The rapidly advancing field of utilizing GCaMP reporter constructs represents a major step forward in our ability to monitor intracellular Ca2+ dynamics. With the use of these genetically encoded Ca2+ sensors, investigators have studied activation of endogenous Gq types of G protein-coupled receptors (GPCRs) and subsequent rises in intracellular calcium. Escalations in intracellular Ca2+ from GPCR activation can be faithfully monitored in space and time as an increase in fluorescent emission from these proteins. Further, transgenic mice are now commercially available that express GCaMPs in a Cre recombinase dependent fashion. These GCaMP reporter mice can be bred to distinct Cre recombinase driver mice to direct expression of this sensor in unique populations of cells. Concerning the central nervous system (CNS), sources of calcium influx, including those arising from Gq activation can be observed in targeted cell types like neurons or astrocytes. This powerful genetic method allows simultaneous monitoring of the activity of dozens of cells upon activation of endogenous Gq-coupled GPCRs. Therefore, in combination with pharmacological tools, this strategy of monitoring GPCR activation is amenable to analysis of orthosteric and allosteric ligands of Gq-coupled receptors in their endogenous environments.The family of GCaMPs are engineered proteins that contain Ca This is justified as their interacting, coupled receptors have been an established source of clinically active therapeutics. Several G proteins contain lipid modifications on one or more of their subunits to enable targeting to the plasma membrane while facilitating protein interactions. The precise arrangement and targeting of subunits in heterotrimeric G proteins affects not only which receptor with which it can interact, but also the downstream effector target. This general scheme of extracellular signal transduction has been selected for across evolution and has been repeated in nature abundantly. Built-in flexibilities originating from unique receptors, G-proteins and effectors provide the means to distribute distinct physiological response pathways to external stimuli ranging from photons to complex protein hormones (Guanosine nucleotide-binding proteins (G proteins) are intracellular proteins involved in transmitting signals from outside a cell to the inside of the cell . Since thormones .α which belong to a larger group of enzymes called GTPases. The Gα subunit of heterotrimeic G proteins is highly controlled by factors that influence its ability to bind to guanosine triphosphate (GTP). The GTPase activity of Gα proteins hydrolyze GTP to guanosine diphosphate (GDP). When bound by GTP, Gα is considered in an active state and when bound by GDP, Gα is in a less active state  by activation of adenylyl cyclase. Another prominent branch of this family of biological signaling tools includes Gαq.There are ∼16 genes found in human that encode different forms of Gve state . The timαq and a closely related gene Gα11, are broadly expressed and maintain homeostatic processes in digestive, urinary, cardiovascular and central nervous systems (CNS). It is critical to appreciate that activated Gq/11 results in several parallel signaling pathways that include mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K)/AKT pathways. However, the pathway in which GTP bound Gq/11 (as well as some combinations of Gβγ) can stimulate the activity of the effector protein phospholipase Cβ (PLCβ) is the most studied (2) to diacyl glycerol (DAG) and inositol trisphosphate (IP3). An increase in intracellular IP3 rapidly gates ionotropic IP3 receptors embedded in membranes of endoplasmic reticulum (ER). IP3 receptor activation results in the flux of calcium ions (Ca2+) from highly concentrated internal ER stores to low concentration intracellular cytoplasmic regions via channel gating  that preferentially interact with Gq/11 include the group I metabotropic glutamate; M1, M3, and M5 muscarinic acetylcholine; 5-HT2 serotonergic, α1 adrenergic, vasopressin, angiotensin II and histamine H1 receptors among several others.A vast amount of research has recognized an enormous array of extracellular and intracellular stimuli that dictate changes in the intracellular second messenger Ca kingdom . Cells dq/11-coupled GPCRs were inserted into recombinant, mammalian directed expression vectors and subsequently driven to be transcribed by strong promotors into a variety of host cells. Some of these expression studies have greatly contributed to our atomic level structural understanding and knowledge of these critical receptor subtypes. Expression strategies have served many other useful purposes including pharmacological profiling, detailed determination of signal transduction pathways as well as site-directed mutagenesis studies of critical amino acids involved in structure, function and ligand binding. However, one caveat with this general paradigm is the issue of over-expression of the receptor.A great deal of our knowledge base of these receptor subtypes has its origins in the cloning era of these genes. The coding regions of many Gαq/11-coupled GPCRs in their endogenous states, which more closely reflect the natural environment, are becoming sharper and increasingly more available. The focus of this review article will address combining methods and paradigms from the fields of calcium imaging, mouse genetics and pharmacology to uncover endogenous Gαq/11-coupled GPCRs and their responses to acute or sustained stimulation at the molecular and cellular levels.Does placing too many of the same receptor type bias a signal transduction pathway through mass action relationships? Can over-expression lead to too many spare receptors and lead to aberrant constitutive activity? Questions like these have been addressed and will need to be monitored heading into the future utilizing this set of critical tools and methodologies. Nevertheless, methods examining Gq/11-coupled GPCRs in selective cells has become more prevalent using the power of transgenic animals. Temporally and spatially regulated genes can be monitored with fluorescent microscopy in mice by utilizing a DNA recombination system based on Cre recombinase. Cre based systems using P1 bacteriophage Cre recombinase which catalyze the excision of DNA located between flanking loxP sites, has been widely used since its first application in eukaryotic cells and transgenic mice  transgenes. It is now routine to accurately drive the expression of genetically programmed fluorescent reporters  or Cre recombinase in specific cellular populations with these large (150–350 kbp) transgenes. The GENSAT (Gene Expression Nervous System Atlas) project has used this technology to generate mouse lines with targeted cellular expression of eGFP or Cre recombinase . One advdrd2-eGFP BAC transgene had an altered phenotype  can represent a fundamental change of state in many cell types. Biological processes ranging from cardiac muscle contraction, insulin secretion, cell adhesion, proliferation or cell death represent cellular and molecular reactions dependent upon [Ca2+]i. Notably, these signals vary with a time course of milliseconds in the case of muscle contraction, to minutes in the case of sustained insulin secretion, to hours or days in some cases of programmed cell death. Therefore, it is critical that the period of time in which [Ca2+]i changes occur, can be reliably monitored in an endogenous environment. By measuring the kinetics of calcium transients, important information can be inferred such as properties of ligand kinetics, receptor reserve and amplification of signaling  ester forms of these compounds by adding the dissolved indicator to various types of cell preparations. Endogenous, ubiquitous esterases cleave ester bonds and “trap” the now membrane impermeant Ca2+ sensitive dye intracellularly. The Ca2+ dependent amount of light emitted from these cells is generally measured using fluorescence microscopy, fluorescence microplate assays, or flow cytometry in combination with photon detection. The pharmacological and biophysical properties of these organic dyes have been reviewed  gene  gene . GFP is FP) gene . One gencapacity . Additioty . Mitochondria are other vital intracellular organelles that can serve as critical sources of calcium upon proper stimulation. These two examples represent significant reservoirs of calcium that facilitate a local rise in [Ca2+]i by a subcellular dependent fashion. As an example of the advancing technology integrating genetics and [Ca2+]i imaging, 2+ from mitochondria ([Ca2+]m) in astrocytes using improved and compartmentalized GCaMP probes while q/11 calcium mobilization systems.However, what seems to be under-utilized by GCaMP functionality in the literature to date is the versatility to monitor increases in intracellular calcium from ignaling . It is nin utero electroporation -3,5-dihydroxy-phenylglycine (DHPG). DHPG has been utilized as one of the most selective Group I mGluR orthosteric agonists. Recently, brain slice preparations of the striatum detected rises in [Ca2+]i from genetically identified neurons following acute DHPG application using either organic dye loading methods (drd1) or D2 (drd2) gene driven Cre recombinase provided a scaffold to monitor DHPG mediated changes in [Ca2+]i. The acute application of DHPG did not affect the basal fluorescence of GCaMP3 in most of the imaged D1+ or D2+ striatal neurons  would be one requirement of such a sensor. In fact, GPCR activation has been observed with several imaging probe techniques including intramolecular and intermolecular Förster resonance energy transfer (FRET)-based genetically encoded indicators (Another goal moving forward in the GPCR field is to develop a “universal” detector of endogenous GPCR activation. The detection of protein–protein interactions (e.g., receptor-G-proteins, liberation of Gdicators . Howeverdicators . By combin vivo preparations (in vivo. This appears to be a reasonable challenge moving forward as detection of small but reliable Ca2+ increases can be detected in very fine mouse astrocyte processes both in vitro (in vivo (Improving GCaMP fluorescent signals have been detected using arations , even inarations . These ein vitro  and in v(in vivo . These oThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Scientific Reports6: Article number: 18732; 10.1038/srep18732 published online: 01072016; updated: 02152016The original version of this Article contained typographical errors in the spelling of the authors Xiaoyong Qiao, Yaxian Ma, Weiyao Yin, Wenming Xu and Liangzhi Xu which were incorrectly given as Xiao Yong Qiao, Ya Xian Ma, Wei Yao Yinrg, Wen Ming Xu and Liang Zhi Xu respectively.In addition, there was a typographical error in the Abstract.2 expression and ALP activity.”“Inhibition of p38 MAPK by SB023580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on Runxnow reads2 expression and ALP activity.”“Inhibition of p38 MAPK by SB203580 or pERK by U0126 abolished the proliferation and up-regulatory effects of irisin on RunxThese errors have now been corrected in the PDF and HTML versions of the Article.


