Nucleus is a highly compartmentalized part of the cell where the key processes of genome functionality are realized through the formation of non-membranous nuclear domains. Physically nuclear domains appear as liquid droplets with different viscosity stably maintained throughout the interphase or during the long diplotene stage of meiosis. Since nuclear body surface represents boundary between two liquid phases, the ultrastructural surface topography of nuclear domains is of an outstanding interest. The aim of this study was to examine ultrathin surface topography of the amphibian and avian oocyte nuclear structures such as lampbrush chromosomes, nucleoli, histone-locus bodies, Cajal body-like bodies, and the interchromatin granule clusters via low-voltage scanning electron microscopy. Our results demonstrate that nuclear bodies with similar molecular composition may differ dramatically in the surface topography and vice versa, nuclear bodies that do not share common molecular components may possess similar topographical characteristics. We also have analyzed surface distribution of particular nuclear antigens  using indirect immunogold labeling with subsequent secondary electron detection of gold nanoparticles. We suggest that ultrastructural surface morphology reflects functional status of a nuclear body. Nuclear structures are complex and dynamic parts of the cell that lack a surrounding membrane1358911Giant nuclei of growing avian and amphibian oocytes represent a useful system for exploration of the surface ultrastructure of a number of nuclear bodies as well as transcriptionally active chromosomes. The main advantage here is the large size of the nucleus which allows manual isolation of intact nuclear structures into distinct preparations. Growing oocyte nucleus houses actively transcribed chromosomes in the lampbrush form and, in the case of amphibians, diverse extrachromosomal and chromosome associated nuclear bodies , interchromatin granule clusters (IGC) or B-snurposomes and other nuclear organelles)1415161720212123The inner ultrathin organization of the amphibian and avian oocyte nuclear structures has been investigated in depth using transmission electron microscopy (TEM)2325262728293029323332Low-voltage scanning electron microscopy (LV–SEM) is one of the techniques for analyzing surface topology of various biological samples with high resolution. Compared to conventional SEM, LV–SEM with the secondary electron detection allows for analysis of the surface topography of uncoated, non-osmicated biological samples. Moreover, only in case of LV–SEM one has the opportunity to apply immunogold labelling techniques36In this work we have aimed to visualize with high resolution the surface of microsurgically isolated nuclear bodies and giant lampbrush chromosomes from avian and amphibian oocytes. Our results demonstrate that LV–SEM without osmium fixation and conductive coating allows for the identification of extrachromosomal and chromosome-associated nuclear bodies in the oocyte nuclear content preparations. Moreover, individual lampbrush chromosome regions such as chromomeres, lateral loops with specific morphology of RNP-matrix, transcribed and untranscribed looped-out DNA regions are easily recognizable. Although usage of hypotonic treatments, chemical fixation, dehydration, and air-drying for samples preparation disturb liquid properties of nuclear structures, surface ultrastructure is adequately preserved and generally reflects the state of liquid-liquid phase border. Ultrastructural topography of amplified nucleoli, HLB and IGC from amphibian oocytes has been characterized for the first time. We have adapted standard immunogold labelling technique with subsequent secondary electron detection of gold nanoparticles to reveal superficial distribution of certain nuclear antigens namely coilin, small nuclear RNAs (snRNA) and double-stranded DNA (dsDNA) on the oocyte nuclear structures. Based on the results obtained we argue that in spite of similar molecular compositions by surface ultrastructure the oocyte nuclear bodies may differ significantly, although similar surface topological characteristics can be exhibited by structures with different molecular composition. We discuss how the surface morphology may reflect functional status of a nuclear body.Gallus gallus domesticus), Japanese quail (Coturnix coturnix japonica), rock pigeon (Columba livia) and African clawed frog (Xenopus laevis) were used in this study. All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory AnimalsOocytes of the domestic chicken  and incubated at +18 °C overnight. Avian ovaries were dissected, placed in a humidity chamber at +15 °C and used for preparation of nuclear spreads within the next two hours. Both avian and frog oocyte nuclei were isolated one by one using precision forceps or tungsten needles in an embryo dish with 5:1 medium . Isolated avian oocyte nuclei were immediately washed from the remaining yolk in a hypotonic 1/4 solution  and transferred to individual isolation chambers filled with 1:4 solution. To release nuclear content the nuclear envelopes were broken using thin tungsten needles inside isolation chamber. Isolated frog nuclei were left in the 5:1 solution for approximately 10 seconds and then transferred to the embryo dish filled with 1:4 solution, where nuclear envelopes were separated from the nuclear content using thin tungsten needles. All manipulations with the oocyte nuclei were performed under stereomicroscope ; nuclei and nuclear content were transferred individually using standard micropipettes. To disperse nuclear content, preparations were left on ice or at +4 °C for 20 minutes and then centrifuged at 3500 g at +4 °C for 30 minutes.Fragment of frog ovary was dissected, placed in OR2 medium  for 30 minutes at room temperature, then dehydrated in a series of ethanol-water solutions of increasing concentration  and dried in air. A single drying technique was used for all preparations. Isolation chambers were removed from the slides during transition from 70% to 96% ethanol. Preparations were analyzed in the wide-field microscope–Leica DMR4000B; selected preparations were imaged at low magnification for easier orientation under LV–SEM.After the centrifugation step preparations of nuclear content destined for topography analysis with low voltage scanning electron microscopy were fixed in the mixture of 2.5% glutaraldehyde and 2% formaldehyde in 1 × PBS  regime. Detector of secondary electrons (In-lens SE) was used for viewing and imaging the surface topography. Following settings were used: working distance −0.4–0.6 mm, chamber pressure in the 10The following primary antibodies were used: mouse monoclonal antibodies (mAb) against double-stranded DNA (dsDNA) (HYB331-01) (Abcam), mAb against 2,2,7-trimethylguanosine cap of snRNA (K121) (Santa-Cruz Biotechnology); rabbit polyclonal serum H-300 against coilin (Santa Cruz Biotechnology). Corresponding secondary antibodies were used: goat anti-mouse IgG (H + L) conjugated with 18 nm colloidal gold nanoparticles (Jackson Immunoresearch Lab); goat anti-rabbit IgG conjugated with 10 nm colloidal gold nanoparticles (Sigma).For immunogold labeling, preparations of the oocyte nuclear content were made as described elsewhereNuclear content preparations selected for immunogold labeling were rehydrated in a series of ethanol-water solution of decreasing concentration  and then transferred to PBS for 10 minutes. Rehydrated preparations were then incubated with blocking solution (0.5% blocking reagent (Millipore) in PBS), followed by primary and secondary antibodies, both diluted in blocking solution. All incubations were made in a humidity chamber at room temperature, for one hour each. After every antibody incubation step, preparations were washed three times with PBS at room temperature. For double immunolabeling, two antigens were detected sequentially. Upon completion of the first antigen immunolabeling, preparations were fixed in 2% formaldehyde in PBS for 15 minutes, washed in three changes of PBS, and then the second antigen was immunolabelled. Antigens detected with larger (18 nm gold) conjugates were immunolabeled first. After immunogold labeling the preparations were fixed in a 2.5% glutaraldehyde solution in PBS (pH 