Dorsal root avulsion results in permanent impairment of sensory functions due to disconnection between the peripheral and central nervous system. Improved strategies are therefore needed to reconnect injured sensory neurons with their spinal cord targets in order to achieve functional repair after brachial and lumbosacral plexus avulsion injuries. Here, we show that sensory functions can be restored in the adult mouse if avulsed sensory fibers are bridged with the spinal cord by human neural progenitor (hNP) transplants. Responses to peripheral mechanical sensory stimulation were significantly improved in transplanted animals. Transganglionic tracing showed host sensory axons only in the spinal cord dorsal horn of treated animals. Immunohistochemical analysis confirmed that sensory fibers had grown through the bridge and showed robust survival and differentiation of the transplants. Section of the repaired dorsal roots distal to the transplant completely abolished the behavioral improvement. This demonstrates that hNP transplants promote recovery of sensorimotor functions after dorsal root avulsion, and that these effects are mediated by spinal ingrowth of host sensory axons. These results provide a rationale for the development of novel stem cell-based strategies for functionally useful bridging of the peripheral and central nervous system. Avulsion of spinal roots results in a seriously disabling condition, involving paralysis, loss of sensation and often chronic, intractable neuropathic pain. Partial function of paralyzed proximal muscles can sometimes be achieved by implantation into the spinal cord of the avulsed ventral roots, which offers the injured motor neuron axons access to a peripheral nervous environment2Numerous attempts have been made over the years to overcome the challenge presented by the central nervous system (CNS) to axonal regeneration from dorsal roots into the spinal cord. Thus, transected postganglionic sympathetic axons, ventral root motor axons or peripheral axons of dorsal root ganglion (DRG) cells, all of which are known to have a powerful regeneration capacity, have been anastomosed to the cut proximal portion of a dorsal root45The first demonstration that fibers were able to grow from the periphery into the adult mammalian spinal cord and make functional connections there, came with a novel experimental approach, transplantation of human fetal sensory neurons to the cavity of extirpated, native, adult rat DRG7810121415These studies have all been carried out after cut (rhizotomy) or crush injuries, rather than after true avulsion (tearing) of dorsal roots. Recently, we developed a clinically more applicable dorsal root avulsion injury model1819in vitro differentiation assays  line was generated according to a previously published protocoln assays . At thisn assays . For difAfter ten days, 42.4 ± 2.0% of all cultured cells expressed the neuronal marker HuC/D, 41.3 ± 3.3% expressed the ventral progenitor marker Nkx6.1, 22.8 ± 7.6% expressed the interneuron marker Chx10, 4.7 ± 0.9% expressed the motor neuron progenitor marker Islet1 and 11.97 ± 2.5% expressed glial fibrillary acidic protein (GFAP) . After 2The survival and localization of transplanted cells, their differentiation and potential axonal growth into the spinal cord in DRA animals was analyzed three months after transplantation.In all animals receiving transplants, GFP-expressing cells were detected on the surface of the spinal cord and in direct vicinity to the areas of DRA, where some invasion into the spinal cord surface could be observed . The perDue to variability in the GFP expression of transplanted human stem cells, we used human-specific heat-shock protein 27 (Hsp27) and human nuclear protein (HuNu) as additional markers to label human cells for the analysis of transplanted human progenitor cell differentiation. Virtually all hNPs had differentiated into neuronal or glial phenotypes . 46.2 ± No immunoreactivity for vesicular glutamate transporter 2 (VGlut2) was observed in the transplants . In contThree months after DRA, sensory fibers were traced by injection into the left sciatic nerve with either IB4, which labels a subpopulation of unmyelinated axonsIn contrast, all animals that received an hNP transplant after DRA showed CTB labeled sensory axons projecting into the dorsal horn . No CTB To further examine the distribution of subpopulations of sensory axons at the dorsal root-spinal cord junction, we used antibodies against neurofilament 200kD (NF200) which labels myelinated axons, and calcitonin gene-related peptide (CGRP) which labels peptidergic, non-myelinated axons.In parts of the transplant that did not have access to the spinal cord, we detected extensive ingrowth of NF200 positive fibers into the transplant. These fibers were located next to human Hsp27-expressing fibers that grew in parallel to the spinal cord surface .At the sites of avulsions, NF200 fibers were observed to traverse the spinal cord surface and project into the dorsal horn . CGRP laSensorimotor and somatosensory functions were examined in order to determine whether hNPs transplanted to the site of DRA were able to promote functional recovery, and whether this potential recovery was associated with mechanical hypersensitivity , as an indication of abnormal pain processing.Locomotor performance analyzed using CatWalk analysis was found to be completely unaffected after DRA. Neither interrelated paw parameters (coordination-related) nor individual paw parameters  were different between sham-operated, DRA-operated and hNP treated DRA-operated animals (data not shown).Two mechanosensitivity assays were included in the behavioral monitoring of animals using von Frey hair filaments. First, the paw withdrawal frequency (PWF) to a noxious tactile stimulus was determined as a measure of nociceptive sensitivity . This assay revealed a marked reduction in nociceptive sensation in DRA animals one month after surgery. Nociceptive function was tested by applying a 5.5 g von Frey hair filament five consecutive times to the mid-plantar territory of the ipsilateral and contralateral hind paws. While untreated avulsed mice showed a permanent reduction in PWF of 80% as compared to sham-operated mice, this reduction was improved to as little as 20% for mice which received a transplant . In the In all animals of the 5 month transplant group GFP-expressing cells were detected on the surface of the spinal cord and in direct vicinity to the areas of DRA similar to the three month group. The percentage of detected cells in 5 month old transplants was 1.2 ± 0.5% of the total number of transplanted cells.In five months transplant tissue, 46.1 ± 4.0% of all cells expressed MAP2, whereas only 16.4 ± 2.4% expressed DCX. The observed change in DCX expression over time and the stable expression of MAP2 shows the progressive but incomplete maturation of human neurons from three to five months . The occTo substantiate the interpretation that sensory fibers have regenerated into the spinal cord, long term hNP transplanted animals were tested for nociceptive sensitivity and for sensorimotor function. The functional tests were performed before and after the animals were subjected to section of the L3-5 dorsal roots adjacent to the transplant. For nociceptive sensitivity, the PWF was determined as described for the three months transplant group. For sensorimotor function, animals were subjected to the grip strength test. The latter test measured the tensile force at which the mice released their hind paw’s grip of the apparatus’ grid. Non-transplanted DRA animals demonstrated a strong sensorimotor disturbance and reduced hind paw nociceptive functions . While sIn this study, we explored the potential of transplanted human ESC-derived neural progenitors to assist in restoring a sensory conduit from the periphery into the spinal cord in a dorsal root avulsion injury model and to investigate their ability to generate new neurons which may serve as replacements for avulsion induced cell loss in the spinal cord. Our findings support the conclusion that such transplants contribute to partial functional recovery by supporting sensory axon growth into the host spinal cord.Morphological analysis revealed survival of all transplants during the five months post-operation period, absence of tumor formation and extensive generation of human neurons and glial cells. In all animals, the transplanted cells were located on the surface of the spinal cord with some invasion into the superficial white matter affected by the avulsion injury. However, migration of hNPs into the host spinal cord gray matter was not observed. Thus, transplantation of hNPs at the reattachment site between the gently avulsed dorsal root and spinal cord appear not to provide a source of spinal cord neurons for the replacement of dorsal horn neurons lost due to an avulsion injury.Transplanted hNPs continued gradual maturation to neural phenotypes between three and five months after transplantation, although even at five months, immature cells still occurred, evidenced by the presence of DCX expressing cells. Interestingly, virtually all neurons in the transplants acquired an inhibitory phenotype and no hNP-derived cells expressing glutamatergic marker Vglut2 were observed. Furthermore, neurons in the transplant did not extend neurites into the host spinal cord gray matter, indicating that they were unable to reach the intrinsic spinal cord circuitries. These findings suggest that the transplant did not act as a relay between host sensory axons and the spinal cord.en route into the spinal cord. Previously, we showed that embryonic sensory neurons placed into the adult rat DRG cavity extended their fibers into the host spinal cord along blood vessels as bridges to reach the gray matter where they left the blood vessels and extensively arborized and made synaptic connections in the gray matter area25At the interface between the transplant and the spinal cord, some areas appeared to be open “gates” between the peripheral and central nervous system, with a reduced concentration of GFAP expressing astrocytes compared to the dense lining of astrocytes at the dorsal root-spinal cord junction after DRA in stem cell untreated animals. These sites were associated with host sensory fibers that traversed the transplant Previous studies have shown that transplanted neural stem cells are able to release a variety of growth promoting molecules, including NGF, brain-derived neurotrophic factor (BDNF), NT3, GDNF and ciliary neurotrophic factor (CNTF)272829301011141516CTB tracing and NF200 labeling provided evidence for the regeneration of host sensory axons into the spinal cord. CTB is widely used for tracing myelinated sensory axons3323To assess the effect of hNP