Mycobacterium tuberculosis (Mtb) remains a global cause of considerable morbidity and mortality [Tuberculosis (TB) caused by the bacteria ortality . One of ortality .Mtb antigens and possibly contribute to a higher incidence of active TB disease [Helminth infections (soil transmitted and vector-borne) exhibit broad geographic overlap with areas of TB endemicity . These c disease . In this disease  in a giv disease  comparedThe question of what constitutes protective immunity in human TB is an evolving issue. A few well-defined risk factors such as advanced HIV disease and older age have been established; in addition, the pivotal protective role of a CD4+ response involving primarily interleukin 12 (IL-12), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) (Th1-like) has been established from human genetic and animal model studies . There iMtb entry into DCs, was seen on exposure to live microfilariae [Mtb was noted in a mouse model of infection with the intestinal helminth Nippostrongylus brasiliensis mediated through IL-4 receptor–mediated alternative macrophage activation [Studies have shown direct and indirect effects of helminths on APCs. Decreased viability and function of dendritic cells (DCs)  as well filariae . In additivation . Finallytivation .It is well established from in vitro and neonatal priming studies in animals that the cytokine/chemokine milieu in which a T cell has its primary encounter with antigen determines the response (Th1/Th2) and the eventual outcome of infection . It is aThe association between active TB disease and coincident helminth infection hasbeen investigated primarily in cross-sectional studies , as prosHelminths have evolved complex mechanisms for immune subversion, with effects on both adaptive and innate immune responses that lead to their long-term persistence. Spillover effects on mycobacterial antigen responses have been seen in both in vitro and in vivo studies. A possible mechanism of interaction between the two infections is outlined in


Background. Endothelial function is impaired in chronic heart failure (CHF). Statins upregulate endothelial NO synthase (eNOS) and improve endothelial function. Recent studies demonstrated that HDL stimulates NO production due to eNOS phosphorylation at Ser1177, dephosphorylation at Thr495, and diminished phosphorylation of PKC-βII at Ser660. The aim of this study was to elucidate the impact of rosuvastatin on HDL mediated eNOS and PKC-βII phosphorylation and its relation to endothelial function. Methods. 18 CHF patients were randomized to 12 weeks of rosuvastatin or placebo. At baseline, 12 weeks, and 4 weeks after treatment cessation we determined lipid levels and isolated HDL. Human aortic endothelial cells (HAEC) were incubated with isolated HDL and phosphorylation of eNOS and PKC-βII was evaluated. Flow-mediated dilatation (FMD) was measured at the radial artery. Results. Rosuvastatin improved FMD significantly. This effect was blunted after treatment cessation. LDL plasma levels were reduced after rosuvastatin treatment whereas drug withdrawal resulted in significant increase. HDL levels remained unaffected. Incubation of HAEC with HDL had no impact on phosphorylation of eNOS or PKC-βII. Conclusion. HDL mediated eNOS and PKC-βII phosphorylation levels in endothelial cells do not change with rosuvastatin in CHF patients and do not mediate the marked improvement in endothelial function. Patients with chronic heart failure (CHF) are characterized by endothelial dysfunction which is associated with worse prognosis . DiffereHigh-density lipoprotein (HDL) plasma levels are of prognostic relevance for cardiovascular diseases. Nevertheless, several pharmacological approaches to increase HDL quantity failed to reduce cardiovascular events . TherefoβII (PKC-βII) results in pronounced phosphorylation of eNOS at its inhibitory site Thr495 whereas the eNOS activating phosphorylation at Ser1177, mediated by PI3K and Akt, is reduced. Thereby, the stimulating effect of isolated HDL on NO production in endothelial cells is blunted. However, this process seems partially reversible with exercise training [In CHF, functional properties of HDL are altered. With increasing disease severity, more malondialdehyde is bound to HDL particles and subsequent activation of protein kinase-Numerous studies have shown that statins improve endothelial function mediated by increased eNOS expression, reduced oxidant stress, and restored endothelial repair mechanisms via circulating endothelial progenitor cells –12.1177 and increased phosphorylation at Thr495 in cultured endothelial cells. This was associated with reduced eNOS-dependent NO production and increased superoxide generation and resulted in impaired vasodilation of murine aortic rings in response to acetylcholine. Interestingly, a short time treatment with simvastatin partially corrected these dysfunctional properties of HDL [Recently, Chang et al. found that HDL isolated from patients with valvular heart disease in comparison to healthy controls inhibited phosphorylation of eNOS at Sers of HDL .These findings indicate that the statin related improvement in endothelial function in humans might be partially mediated by functional properties of HDL.βII and eNOS at Ser1177 and Thr495 in cultured endothelial cells. Furthermore, we report for the first time the effect of statin withdrawal on blood lipids and endothelial function in CHF patients.Previously, we reported the effects of a high-dose rosuvastatin therapy for 12 weeks in patients with CHF on flow-mediated dilatation (FMD) of the radial artery and endothelial repair mechanisms . To furthttps://clinicaltrials.gov/ with the following number: NCT00176332.The main clinical trial is registered at The study protocol was approved by the ethics committee of the University of Leipzig and written informed consent was obtained from all patients.n = 9, placebo group n = 9). Patients with CHF as a result of either ischemic heart disease or dilated cardiomyopathy, clinically stable at New York Heart Association class II or class III for at least one month, left ventricular-ejection fraction <40%, end diastolic left ventricular diameter >55 mm, and peak oxygen uptake <20 mL/min/kg body weight were included. Significant valvular disease and ongoing nicotine abuse served as exclusion criterions. Patients were randomly assigned in a double blind manner to an intervention group receiving 40 mg rosuvastatin daily or a placebo group. At the beginning of the study (bsl), after 12 weeks of active treatment (12 wk), and 4 weeks after discontinuation of the study drug (16 wk), blood samples were taken from all patients to determine blood lipid levels and isolate HDL particles.In a subgroup of 18 patients from the main study cohort, further evaluation of HDL function was performed (rosuvastatin group Flow-mediated dilatation (FMD) of the radial artery was measured using a high-resolution ultrasound scanning echo-tracking angiometer  as described previously .d = 1.006–1.21 g/mL) as recently described in detail [HDL was isolated from serum by sequential density ultracentrifugation  as recently described [Human aortic ECs  were cultured in EGM-2 cell culture medium : Cells were incubated for 0, 5, 10, 15, 30, or 60 minutes with 50 escribed , 14, 15.1177 and antiphospho-eNOS-Thr495 , and anti-PKC-βII and antiphospho-PKC-βII-Ser660 (both Santa Cruz). For the evaluation of HDL-induced phosphorylation of the respective protein, the maximal stimulation was used as recently described [Ten micrograms of total protein was separated on a denaturing polyacrylamide gel and transferred to a PVDF membrane. To detect specific proteins, the following antibodies were applied: anti-eNOS (Santa Cruz), antiphospho-eNOS-Serp value of less than 0.05 was considered statistically significant.Data were analysed using SPSS version 22 (IBM Corp.). For the descriptive statistics of clinical parameters, median and interquartile range were calculated. Mean values ± standard error (SEM) was calculated for all other variables. Repeated measures ANOVA were used to test for change over time. Comparisons from baseline were performed using an analysis of covariance. Categorical variables were tested applying Fisher's exact test. Correlation between selected variables was estimated by Spearman's rank correlation coefficient. A The characteristics of the study patients are depicted in p < 0.001 for bsl versus 12 wk and 12 wk versus 16 wk). However, HDL levels remained unaffected   .As already shown in an earlier publication, the flow-mediated dilatation of the radial artery significantly improved by 183% with rosuvastatin treatment without any change in the placebo group . The FMD1177  or Thr495  and does not influence phosphorylation of PKC-βII at Ser660 . In the placebo group, no change in these parameters was evident  nor at Thr495  or PKC-βII at Ser660 .In the rosuvastatin group, we found no correlation between flow-mediated dilatation and phosphorylation of eNOS neither at SerβII at Ser660 and the phosphorylation of eNOS at its activity regulating sites Ser1177 and Thr495 in cultured endothelial cells. (ii) Four weeks after rosuvastatin withdrawal, the treatment effect on LDL cholesterol level and endothelial function is completely abolished.The following findings emerge from this study: (i) Treatment with rosuvastatin 40 mg for 12 weeks in patients with chronic heart failure does not affect the ability of isolated HDL to change the phosphorylation of PKC-1177 and to inhibit eNOS phosphorylation at the Thr495 site in cultured human umbilical vein endothelial cells, resulting in enhanced NO production. Vasodilation of murine aortic rings significantly increased when incubated with HDL from simvastatin treated patients compared to HDL from untreated patients in this study [Our data are in contrast to findings by Chang et al. who demonstrated that low-dose simvastatin treatment with 20 mg per day even for the short period of 5–7 days in patients with valvular heart disease was sufficient to induce HDL-mediated phosphorylation of Akt and eNOS at Seris study .Beside different statins used with different dosages and treatment periods, the patient groups studied differ significantly. We selected heart failure patients with reduced left ventricular-ejection fraction  and excluded those with more than mild-to-moderate valvular disease. Additionally, more than one-third of patients in our group suffered from ischemic heart disease due to coronary artery disease whereas Chang et al. excluded patients with coronary artery disease. A previous study from our group indicated that HDL dysfunction in patients with reduced LV-EF is related to disease severity with lower HDL-induced NO production in patients according to New York Heart Association Classification class IIIb compared to those in class II . InteresβII and downstream eNOS phosphorylation at Thr495 resulting in diminished NO production and endothelial dysfunction [βII phosphorylation remained totally unchanged in response to rosuvastatin even though serum markers of oxidant stress including oxidized LDL and lipid peroxide concentration were reduced as already reported [The amount of malondialdehyde (MDA) bound to HDL particles has been shown to be crucial for HDL-mediated activation of PKC-function , 16. Parfunction . Also infunction . It has function , 19. In reported . We therα dependent mechanism and thereby enhanced downstream effects on eNOS [However, results from cell culture experiments indicate that statin treatment