7.5) for 15 minutes, dehydrated in a series of ethanol-water solution of increasing concentration  and air dried.Immunogold labeled preparations were analyzed using Zeiss Merlin scanning electron microscope in a low voltage regime. Acceleration voltage of 0.4 kV was used for the imaging of gold nanoparticles; other parameters were the same as for the surface topography analysis. In the case of 0.4 kV acceleration voltage, the voltage signal of secondary electrons contains more SE2 electrons excited by back-scatter compared to the of secondary electron voltage signal with the 0.2 kV acceleration voltage4 electrons per pixel, or from 1014 to 1017 electrons per cm2 depending on magnification. No posterior filtering or noise reduction was used. The radiation damage rate was quite low due to low energy of the electron beam, so it was not observed after ten scans at high magnification; whereas electron beam induced hydrocarbon contamination appears after the second image acquisition. Measurements of ultrastructural elements were performed using tools of SmartTiff (Ziess) software; at least five examples of the same ultrastructural element were measured. For more explicit presentation of nanoparticle distribution layer masks were created in Adobe Photoshop. The mask is a separate image which mirrors contours of a nuclear structure and distribution of gold nanoparticles.Two modes of the image acquisition were used: scanning of a single frame with a high pixel dwell time, or scanning of a series of 10 frames with subsequent averaging. The modes were changed to overcome the charging of the surface of the sample. The pixel size varied from 3 nm to 150 nm for different magnifications, and a pixel dwell time was in a range of tens of microseconds (up to 100 μs) for a single scan or around 10 μs for scanning with subsequent averaging. Total acquisition time was in a range of 1–5 minutes. For all images electron dose was of about 2–4 × 10Previous high-resolution SEM images of loops with specific morphology on avian and amphibian lampbrush chromosomes were obtained after gold or palladium coating and osmium fixation29The surface of the following lampbrush chromosome regions was examined with LV-SEM: chromomeres, interchromomeric regions, transcriptionally active simple lateral loops, certain complex loops and chromosome associated structures.in vivoAxes of lampbrush chromosomes consist of the chromomeres connected by relatively short interchromomeric regions linking individual chromomeres. The morphology of chromosome axis was analyzed on an example of a compact W-chromosome which carries less lateral loops –c. On thThe organization of centromere regions in the lampbrush chromosomes was described in detail in our recent reviewLampbrush chromosomes carry lateral loops of two major morphological types: one, the prevailing type of lateral loops, is called simple; another type is rare, loci-specific loops called complex loops or loops with complex morphology16Individual transcription units are separated from each other by short regions of “naked” loop axis 10 to 20 nm thick . The RNP281155Organization of the RNP-matrix in complex loops differs from that found in simple loops. LV-SEM revealed that the RNP-matrix of dense loops arising from the pericentromere region of pigeon lampbrush chromosome W consists of compactly packed RNP-fibrils forming thick cylindrical structures of 600 to 700 nm in diameter . Large v27Besides complex loop-based structures, lampbrush karyotypes of many avian species are characterized by formation of giant terminal RNP-aggregates (GITERA)59Xenopus oocyte nucleus can be seen in LV-SEM revealed distinctive ultrastructural topography of the oocyte nuclear bodies. Numerous and diverse extrachromosomal bodies  in the Xenopus oocyte demonstrated high-relief and structured surface with furrows and ridges consisting of granules 40 to 60 nm in size clustered around pores  on the surface rich with cavities and pores . The surXenopus oocytesXenopus and newt) oocyte revealed that IGC are composed from 20–30 nm sized granules14Pleurodeles B-snurposomes16HLB can possess from zero to dozens of spherical structures called B-snurposomes–analogues of IGC in Acheta domesticus68Coilin-containing spherical bodies in the avian and amphibian oocytes demonstrated quite different topography –h. ExtraXenopus HLB. However, the surface of the centromere protein bodies is smoother, does not contain patches, with numerous grains on their surface being smaller than those on HLB. At the same time, the surface of the centromere protein bodies dramatically differs from the surface of CB-like bodies from pigeon oocytes.Centromere protein bodies, typical for oocytes of certain avian species, are coilin-free and do not accumulate any RNPs2431Summarizing morphological data it should be noted that in our study RNP-particles , in contrast to other measured ultrastructural elements, appeared to be about 10 nm larger than in conventional SEM or TEM studies. We suggest that size increase is caused by hydrocarbon contamination after electron beam damage of uncoated preparations during image acquisition. Moreover, different ultrastructural elements may have different sensitivity to radiation damage, namely RNP-particles are more sensitive than chromatin or coilin fibrils. Indeed, in our recent atomic force microscopy study of similarly prepared lampbrush chromosomes nascent RNP-particles of lateral lampbrush loops were evaluated as 25 nmThe immunogold labeling technique was applied to analyze surface distribution of the components typical of nuclear content. We have analyzed the distribution of double-stranded DNA (dsDNA), coilin and snRNAs exposed to the surface of nuclear structures isolated. To this purpose we have adapted the fixation protocol used for oocyte nuclear content preparations with only formaldehyde fixation step followed by ethanol dehydration. The diameter of the entire labeling complex was about 26 nm for 10 nm and 34 nm for 18 nm colloidal gold conjugates correspondingly, which only allows binding to the surface antigens.The DNA exposed on the surface of the chromatin domains was revealed using immunogold labeling with antibodies against dsDNA  on exampWithin transcriptional units of lateral loops the colloidal gold nanoparticles were evenly distributed along regions of “naked” loop axis ,c. The tIt is known that heterogeneous group of bodies to which CB, HLB, extrachromosomal CB-like bodies and “pearls” belong contain protein coilin and snRNPs 2373747572373747576Coilin was evenly distributed across the surface of the HLB . The disXenopus HLB. At the same time density of superficial snRNAs were comparable for both nuclear bodies. Higher concentration of coilin may be associated with lower functional activity of CB-like bodies.On the surface of pigeon CB-like bodies, coilin and snRNAs were also evenly distributed ,c,e. It Xenopus HLB .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Using an optimized method that can accurately assess either severe or mild pdMCAO-induced sensorimotor deficits, neurobehavioral tests were performed over 28 days. The progression of lesion volume and the evolution of astrocytic and microglial activation were determined in the acute stage of ischemic stroke after pdMCAO (0–3 days). Nrf2-downstream target antioxidant genes expression levels was assessed by Western blot. We found that Ginseng pretreatment ameliorated acute sensorimotor deficits and promoted long-term functional recovery, prevented the acute enlargement of lesion volume (36.37 ± 7.45% on day 3), attenuated reactive astroglial progression but not microglia activation, and enhanced the induction of Nrf2-downstream target proteins after ischemic insult in WT mice, an effect which was lost in Nrf2 knockouts. The spatiotemporal pattern of reactive astrogliosis evaluation correlated well with acute ischemic damage progression in an Nrf2-dependent fashion during the acute phase of ischemia. In contrast, Nrf2 deficiency mice exhibited exacerbated ischemic condition compared to WT controls. Together, Ginseng pretreatment protects against acute sensorimotor deficits and promotes its long-term recovery after pdMCAO, at least partly, through Nrf2 activation, highlighting the potential efficacy of oral consumption