transplants on sensory functions after DRA, we used a battery of behavioral tests. These tests included the CatWalk gait analysis, which has been reliably and repeatedly used to assess locomotor deficits in animal models of neurotrauma including spinal cord injury and peripheral nerve lesions3738The mouse sciatic nerve is innervated primarily by spinal nerves L3 and L4The central distribution of CTB labeling in transplant-treated mice is in agreement with the observed behavioral recovery. CTB labeled profiles were detected in deep as well as superficial parts of the spinal cord dorsal horn. CTB labels both high and low-threshold cutaneous mechanoreceptors4344Previous studies have shown that cell transplantation to the injured spinal cord increases the risk for the development of mechanical allodynia, thereby limiting the usefulness of such repair strategies46This study shows that stem cell transplants improve sensorimotor outcomes following DRA in mice. Although cell transplantation techniques have shown promising results in models of dorsal root injury, to our knowledge, this is the first study using an injury lesion similar to the avulsion injuries encountered in manhttp://www.wicell.org.The CAG-hrGFP hESC line was established by the TALEN technology to insert hrGFP  reporter driven by the CAG promoter (chicken beta-actin promoter with CMV enhancer) in the AAVS1 siteThe CAG-hrGFP hESCs maintained on MEFs were differentiated into primitive neuroepithelial cells under a chemically defined culture system for seven days as described previouslyPre-differentiation towards spinal neuronal phenotypes was induced by gently blowing off human cells from the feeder layer, followed by the transfer to a fresh culture dish for sphere formation in the presence of neural differentiation medium supplemented with 0.1 μM RA and 0.2 μM Shh agonist Ag1.3 (Curis). 50% of the medium was changed every second day. After seven days of pre-differentiation, hNP spheres (around one million cells) were placed in polystyrene tubes in culture medium supplied with 10 ng/ml of mimetics of CNTF (Cintrophin) and GDNF (Gliafin)in vitro differentiation, spheres were enzymatically dissociated with the help of TrypLE™ Express (Gibco) and seeded on 0.0001% poly-l-ornithine (Sigma) followed by 20 μg/ml laminin (Sigma) pre-coated coverslips at a density of 1.25 × 104 cells/cm2 in ES cell medium supplemented with 1x antibiotic-antimycotic (Invitrogen) and 10 ng/mL of CNTF (Miltenyi Biotec) and GDNF (Miltenyi Biotec). 50% of the medium was changed every other day until cells were fixed in 4% paraformaldehyde in phosphate buffered saline  after 10 or 21 days of terminal differentiation.For 3) in PBS) for 45 mins at room temperature and then incubated overnight at 4 °C with different combinations of primary antibodies: HuC/D, GFAP, Nkx6.1, Chx10, Islet1, GFP, calbindin, ChAT or VIAAT  and corresponding secondary antibodies; Alexa350, Alexa488, Alexa555, Cy3 (Table1) in blocking solution for 1 h at room temperature. After removing the secondary antibody followed by one wash with PBS, cells were stained with Hoechst 33342 in PBS for 5 minutes, washed twice with PBS, once in distilled water and embedded in 8 μl of mounting medium  on a glass slide.All animal experiments were approved by the Uppsala Regional Ethical Committee for Animal Experiments, as required by Swedish Legislation and in accordance with European Union Directives.Surgery was performed on adult male nu/nu NMRI mice  according to the previously described procedureFor non-transplanted animals, roots were directly re-positioned after the avulsion performed in similar way. In sham-operated animals, the dura and the spinal roots were left untouched after the laminectomy. The muscles and the skin were then sutured in layers using single silk  sutures. Animals were allowed to recover in a heated cage and were given 3μg buprenorphine  subcutaneously twice daily for three days.Griffonia Simplicifolia Agglutinin isolectin B4  antibody was incubated overnight at 4 °C . The left L3–L5 spinal cord segments with attached dorsal roots as well as DRGs were removed, post-fixed at 4 °C for 4 hours and then cryo-protected overnight in PBS containing 15% sucrose. Serial coronal sections (14 μm) of the spinal cord, of DRGs and dorsal roots were cut on a cryostat, placed on SuperFrost® Plus glass slides  and processed for immunohistochemistry (see above) with mixed primary antibodies for laminin, GFAP, HuNu, MAP2, DCX, human-specific GFAP (STEM123), VGlut2, VIAAT, TH, calbindin , Hsp27, NF200 or CGRP. The anti-cholera toxin subunit B  antibody was incubated for 4 h at room temperature and the anti- at 4 °C . A minimGriffonia Simplicifolia Agglutinin isolectin B4  that labels unmyelinated fibersTransganglionic tract tracing methods were used in animals surviving three months after operation to label and chart the course and projections of sensory fibers in the avulsed dorsal root and corresponding spinal cord segments. We used CTB  that labels myelinated sensory afferentsImages of cells on coverslips were captured with a Plan-Apochromat 20x objective (NA 0.75) and images for CTB quantification were taken using a Plan-Apochromat 10 x objective (NA 0.45) attached to a Nikon Eclipse E800 epifluorescence microscope equipped with a Nikon DXM1200F CCD camera. Overlays of multichannel pictures were prepared with Fiji ImageJ2.0.0-rc-2Immuno-labeled sections were analyzed using a Zeiss LSM710 confocal laser scanning microscope. Single images were captured using a LD LCI Plan-Apochromat 25x objective (NA 0.8), for orthogonal projections of human GFAP, MAP2 or DCX labeled human cells, images were taken with a Plan-Apochromat 40x objective (NA 1.3) and for orthogonal projections of areas inside the region of the transplant a Plan-Apochromat 63x objective (NA 1.4) was used. Z-stacks were taken with an optical slice thickness of 1 μm at an interval of 1 μm.th slide of 3 animals, respectively. Differences in the MAP2/DCX distribution between the two time points were analyzed using a two-way ANOVA with Sidak’s multiple comparisons test, p < 0.05. The proportion of anti-human GFAP labeled cells of all human cells was quantified by counting all hGFAP/Hoechst cells and HuNu/Hoechst cells in adjacent slices but otherwise identical to MAP2 and DCX quantifications.The proportion of MAP2/HuNu/Hoechst or DCX/HuNu/Hoechst triple labeled human cells to the total number of HuNu/Hoechst positive cells per slide was used to analyze the differentiation of transplanted hNPs to neurons and to estimate their degree of maturity over time. The cells were counted using z-stacks covering a spinal cord segment of 14 μm following semi-stereological principles in three and five months old transplants from every 10th slide of 3 animals for DRA and DRA+hNP conditions from animals three months after surgery was analyzed. Images for CTB quantification were taken under identical image acquisition settings for both conditions using a Plan-Apochromat 10 x objective (NA 0.45) attached to the epifluorescence microscope setup. For quantification, images were preprocessed in Fiji Image2 by subtracting background using the 488 nm channel and the implemented rolling ball algorithm, followed by setting a manual threshold for CTB positive staining. The dorsal horn was outlined in the raw image and transferred onto the threshold set image, the total area of CTB immunoreactivity in the ipsilateral dorsal horn was measured and differences in the total area of immunoreactivity were analyzed using a t-test, p < 0.05. No quantification of IB4 tracer injection was performed as no labeling in the spinal cord of both avulsion groups (treated and untreated with stem cells) was found.The area of CTB immunoreactivity in the dorsal horn of every 10th section through the transplants using z-stacks covering a spinal cord segment of 14 μm following semi-stereological principles. The mean number was calculated for each animal and then for all experimental groups with three and five months survival after transplantation.We roughly estimated the rate of cell survival in the transplants after three and five months. The amount of HuNu positive nuclei was counted on every 10All statistical analyzes were performed in GraphPad Prism 5.04.Behavioral testing was done to assess locomotor performance and sensorimotor function  and nociceptive sensory function . In addition, the putative development of mechanical allodynia as a sign of abnormal pain processing was determined with the paw withdrawal threshold, PWT. Behavioral assessments were performed in three groups of animals: sham-operated (n = 9), DRA-operated (n = 8), and DRA-operated with transplants (n = 9).CatWalk gait analysis was selected for its ability to detect locomotor deficits in a range of neurotrauma models3738Two mechanosensitivity assays were included in the behavioral monitoring of this investigation. These assays both relied on von Frey hair filaments. In the first assay, PWF to a suprathreshold mechanical stimulus was determined. The PWF was used as a measure of nociceptive sensory function using a 5.5 g-filament. This filament was applied five consecutive times to the glabrous mid-plantar hind paw surface with an inter-stimulus interval of at least 5 seconds. The ratio of positive paw withdrawal out of the five filament applications was determined. In the second assay, a set of seven hair filaments was used  to determine the PWT using the up-down methodNon-ambulatory sensorimotor function was assessed using the grip strength test. In this test, each mouse was gently held upright by the scruff of the neck and then positioned above the lateral aspect of the grid facing towards the meter of the apparatus until the animal gripped the grid with either one of the hind paws. The animal was then gently pulled back horizontally to the meter and the tension force at which the grip was released by the animal was measured. Measurements were repeated two more times and the average grip strength (in grams) was calculated for each hind paw of each animal at each time point of investigation. In order to reduce stress of the animal during the procedure, the animal was habituated to the procedure before taking the actual measurements. Data was expressed in the gram-force required for the release of the hind paw from the grid.After the behavioral assessment at five months, DRA animals with transplants underwent a second surgery at which L3-L5 dorsal roots were cut distal to the transplant. Two weeks later, the animals were re-assessed in the behavioral assays.How to cite this article: Hoeber, J. et al. Human Embryonic Stem Cell-Derived Progenitors Assist Functional Sensory Axon Regeneration after Dorsal Root Avulsion Injury. Sci. Rep. 5, 10666; doi: 10.1038/srep10666 (2015).