might improve HDL-mediated eNOS activity via changes on the receptor site: Apolipoprotein A1, a key lipoprotein in HDL particles, binds to the scavenger receptor-BI on endothelial cells . The sti on eNOS .1177 in a PI3K/Akt-dependent manner [Additionally, another signalling pathway evolved in HDL-mediated eNOS activation was found to be modified by statins: sphingosine 1-phosphate (S1P), which is enriched in HDL fractions, binds to G-protein-coupled S1P receptors on endothelial cells and regulates eNOS activation . The inct manner , 23, 24.To demonstrate such treatment effects in humans, endothelial cells would have to be harvested before and after oral statin treatment. This has not been performed in the present study.Recent analysis of the West of Scotland Coronary Prevention Study identified a long-term legacy benefit from five years of cholesterol lowering therapy with pravastatin in middle aged men at increased risk for atherosclerosis through decreased cardiovascular event rates and death from cardiovascular causes even 15 years after the active treatment period . HoweverThere are some limitations: First, the sample size for the analysis of HDL function is quite low. However, we found not even a trend towards altered eNOS phosphorylation levels after rosuvastatin therapy, whereas lipid levels and endothelial function clearly changed with therapy and drug withdrawal. Second, we did not evaluate the effect of rosuvastatin on SR-BI or S1P receptor expression and Pon activity and MDA. Nevertheless, primary goal of the study was to investigate the influence of rosuvastatin on eNOS activity. Third, NO generation in endothelial cells in response to HDL was not quantified, and we cannot rule out the possibility that rosuvastatin ameliorates NO generation independent of the evaluated phosphorylation levels.βII and eNOS at Ser1177 and Thr495 in cultured endothelial cells. We did not find any relation between FMD and HDL-induced PKC-βII and eNOS phosphorylation with or without rosuvastatin treatment. This indicates that HDL function, at least its effect on eNOS phosphorylation, does not mediate the marked improvement in endothelial function with rosuvastatin treatment in CHF patients.Rosuvastatin treatment in CHF patients does not alter the ability of isolated HDL on the phosphorylation levels of PKC-


Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration.  Applying these techniques for 3D tissue analysis is seriously limited by the need to section the tissue, image each section, and then reassemble the images into a 3D representation of the structures of interest. Here we present a fundamental shift from the two dimensional plane to that of three dimensions whilst retaining molecular structures of interest without the need to section the plant tissue. Recent advances in fixation and ‘clearing’ techniques such as SeeDBleA2Fixation and embedding of plant tissue for molecular interrogation using techniques such as histological staining, immunohistochemistry or A. thaliana apical meristems however with harsh enzymatic degradation, the tissue lost structural integrityThree dimensional imaging of plants using confocal microscopy has been limited to already semi-transparent tissue types such as root tips or meristems but resolution becomes limiting in cells deeper within tissues7A. thaliana and N. tabacum  and calcofluor white  to assess the retention of DNA and cellular structure . Using CA. thaliana lines stably transformed with GFP localised to the endoplasmic reticulum  or with CFP localised to peroxisomes A. thaliana leaves, including the internal leaf phloem at a depth of ~80 μm  0.05% aqueous calcofluor white for 20 mins in dark before being washed with 0.001% NaN3 in PBS pH 7.4 with at least 3 changes to remove all SDS (SDS inhibits enzymatic activity). Once all SDS was removed samples were carefully placed into the enzyme cocktail mix containing cellulase, xylanase, arabinofuranosidase, pectate lyase and α-amylase .


Wolbachia pipientis is a ubiquitous, maternally transmitted bacterium that infects the germline of insect hosts. Estimates are that Wolbachia infect nearly 40% of insect species on the planet, making it the most prevalent infection on Earth. The bacterium, infamous for the reproductive phenotypes it induces in arthropod hosts, has risen to recent prominence due to its use in vector control. Wolbachia infection prevents the colonization of vectors by RNA viruses, including Drosophila C virus and important human pathogens such as Dengue and Chikungunya. Here we present data indicating that Wolbachia utilize the host actin cytoskeleton during oogenesis for persistence within and transmission between Drosophila melanogaster generations. We show that phenotypically wild type flies heterozygous for cytoskeletal mutations in Drosophila profilin (221chic/+ and 1320chic/+) or villin (6-396qua/+) either clear a Wolbachia infection, or result in significantly reduced infection levels. This reduction of Wolbachia is supported by PCR evidence, Western blot results and cytological examination. This phenotype is unlikely to be the result of maternal loading defects, defects in oocyte polarization, or germline stem cell proliferation, as the flies are phenotypically wild type in egg size, shape, and number. Importantly, however, heterozygous mutant flies exhibit decreased total G-actin in the ovary, compared to control flies and 221chic heterozygous mutants exhibit decreased expression of profilin. Additionally, RNAi knockdown of profilin during development decreases Wolbachia titers. We analyze evidence in support of alternative theories to explain this Wolbachia phenotype and conclude that our results support the hypothesis that Wolbachia utilize the actin skeleton for efficient transmission and maintenance within Drosophila. Wolbachia pipientis, infects 40% of insect species and is currently used to prevent transmission of Dengue by mosquitoes. The bacterium targets the germline of insects, where it is faithfully transmitted to the developing oocyte and the next generation. Here we identify host cytoskeletal proteins required by Wolbachia in order to be efficiently transmitted between Drosophila melanogaster generations. We show that after only two generations in a phenotypically wild type, heterozygous mutant fly, Wolbachia infections are cleared or reduced in titer. Characterization of the mutants suggests that Wolbachia is sensitive to the regulation of actin in the ovary and that actin may be used by Wolbachia to both target and proliferate within host tissues and to be faithfully, maternally transmitted.The world’s most common intracellular infection,  Wolbachia pipientis is an intracellular α-proteobacterium that forms symbioses with an extremely broad array of hosts, including isopods, nematodes, and insects [Wolbachia were first noted in the tissues of the mosquito, Culex pipiens, by Hertig and Wolbach in 1924, but subsequently, many more insects were found to harbor Wolbachia. Current estimates suggest that upwards of 40% of insect species may be infected by the parasite, making Wolbachia one of the most common intracellular bacteria on the planet [Wolbachia are well known for the reproductive effects induced in the host, which range from the exotic  to the most common of reproductive effects, cytoplasmic incompatibility (CI) [Wolbachia are heavily studied as potential drug targets for filarial nematode infection [Wolbachia may be one answer to controlling some vector borne human diseases—indeed mosquitoes harboring a virus-blocking strain of Wolbachia are presently being released in underdeveloped parts of the world with this hope in mind [Wolbachia in the insect world, and its relevance to human health, it is essential to understand the biological basis of transmission of the symbiont between host generations. insects . Wolbachnfection ,4 and arnfection ,6. Wolba in mind . Given tWolbachia are maternally transmitted bacteria that infect the germline of their hosts such that their transmission fidelity in wild populations is extraordinarily high. Although physiologically stressful conditions are known to induce the loss of superinfections [Drosophila populations as well as in insects harboring transferred Wolbachia infections [Wolbachia’s maternal transmission and in addition, densities in the embryo, and posterior localization, are correlated with reproductive phenotype (e.g. CI) [fections , perfectfections –12. Locae.g. CI) ,14.Wolbachia interactions with host cytoskeletal elements. Specifically, in Drosophila, Wolbachia require host microtubules and the motors Dynein and Dynactin for anterior localization early in development and Kinesin-1 for posterior localization in mid oogenesis, positioning them for inclusion in the germline [Wolbachia use astral microtubules during asymmetric divisions in the developing embryo, leading to the widespread, but uneven, pattern of localization of the bacteria in adult tissues [Wolbachia undergo somatic cell to germline transmission, suggesting an ability for the bacterium to alter the host actin cytoskeleton to facilitate uptake by germ cells [Wolbachia proteins from the Brugia malayi symbiont and host actin [Wolbachia ultrastructure in Brugia does not reveal any obvious mechanism  [in vitro biochemistry and until now, no genetic evidence of interaction between Wolbachia and actin has been reported.Previous studies have provided some support for germline ,16. This tissues . In bothrm cells ,19. Morest actin , althougtsiales) . These pWolbachia persistence and transmission within Drosophila melanogaster is sensitive to mutations affecting the actin cytoskeleton. The importance of actin during Wolbachia infection was investigated by acquiring Drosophila mutants in actin binding proteins, both involved in the regulation of F-actin filaments: the homologs of profilin (chickadee), which regulates the formation of filamentous actin, and villin (quail), which bundles actin filaments. We show that flies heterozygous for mutations in profilin (chic221/+ and chic1320/+) or villin (qua6-396/+) lose Wolbachia infection after only a few generations. Importantly, the effect is due to both an inability of Wolbachia to efficiently colonize germaria in heterozygous mutant hosts and by a reduction in titer when the host is infected. Importantly, both the less severe chic allele (chic1320), known to decrease an oocyte specific isoform of Drosophila profilin chickadee [chic allele (chic221) produced a Wolbachia titer phenotype. We identified two different actin binding proteins (profilin and villin) that affect Wolbachia transmission and maintenance, supporting the conclusion that Wolbachia persistence within the host is sensitive to actin.Here we present data showing that http://flystocks.bio.indiana.edu/): stock number 145, which carries w1 was used as the Wolbachia infected control line. Two chickadee mutant fly stocks were used in this study. The chic221cn1/CyO; ry506 flies carry a null recessive allele resulting from the deletion of 5’ non-coding and some chic-coding sequences [P{PZ}chic01320cn1/CyO; ry506 flies carry a strong homozygous infertile loss-of-function allele in chickadee, generated by P-element insertion [quail mutant flies, qua6-396/SM1, carry a female sterile, recessive mutation induced by ethyl methanesulfonate [w1118; Df(2L)BSC148/CyO, is a chromosomal deletion of segments 36C8-36E3, covering the region containing the quail locus. The second of these stocks #24377, w1118; Df(2L)BSC353/CyO, covers segments 26A3-26B3, the region