of Ginseng for stroke preventative intervention in patients who are at great risk of recurrent stroke or transient ischemic attack. The attenuated reactive astrogliosis contributes to the Nrf2 pathway related neuroprotection against acute ischemic outcome and substantially long-term sensorimotor deficits in the context of ischemic stroke under pdMCAO.Endogenous neuroprotective mechanisms by which the brain protects itself against noxious stimuli and recovers from ischemic damage are key targets of stroke research, ultimately facilitating functional recovery. Transcriptional factor Nrf2, enriched in astrocytes, is a master regulator of endogenous defense systems against oxidative stress and inflammation. Korean Red Ginseng (Ginseng), one most widely used herbal medicine, has exhibited promising potentials in neuroprotection. Our study aimed to determine whether the standardized Ginseng extract pretreatment could attenuate acute sensorimotor deficits and improve long-term functional recovery after ischemic stroke though Nrf2 pathway and whether reactive astrogliosis is associated with such effect. Adult Nrf2 Ischemic stroke, more often disabling than fatal, is the leading cause of severe and long-term disability in the United States  is derived from the peeled and dried root of Panax ginseng, and its major active components are ginsenosides  mouse model generates highly reproducible ischemic cortical lesion, inducing a definable behavioral deficit that closely mimics human ischemic stroke. It is therefore believed to be one of the most predictable and potentially useful stroke translational models, allowing us to look at longer time points with most high survival rate  to demonstrate the contribution of Nrf2 in the effects of Ginseng pretreatment by examining the induction of Nrf2 downstream target cytoprotective and antioxidative proteins; (4) to assess the contributions of the spatiotemporal reactive astrogliosis and microgliosis in the Ginseng effects in the acute phase of ischemic injury; and (5) to provide the in vivo evidence addressing the functional contribution of Nrf2 and its effects on reactive gliosis after focal permanent stroke.In the present study, we hypothesized that the standardized Ginseng extract pretreatment protects against acute sensorimotor deficits and improve functional recovery after ischemic stroke potentially through the Nrf2 pathway; preferential alteration of the spatiotemporal reactive astrogliosis is associated with such effect. Accordingly, we had five goals in this study: (1) to establish a novel method that can accurately evaluate mild sensorimotor deficits in ischemic stroke mouse models; (2) to evaluate the effects of Ginseng pretreatment on acute sensorimotor deficits and anatomical outcomes and functional recovery following pdMCAO and determine whether these effects are lost in Nrf2−/− mice were generated as previously described  Cohort 1: Twenty WT mice were used for the establishment of long-term sensorimotor assessment; (2) Cohort 2: Forty-five WT mice and 46 Nrf2−/− mice were used for long-term sensorimotor evaluation of Ginseng pretreatment on ischemic stroke; (3) Cohort 3: Forty WT mice and 40 Nrf2−/− mice were used for analysis of immunohistochemistry staining and Western blot. For each cohort in this study, mice were randomly distributed into groups of both genotypes and treatments.All procedures on animals were performed according to the NIH Guide for the Care and Use of Laboratory Animals and approved by the University of Florida Institutional Animal Care and Use Committee. The Nrf22 bone above the artery was carefully removed with a micro forceps. The distal MCA branches were occluded permanently using a bipolar electrocoagulation forceps . The occlusion was checked 30 s later, and, if needed, the coagulation was repeated to achieve complete occlusion. The temporal muscles were repositioned and the skin incision was closed with wound clips. Body temperature was maintained at 37°C by a thermostatically controlled heating pad during surgery, and mice were allowed to recover in a temperature- and humidity-controlled chamber after surgery. The sham-operated (sham) control mice were subjected to the same surgery procedure, except that the distal MCA was not occluded. Mice that died during surgery or experiments were excluded from the final analyses. No apparent evidence of hemorrhages was observed during or after surgery. Mice that died during surgery or experiments were excluded from the final analysis. As for all of our animal colonies, fresh food and water were provided to the animal ad libitum.Ischemic stroke was induced by pdMCAO as described previously  for 7 days prior to pdMCAO  total forelimb use  = total number of contacts with left side of forelimb/total number of contacts with each side of forelimb × 100; (2) first contact events  = total number of first contact events /total number of full rearing with any forelimb contact × 100; and (3) both forelimbs use events (%) and single forelimb use events  = total number of both forelimbs use events or single forelimb (left/right) use events/total number of full rearing with any forelimb contact × 100.The corner test was performed to determine overall sensorimotor and postural asymmetries  or cresyl violet staining on day 1 and day 3 after ischemic stroke, respectively. The brains were quickly removed after euthanasia and sliced into 1 mm coronal sections using a brain matrix. Slices were incubated for 20 min at 37°C in 2% TTC (Sigma-Aldrich) dissolved in phosphate-buffered saline  for vital staining  for 15 min at room temperature to remove endogenous peroxidase activity. After washing and blocking with 2% normal horse serum (V/V), sections were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: rabbit polyclonal glial fibrillary acidic protein , rabbit polyclonal ionized calcium-binding adapter protein 1 , mouse monoclonal glutamine synthetase , and rabbit polyclonal aquaporin 4 . On the following day, sections were washed and incubated with appropriate secondary antibodies and visualized with 3,3-diaminobenzidine (DAB) substrate (Vector Laboratories). We counted the total numbers of GFAP-positive astrocytes, GS immunointensity, APQ4 immunointensity and the total number of Iba1-positive cells in indicated regions. The percentages of GFAP-positive reactive astrocytes were also measured, featured by hypertrophic cell bodies and intensely stained processes. Three consecutive sections from each brain were analyzed and provided a single value for this mouse. The images were captured using ScanScope CS and analyzed using ImageScope Software (Aperio Technologies). To avoid potential bias, two independent analyses were performed by two trained observers blinded to pretreatment and genotype.At indicated time points, mice were anesthetized and transcardially perfused with PBS (pH 7.4) followed by 4% paraformaldehyde  in PBS. The brains were collected, post-fixed and cryoprotected in 30% (w/v) sucrose and cut into 30-μm-thick coronal slices on a Leica rotary microtome cryostat. Immunostaining was performed using standard protocols. Sections were washed and then treated with 3% HWestern blot analyses were performed as previously described  with assistance from a biostatistician. The number of mice in independent experiments is stated in each figure or figure legend. Behavioral tests were analyzed by Repeated Measures Linear Mixed Modeling to account for identified baseline and post-ischemic outcome differences between groups. Multi-group comparisons were carried out using one-way or two-way ANOVA followed by Bonferroni P < 0.05), but not at the later stages (7–28 days). No animal died during these experiments. These results indicated that pdMCAO led to the reduction of early-stage locomotor activity without affecting weight change dynamics.To optimize a method that can accurately evaluate sensorimotor deficits in pdMCAO mice model, a typical mild ischemic stroke model, we performed a behavioral test battery over 4 weeks Figure . Weight P < 0.05 and P < 0.01, respectively), but not at the later time points ; this difference was not detected between days 21 and 28 when compared with shams . The corner test revealed that Ginseng pretreatment significantly preserved the sensorimotor function