The molecular pathways underlying the pathogenesis after subarachnoid haemorrhage (SAH) are poorly understood and continue to be a matter of debate. A valid murine SAH injection model is not yet available but would be the prerequisite for further transgenic studies assessing the mechanisms following SAH. Using the murine single injection model, we examined the effects of SAH on regional cerebral blood flow (rCBF) in the somatosensory (S1) and cerebellar cortex, neuro-behavioural and morphological integrity and changes in quantitative electrocorticographic and electrocardiographic parameters. Micro CT imaging verified successful blood delivery into the cisterna magna. An acute impairment of rCBF was observed immediately after injection in the SAH and after 6, 12 and 24 hours in the S1 and 6 and 12 hours after SAH in the cerebellum. Injection of blood into the foramen magnum reduced telemetric recorded total ECoG power by an average of 65%. Spectral analysis of ECoGs revealed significantly increased absolute delta power, i.e., slowing, cortical depolarisations and changes in ripples and fast ripple oscillations 12 hours and 24 hours after SAH. Therefore, murine single-blood-injection SAH model is suitable for pathophysiological and further molecular analysis following SAH. Today, aneurismal subarachnoid hemorrhage (SAH) accounts for about 5% of stroke cases affecting more than 600.000 patients each year worldwide Therefore, goal of the present trial was to describe the pathophysiology of EBI and DCI after experimental SAH in a murine single injection model in great detail by analyzing brain perfusion, ischemic events in the CNS, quantitative ECoG parameters and neurologic outcome, as well as cardiac disturbances.All animal procedures were approved by the “Landesamt für Naturschutz, Umwelt und Verbraucherschutz” of the federal state of North Rhine-Westphalia, Germany (file number: 87–51.04.2010.A236).To study effects of SAH, 50 µl of freshly drawn blood obtained from the tail vein  was injected into the cisterna magna with a 30-gauge needle in order to induce SAH. Surgery was performed on 83 male C57BL/6J mice in the age range of 15–20 weeks . Perfusion and cortical activity was assessed in 69 mice of which 18 were sacrificed 6 hours after injection, 11 mice 12 hours after injection, 15 mice 24 hours after injection and 25 mice 72 hours after injection. ICP was measured in 11 mice and micro CT imaging in SAH animals was performed on 3 mice.General anesthesia of C57BL/6J mice was induced by intraperitoneal injection of ketamine (100 mg/kg bw i.p.), xylazine (10 mg/kg bw i.p.) and midazolam (5 mg/kg bw i.p.). Mice underwent surgery on a heating pad maintaining a consistent body temperature of 37°C. After skin incision and preparation of the skull, mice were positioned in a stereotactic frame and bilateral burr holes were placed into the scull above the S1  and the cerebellar cortex  according to Paxinos Mouse Brain Atlas.(Paxinos and Franklin) Telemetric biopotential transmitters were implanted as previously described In order to verify the precision of the intra-cisternal injection, to rule out intra-cranial hemorrhage and to assess the extent of dispersion of blood in the subarachnoid space and ventricle system, micro CT imaging was performed on three mice of the SAH group. 50 µl of the CT non-ionic contrast agent Imerone 400 was added to the 50 µl blood drawn from the tail vain before injecting 50 µl of the mixture into the cisterna magna of the anesthetized mice of the SAH group. The mice were instantly placed in an ALOKA LaTheta Laboratory CT  and scanned at 80 kV producing CT images with a pitch 0.3 mm.Before anesthesia and perfusion fixation, mice were blindly evaluated for neurologic deficits by one author (M.D.), using a scale previously described by Parra et al. rCBF monitoring was performed using a Laser Doppler device  which measured relative rCBF (Flux) as well concentration (Conc) of moving blood cells. Laser Doppler probes were inserted into burr holes above the S1 and the cerebellar cortex and data collected 10 minutes before and for 30 minutes after SAH induction as well as at each of the following time points until euthanization: 6, 12, 24 and 72 hours after SAH . Five minutes of these recordings were evaluated and parameters expressed as relative to baseline. All Laser Doppler measurements were performed under general anesthesia. Analysis of cerebral perfusion data was performed blinded.Intra-cranial pressure (ICP) was measured in the cisterna magna using a LogiCal disposable pressure transducer neonatal monitoring kit  connected to an M Series CCT monitoring system . The tip of a saline-filled 20 G needle, connected to the pressure transducer by saline filled tubing was inserted into the cisterna magna of the anesthetized mice. The baseline was recorded before either blood or saline injection was carried out, and ICP was measured for one minute after injection.Telemetric data from the implanted TL11M2-F20-EET transmitters were continuously collected using Dataquest A.R.T 3.1 software . Neuroscore 2.1.0  was used to calculate absolute and relative power of frequency bands (Fast Fourier Transform based using a Hamming window). The frequency spectrum was defined as follows: Delta (0.5–4 Hz), Theta (4–8 Hz), Alpha (8–12 Hz), Sigma (12–16 Hz), Beta (16–24 Hz), Gamma (30–80 Hz), Ripples (80–200 Hz), Fast Ripples (200–500 Hz).Continuous electrocardiograms (ECG) could be recorded by the implanted transmitter (in addition to ECoG data) in 11 mice  during the acute phase. Electrodes collecting the ECG signal, were implanted along with the transmitter dorsally above the left hindlimb , and thus were able to record a robust ECG signal. NEUROSCORE 2.1.0  was used to perform frequency domain analysis of the ECG in order to calculate LF/HF ratios immediately before (control), during and 1 minute after SAH or saline injection. FFT of the control ECG produced a periodogram with two peaks. Accordingly, for frequency domain analysis, the low frequency ECG band (LF) was defined as 0.1–1 Hz and the high frequency band (HF) as 1–5 Hz. The R-peak of the ECG QRS complex was detected automatically by Neuroscore and used to calculate heart rate and R-R intervals. The coefficient of variation (CV) was calculated from R-R intervals. Artifact-free thirty second epochs were evaluated before injection (control), immediately after injection, and 1 minute after injection.Anesthetized mice were intracardially perfused with 10% formalin followed by india ink with 10% formalin at 70 mmHg. In contrast to other authors, we did not include gelatin in the india ink-formalin mixture, because in pre-experiments we found that due to the viscosity of the solution, there was little perfusion of smaller blood vessels and high perfusion pressure was required to perfuse the larger vessels which dilated them rendering measurements of diameter pointless. To lower viscosity we also attempted to heat the solution, however the temperature required was so high i.e. supra-physiological that one must consider that infusing such a hot solution can cause severe vascular and cellular responses interfering with results. Without gelatine we were able to visualize vasculature that was not visible with the gelatin casting method and did not require high-perfusion pressure.http://rsbweb.nih.gov/ij/) and relating these measurements to an internal standard (0.5 mm marking) in the image. Morphological analysis was performed on brains from 10 mice . Brains were first cut into parasagittal sections of 1 mm thickness using a commercially available rodent brain matrix  and were subsequently paraffin-embedded. Four µm-thick coronal sections were serially cut and stained with H&E and Gomori trichrome using standard protocols. For the detection of potential brain lesions injury an immunohistochemical analysis with antibodies against Iba1  and GFAP , respectively, was performed similarly as recently described . Briefly, after dewaxing, sections were incubated with a target retrieval solution  at 95°C. Then, endogenous peroxidase was blocked by 3%H2O2 solution  and sections were treated with FICIN  at 37°C for digestion of formalin-fixed paraffin-embedded tissues. Non-specific biotin binding was blocked using a commercially available kit . Subsequently, sections were incubated with primary antibodies for 60 min at 21°C in a humidified chamber using the following dilutions: 1∶200 (Iba-1) and 1∶100 (GFAP). Immunoreactivity was visualized by the avidin-biotin complex method. Sections were developed in diaminobenzidine  with 0.03% hydrogen peroxide and counterstained with Mayers Hematoxylin. GFAP staining was performed using an immunostainer . Endogenous peroxidase was blocked by peroxidase blocking solution  Immunoreactivity was visualized by universal immuno-enzyme polymer method . Finally, sections were developed in diaminobenzidine . Omission of the primary antisera in a subset of control slides resulted in no immunostaining at all Mouse brains with ink-stained basal vessels were photographed with a 10-fold magnification using a stereoscope . Diameter of main cerebral vessels  were determined by measuring digital images with ImageJ . Normal distribution of absolute ECoG power was assessed using the Shapiro-Wilk test of normality. Due to the mostly non-normal distribution absolute power values were log transformed to obtain a more Gaussian distribution Micro CT imaging verified that blood (mixed with contrast agent) was successfully delivered into the cisterna magna in all scanned animals of the SAH group. Within the 20 minute scanning period, blood could be detected in all ventricles and the entire subarachnoid space , confirmFive of the 41 mice in the SAH group died due to the subarachnoid blood injection . Thereof, four mice died within 15 minutes after blood injection whereas one mouse died about one hour later. There were no deaths in the saline injection group and in the sham group.Neurologic integrity (motor and sensory) was assessed 12 hours, 24 hours and 3 days after SAH .. SAH imAn acute impairment of regional cerebral perfusion (rCBF) and concentration of the moving blood cells (Conc) measured by Laser Doppler Flowmetry above the somatosensory cortex (S1) and the cerebellar cortex was observed immediately after injection in the SAH group and in about half of animals of the saline injection group but not in sham operated animals , S2 Fig.Quantification of maximal acute cerebral hypoperfusion following injection measured at the rCBF nadir revealed significant differences of the absolute values of cerebral and cerebellar rCBF and cerebellar Conc between the SAH and saline injection group . As perforation of the atlanto-occipital membrane in the sham group did not lead to changes in cerebral and cerebellar perfusion, nadir values of the SAH and saline-injection group were compared to the values immediately after perforation of the atlanto-occipital membrane: Comparison of these values revealed significant differences between the sham group and the SAH or the saline-injection group in cerebral rCBF  and between the sham operated and SAH group for the cerebellar rCBF .Relative perfusion values may give a better insight in the mechanisms following SAH : For cerUpon injection, ICP immediately surged from baseline  to similar values in both SAH and saline groups . Both groups display statistically significant elevation of ICP in the first 20 seconds