containing the chic locus. Both of these chromosomal deletions are part of the aberration stock collection and were created by FLP-mediated recombination between FRT-bearing transposon insertions [Wolbachia were introduced into the heterozygous mutant backgrounds through crosses between w1 infected females (stock 145) and uninfected heterozygous males (mutant/CyO). In order to control for genetic background, we also created isogenized lines by backcrossing stock 145 and each mutant line to an uninfected w; Sco/Cyo stock for three generations  at Indiana University  [w; P{w+, Act GAL4} /TM3 males. In order to knock down profilin, we then crossed homozygous females from this line to a homozygous Hsp70:Gal4 driver . An additional control for expression from the Hsp70:Gal4 driver included a UAS:GFP stock . Flies were shocked at 37C for 10 minutes to induce the short hairpin. Wobachia infection status for stocks acquired from the BDSC was determined via PCR and Western blot targeting the gene wsp or its product (see methods below). All flies examined for Wolbachia infection in the experiments below were age matched in order to avoid confounding correlations between fly age and Wolbachia titer.In addition to these isogenized lines, and to examine the effect on 0}attP2) . In ordeWolbachia Surface Protein (WSP), NR-31029, and used at a dilution of 1:1000. Additionally, we used anti-actin monoclonal at 1:10,000 (Seven Hills Bioreagents) as a loading control as well as secondary antibodies: HRP enzyme conjugates (Invitrogen) at 1:5000. Densitometry measures were made in ImageJ using scanned film with same exposure times across multiple experiments. Control and experimental flies were included on the same blot in order to ensure consistencies in measured ratios.Flies were ground in 1.5ml centrifuge tubes using an electric hand drill and disposable pestle in lysis buffer: 150mM NaCl, 1% Triton X-100, 50mM TrisHCl (pH8) containing HALT protease inhibitor cocktail (Thermo Scientific) and 5 mM EDTA. The lysates were centrifuged for 1 minute at 8000 X g to pellet debris. Samples were heated for 5 minutes at 95°C in Laemmli sample buffer containing 5% β-mercaptoethanol (Bio-Rad) prior to SDS-PAGE electrophoresis. Proteins were separated on 4–20% Tris-Glycine NB precast gels (NuSep) in 1X Tris/Glycine/SDS running buffer (Bio-Rad) and transferred to PVDF membrane in Tris-Glycine transfer buffer with 15% methanol at 40v on ice for 3–4 hours. The membrane was blocked for 5 minutes in Starting Block T20 (TBS) Blocking Buffer (Thermo Scientific), followed by incubation in primary antibody (for 1 hour at RT or O/N at 4°C) according to standard protocols. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) was used according to the manufacturer’s instructions to detect HRP (after incubation with secondary antibodies) on the immunoblots. Blots were re-probed after stripping in 100mM Glycine, 0.15 ND-40, 1% SDS, pH 2 for 1 hour at RT, then overnight at 4°C. PageRuler prestained protein ladder (Thermo Scientific) was used as a molecular mass marker. The following antibody was obtained through BEI Resources, NIAID, NIH: Monoclonal Anti-Wolbachia FtsZ was diluted 1:150 in PBST with 1% BSA. Cy3 conjugated to goat anti-mouse secondary antibody (Jackson Immunoresearch) or rabbit secondary antibody (Jackson Immunoresearch) diluted 1:250 in PBST + BSA was used to detect the primary antibody. For F-actin detection we used Acti-stain 488 Fluorescent Phalloidin . Tissues were mounted in Slow Fade “Gold” antifade reagent (Invitrogen) and stored at 4°C.Immunohistochemistry was performed as follows: ovaries for immunolocalization were dissected in Ringer’s solution 3–5 days after fly eclosion, then fixed as previously described  with folin situ hybridization, following published protocols [°C. In the morning, washes in DEPC-PBST preceded a 5 minute proteinase K treatment (0.05 mg/mL) at 37C before prehybridization in hyb buffer . Universal bacterial probe EUB338 conjugated to Alexa488 (Molecular Probes) was used to detect Wolbachia in the ovarioles. Hybridized ovaries were mounted in Slow Fade “Gold” antifade reagent (Invitrogen).To confirm staining by immunohistochemistry, we also used fluorescent rotocols  with theWolbachia and F-actin within the germarium z-sections maximum projections were used and regions of the germarium demarcated using masks . Care was taken such that exposure times were normalized across all experiments. For quantification of ng masks . We wereWolbachia titer (with reference to the host) using an Applied Biosystems StepOne Real-time PCR system and SybrGreen chemistry (Applied Biosystems). We used wsp primers for Wolbachia  and Rpl32 primers for the host  at the following temperatures: 95°C for 10 min, then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. To detect number of profilin transcripts we utilized the RNA extracted from these flies and the SensiFAST SYBER Hi-ROX One-step RT mix (Bioline) and the following primer set: chicF: TGCACTGCATGAAGACAACA, chicR: GTTTCTCTACCACGGAAGCG . Reactions were performed in a 96-well plate and calibration standards were used in every run to calculate primer efficiencies. These efficiencies, along with the CT values generated by the machine, were used to calculate the relative amounts of Wolbachia using the ΔΔ Ct (Livak) and Pfaffl methods [DNA was extracted from flies utilizing the Qiagen DNeasy Blood and Tissue Kit (Qiagen) according to directions with the following modification. Flies were ground in a 1.5ml centrifuge tube using a disposable pestle and an electric hand drill in 180 ul PBS, 200 ul ALT buffer, and 20 ul Proteinase K solution. The samples were incubated at 56°C for 10 minutes with vigorous shaking and then centrifuged briefly to pellet debris before continuing with the ethanol precipitation in the kit protocol. DNAs were quantified by measuring absorbance at 260nm using an Epoch spectrophotometer (Biotek). Semi-quantitative PCR was performed by standardizing the amount of DNA in each reaction. We utilized Phusion High Fidelity PCR Master Mix with HF buffer (New England Biolabs). The protocol for amplification was: 98°C for 3 minutes, followed by 25 cycles of 98°C for 10 seconds, 56°C for 45 seconds, 72°C for 1 minute 30 seconds with a final 10 minute extension at 72°C. Primers were as follows: wsp F1 5’-GTC CAA TAR STG ATG ARG AAA C—3’ and wsp R1 5’- CYG CAC CAA YAG YRC TRT AAA -3’ . RNA andin vivo F/G actin assay kit . Age-matched, virgin female flies from chic221/Cyo or control (stock #145) were dissected in LAS2 buffer at 37°C and incubated for 10 minutes at 37°C. A brief 300 g centrifugation step (5 minutes) was followed by a 1 hour ultracentrifugation at 100,000 g at 37°C. Supernatants containing globular actin were removed and pellets resuspended in actin depolymerization buffer on ice, by pipetting up and down every 15 minutes for 1 hour. Pellets containing F-actin fractions and supernatants containing G-actin fractions were run on an SDS-PAGE gel and Western blots performed (as above) using a primary mouse monoclonal anti-actin antibody. Bands were quantified using densitometric analysis in ImageJ (as above).In order to identify the ratio of filamentous to globular actin in ovaries from age matched flies, we used ultracentrifugation coupled to SDS-PAGE and Western blots using an Wolbachia upon receipt from the Bloomington Drosophila Stock Center. In order to establish an infection in the flies, infected control females were crossed with mutant uninfected males to generate F1 progeny, half of which carried the mutation, and half of which carried the Cyo balancer (a second chromosome containing inversion breakpoints and a dominant visible mutation of curly wings). F1 heterozygous mutants for the actin binding protein alleles were then back-crossed to the paternal mutant line (mutant/Cyo) and F2 progeny from that cross, carrying the mutation and harboring straight wings, were collected. We screened both the F1 and F2 progeny for Wolbachia infection using PCR against the Wolbachia surface protein gene (wsp)  compared to the control lines . As additional support for the importance of the chic and qua loci in the Wolbachia titer defects we observed, we also quantified the amount of Wolbachia within two chromosomal deficiency stocks (deletions in the same region as either chic or qua in isogenic backgrounds)[chic and qua mutants, supporting our observation that these genomic loci are responsible for the Wolbachia titer defect .All three actin binding protein mutant fly stocks used in this study were uninfected with ne (wsp) . We obse progeny . In ordeol flies . Progenykgrounds). These dr defect . In addiWolbachia titer, we created isogenized lines from the control stock (145) and each of the mutant stocks by backcrossing to an uninfected w; Sco/Cyo line for three generations. We then crossed these Wolbachia infected F3 females  to Wolbachia uninfected w; mutant/Cyo males . This result suggested to us that the reduction in titer was at least partially due to a result of a developmental defect in Wolbachia maintenance and persistence within the heterozygous mutant hosts.In order to control for effects of host genetic background on yo males . In the y1sc* v1; P{TRiP.HMS00550}attP2) [w; P{w+, Act GAL4} /TM3 males. From this cross we only recovered stubble progeny, suggesting that this particular RNAi line, which hadn’t previously been utilized in a publication to knock down profilin expression, is effective. In order to test the effect of induction on fly development we crossed homozygous females  to a homozygous Hsp70:Gal4 driver [y1sc* v1; P{TRiP.HMS00550}attP2 with or without Hsp70:Gal4 and with or without heat shock). In the maternal y1sc* v1; P{TRiP.HMS00550}attP2 background, heat shock did not affect either Wolbachia titers  nor profilin expression . In contrast, profilin expression was statistically significantly reduced in flies expressing the RNAi construct compared to non-heat shocked siblings . In addition, knockdown of profilin did have a significant and measurable effect on Wolbachia titers in these same flies; the fly Wolbachia titers were reduced by 1/3 compared to their non-heat shocked siblings .As an additional control for host genetic background and to explore direct effects on profilin knockdown during development, we took advantage of an infected fly stock carrying a UAS inducible profilin-specific short hairpin silencing trigger  . In ordeWolbachia transmission was imperfect in the mutant flies . Additionally, we could detect a statistically significant reduction between the F1 and F2 heterozygous mutant flies (p = 0.012). As observed in our results based on PCR, Wolbachia titer (based on quantity of protein on a Western blot) is also reduced, with some variability, in the F2 progeny .To provide additional support for the reduction in titer observed via PCR, we probed Western blots of pooled or individual fly lysates produced from the F1 and F2 progeny and their parents for Wsp Fig  and 1E. lies Fig  and 1B. ted line . Indeed, progeny . We hypoWolbachia occupy the germline and somatic stem cell niches (SSCN) in their hosts [Wolbachia can achieve this localization after injection into the fly abdomen, suggesting that the stem cell niche targets are essential for Wolbachia infection [Drosophila ovariole provides an opportunity to view oocyte development and Wolbachia localization within each progressive stage. Wolbachia concentrate preferentially in the somatic stem cell niche, which is thought to serve as a source