflammation and diabetes mellitus. These comorbidities can significantly influence the course of OA, and pain research just began to study the significance of such factors in pain generation. In addition, psychologic and socioeconomic factors may aggravate OA pain, and in some cases genetic factors influencing OA pain were found. Considering the local factors in the joint, the neuronal processes and the comorbidities, a better definition of OA pain phenotypes may become possible. Studies are under way in order to improve OA and OA pain monitoring. Pain due to osteoarthritis (OA) is one of the most frequent causes of chronic pain  the local pathological processes in the OA joint leading to OA pain; (ii) the neuronal mechanisms and alterations of pain processing which are involved in OA pain; and (iii) general factors such as genetic and metabolic factors which may play a role in OA pain. The review will refer to human OA as well as to experimental OA models. It has to be noted that the vast majority of the pain studies has been made for knee OA. It should be kept in mind that there might be differences in OA of other joints.While the hallmark of OA is the progressive destruction of cartilage, OA is now considered a disease of the whole joint Felson, . The subper se is now considered a risk factor for OA progression  model of OA is used. The injection of MIA inhibits glycolysis and is toxic for chondrocytes. The pathology develops very rapidly. One day after MIA injection into the joint chondrocytes are shrunken and show fragmented pyknotic nuclei, the synovial membrane is expanded by fibrin proteinaceous edema fluid, and the joint is mildly infiltrated by lymphocytes, macrophages and plasma cells. Some days later, the inflammatory response in synovium subsides, necrotic cartilage collapses and chondrocytes are lost. Osteoclastic activity is increased, the subchondral bone collapses, and fragmentation of bony trabeculae surrounded by osteoclasts and some replacement by fibrous tissue and newly laid trabecular bone appear , and this pain may be evoked by specific activities Felson, . These aIn the clinical setting pain and other symptoms of OA are usually assessed by questionnaires such as WOMAC  or by A∂- and C-fibers which show a small response to movements to the limit of the normal movement range and much stronger responses upon noxious stimulus intensities . A long-lasting sensitization  of mechanosensitive A∂- and C-fibers is observed during experimental models of inflammation, and in addition, primarily mechanoinsensitive (silent) nociceptors begin to respond to mechanical stimuli  at 8 and 14 days of MIA-induced OA  and express iNOS they may damage neurons. If they are less aggressive, e.g., after TNF stimulation, they release cytokines and can stimulate neurons without destroying them  of the peripheral nerve fibers neuropathic changes are induced. The changes in the DRG neurons may represent responses of the cell bodies to damaging stimuli to nerve fibers in the peripheral tissue or in the peripheral nerve. However, under pathological conditions macrophages may invade the DRGs. An invasion of macrophages into the DRGs is typically found after nerve lesion  and lowering of the excitation threshold of high-threshold spinal cord neurons. Furthermore, the neurons begin to show increased responses to stimuli applied to regions adjacent to and remote from the joint, and the total receptive field can exhibit an enlargement. These changes are the neuronal basis of primary hyperalgesia (at the site of disease) and secondary hyperalgesia , the spinal content of substance P and CGRP is enhanced , is out of order in patients with severe OA pain, but importantly, it is restored under pain-free conditions after joint replacement  study patients with osteoarthritic knees showed increased activity in the cingulate cortex, the thalamus and the amygdala upon their accustomed physical activities, and this increase of activity was higher than during the application of experimental thermal pain stimuli applied to the knee region in pain free intervals , voltage-gated K+ and Ca2+ channels for the regulation of excitability, and ion channels and membrane receptors for numerous mediators, and receptors for damaging-associated molecular patterns (DAMPs) and pathogen-associated molecular pattern (PAMPS) such as Toll-like receptors  and thus evoke pain, or mediators bind to metabotropic membrane receptors which activate second messenger systems. The latter can influence ion channels of transduction and voltage-gated ion channels and thus change the sensitivity of the neuron for mechanical and thermal stimuli, and they change the excitability by influencing voltage-gated ion channels. Among the classical mediators are the prostaglandins and bradykinin. These mediators can cause a transient excitation and/or sensitization of the neurons. Of particular interest are mediators which cause a long-lasting sensitization for mechanical stimuli, and this group includes NGF and cytokines.An intriguing question is whether key mediators can be identified in the joint which are particularly involved in the generation and maintenance of OA pain. Such mediators may be targets for pain treatment because they are key molecules in the process of sensitization of nociceptive sensory neurons. It is important to note, therefore, that most of the nociceptive sensory neurons are