after injection compared to baseline in paired-sample t-test . Twenty To determine intra-individual perfusion changes after SAH, the relative change from baseline was determined for Laser Doppler parameters. In S1, injection of saline induced a robust and long-lasting increase in rCBF (maximum: 580% from baseline after 24 hours) and Conc (maximum: +202% from baseline after 24 hours) lasting from 6 to 24 hours after saline injection, whereas SAH decreased rCBF (minimum: −43% from baseline after 6 hours) for 12 hours and Conc (minimum: −27% from baseline after 6 hours) for 24 hours after SAH . Sham opTelemetric electrocorticograms and electrocardiograms were continuously recorded before induction of SAH and until animals were sacrificed. Spectral analysis of electrocorticograms (ECoGs) was performed to evaluate specific differences in cortical activity among the different animal groups. There were no differences in spectral distribution between the 3 groups before injection. Injection of blood into the foramen magnum instantly reduced total ECoG power by an average of 65% (from 2.6 mV±0.7 mV to 0.9 mV±0.3 mV), whereas injection of saline (or sham injection) did not reduce total ECoG power at all , which is also reflected by a reduction in alpha/delta ratio (ADR) of SAH animals compared to saline-injected animals 24 hours after SAH . Periodically recurring short episodes of depression of activity within the 0.5–2 Hz range lasting 2–3 seconds were observed in some SAH animals but not in saline-injected or sham-operated animals , likely Continuous telemetric electrocardiograms (ECG) could be recorded in 11 mice  during the acute phase. During the control condition heart rate and squared coefficient of variation (SCV) did not differ between SAH and saline groups . One minute after blood injection, heart rate dropped from 145±10 bpm to 113±11 bpm in the SAH group, however this reduction did not reach significance (p = 0.07). Heart rate was not affected by saline injection (159±36 bpm vs. 151±21 bpm). The SCV was significantly increased immediately after blood (p<0.0001) and saline (p = 0.027) injection compared to the control condition . The LF/Analysis of vessel diameter of basal cerebral arteries in ink-perfused whole brain preparations revealed no differences between the SAH and the control groups at 6, and 24 hours .. AlthouThe murine single-blood-injection SAH model elicits early impairment of cerebral perfusion and cortical signaling and delayed neurologic deficits and ECoG slowing. SAH models are well-documented in other rodents than mice such as rats Our present model analyses two different aspects of the pathophysiological changes after SAH which are the EBI with an early global brain injury initially after aneurysm rupture and DCI as a pathology manifesting in the subacute phase after SAH as an ischemia related clinical syndrome with focal neurological and cognitive deficits. DCI was evaluated for the first time in a murine SAH filament perforation model evaluating the constriction of in the late 1990′s Initial responses and EBI after SAH has been modeled in a murine filament perforation model in which the ICP increased sharply and the rCBF drops by about 80% in response to SAH The present study aimed to analyze impairment of cerebral perfusion and neuronal signaling measured by ECoG following SAH in a murine blood injection model. Mortality rate was 12.2% in the SAH group but there were no deaths in the saline injection and the sham group. ICP curves of both saline and blood injected animals were similar, displaying a robust surge upon injection lasting 20 seconds corresponding to the acute impairment of regional cerebral perfusion observed immediately after injection in the SAH group and in most animals of the saline injection group but not in sham operated animals. Quantification of acute absolute and relative hypoperfusion following injection revealed significant differences of cerebral and cerebellar rCBF and cerebellar Conc between the SAH and saline injection groups. The degree of cerebral hypoperfusion significantly differed between the SAH and both control groups. An acute hypoperfusion after SAH is in line with various previous studies. In part, increasing volume in the subarachnoid space or shifting of the subarachnoid milieu appears to impair cerebral perfusion as it also dropped in some animals of the saline-injection group. However, absolute and relative cerebral perfusion values were significantly lower after SAH compared to the saline-injection indicating a specific role of subarachnoid blood in the genesis of cerebral hypoperfusion. Furthermore, injection of blood into the foramen magnum reduced total ECoG power by an average of 65%, whereas injection of saline (or sham injection) did not reduce total ECoG power. Although it did not reach significance, fast ripples, pathological high frequency oscillations known to precede epileptic seizures, were barely detectable prior to SAH but became robustly detectable in several mice after blood but not after saline injection. To our knowledge, the present study is the most comprehensive analysis of acute ECoG changes following SAH and the only one using quantitative ECoG methods in an animal model. Previously, suppression of EEG delta activity in the electrical cortical activity has been described in a rat animal model immediately after SAH which is supported by our findings Furthermore, the present study analyses delayed cerebral ischemia after SAH in great detail. Significant impairment of rCBF in the S1 occurred between 6 and 12 hours after induction of SAH in comparison to both sham-operated and saline injected animals which was accompanied by a reduction of the concentration of moving blood cells at 6 and 12 hours. In the SAH group, rCBF and Conc were within the pre-injection range after 24 hours, whereas saline-injected mice exhibited significant hyperperfusion. This hyperperfusion may represent a late response of cerebral autoregulation to increased ICP after saline-injection and the consequential shift of the subarachnoid milieu. If so, the absence of reactive hyperperfusion in the SAH group would be pathological and could either reflect persistent cerebral vasospasms, or disturbed cerebral autoregulation or both. These results are in line with the results from a previous study using the blood injection model, in which significant MCA constriction between 6 and 36 hours was reported Furthermore, cerebellar perfusion was found to be significantly reduced 6 and 12 hours after SAH compared to both sham and saline-injection groups. Therefore, cerebellar hypoperfusion occurs in parallel to perfusion impairment in the cerebrum. However, cerebellar hypoperfusion may be promoted by the distribution of subarachnoid blood injected into the cisterna magna. The impact of hypoperfusion of the basilar supply area for human patients suffering from SAH must be investigated in further studies.During DCI, we observed no significant differences in diameter of basal cerebral arteries in ink-perfused brains. In principle, cerebral vessels may be dilated by high pressure during perfusion, especially by viscous fluids. As we performed pre-experiments, kept the perfusion pressure constant at 70 mmHg and did not include gelatin in the perfusion mixture, we could exclude this possibility. More likely, impairment of cerebral perfusion during DCI could be caused by vasospasms of the microvasculature as opposed to narrowing of main basal cerebral arteries.However, the present model gives additional insights into neuronal signaling after SAH: SAH animals displayed significantly increased absolute delta power i.e. ECoG slowing, 12 hours and 24 hours after SAH, which is also reflected by a reduction in ADR of SAH animals compared to saline-injected animals 24 hours after SAH, indicating ischemic injury. As rCBF is reduced in the SAH group at the same time and differences resolve after 72 hours parallel to normalized rCBF, slowing and decreased ADR most likely correlates to impaired cerebral perfusion. These results are in line with previous reports from patients. Recently, Stuart and colleagues reported a correlation between the extent of intracortical EEG-measured ADR decrease and the degree of cerebral vasospasms in poor-grade SAH patients Parallel to the rCBF reduction in the S1, sensory performance of mice in neurological tests was significantly impaired 12 and 24 hours after SAH, possibly directly linking rCBF reduction to corresponding deficits. Sensory performance of mice was impacted to a greater degree than motor performance after 12 and 24 hours. Neurological performance was not tested after 6 hours, as mice were still recovering from anesthesia and neurological impairment due cerebral vasospasms would not have been reliably distinguishable from narcosis effects. Interestingly, sensory deficits were only transient because after 72 hours sensory performance was no longer significantly different between all groups. However, SAH animals displayed significantly poorer total neurologic integrity compared to both control groups after 72 hours, indicating other persisting deficits. Also these results are in line with a previous report which described poorer neurological performance of SAH animals tested 72 hours after blood injection 2) and arterial blood pressure. Analysis of these parameters in addition to the performed evaluations would require additional equipment implanted into the mice and therefore additional surgeries e.g. craniotomies or surgeries to implant the transmitters. Subsequently, stress of the mice would be increased affecting the animal's health and quality of the data. Also, the size of mice might be a limitation. However, the impact of variables like pCO2 or arterial blood pressure must be evaluated in further studies. Furthermore, although bent ECoG electrode tips are fixed over the dural mater, micro lesions of underlying cortical tissue can occur due to pressure through the thin murine dural mater. However, neither neurological deficits nor ictal waveforms due to implantation of ECoG electrodes have been observed in past studies rendering this telemetric ECoG method popular and reliable Finally, we acknowledge some limitations of the present study: Some parameters could not been assessed in the present study such as blood gases Click here for additional data file.S2 FigRelative rCBF impairment following acute SAH. A: relative rCBF impairment in the somatosensory cortex following acute SAH. B: relative rCBF impairment in the cerebellar cortex following acute SAH. Blood injection caused instant severe hypoperfusion, whereas saline injection produced mild rCBF impairment in some animals or no impairment at all. No impairment was found in the sham group.(JPG)Click here for additional data file.S3 FigIndia ink stained cerebral vessels. A: Brain of a mouse transcardially perfused with india ink. B: Vessel diameter at 6 hours post injection. C: Vessel diameter at 24 hours post injection .(JPG)Click here for additional data file.S1 TableScale used for neurological examination. Neurologic deficits were examined using a scale previously described by Parra et al. (2002).(XLSX)Click here for additional data file.S2 TableRelative cerebral perfusion values derived from Laser Doppler measurements.(XLSX)Click here for additional data file.S3 TableCerebral and cerebellar perfusion values during DCI.(XLSX)Click here for additional data file.