of infection for the germline. As germline development progresses from regions 2a to 2b, Wolbachia are thought to infect via the somatic stem cell niche, increasing the numbers of bacteria found within the germline after association with the SSCN [Wolbachia in the germarium of our flies, producing localizations expected based on previous publications [Wolbachia infection within the entire germarium is significantly reduced in heterozygous mutant flies  is also significantly reduced, compared to the control maternal line  is significantly reduced in all heterozygous mutant flies . The reductions in infection in the germaria suggest two things: (1) that Wolbachia has difficulties in transiting or maintenance in a population within the germarium during development in the heterozygous mutant flies and (2) even when region two, the location of the SSCN, is occupied by Wolbachia, the bacteria are deficient in colonization of the early egg chamber in the heterozygous mutant flies  and villin (quail) are important in the regulation of F-actin during oogenesis. Because profilin promotes the polymerization of F-actin filaments and villin stabilizes these filaments through bundling, we were curious to know whether or not the heterozygous mutant flies differed in the quantity of F-actin found in the germarium, when compared to control flies. In addition, visualization of the F-actin cytoskeleton allowed us to examine the actin ring canals in the heterozygous mutant flies at all stages of oocyte development. At no point were ring canals occluded by nuclei, supporting our finding that cytoplasmic streaming and maternal dumping are unaffected in heterozygous mutant flies . Using quantified fluorescence of F-actin in the images we were unable to detect a statistically significant difference between median levels of phalloidin staining in control flies compared to the heterozygous mutants , the Wolbachia titer phenotype observed in these flies may not be directly related to the F-actin network in the germarium. We therefore examined the in vivo amounts of filamentous and globular actin in ovaries from control flies and compared this to that seen in heterozygous mutant chic221 flies. Using ultracentrifugation coupled to Western blot, we found that we could consistently detect globular actin in the ovaries of control flies. In contrast, we found a statistically significant decrease in total amount of globular actin detected in the heterozygous mutant lines  and Wolbachia surface protein  relative to Rpl32. Control flies express, on average, 2x as much profilin as heterozygous mutant F1 progeny . Additionally, although we could detect Wolbachia in each of the wild type flies included (N = 10), we were only able to detect a Wolbachia infection in three of the heterozygous mutant F1 flies  produce viable progeny, and we found no occluded ring canals in any of these backgrounds, a subtle defect in maternal cytoplasmic dumping could alter the ability of Wolbachia to be transmitted faithfully to the oocyte. Wolbachia has been suggested to utilize cytoplasmic dumping to increase titer in the oocyte (as compared to the nurse cells) [chic mutants fail to localize STAUFEN and oskar mRNA [Wolbachia utilizes these posterior determinants to localize in the oocyte, as disruption of osk and stau results in mislocalization of Wolbachia in D. melanogaster [Wolbachia in these heterozygous mutants occurs in the germarium, before defects would begin to affect Wolbachia titers. Therefore, although our fly mutants could conceivably exhibit subtle polarization defects, these defects alone would not entirely explain the observed phenotype.The developing oocyte is loaded with maternal determinants (e.g. mRNA and protein), a process which begins early (stage 1), and continues until about stage 10 when maternal nurse cells dump their remaining cytoplasmic contents into the oocyte . The acthenotype ,40. In qg canals . We rease cells) . In addikar mRNA . Wolbachnogaster . If hetechickadee mutants fail to maintain germline stem cell number. However, chic221/+ flies are equivalent to wild type [Wolbachia are known to alter germline stem cell proliferation [Wolbachia colonize the germline stem cell niche [wMel colonizes the somatic stem cell niche in Drosophila melanogaster (Wolbachia proliferation in these mutant flies. We therefore counted the number of viable progeny (a measure of fertility) for each of the mutant lines. No statistically significant difference was observed for any of the heterozygous, mutant flies, when compared to the control  maintenance and germ cell enclosure by somatic cyst cells ,33—homozild type ; that isferation  and somell niche , wMel conogaster . Regardl control . We therWolbachia colonize the primordial germ cells and the posterior pole of developing embryos in numerous insect hosts. In D. melanogaster, for example, strain wMel concentrates at the posterior pole in a poleplasm dependent fashion [Wolbachia is not universal in insects nor in Drosophila. Wolbachia strain wRi infects the entire embryo uniformly while B group Wolbachia actually show exhibit anterior localization [Drosophila species there are different patterns of Wolbachia colonization: although wWil infects primordial germ cells, wAu infects the entire embryo [Wolbachia in the posterior of the embryo [Drosophila species and quite low for others [There is significant evidence that  fashion ,42,43. Hlization . Similare embryo . This poe embryo . Howeverr others –47. ThisWolbachia use to eventually colonize the germline? Wolbachia colonization of somatic tissues has been known for some time [Wolbachia infection of the soma may serve as a reservoir for germline infection. In the terrestrial isopod, Armadillidium vulgare, Wolbachia is absent from many early oocytes and infects the older oocytes late in development, an enrichment that is thought to come from a somatic reservoir (the follicle cells) [Wolbachia initially are concentrated in the posterior of the P2 blastomere, the precursor of the adult germ line. However, Wolbachia are subsequently excluded from the germ line in the next cell division and instead, invade the germ cells later, from the surrounding somatic gonadal cells [Brugia is correlated with a disruption in polymerized actin at those foci [Wolbachia require actin for soma to germline transmission. Importantly, however, we did not observe actin disruptions (similar to those seen in Brugia) within Drosophila germaria.What other ways might ome time  but recee cells) . In nemaal cells . This soose foci . BecauseWolbachia rely on the actin cytoskeleton to achieve adequate titer in the Drosophila host during development. First, we observe reductions in titer of Wolbachia in heterozygous mutants compared to both their non-mutant sibling controls as well as parental controls , are known to increase the amount and stability of F-actin in the us actin ,50. One ogenesis ,34. Becaolbachia . These dWolbachia infection. Perhaps this reproductive parasite secretes proteins that interact directly with eukaryotic actin or host actin binding proteins. Indeed, other members of the Rickettsiales are known for their striking coopting of host actin in the production of comet tails [Wolbachia persist within membrane-bound compartments and no such comet-like structures have been observed to be associated with the vacuole [Wolbachia is able to enter and exit eukaryotic cells; Wolbachia transit to the germline from the fly abdomen and are loaded into the germ cells from surrounding somatic cells [Wolbachia’s success likely depends upon an ability to secrete proteins that modify host actin to promote internalization by non-phagocytic cells. Recently, in vitro biochemical associations between the filarial nematode Wolbachia (wBm) PAL-like protein wBm0152 and actin have been observed, although results do not conclusively implicate this particular protein in interactions with host actin during infection [Wolbachia infection depends on the actin cytoskeleton. Therefore, future work to identify and characterize Wolbachia proteins that bind to or alter host actin dynamics will be important for understanding the molecular basis of the interaction between the host and the symbiont.We have shown that the host actin cytoskeleton is clearly important for the maintenance of a et tails . However vacuole . That saic cells ,26,31. Wnfection . RegardlWolbachia achieves this through a specific infection of the somatic stem cell niche in the germarium of Drosophila melanogaster [Wolbachia is extraordinarily sensitive to the regulation of actin, such that phenotypically wild type heterozygous mutant flies cannot faithfully transmit the bacterium to their progeny. Our results, particularly that titer is significantly reduced in the germaria of chic221/+, chic1320/+, and qua6-396/+ flies, suggest that Wolbachia utilize host actin to enter and persist within host tissues during Drosophila development. Additionally, our finding that these heterozygous mutant flies cannot transmit the infection suggests that Wolbachia titers within a host are reduced when actin regulation is disrupted, impacting transmission efficiency.In order for intracellular, maternally transmitted symbionts to successfully infect the next generation, the bacteria must target the oocyte. nogaster . Here weS1 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigBlue = entire germarium; Green = region 2; Red = Stage 1 egg chamber.(TIF)Click here for additional data file.S3 Figin situ hybridization (using the Eub338-Alexa488 probe) as well as staining with another anti-body (custom anti-FtsZ). Note strong staining in the germarium and in the presumed early oocyte.This staining is recapitulated by fluorescence (TIF)Click here for additional data file.S4 Figqua6-396/+, chic1320/+, and chic221/+ F1 Wolbachia infected progeny with regards to amount of F-actin staining . Bars = minimum and maximum values. Box = first and third quartiles while the median is shown as a band through the box. Although the 95% confidence intervals overlap for all genetic backgrounds, the distributions of values for the mutants are much more variable than found in the control flies . (B) Quantification of G-actin in the ovaries of control and chic221/Cyo female flies. Densitometry measures using western blots (a-actin) showed statistically significant reductions in actin in heterozygous mutant female flies  (C) Relative quantification of profilin transcripts within individual chic221/+ F1 female flies as well as wild type, control flies (stock #145). A statistically significant decrease in profliin expression was observed in the heterozygous mutant flies compared to controls . Importantly, Wolbachia was only detected in three of the twenty heterozygous mutant flies but consistently found in all of the wild type flies (using qPCR on wsp).Maximum projections, generated from z-stacks, were utilized to compare between control flies and (TIF)Click here for additional data file.S5 Figqua6-396and profilin mutants chic221 and chic1320). In each case, 50 single pair crosses between virgin females and males from the same background were performed, parents were transferred to new vials and the offspring counted every four days. Median, quartiles and minimum and maximum number of progeny shown for each. The 95% confidence intervals overlap for all genetic backgrounds and comparisons of means are not statistically significant .Number of viable progeny produced by control and mutant lines (villin mutant (TIF)Click here for additional data file.