polymodal. They respond to noxious mechanical stimuli, proportions of them respond in addition to noxious thermal stimuli such as heat and cold, and they exhibit chemosensitivity for mediators. A simplified model of a sensory ending of a nociceptor is displayed in Figure With respect to OA, the current situation is very complex for several reasons. Firstly, many mediators have been identified which are involved in the pathological process of OA. Only for some of them it has been tested whether they are directly relevant to pain by acting on nociceptive neurons. Secondly, many mediators were identified which may activate and/or sensitize joint nociceptors in experiments , and reduces the threshold of thermal excitation. Long-term exposure to NGF increases the expression of TRPV1, bradykinin receptors, purinergic P2X receptors, and the synthesis of putative nociceptive transmitters such as substance P and CGRP in DRG neurons .There is some indication of an association between the concentration of IL-6 in SF and the pain intensity of OA patients at various OA stages. The concentration of IL-6 was higher in patients with symptomatic knees compared with asymptomatic knees, and the patient-reported pain score was positively correlated with the IL-6 concentration  are of particular interest because they further synovial inflammation by stimulating leukocyte migration , IL-6, NO from chondrocytes after stimulation with IL-1β . The KOOS pain score of both groups were comparable, only standing upright was significantly more painful in patients with high BMI. By contrast, diabetic patients exhibited significantly increased pain intensity during loading the joint  and also during sitting and lying, and at night while in bed . Whereas Reimann et al.  was developed to detect cartilage degeneration at an early stage. NIRS reflects changes in the cartilage composition, water-binding properties and mechanical features of cartilage  levels were significantly associated with OA pain and decreased physical function, whereas no significant associations were found between hs-CRP levels and radiographic OA  and serum HA were significantly increased in patients with knee pain , NSAIDs, opioids and glucocorticoids  in the MIA and meniscal transsection model of OA  was developed to treat OA and cartilage injuries. PRP is prepared by centrifugation of autologous blood and includes a variety of growth and coagulation factors, adhesion molecules, cytokines, chemokines and integrins. Many of these substances can stimulate chondrocytes and multi-potent mesenchymal stem cells, induce synthesis of aggrecan and collagen type II, inhibit apoptosis and reduce the catabolic effects of inflammatory cytokines. PRP can release IL-1 receptor antagonist and soluble TNF receptor I and II, interferon γ as well as anti-inflammatory cytokines IL-4, IL-10 and IL-13 . Injection of A-803467 into the knee joint reduced the incapacitance and secondary hyperalgesia in MIA rats (Schuelert and McDougall, v1.8 is one of the sodium channels with a quite selective expression in nociceptive neurons and it is thought to contribute significantly to the generation of APs at the sensory endings (Waxman and Zamponi, v1.8 are increased by PGE2 (England et al., Other principles may become of interest. Approaches are either to target specific molecules in nociceptive neurons, such as specific ion channels, or to inhibit processes in the tissue which cause nociception. An example of the first approach is the use of sodium channel blockers. In the MIA model the intraarterial injection of A-803467, a blocker of the sodium channel NaAs an option for pain therapy, cannabinoids are discussed. Cannabinoids can act at CB1 and CB2 receptors. In the rat, CB1 receptor agonists cause antinociception in nociceptors of both the normal knee joint and MIA-induced OA knee joints, but the CB2 receptor agonist GW405833 reduced nociception in the normal knee joint but enhanced mechanonociception in OA knee joints (Schuelert et al., inhibition of osteoclast activity. In the MIA model of rat pre-emptive chronic zoledronate treatment prevented pathological changes but in rats with advanced OA this treatment only partially restored bone mineral density and had a significant, but limited effect on pain (Strassle et al., An approach targeting processes in the tissue is the In order to improve the perspective of OA patients towards a life with less pain and restrictions, considerable efforts are necessary. The review tried to address the areas which are relevant for pain in OA patients: local processes in the joint, alterations of the nociceptive system, general factors such as comorbidities. The fact that OA is now in the focus of different disciplines will hopefully further a better understanding of OA pathology and the OA pain mechanisms and ultimately lead to better treatment options. The authors believe that research on OA pathology and OA pain would benefit from more integrative approaches to identify intersections between OA pathology and OA pain mechanisms. It remains to be determined whether mechanisms of OA pathology and of OA pain can be treated in a convergent manner or whether the disease and the pain remain entities which have to be addressed separately.AE and H-GS revised literature, wrote the article, prepared figures. GOH revised literature, wrote the article.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.