from day 3 to day 28 in WT but not in Nrf2−/− mice , Ginseng pretreated WT mice exhibited remarkable attenuated sensorimotor deficits compared to vehicle-pretreated WT controls, reflected by significantly increased percentages of both total forelimb use and first contact events with both forelimbs on day 3 after pdMCAO . In contrast, compared to vehicle-pretreated WT controls, vehicle-pretreated Nrf2−/− mice group displayed significantly severe infarct volume on day 3 but not on day 1  after pdMCAO. These results indicated that Ginseng pretreatment protected against ischemic lesion, an effect which was diminished by the loss of Nrf2, suggesting the potential involvement of Nrf2 activation in the neuroprotection of Ginseng pretreatment.Functional deficits caused by acute ischemic stroke are often associated with the extent of histological damage in affected brain regions. Thereafter, with the third cohort of mice, we examined whether Ginseng pretreatment could protect against acute ischemic lesion size and whether Nrf2 contributes to this process. Consistent with the behavioral findings above . On day 3, Ginseng pretreatment preserved more astrocytes and smaller proportions of reactive astrocytes in WT mice, but not in Nrf2−/− mice , while the Nrf2 deficiency significantly exacerbated the astrocyte loss (P < 0.05). Additionally, much more degenerated astrocytes with breakdown cell bodies were found in Nrf2−/− mice groups than WT mice groups. These results indicated that the Nrf2 activation from Ginseng pretreatment may attenuate the early stage of progression of reactive astroglosis that helped prevent the expansion of ischemic infarct volume  but not day 3 after pdMCAO in WT mice; this effect was absent in Ginseng pretreated Nrf2−/− group. Ginseng pretreatment reduced the rapid increase of AQP4 on day 1 (P < 0.05) but did not significantly affect the loss of AQP4 on day 3 after pdMCAO in WT mice . Our data indicated that, under our experimental condition, microglia activation would not have a substantial role in Ginseng’s neuroprotection against ischemic damage at indicated time points, while Nrf2 absence exacerbated the microglial activation.Finally, we also investigated whether microglial activation contributes to the neuroprotection of Ginseng by Iba1 immunostaining. As shown in Figure −/− mice, we found that 7 days of Ginseng pretreatment dramatically attenuated acute sensorimotor deficits and promoted long-term recovery over 28 days after ischemic injury, which was accompanied by the reduction of acute brain lesion volume (36.37 ± 7.45% on day 3). Such neuroprotection was lost in Nrf2−/− mice. Meanwhile, we found that Ginseng pretreatment significantly enhanced expression levels of Nrf2 downstream cytoprotective and antioxidative proteins including NQO1, HO1, SOD2 and GPx1 at the early stage of ischemic stroke onset in WT, and this was completely abolished in the Nrf2−/− mice. Such Nrf2-mediated effects might attenuate spatiotemporal reactive astrogliosis, but they would not attenuate the microglia activation, that eventually contributed to the neuroprotection of Ginseng against ischemic damage. In addition, such insights concerning the role of Nrf2 deficiency in ischemic stroke progression induced by pdMCAO offered further understanding of the natural properity of Nrf2 in neuroprotection.In the present study, we optimized a method that can more accurately assess both severe and mild sensorimotor deficits in pdMCAO-induced ischemic stroke mice model. By using this method and Nrf2Functional assessment is a key component for testing the efficacy of potential therapeutics in translational stroke research and sensitive behavior tests, especially in mice, are known to be challenging. The cylinder test was initially designed to assess the side-biased forelimb functional deficit in the Parkinson’s disease rat model, and then was extended to various rodent stroke models indicated by the rate of total forelimb use (contralateral side to brain lesion; Schallert et al., Ginseng has been shown to direct stimulation of cell defense mechanisms and alter specific antioxidant enzyme activities that are necessary for eliminating free radicals and reducing by-products of tissue peroxidation reactions Gillis, . The majAstrocytes are the most abundant cells in the CNS, sending out thin branches enwrapping neuronal synapses and covering adjacent brain capillaries within their anatomy domains. They play vital roles in maintaining brain homeostasis including brain energy metabolism, synaptic transmission regulation, potassium buffering, extracellular glutamate level control, and interstitial volume regulation. Neurons critically rely on astrocytes for their supportive properties and intrinsic protection in many CNS disorders, including ischemic stroke (Sims and Yew, It is known that Nrf2-target genes are preferentially activated in astrocytes, which consequently have more efficient antioxidant defense and detoxification than neurons (Vargas and Johnson, in vivo research will not only allow us to clarify that Ginseng is able to rapidly elicit or boost endogenous cellular neuroprotection after stroke, but also provide new insight into the nature of Nrf2 in the context of ischemic stroke. This may open a window of opportunity to utilize these endogenous neuroprotection mechanisms in patients with stroke and other neurological diseases in clinic. To our knowledge, our study is the first to show evidence for the role of Nrf2 deficiency in long-term sensorimotor deficits and neuropathological outcome in pdMCAO-induced ischemic stroke in mice. The loss of Nrf2 exacerbated the acute sensorimotor deficits, the deterioration of ischemic damage and the progression of reactive astrogliosis, and delayed the long-term functional recovery from ischemic damage. These data further highlighted the vital role of Nrf2 in ischemic stroke pathogenesis.Our in vivo work assessed the unique properties that Ginseng can provide the brain cells with resistance against acute and potentially chronic debilitating neurodegenerative conditions like ischemic stroke, and it highlighted the contribution of Nrf2 to Ginseng’ protective effect.In conclusion, the optimized method of the cylinder test depicts a more sensitive and reliable mean to detect sensorimotor deficits, which could be most instrumental for ischemic stroke research. Ginseng pretreatment protects against acute sensorimotor deficits and promotes its long-term recovery after pdMCAO, at least partly, through Nrf2 activation, highlighting the potential efficacy of oral consumption of Ginseng for stroke preventative intervention in patients who are at great risk of recurrent stroke or transient ischemic attack, that impedes the ischemic cascade and ultimately facilitates functional recovery. The attenuated spatiotemporal reactive astrogliosis contributes to the Nrf2 pathway related neuroprotection against acute ischemic outcome and substantially long-term sensorimotor deficits in the context of ischemic stroke under pdMCAO, providing the new insight into the cellular mechanism of Nrf2 in endogenous neuroprotection. Nrf2 deficiency led to deleterious progression following pdMCAO-induced ischemic stroke, enriching our acknowledgment of the natural properties of Nrf2 in health and neurological diseases. This LL and SD: conceived the project and designed the experiments and prepared the manuscript with input from MKV. LL, MKV, VMF and YD: performed the experiments, data collection and analyzing. HK provided the Korean Red Ginseng. All authors commented and agreed with the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sula nebouxii (BFBO), during reproduction we evaluated whether individual differences in diet  prior to egg laying and during incubation influenced their lipid metabolic profile (measured as triglyceride levels and C:N ratio) and their reproductive performance . Females with higher δ15N values in their blood during the courtship and incubation periods had a higher lipid metabolic profile, earlier laying date, greater clutch size (2–3 eggs) and higher hatching success. Females that laid earlier and more eggs (2–3 eggs) consumed more Pacific anchoveta (Cetengraulis mysticetus) and Pacific thread herring (Opisthonema libertate) than did other females. These