Yersinia pseudotuberculosis transposon mutants for those with increased binding to the AMCs CCL28 and CCL25. Mutants exhibiting increased binding to AMCs were subjected to AMC killing assays, which revealed their increased sensitivity to chemokine-mediated cell death. The majority of the mutants exhibiting increased binding to AMCs contained transposon insertions in genes related to lipopolysaccharide biosynthesis. A particularly strong effect on susceptibility to AMC mediated killing was observed by disruption of the hldD/waaF/waaC operon, necessary for ADP-L-glycero-D-manno-heptose synthesis and a complete lipopolysaccharide core oligosaccharide. Periodate oxidation of surface carbohydrates also enhanced AMC binding, whereas enzymatic removal of surface proteins significantly reduced binding. These results suggest that the structure of Y. pseudotuberculosis LPS greatly affects the antimicrobial activity of AMCs by shielding a protein ligand on the bacterial cell surface.Antimicrobial chemokines (AMCs) are a recently described family of host defense peptides that play an important role in protecting a wide variety of organisms from bacterial infection. Very little is known about the bacterial targets of AMCs or factors that influence bacterial susceptibility to AMCs. In an effort to understand how bacterial pathogens resist killing by AMCs, we screened  Recently a significant number of chemokines and chemokine-derived peptides have been shown to possess direct antimicrobial activity, placing them within the broader family of host-defense peptides (reviewed in reference ). Host din vitro including Gram-negative and Gram-positive bacteria as well as fungi and protozoa, including both human and veterinary pathogens [The chemokines CCL25 and CCL28 both exhibit antimicrobial properties and together their expression profile covers a broad range of mucosal tissues. CCL28  is expressed at high levels in a variety of mucosal tissues, including salivary glands, lactating mammary glands, and the large intestine, with protein levels ranging from 65–232 nM in human parotid saliva –10. The athogens , 13–16.AMCs and defensins share a conserved motif termed the γ-core, comprising an anti-parallel β-sheet with positive charges distributed at the poles of the motif . It is tEnterobacteriaciae [The specific molecular interactions that mediate effective binding to the surface of bacteria and the precise mode of action of AMCs are unknown. While some alpha-helical antimicrobial peptides have recently been shown to target outer membrane lipoproteins such as Lpp in eriaciae , it is nYersinia pseudotuberculosis as a model, we performed a genetic screen to identify genes that influence bacterial resistance to AMC binding. Y. pseudotuberculosis is a food- and water-borne pathogen that infects the gastrointestinal epithelium causing an enteric disease characterized by an acute inflammatory response, fever, diarrhea, and abdominal pain. Disease is usually self-limiting but occasionally sepsis, invasion of internal organs, or post-infection autoimmune sequelae such as reactive arthritis occur [Y. pseudotuberculosis infection depends on proper recruitment and activation of phagocytic cells of the innate immune system that kill bacteria through a variety of mechanisms [Y. pseudotuberculosis likely encounter AMCs at several points during infection as they traverse the oral cavity and the intestinal mucosa.In this study, we sought to identify genes contributing to bacterial resistance to the mucosal AMCs CCL25 and CCL28. Using is occur . Ultimatchanisms , 24. TheY. pseudotuberculosis growing at ambient temperatures. Y. pseudotuberculosis regulates many phenotypes according to temperature. Notably, the LPS O-antigen is maximally produced at temperatures below 30°C [Y. pseudotuberculosis mutants that exhibited increased binding by CCL25 and CCL28, the vast majority of which were affected in some aspect of LPS synthesis. The predicted core and O-antigen structure of the serotype O:1b IP32953 strain is depicted in hldD-waaC operon and other LPS biosynthesis genes in AMC resistance by Y. pseudotuberculosis.In this study we sought to investigate AMC resistance factors that would be relevant when bacteria are transmitted by food or water, involving ingestion of low 30°C  and somelow 30°C , 27. We Y. pseudotuberculosis IP 32953, a clinical isolate [Y. pseudotuberculosis was routinely grown at 21°C unless otherwise specified. E. coli SM10 carrying pRL27 [E. coli DH5α was used as a cloning host.A pYV plasmid cured derivative of ng pRL27  was usedY. pseudotuberculosis was performed by biparental mating with E. coli SM10 harboring pRL27, which contains a Tn5 element encoding kanamycin resistance [Yersinia selective agar containing kanamycin. Approximately 50,000 transconjugants were derived from four separate mating experiments and stored at -80°C in TSA + 15% glycerol. Assuming random insertion of the transposon, this number of mutants would be sufficient for >10X coverage of the Y. pseudotuberculosis genome. Approximately 15,000 random mutants from this library were grown on Congo-Red agar containing kanamycin at 21°C for 48 h, and colonies that were noticeably darker or lighter than the wild type bacteria were selected for further study. High binding mutants were also selected by subculturing samples of the transposon mutant library for 1 h at 21°C followed by washing in phosphate buffered saline (PBS). The washed bacteria were then incubated with 2.5 nM recombinant CCL28 prepared as described [Random transposon mutagenesis in sistance . Mixtureescribed  in PBS oY. pseudotuberculosis IP32953 genome using Geneious software package (Biomatters) to identify the location and orientation of transposon insertions.The locations of transposon insertions in the selected mutants were determined by arbitrary PCR essentially as described by Lauro et al. . For thehldD, hldD/waaF, and hldD/waaF/waaC, including a region of 310 base pairs upstream of hldD. The forward primer was the same for each construct (5’-ACG AGT CGT GTC GAG TTG TTG TTC-3’). The following reverse primers, specific for each construct were used: 5’- CAT GGC CTA ACG GCA TTG GAA TTG-3’ for hldD, 5’- CGT CAG CGC GGG TAA GGT ATG TAA A-3’ for hldD/waaF, and 5’- AGC ACC ACA TTG AGC CGT GGT TAT-3’ for hldD/waaF/waaC. PCR products were cloned using the CloneJET cloning kit (Fermentas) and transformed into DH5α cells. Plasmids were isolated from ampicillin resistant clones using the QIAprep Spin Miniprep Kit (QIAGEN) and then cycle sequenced to confirm amplification of the proper gene and also to ensure that the inserts were in the appropriate hldD/waaF/waaC orientation relative to the lac promoter sequence. Once this was confirmed, complementation plasmids were transformed into the hldD::Tn5 mutant strain background via electroporation.Primers were designed to amplify Y. pseudotuberculosis strains were grown at 21°C (unless otherwise stated) prior to exposure to AMCs in order to simulate the conditions of food or water borne illness. Bacteria grown to mid-logarithmic phase were diluted to 106−107 cfu/ml in filtered PBS supplemented with 0.5 mg/ml bovine serum albumin (BSA). Chemokines  were then added to a final concentration of 250 nM and incubated on ice for 30 min. After three washes in PBS, biotin- conjugated anti-chemokine antibody (R&D Systems) was added and incubated on ice for 30 min and then washed in PBS. Finally, fluorescent streptavidin conjugates (BD Biosciences) were added, incubated for 30 min and washed. Fluorescence was measured using a BD FACSCanto II flow cytometer and analyzed using FACSDiva software (BD Biosciences).To determine the impact of surface carbohydrates and proteins on AMC binding, bacterial cultures were grown as described above. For oxidation of carbohydrates, the bacteria were then incubated in PBS containing varying concentrations (0–80 mM) sodium periodate (Sigma-Aldrich) for 60 min, followed by neutralization with ethylene glycol, and washed (5X) in PBS supplemented with BSA. For removal of surface proteins, bacteria were incubated in PBS containing 0.1 mg/ml proteinase K (Sigma-Aldrich) for 60 min at 37°C, followed by neutralization with excess BSA, and washed in PBS with BSA. Treated cells were then subjected to AMC binding and detection by flow cytometry.www.graphpad.com).Bacteria were grown as described above for AMC binding assays and diluted into filtered 0.1μM potassium phosphate buffer (PPB). After addition of AMCs (250 nM CCL28 diluted in 0.01mg/ml BSA) or BSA (0.01 mg/ml), bacteria were incubated at 21 for 5 h. At the indicated time points, 20 μl of bacteria were removed and put on ice in PBS containing freshly diluted Polybead ® Polystyrene 15 μm Microsphere counting beads diluted 1:62500 (Polysciences Inc). Propidium iodide (PI) (Invitrogen) was added just before reading on the flow cytometer. The number of beads remains constant and the beads can be distinguished from the bacteria on the flow cytometer, which allows the number of viable bacteria to be determined by subtracting PI positive bacteria from the total number of bacteria counted per 30000 beads. Percent survival was calculated by dividing viable bacteria of the sample by viable bacteria in the BSA treated control. Polymyxin B susceptibility assays were carried out in the same manner, except that bacteria were exposed to a range of polymyxin B (Sigma-Aldrich) concentrations (0–20 μM) for 5 h, and then bacterial survival relative to BSA-treated controls were determined by flow cytometry. The statistical significance of specific comparisons of interest was assessed via two-way ANOVA with Dunnett’s correction using GraphPad Prism software . LPS was stained using the Pro-Q Emerald 300 LPS Gel Stain Kit (Invitrogen) according to the manufacturer’s directions. Gels were then visualized using a UV transilluminator.Y. pseudotuberculosis to survive or invade at mucosal surfaces. In this study we focused on the mucosally expressed AMCs CCL25 and CCL28. In order to identify bacterial genes that impact AMC resistance, pools of random transposon mutants were put through two different selection strategies to identify mutants with altered binding to AMCs. In the first approach, we postulated that AMC binding would be influenced by changes in cell envelope structures, which are known to affect adsorption of Congo-Red dye [Y. pseudotuberculosis strain IP32953 produces light pink colonies on Congo-Red agar when grown at 21°C. From a pool of ~15,000 random transposon mutants, we identified 88 mutants that exhibited obvious differences in colony morphology and/or Congo-Red binding. These mutants were then