Two month old ovariectomized C57BL/6 mice were treated with vehicle or 50 mg/kg/day endoxifen hydrochloride via oral gavage for 45 days. Animals were analyzed by dual-energy x-ray absorptiometry, peripheral quantitative computed tomography, micro-computed tomography and histomorphometry. Serum from control and endoxifen treated mice was evaluated for bone resorption and bone formation markers. Gene expression changes were monitored in osteoblasts, osteoclasts and the cortical shells of long bones from endoxifen treated mice and in a human fetal osteoblast cell line. Endoxifen treatment led to significantly higher bone mineral density and bone mineral content throughout the skeleton relative to control animals. Endoxifen treatment also resulted in increased numbers of osteoblasts and osteoclasts per tissue area, which was corroborated by increased serum levels of bone formation and resorption markers. Finally, endoxifen induced the expression of osteoblast, osteoclast and osteocyte marker genes. These studies are the first to examine the in vivo and in vitro impacts of endoxifen on bone and our results demonstrate that endoxifen increases cancellous as well as cortical bone mass in ovariectomized mice, effects that may have implications for postmenopausal breast cancer patients.Endoxifen has recently been identified as the predominant active metabolite of tamoxifen and is currently being developed as a novel hormonal therapy for the treatment of endocrine sensitive breast cancer. Based on past studies in breast cancer cells and model systems, endoxifen classically functions as an anti-estrogenic compound. Since estrogen and estrogen receptors play critical roles in mediating bone homeostasis, and endoxifen is currently being implemented as a novel breast cancer therapy, we sought to comprehensively characterize the  It is estimated that nearly 1.5 million women will be diagnosed with breast cancer this year and nearly 70% of these individuals will have estrogen receptor alpha (ERα) positive tumors. Women with ERα positive tumors are treated with drugs that either suppress the production of estrogen (aromatase inhibitors [AIs]), or block estrogen signaling (e.g. tamoxifen), in order to reduce the risk of breast cancer recurrence. At present, the drugs of choice for postmenopausal women are typically AIs as they have been shown to have equal or improved benefit relative to tamoxifen To address the negative skeletal effects incurred by AIs, clinicians encourage the use of vitamin D and calcium and additionally may prescribe a class of drugs known as bisphosphonates Selective estrogen receptor modulators (SERMs) have provided major therapeutic advances in addressing these issues since they exert both estrogen and anti-estrogen-like actions in a tissue dependent manner Like many drugs, tamoxifen is a parent compound that undergoes significant metabolism in the human body. While 4-hydroxytamoxifen (4HT) is the most commonly studied metabolite, it represents less than 10% of tamoxifen primary oxidation Based on these preclinical data, and the clinical data demonstrating an association between CYP2D6 metabolism and endoxifen concentrations with breast cancer recurrence, a novel formulation (endoxifen hydrochloride) was synthesized and phase I clinical trials designed to evaluate the pharmacokinetics and safety of endoxifen in hormone responsive cancers that are refractory to standard therapies are under way at the Mayo Clinic (NCT ID: NCT01327781) and National Cancer Institute (NCT ID: NCT01273168). Phase II studies of endoxifen in AI refractory breast cancer will commence in 2014.in vivo effects of endoxifen on the mouse skeleton. The potential effects of endoxifen on the skeleton have never been reported and such effects cannot be extrapolated from past studies examining the actions of tamoxifen on bone due to differences in the metabolism of tamoxifen in rodents in vivo impact of endoxifen treatment on bone forming osteoblast and bone resorbing osteoclast precursor cells as well as osteocytes. Our data provide evidence that endoxifen alters the activities of osteoblasts, osteoclasts and osteocytes resulting in compartment-specific alterations in bone mass and architecture relative to vehicle treated mice.Based on the unique mechanism of action of endoxifen in breast cancer cells, its development and implementation as a novel breast cancer therapy and the importance of maintaining healthy bone in cancer patients, we sought to comprehensively characterize the For this study, we utilized 20 two-month-old female C57BL/6 wild-type mice. All mice underwent ovariectomy to induce gonadal hormone insufficiency and were subsequently randomized to two groups of 10 animals each which were treated with either vehicle control (0.375 mg/mL ascorbic acid) or endoxifen-hydrochloride (50 mg/kg/day) via oral gavage. Purified z-endoxifen hydrochloride was synthesized by the National Cancer Institute and obtained through the Developmental Therapeutics Program (NSC 750393). Fresh vehicle and endoxifen-hydrochloride solutions were prepared weekly and stored in the dark at 4°C. Treatments persisted daily for a total of 45 days. During this time, all mice were housed in a temperature controlled room (22±2°C) with a light/dark cycle of 12 hours. Animals had free access to water and were fed standard laboratory chow ad libitum.2 inhalation protocols and all efforts were made to minimize suffering.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Mayo Clinic Institutional Animal Care and Use Committee (IACUC) approved all animal care and experimental procedures described in this paper (Protocol A22112). Bones and tissues were harvested after euthanasia following Mayo Clinic IACUC-approved COFollowing 45 days of treatment, all mice underwent DXA and pQCT scans. For DXA analysis, a Lunar PIXImus densitometer (software version 2.10) was calibrated using a hydroxyapatite phantom provided by the manufacturer . Subsequently, mice were anesthetized and whole body scans were conducted. Data analysis consisted of whole body lean mass, fat mass, bone mineral density and bone mineral content. For pQCT, mice were subsequently placed in a supine position on a gantry using the Stratec XCT Research SA+, software version 6.20C, . Slice images were measured at 1.9 mm  and 9 mm  from the proximal end of the tibia to obtain trabecular and cortical parameters respectively as described previously 2 and left femora and tibiae, as well as 5th lumbar vertebrae (LV5), were removed and fixed in 10% neutral buffered formalin overnight. Formalin fixed bones were transferred to 70% ethanol and stored at 4°C until time of analysis. Micro-CT was used for nondestructive three-dimensional evaluation of bone volume and cortical and cancellous bone architecture. Femora, tibiae and LV5 were scanned in 70% ethanol at a voxel size of 12×12×12 µm using a Scanco Micro-CT40 scanner  set at 55 kVp. The threshold for analysis was determined empirically and set at 245 . In the femur, cortical bone was evaluated in 20 slices (240 µm) in the femoral shaft with the first slice taken 60% distal to the top of the femoral head. Measurements in this region included cross-sectional volume , cortical volume (mm3), marrow volume (mm3), cortical thickness (µm) and intracortical porosity. Cortical thickness was determined using the plate model and intracortical porosity was calculated as the difference between cortical bone volume at a threshold of 245 and a threshold of 0. Volume measurements in the cortical diaphysis were adjusted to a height of 1 mm. Measurements in the femur metaphysis were obtained in a total of 40 slices (480 µm) starting at 45 slices (540 µm) proximal to the growth plate and included bone volume fraction , trabecular number (mm−1), trabecular thickness (µm), and trabecular spacing (µm). The same four parameters were also evaluated in the cancellous compartment  in the femur epiphysis. The cancellous region of interest was delineated manually a few voxels away from the endocortical surface. For the tibia, cortical bone was evaluated in the tibia diaphysis  starting 40 slices (480 µm) proximal to the tibiofibular junction and cancellous bone was evaluated in the proximal tibia metaphysis  starting at 20 slices (240 µm) distal to the growth plate. Volume measurements in the cortical diaphysis in both femur and tibia were adjusted to a height of 1 mm. Cancellous bone was analyzed in both the femur metaphysis and epiphysis. Finally, cancellous bone volume fraction (%), trabecular number (mm−1), trabecular thickness (µm), and trabecular spacing (µm) were determined in the cancellous compartment of the vertebral body .Immediately following DXA and pQCT scans, mice were sacrificed using COth lumbar vertebra were dehydrated in graded increases of ethanol and xylene and embedded in methyl methacrylate. Sections (4-µm thick) were cut with a vertical bed microtome (Leica/Jung 2165) and were stained according to the Von Kossa method with a tetrachrome counter stain  for assessment of cell-based measurements. Osteoblast and osteoclast number and perimeter were measured in the entire cancellous compartment of the vertebral body. Osteoblast and osteoclast number was expressed per bone perimeter (#/mm) and osteoblast and osteoclast perimeter was expressed per total bone perimeter (%), bone area (mm/mm2) and tissue area (mm/mm2). Osteocyte number was also determined in the entire cancellous compartment of the vertebral body and expressed per total bone area (#/mm). Histomorphometric data were collected using the OsteoMeasure System . All histomorphometric data are reported using standard 2-dimentional nomenclature The histological methods used here have been previously described in detail At the time of sacrifice, serum was collected via terminal bleeds from all mice. The serum levels of a bone formation marker, procollagen type 1 amino-terminal propeptide (P1NP), and a bone resorption marker, C-Telopeptide of Type I Collagen (CTX-1), were quantitated using ELISA kits from ImmunoDiagnostic Systems  as described by the manufacturer. All assays were performed in duplicate and averaged among the two treatment groups.2 overnight. Twenty four hours after plating, non-adherent cells were removed and fresh media was added. Cells were allowed to proliferate for approximately 3 days and total RNA was then isolated using Trizol reagent . Following removal of bone marrow from these femora and tibia, the cortical shells were immediately placed in Trizol reagent and homogenized for RNA extraction.At the time of sacrifice, the right femur and tibia of 4-5 vehicle and endoxifen treated mice were collected and cleaned of all muscle tissue. Subsequently, the epiphyses were removed and bone marrow cells were flushed and collected in 1X PBS. Cells were plated in 6 well plates using α-MEM plus 10% fetal bovine serum (FBS) and a 1% antibiotic-antimycotic solution and cultured in a humidified 37°C incubator with 5% COHuman fetal osteoblasts cells expressing estrogen receptor α (FOB/ER9) were cultured as previously described 5 per well in 24-well plates, and cultured in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) (50 ng/ml) and MCSF (25 ng/ml). In parallel, cells were also cultured in the absence of MCSF to verify that there were no contaminating mesenchymal cells in the preparations. Osteoclast precursors were re-fed with the same media on day 3 and cells were fixed in 1% paraformaldehyde 24 hours later. To identify mature osteoclasts, cells were stained with Hoechst 33342 and for TRAP activity as previously described Freshly isolated bone marrow cells were also collected from an additional 4-5 vehicle and endoxifen treated mice using the right femur and tibia of each animal as previously described http://frodo.wi.mit.edu/primer3/) and were purchased from Integrated DNA Technologies . Primer sequences are listed in Total RNA was isolated from adherent marrow stromal cells and mature osteoclasts using Trizol reagent (Invitrogen) as specified by the manufacturer. RNA was quantitated using a NanoDrop 1000 spectrophotometer . Equal amounts of RNA originating from replicate mice in the vehicle and endoxifen treated groups were combined and cDNA was synthesized using the iScript™ cDNA Synthesis Kit . Real-time PCR was performed in triplicate as previously described Prior to analysis, all data were reviewed by a dedicated statistician for normality and equality of variances using a Kolmogorov-Smirnov test for normality. Each of the parameters presented in this manuscript exhibited normal distributions and equal variances among animal groups. For these reasons, a 2-sided unpaired student's T-test was used to evaluate significant differences between vehicle and endoxifen treated mice. Statistical calculations were performed using Microsoft® Office Excel and all data are presented as the mean ± standard error. P-values <0.05 were considered to be statistically significant.As a first step towards elucidating the potential effects of endoxifen on the skeleton, we utilized DXA analyses to measure total body bone mineral density and bone mineral content, as well as lean mass and fat mass following 45 days of vehicle or endoxifen treatment. Total body bone mineral density and content were significantly greater in the endoxifen treated mice relative to vehicle treated control animals . InteresTo further analyze the effect of endoxifen on bone, we next examined a number of trabecular and cortical bone parameters in the tibial metaphysis and diaphysis respectively via pQCT . EndoxifFollowing these studies, we analyzed the micro-architecture of long bones and vertebra from vehicle and endoxifen treated mice using micro-CT. With regard to the proximal tibial metaphysis, endoxifen treated animals exhibited greater bone volume to tissue volume ratio, trabecular number and trabecular thickness, and reduced trabecular spacing relative to vehicle treated mice . In the At the femur epiphysis, the bone volume to tissue volume ratio was significantly greater in endoxifen treated mice relative to vehicle treated controls with no significant differences observed for trabecular number, thickness or spacing . Howeverth lumbar vertebra, the bone volume to tissue volume ratio was significantly greater in endoxifen treated animals relative to vehicle treated controls . Since tamoxifen and other drugs which are classified as SERMs have known effects on the skeleton, we sought to characterize the effects of endoxifen on bone using a mouse model system. As mentioned previously, the potential effects of endoxifen on the skeleton cannot be extrapolated from past rodent studies examining the actions of tamoxifen on bone since the concentrations of endoxifen have been shown to be extremely low in tamoxifen treated murine models There is a substantial amount of data demonstrating that a number of SERMs can protect against bone loss following estrogen depletion in various animal model systems and act to preserve bone mass in post-menopausal women (reviewed in: in vitroth lumbar vertebra revealed tissue level increases in osteoclast-lined bone perimeter. This result is consistent with our observation of increased serum levels of CTX-1, a biochemical marker of bone resorption, in endoxifen treated mice.At the cellular level, SERMs are considered to be primarily anti-resorptive therapies since they repress osteoclast differentiation and activity with lesser effects on osteoblasts. Tamoxifen and raloxifene have been shown to reduce osteoclast differentiation In vitro studies have also demonstrated that tamoxifen and raloxifene can induce the expression of Runx2 in osteoblasts in vitroin vivo, using adherent marrow stromal cells isolated from endoxifen treated mice, as well as in vitro, using the human FOB/ER9 cell line. Additionally, our data suggest that endoxifen's effects on the mouse skeleton may also occur through the actions of osteocytes due to the fact that increased expression of bone marker and osteocyte marker genes were observed in the cortical shells of long bones isolated from endoxifen treated mice. These gene expression studies are consistent with our observation that the numbers of osteocytes embedded within the bone are increased following endoxifen exposure.SERMs are primarily known to function by binding to ERα and ERβ to elicit transcriptional responses. Both ERα and ERβ are expressed in bone, however; their relative expression levels appear to differ based on the bone compartment and cell type. Specifically, ERα is primarily expressed in osteoblasts and osteocytes located in cortical bone while ERβ is more highly expressed in these two cell types in cancellous bone in vivo impact of endoxifen on bone and have revealed that endoxifen increases cancellous as well as cortical bone mass in ovariectomized mice. The mechanisms by which endoxifen elicits these effects on bone appear to occur, at least in part, through regulating the functions of the three major bone cell types. Our data suggest that this novel breast cancer therapy may elicit beneficial effects on bone in post-menopausal breast cancer patients, an effect that is being evaluated in the ongoing endoxifen clinical trials.In summary, these studies are the first to examine the


Pyogenic infections of the lumbar spine are a rare but critical pathology, yet with considerably high mortality rates. In cases indicating surgical therapy, the implantation of titanium cages or autologous bone grafts represent today's gold standard. Although non-metallic implants such as poly-ether-ether-ketone (PEEK) have proven to be advantageous in diverse degenerative conditions, their saftey and practicability in lumbar spine infection remains questionable. Moreover, the efficacy of a single-step radical debridement of the infected disc space with subsequent fusion from a strictly posterior approach continues to be an issue of debate. We therefore sought to evaluate the feasibility, clinical and radiological outcome of a single-step TLIF procedure using oblique PEEK cages in the surgical management of patients with lumbar pyogenic spondylodiscitis.From January 2009 through December 2013, all patients meeting the indication for surgical treatment of lumbar pyogenic spondylodiscitis were included. Patients demonstrating intact cortical bone on preoperative CT received a single-step radical debridement of the infected intervertebral disc space, posterior screw-and-rod instrumentation and implantation of an oblique PEEK cage using the TLIF technique. Oral antibiotics were continued for 12 weeks postoperatively and clinical and radiological results recorded throughout a minimum 1-year clinical follow-up.A total of 104 patients were admitted to receive surgical therapy for lumbar pyogenic spondylodiscitis. Within this patient population, 18 patients met the diagnostic criteria to receive the implantation of an oblique PEEK cage. Pathogens were detected in 13 cases with Staph. aureus being the predominant causative organism. All patients were discharged to recover in their domestic environment. Throughout the first year of clinical and radiological follow-up and beyond, none of the 18 patients demonstrated any signs of residual neurologic deficits or recurrent infection. Furthermore, two-plane conventional X-rays showed no significant implant subsidence or failure at any of the given time-points in up to 5 years postoperatively.In patients meeting the criteria for surgical treatment of lumbar pyogenic spondylodiscitis, the implantation of PEEK cages using a single-step TLIF approach is a safe and feasible procedure. Based on our experience, the concern of a recurrent infection when implanting non-metallic cages may be refuted in carefully selected patients. Pyogenic infections of the spine remain a seldom entity, yet associated with a high