two prey species also had high amounts of lipids (C:N ratio) and caloric content . The quality of food consumed by females at the beginning of reproduction affected their physiological condition, as well as their short-term reproductive performance. Our work emphasizes the importance of determining the influence of food quality during reproduction to understand the reproductive decisions and consequences in long-lived animals.Understanding the role of diet in the physiological condition of adults during reproduction and hence its effect on reproductive performance is fundamental to understand reproductive strategies in long-lived animals. In birds, little is known about the influence of the quality of food consumed at the beginning of the reproductive period and its short-term effects on reproductive performance. To assess the role of diet in the physiological condition of female blue-footed booby,  Food availability and selection has a substantial effect on energetic and fitness costs and performance during reproduction and therefore influences how individuals adjust their reproductive strategies ,2,3. ThuFood availability/quality and age of the breeding birds can significantly affect female reproductive strategies ,13,14,1515N values- of feathers as indicators of dietary trophic level) with laying date and egg volume in cassin's auklet   analyzed during the sample runs were bovine liver , USGS-41 glutamic acid , Nylon 5  and glutamic acid . Hence, the precision of isotope measurements was < 0.24‰ for both nitrogen and carbon stable isotopes.Before isotope analysis, whole avian blood and fish muscle were dried in an oven at 60°C for approximately 24 h and then ground into fine powder. Dried samples were weighed and wrapped in tin capsules. Weights ranged from 700 to 1000 mg. Samples were sent to the Stable Isotope Facility (SFU) at the University of California, Davis, USA for analysis of δ Plasma levels of total triglycerides (triglyceride plus free glycerol) were assayed in a multiparameter chemistry analyzer  with commercial kits  adapted for small sample volumes . The ana15N values), and lipid metabolic profile (triglyceride levels and C:N ratio as an indicator of lipid content) of BFBO females during courtship, with triglyceride levels or C:N ratio as a response variable, and δ15N values as an independent variable. To evaluate the relationship between the trophic level, and lipid metabolic profile of females during incubation we used a general linear model (LM), with triglyceride levels or C:N ratio as a response variable, year (2011 or 2012) as a categorical variable and δ15N values as a continuous variable. To assess whether the isotope composition (δ15N) of whole blood of females during the courtship period was related to reproductive parameters, we used an analysis of simple linear regression for each variable, with laying date and clutch size as response variables and δ15N values as a continuous independent variable. To determine the relationship between the isotopic composition (δ15N) in whole blood during the incubation period and hatching success, we ran a generalized linear model (GLM) with a binomial distribution and logit link function ; year (2011 and 2012) and laying order (1–3) were considered as a categorical variables and δ15N values as a continuous variable. The Wald test was used to test the statistical significance of each coefficient in the model. Statistical analysis used Statistica 10 [We used a simple linear regression to evaluate the relationship between the trophic level  and as a function of clutch size (0–3 eggs). We used the SIAR Bayesian multi-source isotopic mixing model in the R environment [13C and δ15N), Californian anchovy (Engraulis mordax), Blue mackerel  and Mackerel scad (Decapterus macarellus) were grouped as “Group 1”. Isotopic values were pooled prior to incorporation into the SIAR mixing model.We used the δironment  to quantironment . To decr13C: 0.24 ± 0.79‰; δ15N: 2.25 ± 0.61‰), these values have already been used in other study with northern gannets Morus bassanus a closely related species with the BFBO [As there have been no controlled studies to determine boobies-specific trophic enrichment factors, we used whole-blood isotope discrimination factors derived from captive controlled studies of a piscivorous bird to correct for tissue-diet trophic isotope fractionation  and incubation .The lipid metabolic profile of females (as measured by triglyceride levels and C:N ratio) were positively related to δ15N values and laying date  and a positive relationship between δ15N values and clutch size , also there was a positive relationship between δ15N values and hatching success . There were differences in hatching success related to laying order  but not between the two breeding seasons . Hence, high δ15N values were related to early laying date, increased clutch size and better hatching success , which was collected only in 2012. For those species that were sampled during both breeding seasons, the isotopic composition did not differ between seasons (P > 0.05), and so the data were pooled and applying mixing models. C:N ratios in fish muscle were highest in Pacific anchoveta and Pacific thread herring and were lowest in Common halfbeak and anchovy (Anchoa spp)  . The enehoa spp) . Mean caBased on the isotopic mixing models, our results show that the diet composition of female birds had small variations between early and late breeders  and vari15N values in the blood of females during courtship and incubation were related to higher lipid metabolic profile (based on triglyceride levels and C:N ratio), earlier laying date, greater clutch size (2–3 eggs) and higher hatching success. Hence, the quality of the diet consumed by females of BFBO during the early stages of reproduction had short-term consequences for their physiological condition and reproductive performance.Higher δ15N values observed in blood samples could be the result of the consequent retention of the heavy isotope (15N) and excretion of light nitrogen. The lipid component of any given tissue type can be quite variable among individuals and generally is depleted in 13C compared with whole tissues [15N values are not a reflection of protein catabolism; rather, it is due to selectivity in the diet of females. The positive relationship between lipid metabolic profile (triglyceride levels and C:N ratio) and δ15N values during courtship and incubation is consistent with this explanation. Likewise, this particular result might help yield insight into the interpretation of triglyceride levels in birds as indicators of physiological state, given that food quality  was positively correlated with triglyceride levels, but should be interpreted with caution, as other potential confounding factors  were not measured. This is a new approach that can contribute toward the understanding of what plasma triglycerides measure or correlate with in seabirds and whether or not they serve as indicators of lipid metabolic profile. Another possible cause of enrichment in 15N in female blood could be seasonal variations of fish prey species, although more studies are needed to corroborate this.Females nesting early need to obtain nutrients required for egg production . Lipids  tissues . Females tissues . High tr15N values (and hence trophic levels), food quality and lipid metabolic profile (as indicated by triglyceride levels and C:N ratio) and measures of reproductive performance. Females that had a higher lipid metabolic profile and reproductive performance fed mainly on Pacific anchoveta and Pacific thread herring, both were prey species with a high content of available energy  and lipid content (as indicated by C:N ratios). Birds with a high rate of body mass gain have been shown to have high plasma triglyceride levels [15N, including research on magellanic penguin (Spheniscus magellanicus) [Uria lomvia) and black-legged kittiwake (Rissa tridactyla) [Our results show a strong link between prey δe levels . Some stlanicus) , thick-bdactyla) , whereasdactyla) , or weredactyla) . These adactyla) . Also, bdactyla) . Our resIndividual variation in laying date has been associated with body condition, age and/or reproductive experience of the females ,72. Indi15N values (reflecting the trophic level at