subjected to a secondary screen (described below). A second, complementary primary screening strategy approach was employed in which subcultures of the mutant library  were mixed with a limited amount of CCL28 to ensure that those individual bacteria with higher affinity/avidity were preferentially bound with CCL28. These CCL28-coated bacteria were then incubated with anti-CCL28 antibody followed by a secondary antibody conjugated to a magnetic bead. Mutants with increased affinity for chemokine were then enriched by passage over a MACS magnetic column and 120 single colonies were selected.Many of the ~27 chemokines that possess antimicrobial activity could impact the ability of -Red dye , 33. NorFor secondary screening, the mutants derived from these two initial selection strategies were grown at 21°C, incubated with CCL25 followed by fluorochrome labeled antibodies, and screened by flow cytometry to measure the amount of AMC bound to the surface of each cell. Both of the initial selection methods yielded mutants with increased AMC binding compared to wild type bacteria. Secondary screening resulted in the identification of 27 of 88 (31%) of mutants derived from the Congo-Red screen and 86 of 120 (72%) of mutants selected through MACS column with increased AMC binding than the wild type strain.Y. pseudotuberculosis serotype O:1b LPS molecule is presented in manC gene, all of which gave very similar binding levels to each other. In addition to the identification of novel resistance genes, this selection approach yielded several genes previously identified as important in resistance to other AMPs, including those coding for phosphoglucomutase (pgm) and aminoarabinose modification of lipid A (pmr), validating the utility of our approach [Arbitrary PCR was then used to map the transposon insertion sites of the individual mutants selected during the secondary screen. We were unable to map the transposon insertion sites for 21 of the high-binding mutants (possibly due to multiple insertion events). A further 24 mutants from the MACS selection pool were not considered further because sequencing revealed that they represented clones of the same transposon insertion event. In the remaining 68 unique mutants, the majority of the insertions were found in genes predicted to be necessary for LPS synthesis, including both core oligosaccharide and O-antigen production . A schemapproach , 35.manC resulted in much more CCL25 and CCL28 binding than wild-type bacteria but the effect of hldD was stronger . These mutants were among the highest binders of all mutants screened, both in terms of fluorescence intensity and numbers of cells bound . Both are predicted heptosyltransferases, with WaaC likely responsible for transferring the first L, D-heptose residue onto the Kdo residue and WaaF transferring the second [hldD mutant strain than wild type ..hldD genild type .hldD could prevent expression of downstream waaF and waaC genes in the operon. Therefore, we sought to determine the minimum portion of the operon sufficient to complement the wild-type phenotype. This was done through the creation of multi-copy plasmids containing either the hldD, hldD/waaF, or hldD/waaF/waaC genes and transforming these into the hldD::Tn5 mutant background. Addition of the hldD gene alone had no effect on CCL28 or CCL25 binding to bacteria. Conversely, the addition of the hldD/waaF portion of the operon partially restored, and the addition of the entire hldD/waaF/waaC operon completely restored the wild type binding patterns for both CCL28 and CCL25 . The inability of the hldD mutant strain to produce core oligosaccharide would also preclude the addition of O-antigen. In order to verify the contribution of the core oligosaccharide to resistance to AMC binding, we cultured the wild-type and the hldD::Tn5 mutant at 37°C, conditions which are expected to strongly reduce the production of O-antigen (see below). At this growth temperature, the effect of hldD mutation on CCL28 binding was also significant, demonstrating that the core oligosaccharide and not just the O-antigen strongly affect binding . In these assays, bacteria were treated with CCL28, and their survival relative to BSA-treated controls was measured at 1, 3, and 5 hours of exposure. hldD::Tn5 mutant is significantly more sensitive to CCL28 mediated killing than the wild type strain. A CCL28 concentration that is unable to kill wild type bacteria during a five-hour incubation resulted in approximately 50% mortality in the hldD::Tn5 mutant. Addition of the plasmid containing the hldD gene alone had no effect on survival of the hldD::Tn5 mutant. Bacteria complemented with the hldD/waaF portion of the operon exhibited enhanced survival at 3 and 5 hours of treatment compared with the hldD::Tn5 mutant strain. Similar to the AMC binding results seen in hldD/waaF/waaC operon was required to fully restore the hldD::Tn5 mutant strain to wild type levels of resistance to AMC mediated cell death.In an effort to determine whether the increased binding caused by hldD/waaF/waaC operon is important for Y. pseudotuberculosis resistance antimicrobial peptides other than AMCs. Polymyxin B is a well-characterized antimicrobial protein that disrupts the bacterial membrane and is commonly used as a prototype for assessing susceptibility to host defense peptides. We next tested bacterial survival during a 2 hour exposure to varying concentrations of polymyxin B for the wild type, hldD::Tn5 mutant, and mutant bacteria containing portions of the operon   is able to provide some protection to the bacteria when treated with polymyxin B, despite the absence of WaaF or WaaC heptosyltransferase activity.We next sought to determine if the aF/waaC) . As expehldD::Tn5 mutant, and mutant bacteria containing portions of the operon . In these experiments LPS was isolated and analyzed on polyacrylamide gels to measure the impact of these genes on the size of LPS components produced by each strain  produced abundant O-antigen similar to the wild type strain, but small amounts of the truncated core molecule were still detectable. This result suggests that expression of the operon via plasmid greatly enhanced but did not entirely restore production of full length LPS to the entire population of bacteria. This correlates with the absence of full protection against polymyxin B  are necessary to confer full resistance to CCL25 and CCL28 binding. We observed slight increases in core LPS structures with a larger molecular weight on Tris-Tricine gels, and marginal binding/killing resistance when hldD and waaF were expressed together in the absence of waaC. This was somewhat surprising since in E. coli the function of WaaF (heptosyltransferase II) cannot substitute for the function of WaaC (heptosyltransferase I) [Y. pseudotuberculosis transferases are less specific than in E. coli and that WaaF alone may be capable of adding heptose I and heptose II to the Kdo sugar, albeit with much less efficiency.We show here that HldD, which provides the L, D-Heptose substrate for the transferases WaaC and WaaF, is crucial for erase I) . This suhldD alone restores partial resistance to polymyxin B. It is possible that the transposon insertion in hldD may not completely abrogate transcription of the downstream genes waaF and waaC. Although expression of hldD alone did not detectably alter the core structure, the sensitivity of our gel assay may not be sufficient to visualize extremely low levels of full-length core oligosaccharide in this strain and more sensitive analysis methods like mass spectrometry would be necessary to confirm this result. Full resistance to polymyxin B mediated killing was not restored even when all three (hldD/waaF/waaC) were expressed in trans. Gel analysis of LPS from these bacteria showed a small amount of truncated core. Exposing bacteria to polymyxin B may reveal subtler differences among bacterial populations because of the relatively greater potency of polymxyin B compared to CCL25 or CCL28.The pattern of resistance conferred by HldD, WaaF and WaaC was not identical for polymyxin and CCL28. We found that Y. pseudotuberculosis IP32953 (serotype O:1b) has a strong effect on its interaction with AMCs. Y. pseudotuberculosis produces higher levels of O-antigen at 22°C but less at 37°C  and an ABC-ribose transport permease subunit (YPTB3806). These genes have not previously been implicated in the evasion of host defense peptides. Two additional mutants had insertions that mapped to intergenic regions. These mutants displayed more moderate increases in their AMC binding compared with the LPS biosynthesis mutants and require further investigation.Yersinia sp. to resist host defense peptides, which confirms the utility of our approach in finding novel resistance genes. For instance, the phosphoglucomutase (pgm) gene of Y. pestis is required for maximum resistance to polymyxin B but not human defensins or LL-37 [pmrF operon is required for aminoarabinose modification of lipid A and confers a high level of polymyxin B resistance, as well as enhancing bacterial survival in phagocytic cells [In our screens we recovered bacteria with transposon insertions in genes already known to be involved in the ability of or LL-37 . Pgm is ic cells , 53. TheY. pseudotuberculosis provides protection against cell death. This protection is not due to sequestration of peptide from the binding target, but rather it appears that a full length LPS structure repels, or sterically hinders AMCs from reaching and binding the cell surface. Modification of the LPS through chemical or genetic disruption abrogates this protection. Furthermore, preliminary findings suggest that the target of AMC binding may be a surface protein, as their enzymatic removal from bacterial cells significantly reduces the efficiency of CCL28 binding. This hypothesized protein binding by the AMC is reminiscent of alpha-helical host defense peptides [It is still unclear precisely how AMCs target and kill microbial pathogens. However, these studies make clear that full length LPS of peptides  and intepeptides  which bi