mortality of up to 17 % . Mild caIn contrast to the wide-spread use of poly-ether-ether-ketone (PEEK) as a biocompatible alternative to metal implants in various degenerative spinal disorders, its implementation within the primary surgical treatment of pyogenic spondylodiscitis remains an issue of debate. Although initial observations in recent small cohort studies including patients with cervical and or thoracolumbar/lumbar spondylodiscitis –13 have Patients presenting with pyogenic spondylodiscitis requiring surgical treatment were prospectively enrolled into the study upon admission to the Departments of Orthopaedic Surgery of the University Hospital Leipzig and the Department of Spine Surgery at the HELIOS Park Hospital Leipzig from January 2009 through December 2013. Indication for surgery included either failure of conservative treatment with image verified disease progression, secondary instability due to critical osseous lesions of the bony endplates or cortical bone, secondary lumbar kyphosis, immobilizing pain and/or neurological impairment. Patients demonstrating intact cortical bone of the adjacent vertebral endplates on CT were selected to receive an oblique PEEK cage as described below. All other patients received a standard titanium cage using the same surgical approach. Patients presenting with an infectious pathology of more than two respective segments were excluded from this study.Laboratory chemical parameters included a routine blood count, C-reactive protein (CRP) serum levels and, in cases of systemic inflammatory response syndrome (SIRS) or sepsis, plasma levels of procalcitonin (PCT). The neurologic state was evaluated according to the American Spinal Injury Association (ASIA) impairment scale. In patients presenting a state of critical illness prior to surgery, multiple organ dysfunction was rated according to the cumulative component. The Multiple Organ Dysfunction Score (MODs) was applied as described by Marshall and colleagues  . SIRS anFollowing the mandatory clinical evaluation, all patients were diagnosed by two-plane radiographic imaging of the respective spinal region and subsequent magnetic resonance imaging (MRI) of the complete spine with contrast medium. MRI was performed on 1.5-T imagers  using a surface coil or spine coil. Axial and sagittal T1-weighted MR images  and fast spin-echo or turbo spin-echo T2-weighted images  were obtained. In addition, axial and sagittal fat-suppressed T1-weighted images (350–800/11–30) were obtained after IV infusion of 0.1 mmol/kg of gadoteric acid . Presence or absence of individual imaging criteria was evaluated to make an overall assessment of the type of spondylodiscitis, respectively. The signal intensity in the marrow of abnormal vertebrae was considered hypointense, isointense, or hyperintense by comparison with the signal intensity of normal vertebrae in the same patient on T1- and T2-weighted images. Based on the indication for surgery, patients were subject to an additional 1 mm thin-film computer tomography (CT) scan of the affected vertebral level to evaluate the integrity of the bony structures. Significant osseous lesions of the cortical bone were considered inapt for the implantation of an oblique PEEK cage due to the risk of deficient bridging stability of the implant.In all cases meeting the above-mentioned criteria for surgery, the surgical strategy included a single-stage posterior pedicle screw-and-rod instrumentation, followed by the radical resection of the affected intervertebral disc, bony debridement and intervertebral fusion.2 Genta-Coll®, Resorba Medical GmbH, Nuernberg, Germany) and autologous bone graft and inserted oblique to bridge the complete diameter of the bony endplates in a transforaminal-lumbar-interbody-fusion (TLIF) technique. Each autologous bone graft was obtained from the respective facetectomy and laminotomy as part of the TLIF procedure. No additional iliac crest bone harvesting, allograft or bone substitute material was required. Thereafter, the wound was closed in a standard single-layer fashion, thus completing the surgical procedure.The surgical approach was performed exclusively from posterior, thereby complementing the conventional open posterior pedicle screw-and-rod instrumentation with a radical resection of the infected disc and isochronal debridement of the intervertebral disc space and bony endplates. After removal of the infected tissue, the disc space was irrigated with an antiseptic solution , followed by normal saline. Subsequently, the intervertebral PEEK cage  was filled with a blend of gentamycin foam  and clinical follow-up  were performed 6 weeks, 3 months, 6 months and 1 year postoperatively. Thereafter, clinical and radiographic follow-up were continued on an anual basis. In addition, pain on a Visual Analogous Pain Scale (VAS) and the Oswestry Disability Index (ODI) were recorded. Postoperative bracing was not applied.n = 18) had a past medical history of acute bacterial infection involving either the nasopharynx or the respiratory tract but none of the patients presented with sepsis. Neurologic impairment was evident in two cases (n = 2), demonstrating lumbar radiculopathy with mild unilateral sensomotoric dysfunction. The average postoperative stay on the intensive care unit was 1.9 days (0–3 days). One patient (n = 1) demonstrated immobilizing pain (VAS 8/10) despite appropriate conservative treatment and an additional abscess formation within the right psoas muscle at the level of infection. The surgical procedure included drainage of the abscess via the ipslateral disc space by perforating the lateral anulus and carefully advancing a soft infant feeding tube into the respective area under fluorscopic guidance  presented with a suspected bisegmental infection at the L3/4 and L4/5 level with secondary kyphosis and immobilizing pain (VAS 10/10). The surgical procedure consisted of a two level posterior decompression, removal of the purulent L3/4 disc, debridement of L4/5 and bisegemental fusion of L3-5 in TLIF technique  with a mean age of 75.0 ± 8.3 years presented with lumbar pyogenic spondylodiscitis requiring surgical therapy. Eighteen of these patients  were prospectively enrolled into the study by means of the above mentioned criteria  were continued for a 7-day minimum postoperatively. Thereafter, antibiotics were continued in adjustment with bacterial resistance and applied orally with a single drug antibiotic treatment (primarily clindamycin) by the time of discharge. In all cases, full ambulation was achieved within ten days after surgery and all patients discharged to recover in their domestic environment at an average of 16.0 ± 6.2 days postoperatively . The two patients with mild radiculopathy showed complete recovery and full ambulation at the 3 month clinical and radiographic follow-up without any signs of residual neurologic deficits (ASIA grade E). In addition, the average VAS decreased to 5.0 ± 1.8 with an average ODI (%) of 46.0 ± 14.3 with all inflammatory parameters back to normal levels, including full premorbid functional recovery at 3 months postoperatively.Overall, microbiological analyses of specimens obtained during the surgical debridement and lavage identified the pathogen in 13 cases  and may lead to significant issues in patient compliance. In addition to clinically evident and image verified disease progression, failure to respond to conservative therapy and/or uncontrollable pain have therefore been considered relative indications for surgical therapy , 16–18. However, considering the asserted advantages of PEEK over metal implants with regard to cage subsidence, radiolucency and improved biomechanical behaviour (e.g. elastic modulus) , we sougIn summary, we believe that minimizing operation time and surgical exposure by equally addressing all critical issues of the respective pathology may certainly benefit the overall outcome, particularly in patients with significant comorbidities. We hereby acknowledge, that this assertion is, at least in part, based on the comparison with other reports , 32, butAlthough today's literature still owes relevant data and long-term experience from larger cohorts on a high evidence based level to permit reliable guidelines, we believe that our data underlines two fundamental treatment principles in pyogenic lumbar spondylodiscitis:1) a safe and radical debridement of the infectious site and 2) warranting segmental stability by reconstructing the physiological profile to achieve complete recovery and subsequently the best possible outcome.Both clinical and radiological results of our study prove the use of PEEK interbody devices to be successful, thereby allowing a stable and solid bony fusion with sufficient decompression of neurological structures via the posterior TLIF approach. With minimally invasive surgical techniques gaining in importance, future studies using minimal access-TLIF techniques for pyogenic lumbar spondylodiscitis may promise equally beneficial outcomes. In addition, the future trend to generate an optimal bioactive implant-bone interface by surface modification  may also add to the advantages previously outlined by Rao and colleagues . This inOur results demonstrate that the single-step TLIF approach sufficiently addresses all aspects indicating the surgical treatment of pyogenic lumbar spondylodiscitis. Concomitantly, the use of an intervertebral oblique PEEK cage and autologous bone provide optimal stability via bridging of the bony endplates in cases where the majority of cortical bone is intact. In addition, this technique allows a minimally-invasive approach, which may further reduce iatrogenic morbidity in these high risk patients without the fear for recurrent infection. With today's advances in implant surface modification, the future use of titanium coated PEEK implants may significantly benefit the management of these detrimental conditions.