which feeding occurred). Females that had not nested or had laid a single egg presented lower δ15N values and lipid metabolic profile and the results of the mixing model and C:N ratios indicated they had fed on lower-quality prey species. The fact that some females did not nest and/or laid a single egg, may suggest a physiological limitation that prevents egg formation, thereby restricting reproductive investment. A disproportionate number of females with smaller clutch size may be young adults that are breeding for the first time with less experience in foraging. Younger birds have been shown to have a lower body mass at the beginning of the breeding season and lower success with prey capture [Egg volume reflects the ability of females to assign nutrient reserves to egg formation  and is a capture , which cBody condition has been widely used as a predictor of reproductive success in birds and it has also been postulated that the amount of reserves at the beginning of reproduction, regulated by environmental conditions, influences the expenditure of energy on reproduction . This imhttp://www.nws.noaa.gov), so females should have experienced favorable breeding conditions due to higher prey availability [Overall, our results suggest that individual differences in prey consumption prior to breeding influenced the short-term lipid metabolic profile and reproductive performance of female BFBO. Some studies have shown that the reproductive performance can be influenced by environmental factors and individual physiology during the reproductive and non-reproductive periods ,87, but lability , which p15N, with greater available energy and lipid content) that allows them to maximize their reproductive effort. However, environmental changes that modify the regional food web trophic structure  can cause a decrease in the availability of small pelagic fish, which would mean that only females with better individual foraging skills would be able to breed. Adjustments by females in reproductive investment based on their individual body condition suggest that self-maintenance  is prioritized at the expense of reproductive performance; this is consistent with the life-history theory for long-lived organisms such as seabirds.BFBO females seem to depend on specific prey (enriched in δS1 Data(XLSX)Click here for additional data file.S2 Data(DOCX)Click here for additional data file.S1 Fig13C) and stable nitrogen (δ15N) isotope values before the chick-rearing diet  and Anchovy, Pacific anchoveta, Pacific thread herring, Common halfbeak, Longfin halfbeak, Group 1 . Diet items are from Isla El Rancho, Sinaloa, Mexico.Mean (± SD) stable carbon (δ(TIF)Click here for additional data file.


Discovery and use of a chemical tool. in silico screen coupled with chemical optimization provided Liddean, a nucleotide-mimetic which modulates reptin's oligomerization status, protein-binding activity and global conformation. Combinatorial peptide phage library screening of Liddean-bound reptin with next generation sequencing identified interaction motifs including a novel reptin docking site on the p53 tumor suppressor protein. Proximity ligation assays demonstrated that endogenous reptin forms a predominantly cytoplasmic complex with its paralog pontin in cancer cells and Liddean promotes a shift of this complex to the nucleus. An emerging view of PPIs in higher eukaryotes is that they occur through a striking diversity of linear peptide motifs. The discovery of a compound that alters reptin's protein interaction landscape potentially leads to novel avenues for therapeutic development.Developing approaches to discover protein–protein interactions (PPIs) remains a fundamental challenge. A chemical biology platform is applied here to identify novel PPIs for the AAA+ superfamily oncoprotein reptin. An  Rate-limiting protein nodes need to be identified as they can serve as a focus for novel diagnostic and/or therapeutic advances. The current view is that drugging PPIs remains an untapped landscape in the drug discovery field.Discovering protein–protein interactions (PPIs) remains a major challenge.2 and mass-spectrometry-based sequencing of multi-protein complexes using tagged-bait proteins.3 Although powerful, their limitations are that they are done outside an authentic cellular context, require artificial tagging of the bait protein and are unable to capture weak, or dynamic interactions. One advance in the study of PPIs is the idea that a large proportion of the polypeptide sequence information in higher eukaryotes is intrinsically disordered thus providing a template for “weak” regulatory, combinatorial and specific PPIs to occur in signal transduction.4 A second advance is the realization that a number of PPIs occur via a linear amino acid motif1 that provides opportunities for sequence based hotspots to be identified. Developing methods to capture such consensus linear motifs of a target protein would complement the technology currently used to discover PPIs.The main approaches used to define the PPIs of a target protein include yeast two-hybrid methods5 Members of this family exist as oligomers and form compelling targets in understanding allosteric control of protein function.6 Reptin and pontin represent two highly conserved members that are now viewed as model systems to define fundamental aspects of AAA+ superfamily function in eukaryotes.7 Reptin is an important regulator of key cellular functions through a range of PPIs.19 The different oligomeric forms of reptin and its ability to form a range of complexes with different compositions could underpin its functional diversity.22 The composition of these oligomeric complexes must be tightly regulated and this has been linked to reptin's bound ligand ATP/ADP.24 Development of a synthetic ATP/ADP mimetic to probe the intrinsic oligomerization properties of reptin and its ability to form diverse PPIs could provide insight into the regulation and function of reptin and the wider AAA+ family.The AAA+ (ATPase associated with various cellular activities) superfamily of proteins is present within all kingdoms of life.in silico screening of virtual libraries to identify novel ATP mimetics; (ii) optimization of a hit as a PPI and oligomerization modifier giving the novel chemical tool, Liddean; (iii) use of combinatorial-peptide libraries and next generation sequencing to identify novel responsive PPIs; and (iv) cell based validation of ligand-activated PPIs using proximity ligation assays. We also demonstrate that our chemical tool Liddean (an ATP-mimetic) can be used to discover and manipulate the PPIs of the AAA+ protein reptin.Here, a chemical biology platform is used to deliver a novel tool to dissect the function of reptin. This approach involves; (i) in silico screen was used to identify small molecules that might bind at the Walker A site on reptin. The rigid-body docking program LIDAEUS25 was used to dock a conformer virtual library of 4.4 million compounds. The results were ranked based on the LIDAEUS score and the top 49 971 compounds were redocked using Vina and Autodock  electrophoresis in the presence of varying amounts of SDS  HSP90 was captured on solid phase followed by the addition of either apo-, ADP- or Liddean-bound reptin. Liddean (and to a lesser extent ADP) increased the stability of the reptin–HSP90 complex . Encouramer peptide library, and peptide pools were sequenced  that is a characteristic feature of oligomeric AAA+ proteins. The identified hit (compound 1) was subjected to SAR analysis guided by an ELISA based PPI assay to generate the chemical tool, Liddean. The SAR data and results from hydrogen–deuterium (H–D) exchange experiments supported our proposal that Liddean binds in the Walker A site of reptin. In addition, Liddean stabilised higher order oligomers of reptin as evidenced by (i) the estimated mass using a denaturing gel electrophoretic screen and (ii) the use of HDX experiments that revealed suppression of deuterium incorporation at the previously reported dimer interface.The AAA+ proteins, including reptin, are known for their nucleotide binding sites, oligomeric propensity, and wide range of functions.33 More recently, based on genetic screens, reptin has been linked to the assembly of cilia structures on the plasma membrane.28 