FST and two extended haplotype homozygosity-based (iHS and Rsb) analyses 24 candidate genome regions within 14 autosomes and the X chromosome were revealed, in which 18 and 4 were previously identified in tropical-adapted and commercial breeds, respectively. These regions overlap with 340 bovine QTL. They include 409 annotated genes, in which 37 were considered as candidates. These genes are involved in various biological pathways . Our results support that different selection pressures  have shaped the genome of EASZ. We argue that these candidate regions represent genome landmarks to be maintained in breeding programs aiming to improve sustainable livestock productivity in the tropics.The small East African Shorthorn Zebu (EASZ) is the main indigenous cattle across East Africa. A recent genome wide SNP analysis revealed an ancient stable African taurine x Asian zebu admixture. Here, we assess the presence of candidate signatures of positive selection in their genome, with the aim to provide qualitative insights about the corresponding selective pressures. Four hundred and twenty-five EASZ and four reference populations  were analysed using 46,171 SNPs covering all autosomes and the X chromosome. Following  Bos taurus indicus - domesticated in South AsiaBos taurus taurus - domesticated in the Near EastB. primigenius africanus into some African cattle populations remains possibleth century5The history of African cattle is complex, with two cattle subspecies having contributed to the genetic make-up of the majority of today’s African indigenous cattleRhipicephalus appendiculatus ticks infestationAnaplasma marginale, Babesia bigemina, Haemonchus placei and Theileria parva910The indigenous small East African Shorthorn Zebu (EASZ) is commonly found in Western Kenya where they represent the main type of cattleAt the genome level, EASZ has now been shown to be an ancient stabilized admixed zebu x taurine type of cattle13Several studies using genome-wide SNPs have been conducted exploring the genomes of sheep, pigs and cattle to identify signatures of selection following domestication1516171819FST) between EASZ and four reference populations , as well as through the identification of regions showing extended haplotype homozygosity within EASZ (iHS), and between EASZ and the reference populations combined (Rsb). We compare our finding with previous studies on tropical cattle and commercial breeds. We identify candidate regions of positive selection unique to EASZ as well as previously reported regions in other tropically adapted cattle and commercial breeds. Moreover, several of these overlap with Quantitative Trait Loci (QTL) previously identified through genome-wide association studies.Through three separate genome-wide SNPs analyses, we report here the identification of candidate signatures for positive selection in the genome of EASZ both on the autosomes and the sex chromosome X. These were identified through the analysis of genetic differentiation (check.marker function of the GenABEL packageNon-European taurine introgressed EASZ (n = 425), from 20 randomly selected sub-locations, covering 4 distinct ecological zones in Western and Nyanza provinces of Kenya10Additional QC criteria included a minimum sample call rate of 95% and a maximum pairwise identity-by-state (IBS) of 95%, with the lower call rate animal being eliminated from the high IBS pair. From the autosomal SNPs, one EASZ sample was excluded for having a low call rate, whilst one EASZ and one Holstein-Friesian sample were excluded following the IBS criterion. As possible duplicate samples had already been removed following the autosomal QC steps, only the criterion of low call rate was applied for the BTA X analysis. It excluded a further two EASZ samples.FSTFSTvalues (weighted by populations sample sizes) were calculated in sliding windows of 10 SNPs, overlapping by 5 SNPs. The upper 0.2% and 3% of the distribution of FST values were arbitrarily chosen as thresholds for the autosomes and BTA X analyses, respectively, taking into account the difference (9032 versus 184) in the number of windows analysed between the two sets of data. Candidate regions were defined if at least two overlapping windows passed the distribution threshold, taking the highest FST window as a candidate region interval.Inter-population Wright’s iHS)Rsbrehh packageiHS analysis, the natural log of the ratio between the integrated EHH for the ancestral (iHHA) and derived allele (iHHD) was calculated for each genotyped SNP with MAF ≥ 0.5% in EASZ. As the standardised iHS values are normally distributed  or derived alleles . Two-sided P-values were derived as −log10(1-2|Ф(iHS)-0.5|), where Ф(iHS) represents the Gaussian cumulative distribution function. The ancestral and derived alleles of each SNP were inferred in two ways: (i) the ancestral allele was inferred as the most common allele within a dataset of 13 Bovinae specieset al.Two EHH-derived statistics, the intra-population Integrated Haplotype Score , African (N’Dama) and Asian (Nellore)) population as well as with all the reference populations combined. The integrated EHHS (site-specific EHH) for each SNP in each population (iES) was calculated, and the Rsb statistics between populations were defined as the natural log of the ratio between iESpop1 and iESpop2. As the standardised Rsb values are normally distributed . One-sided P-values were derived as −log10(1-Ф(Rsb)), where Ф(Rsb) represents the Gaussian cumulative distribution function. A Z-test was not applied to BTA X Rsb values due to their non-normal distribution  = 4, equivalent to a P-value of 0.0001, was used as a threshold to define significant iHS and Rsb values. Candidate regions were retained if two SNPs separated by ≤1 Mb passed this threshold. In case of Rsb analysis, the combined reference analysis was considered to define the candidate regions. A distance of 0.5 Mb in both directions from the most significant SNP within the iHS and Rsb candidate regions was used to define the candidate genome region interval. This distance was chosen based on the rate of change in the mean pairwise linkage disequilibrium statistic (r2), calculated by the r2fast function of the GenABEL package, binned over distance across the EASZ autosomes (2 plateau. This extent of LD has been confirmed in eight cattle breeds (taurine and zebu) in a previous studyInter-population tributed , a Z-tesutosomes . Indeed,via fastPHASE software version 1.4et al.As a prerequisite for these two statistics, haplotypes were reconstructed through phasing the genotyped SNPs Bos taurus taurus genome assembly UMD 3.1, in which genes with boundaries ≤25 kb from the peak position (the most significant SNP in the candidate regions) were considered as candidate genes. Enriched functional annotation clusters were defined using functional annotation tool implemented in DAVID Bioinformatics resources 6.7P-value of 0.05, was used as a threshold for the identification of enriched clusters.Genes within the candidate genome region intervals were retrieved from the Ensembl genome browserhttp://www.animalgenome.org/cgi-bin/QTLdb/BT/index) to obtain the overlapping QTL with the candidate genome regions.A list of all the previously identified bovine Quantitative Trait Loci (QTL) and their coordinates were downloaded from the cattle QTL database  for autosomes, and 0 and 0.04 ± 0.05 (s.d.) for BTA X, respectively . Ten regAmong the 24 candidate regions for positive selection, 18 were previously identified in other tropical cattle populations and 4 in commercial breeds . Six canA total of 340 bovine QTL intersect with the identified candidate regions . These QFST analysis and the two EHH-based analyses (iHS and Rsb), a total of 192, 72 and 145 genes are identified, respectively  relative to the whole bovine genome , mRNA processing regulation (e.g. U5 and U6), and cell cycle regulation (HECTD3). Moreover, the candidate region on BTA 7, which shows an excess in zebu ancestry, contains genes associated with critical biological pathways suggested to be under selection in tropical adapted cattleDNAJC7), and male reproduction (SPATA24).The candidate genome regions with deficiency or excess of zebu ancestry also harbour interesting genes. The five regions with zebu ancestry deficiency  carry geiHS), inter-population Rsb, and FST) with the aim to identify candidate signatures of positive selection in the genome of an indigenous East African cattle population. We pooled all the non-admixed cattle populations into a single reference population in Rsb. As shown in Rsb-specific candidate regions stronger in comparison to their signals in the pairwise analyses. This might be due to a reduction of the effect of population-specific LD caused by genetic drift. Such an empirical haplotype pooling approach has been suggested previously by Gautier and NavesIn this study, we used three analyses , the admixed zebu x taurine genome structure of EASZ (e.g. development and growth pathways) and human selection for specific traits.LOC512672 on BTA 23, IGBP1 on BTA X and BCL6B on BTA 19). For example, LOC512672 is a major histocompatibility complex class I gene. This class of genes is responsible for presenting antigen peptides to cytotoxic T-cells to induce their immunological response1011Several of the candidate genes further support the presence of distinct selective forces . Some ofOR2AP1, OR6C4, RXFP2, KLHL10) have also been identified within candidate regions on BTA 5, 12 and 19. These genes may be associated to superior fertility and semen quality in zebu cattle under heat stress conditions compared to exotic taurineRXFP2 is located within the most significant Rsb candidate genomic region in BTA 12. The protein encoded by this gene is involved in the testicular descent development49RXFP2 has also been identified to be under positive selection in tropically adapted Creole cattleOR6C4 and OR2AP1) identified in BTA 5 can be classified as male reproduction genes. These genes may play a role in guiding sperms towards oocyte during fertilization via the interaction with various chemoattractants secreted by the oocyte-cumulus cells complexCandidate genes associated with male reproduction (ACLY. This gene encodes an enzyme involves in energy production by linking glucose metabolism to lipid synthesisAlso an interesting candidate gene identified in BTA 19 is MYL6 and MYL6B) and SYT10 belong to this category.Several genes identified within the candidate region intervals, but not considered as candidate genes following our criteria of presence within 25 kb of the most significant SNP, are also associated with various biological functions that might be under selection in African cattle. Due to the utilisation of Kenyan zebu cattle in ploughing and transportation by farmersHSPB9, DNAJC7, DNAJC8, DNAJC14 and DNAJC18), or associated with the heat stress response (PPP1R10)PPP1R8) in a positively selected genomic region in the Creole cattle. Also, several coat colour QTL overlap with a candidate region in BTA 5. Brown coats are predominant in EASZKRT and PMEL17)585961Climate stress  is expected to be an important selection pressure acting on EASZ. Several genes associated with the heat shock protein family .