With Liddean in hand, we decided to assess whether it could be used to find and provide details about reptin PPIs. As a proof of principle we confirmed that Liddean-bound reptin modified reptin's known interaction with HSP90. Next we decided to use the stabilized, oligomeric state of Liddean-bound reptin in vitro to search for “consensus peptide motifs” using a combination of next generation deep-sequencing and a combinatorial peptide-phage library. Whilst a very information rich dataset was obtained, our focus turned to a novel reptin-interacting motif in the p53 tumor suppressor protein. Independent screens verified that indeed reptin can bind to p53-derived peptides mainly through a peptide motif that is located in the p53 tetramerization domain . When small molecules were evaluated to the indicated final concentrations , reptin was added immediately to the reaction well to allow binding competition to take place in the presence of the AGR2 (or p53) peptide. The wells were washed17 and bound reptin was detected using a reptin antibody coupled to anti-rabbit secondary antibody and chemiluminescence. Binding activity was quantified by chemiluminescence using a Fluoroskan Ascent FL Labsystems. For measuring the effects of ligands on the SDS-resistant oligomerization state of reptin using SDS gel electrophoresis, reptin protein (1 µg) was added to buffer B  with the indicated amounts of ADP or Liddean. Following incubation at room temperature for 60 minutes, samples were processed for electrophoresis as indicated in the Fig. S8The expression and purification of reptin protein was carried out as previously described–1, 0.1% formic acid in water). Peptides were trapped and desalted on-line on a peptide microtrap  for 1 minute at a flow rate 20 µl min–1. The peptides were eluted onto an analytical column  and separated by a linear gradient. The injection, switching valves, immobilized pepsin column, trap cartridge, and the analytical column was kept at 1 °C in a cool box (within the robotics system). Mass spectrometric analysis was done on an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) with ESI ionization on line connected with a robotic system based on a HTS-XT platform . The instrument was operated in a data-dependent mode for peptide mapping (HPLC-MS/MS). Each MS scan was followed by MS/MS scans of the top three most intensive ions from both CID and HCD fragmentation spectra. Tandem mass spectra were searched using SequestHT against the cRap protein database  containing sequence of reptin protein. Sequence coverage was visualized with Proteome Discoverer 1.4 software (Thermo Fisher Scientific). Analysis of deuterated samples was done in HPLC-MS mode with ion detection in the orbital ion trap and data were processed in HDX Workbench. Graphs showing deuteration kinetics were plotted by DrawHDXPlot (MSTools).Deuteration of the reptin either free or in complex with Liddean was initiated by a sequential dilution into deuterated water with 0.1% DMSO final concentration. The molar ratio between reptin and Liddean was 1 : 5 . The surface panning procedure (direct target coating) was carried out as instructed by the manufacturer's protocol with an additional protein capture step. The micro titer wells were coated as directed with a rabbit anti-reptin polyclonal antibody overnight. The wells were then washed three times with tris buffered saline with 0.1% Tween-20 (TBST). The wells were then blocked with 3% bovine serum albumin in TBS for 1 hour. Washes were once again carried out before the addition of reptin with and without ligand (either ADP or Liddean at 100 µM) and incubated for one hour at RT. Washes were then carried out 6 times with TBST, containing ligand in the ligand treated wells. The phage pool was then added, again containing ligand if required, and incubated for 1 hour. Non-binding phage was removed and the wells were washed 10 times with PBST prior to the elution of the phage as directed by the manufacturer. Amplification and titering of the phage was also carried out with each round of panning as per the manufacturer's instructions. Titering was carried out to ensure that the phage pool being panned was not greater than that of the original pool once it had been amplified and to check for white-type lytic phage contamination. Polymerase chain reaction (PCR) and deep sequencing of phage was carried out in the following stages; (i) PCR was used to amplify phage DNA from each round of screening using the primer bar codes in table code . Cells were fixed with 4% paraformaldehyde dissolved in PBS and permeabilized using 0.25% Triton X-100 in PBS. Duolink II (green) assay from Olink Bioscience was carried out following manufactures instructions. Briefly, unspecific antibody binding was blocked by the addition of 3% BSA (w/v) in PBS for 30 minutes at room temperature. Primary antibodies were diluted in 3% BSA (w/v) in PBS and added to the cells overnight at 4 °C. Negative controls include no primary or no secondary antibodies which gave rise to no foci in subsequent assay processing (data not shown). PLA probes were added to cells and incubated to 1 hour at 37 °C. Ligation mix was added for 30 minute at 37 °C followed by the polymerase amplification mix for 2 hours at 37 °C. Coverslips were mounted in S3023 Mounting medium (Dako). Results were visualized using a BX51 (Olympus) fluorescent microscope. Antibodies and PLA probes include; anti-reptin/TIP49B/RUVB2 rabbit antibody ab36569 (abcam) (1 : 250 dilution); monoclonal anti-pontin 5G3-11 (Sigma) (1 : 250 dilution); Duolink II PLA probe anti rabbit PLUS (Olink) (1 : 10 dilution); and Duolink II PLA probe anti mouse MINUS (Olink) (1 : 10 dilution).


The experimental group showed large, significant decreases in peak vertical ground reaction force in both landing tasks . Control participants did not demonstrate any significant pre-to-post-test changes in response to the 6-week study period. Results of the study affirm the hypothesis that a 6-week NMT program can enhance landing biomechanics associated with ACL injury in 11–13 year old female netball athletes.The purpose of this study was to examine the effects of a neuromuscular training (NMT) program on lower-extremity biomechanics in youth female netball athletes. The hypothesis was that significant improvements would be found in landing biomechanics of the lower-extremities, commonly associated with anterior cruciate ligament (ACL) injury, following NMT. Twenty-three athletes  completed two testing sessions separated by 7-weeks and were randomly assigned to either a experimental or control group. Thirteen athletes underwent 6-weeks of NMT, while the remaining 10 served as controls and continued their regular netball training. Three-dimensional lower-extremity kinematics and vertical ground reaction force (VGRF) were measured during two landing tasks, a drop vertical jump and a double leg broad jump with a single leg landing. The experimental group significantly increased bilateral knee marker distance during the bilateral landing task at maximum knee-flexion range of motion. Knee internal rotation angle during the unilateral landing task at maximum knee flexion-extension range of motion was significantly reduced ( Adolescent female athletes experience non-contact anterior cruciate ligament (ACL) injuries at a 4–6 times higher rate than males participating in the same sports  female athletes land with greater knee extension, and greater extension moments and powers  following the training intervention.n ≤ 12, n ≤ 14, and n ≤ 17 respectively. Participants verbally reported no previous history of lower limb injury and no previous experience partaking in NMT. All participants were instructed to maintain their normal netball training regime. Participants were recruited for participation in this randomized control trial study and divided into either an experimental  or control group  in a blinded fashion following the first battery of testing . All participants had participated in competitive netball for more than 4 years. The Human Research Ethics Committee at Edith Cowan University approved all test procedures and written informed parental consent and participant assent was obtained prior to commencement of the study in accordance with the Declaration of