Arabidopsis thaliana expressing a peroxisome targeted YFP and a mitochondrial targeted GFP probe, we observed sustained interactions between peroxules and small, spherical mitochondria. Further, it was observed that the frequency of HL-induced interactions between peroxules and mitochondria increased in the Arabidopsis anisotropy1 mutant that has reduced cell wall crystallinity and where we show accumulation of higher H2O2 levels than wild type plants. Our observations suggest a testable model whereby peroxules act as interaction platforms for ROS-distressed mitochondria that may release membrane proteins and fission factors. These proteins might thus become easily available to peroxisomes and facilitate their proliferation for enhancing the ROS-combating capability of a plant cell.Peroxules are thin protrusions from spherical peroxisomes produced under low levels of reactive oxygen species (ROS) stress. Whereas, stress mitigation favors peroxule retraction, prolongation of the ROS stress leads to the elongation of the peroxisome into a tubular form. Subsequently, the elongated form becomes constricted through the binding of proteins such as dynamin related proteins 3A and 3B and eventually undergoes fission to increase the peroxisomal population within a cell. The events that occur in the short time window between peroxule initiation and the tubulation of the entire peroxisome have not been observed in living plant cells. Here, using fluorescent protein aided live-imaging, we show that peroxules are formed after only 4 min of high light (HL) irradiation during which there is a perceptible increase in the cytosolic levels of hydrogen peroxide. Using a stable, double transgenic line of  Peroxisomes are directly implicated in the scavenging of reactive oxygen and reactive nitrogen species  in plant cells  into the existing peroxisomal membrane . Further, an Arabidopsis mutant any1 mutant background by crossing with the respective wild type lines. The any1 phenotype of isotropic expanded hypocotyl cells and trichomes as well as the GFP and YFP fluorescence were confirmed through light and epi-fluorescent microscopy. Double transgenic lines of any1-mitoGFP X YFP-PTS1 were created by crossing and stabilized over three generations.Peroxisomes and peroxules were visualized in a transgenic Arabidopsis line expressing a yellow fluorescent protein (YFP) with a peroxisome targeting tri-peptide (SKL) appended to the carboxy terminus ; RFP- 540 to 630 nm (red); chlorophyll—650 to 710 nm . In double and triple transgenic plants the YFP fluorescence was picked up in both GFP and RFP channels and appeared yellow in the merged images.2O2 responsive probe  was analyzed immediately after. The ImageJ RGB Profile Plot plugin was used to determine the changes in fluorescence intensity before and after HL treatment.Arabidopsis transgenics expressing cytosolic HyPer, a Hhttp://www.adobe.com) or the ImageJ/Fiji platform (http://fiji.sc/Fiji). Adobe Photoshop was used for annotation of movies.All images were captured at a color depth of 24 bit RGB. Tissue and 7–10 day old seedlings were mounted in tap water on a glass depression slide and placed under a coverslip. All images and movies were cropped and processed for brightness/contrast as complete montages or image stacks using either Adobe Photoshop CS3 (−2 s−1). Respective controls of the effects of light involved growing plants in complete darkness from stratification up to and including observations, unless stated otherwise. To evaluate the responses and the rapidity of the responses of the organelles to HL, plants were given short exposures (1–5 min) of HL (850 ± 50 μmol m−2 s−1). Observations were taken immediately after the light treatments to give a better sense of how quickly responses may be.Plants were all grown at low to intermediate light  for 8 days, transferred to the dark for 24 h and then exposed to light (164 μmol m−2 s−1) for 30 min, 1 h, or 2 h. Cotyledon and hypocotyl tissue were submerged in a solution of 3,3′-diaminbenzidine (DAB) with a metal enhancer  or distilled water (control) and left under vacuum (−50 KPa) for 4 h. The tissue was cleared with ethanol by washing the samples with 100% ethanol, incubating them in 85% ethanol + 15% methanol overnight, and rinsing them in 70% ethanol and then distilled water. The samples were mounted in 50% glycerol and sealed. All images were acquired at the same light intensity and microscope settings to permit direct comparisons between treatments. The DAB stain intensity was measured as the average inverse gray value using ImageJ (http://imagej.nih.gov/ij/), which was subtracted from the background and considered as the average inverse gray values of distilled water treated seedlings. The staining intensity was representative of the amount of H2O2 produced during the relative light intensity treatments.To look at the effects of light-induced production of Hanisotropy1 (any1) mutant and the extent of its anisotropy. Scanning electron microscopy (SEM) of Arabidopsis wild type (ecotype Columbia) and any1 plants grown in soil under 125 ± 10 μmol m−2 s−1 light intensity was carried out using uncoated tissue in a Tabletop Hitachi TM-1000 microscope with an electron beam accelerated at 15 kV. Leaf cross sections of 12 day old seedlings grown in low light (70 μmol m−2 s−1) were stained with TBO to analyse the alterations in cell isotropy and cell-to-cell connectivity.Scanning electron microscopy (SEM) and toluidine blue O (TBO) staining were used to assess the consequences of alteration of the cell wall in the n = 50. Two-tailed t-tests were made to determine the significance of results. Significance was predetermined as having a p < 0.01 .All experiments were carried out at least five times. Observations of mitochondrial length comparisons and their interactions with peroxisomes and peroxules were made with a minimum sample size of 2O2  responsive HyPer-cytosolic probe  plants. The any1 mutant in an Arabidopsis cellulose synthase (CesA) gene has reduced cell wall crystallinity  from 11 to 13 day old WT plants exhibited peroxules after 8–10 min of HL exposure. Comparable cells in any1-YFP-PTS1 plants showed 30 ± 7% (n = 80 cells) peroxules upon exposure for less than 8 min .Peroxisomes in both WT and n Figure . Longer n Figure , made itany1 plants expressing mitoGFP and a comparison of mitochondrial size between the two kinds of plants under dark and light conditions showed significantly smaller mitochondria in the mutant  the ratio of mitochondria to peroxisomes was nearly double while extreme ratios approaching 5:1 were also observed  including PEX11 isoforms can be suggested. Indeed, the overexpression of different PEX11 isoforms over several hours has been shown to increase peroxisome tubulation and clustering  suggests interesting possibilities involving mitochondria and peroxisomes . Following HL exposure, however, there is a perceptible increase in mitochondria-peroxisome clusters both near and away from chloroplasts. In plants, both mitochondria and peroxisomes are linked metabolically with chloroplasts (Douce et al., However, as observed by us the change in ER dynamics and organization around chloroplasts was transient and the ER resumed its dynamic behavior after a few minutes. However, mitochondria and peroxisomes remained clustered around chloroplasts for a much longer period. We speculated that the 3-organelle cluster is maintained due to the presence of one or more proteins that is/are shared between them. FISSION1 represents such a protein. This tail-anchored membrane protein is highly conserved between different eukaryotes, exists with the same membrane topology in both peroxisomal and mitochondrial membranes, and has been shown to help recruit the DRPs to the peroxisomal and mitochondrial membrane for their eventual fission (Schumann and Subramani, Our observations suggest a simple, presently quite speculative but testable model Figure . As showWhile details of this rather simplistic interpretation of HL induced events aimed at combatting ROS stress in a plant cell continue to be assessed critically, the work presented here clearly provides visual proof of sustained interactions between peroxisomes and mitochondria in living plant cells with peroxules acting as transient interaction platforms.any1 mutant. All authors contributed critical comments and corrections and have approved the manuscript.EJ and JM designed experiments and co-wrote the manuscript. NM and FD provided expert help with experiments and plant materials. GW provided seeds of 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.