Helsinki. Participants completed the Physical Activity Readiness Questionnaire (PAR-Q) prior to beginning of the study and a modified Pubertal Maturation Observational Scale (PMOS)  found between the experimental (2.2 ± 0.9) and control groups (2.8 ± 0.4) indicating that both groups are primarily made up of pubertal athletes.Twenty-three youth female netball athletes  were recruited from local netball clubs to participate in the study. The sample size was chosen based upon a power analysis (β = 0.829) indicating a minimum of 16 athletes are needed for the study. Further, samples size was considered based on similar multiple training group studies involving youth participants  on landing biomechanics in youth netball athletes. All participants completed a 1-week familiarization program during 3 × 60 min training sessions comprised of back squats, push ups, static lunges, horizontal pull ups and glute bridges prior to the initiation of any testing. Baseline testing occurred following the familiarization program, 1 week before the initial NMT session. Post-testing was performed approximately 7 weeks after baseline testing on the EG and CG. The 6-week NMT intervention involved 3 × 60 min training sessions per week performed on non-consecutive days, for a total of 18 training sessions. A minimum of 48 h separated each resistance training session to allow for sufficient recovery. The CG participated in both baseline- and post-testing sessions however did not receive any NMT during the study period.A 10 camera MX-F20 Vicon-Peak Motion Analysis system  operating at 250 Hz was used to capture hip and knee kinematics using the validated Vicon Plug-In Gait model, with each participant fitted with 37 retro-reflective markers 14 mm in diameter. A static trial was collected to align the joint coordinate system to the laboratory. Vertical ground reaction force data were collected using a 600 × 900 mm triaxial force platform  recorded at 1,000 Hz with Vicon Nexus Software .Participants were instructed to perform a drop vertical jump (DVJ) task and a broad jump with a single leg landing (SL). The DVJ trials commenced with each participant standing on a box 31 cm in height with their feet positioned 35 cm apart. Each participant was instructed to step off the box onto the force platform with their dominant leg and immediately perform a maximal vertical jump  and maximum knee flexion-extension ROM were calculated from the embedded joint coordinate system. Three successful trials were collected for the DVJ task and the SL task and an average of the three trials for each landing task was used for data analysis. Positive values indicated knee flexion, knee abduction, knee external rotation, hip flexion, hip abduction and hip external rotation. Negative values represented knee adduction, knee internal rotation, hip adduction and hip internal rotation.Bi-lateral knee valgus (abduction) was calculated from the frontal plane by exporting the coordinate data as a text file in Microsoft Excel and calculating the distance (meters) between the right and left lateral knee markers at the point before initial contact (PIC) and maximum knee flexion-extension ROM as described in Ford et al. .Vertical ground reaction force (VGRF) data were derived from the embedded force platform through Vicon Nexus software. During the DVJ task, peak VGRF was recorded immediately following the box drop and immediately following the vertical jump. Peak VGRF was recorded following the jump in the SL landing task. Flight time was determined from the Kistler force plate system.p ≤ 0.05 was set for all statistical analyses. If significant F-values were found, paired comparisons were used to determine differences in conjunction with a Holm's sequential Bonferonni correction to account for Type I errors. Homogeneity of variance was tested with the use of a Kolmogorov-Smirnov test and liliefors correction. If homogeneity of variance was violated the Kruskal-Wallis H test was used to determine statistical significance. One-way ANOVAs were performed on all variables to determine raw difference scores (post-pre) between groups. Effect sizes were calculated as Hedges g and were interpreted as the following: trivial, < 0.2; small, 0.2–0.5; medium, 0.5–0.8; large, 0.8–1.3; and very large, >1.3  repeated measures ANOVA was used. A significance level of Hopkins, . Intra-cp = 0.009; g = 1.20) and knee external rotation  at intitial contact during the DVJ task. Additionally, a group × time interaction was noted for hip abduction angle at maximum knee flexion-extension ROM . Baseline and post-testing lower extremity kinematics for the DVJ task are presented in Table Significant group x time interactions were noted when examining the knee flexion-extension angle  and knee external rotation  at initial contact. Additionally, there were no significant differences noted between the groups for hip abduction  and hip flexion ROM  at baseline testing. When examining the between group differences at post-testing there was a significant difference between the EG and the CG for knee-extension flexion ROM  and knee external rotation  at initial contact. There was a significant difference noted between the EG and CG when examining the hip abduction at maximum knee flexion-extension ROM .Follow-up comparisions revealed no significant differences between groups at baseline testing for knee flexion-extension ROM  and external rotation  at initial contact were noted between the EG and CG. Additionally, there was a significant group x time interaction for knee external rotation  at maximum knee flexion-extension ROM. When examining the change score between pre and post testing the EG demonstrated a significantly greater knee flexion angle at initial contact  when compared to the CG. Additionally, the EG demonstrated a significantly greater  external rotation of the knee at initial contact. At maximum knee flexion-extension ROM the EG demonstrated a significantly greater  external rotation of the knee when compared to the control group  or external rotation  at initial contact or maximum knee flexion angles  during the SL landing task. However, at the post-testing time point the EG demonstrated significantly greater knee flexion angles at initial contact . Additionally, external rotation of the knee for the EG displayed significantly less external rotation at initial contact  and maximal knee flexion  when examining the distance between the right and left lateral knee markers before initial contact (PIC). Additionally, there was a significant group × time interaction  when examining the distance between the right and left lateral knee markers at maximum knee flexion. Follow-up tests revealed that there were no significant differences between the PIC at pre-testing at the initial contact  and max knee flexion . There was however a significant difference  between the PIC at maximum knee flexion with the experimental group displaying a significantly decreased knee abduction in comparison to CG . When examining the post-testing DVJ performance the EG demonstrated a significantly  lower peak GRF when compared to the CG. Similarly the EG demonstrated a significantly  GRF during the landing phase following the vertical jump. Furthermore, the EG also showed very large statistically significant decreases in peak GRF during the SL landing task  mean ± SD and effect sizes for the experimental and control conditions for both the DVJ and SL jump tasks during the pre- and post-testing sessions are presented in Table p > 0.05). When raw-difference scores were examined the EG displayed an increase in their flight time (+0.03 ± 0.05 s) whilst the CG displayed a decrease in their flight time (−0.03 ± 0.07 s) however although these differences were not statistically significant (p > 0.05) they did display a large effect size (g = 0.98).Flight time during the DVJ revealed no significant group × time interaction (The objective of the current study was to determine if a comprehensive NMT program inclusive of plyometric and strength training could improve lower extremity biomechanics in 11–13 year old netball athletes. Results of the current study indicated EG demonstrated significant improvements in knee- and hip-joint biomechanics and decreases in peak GRF in both a DVJ and SL landing tasks in comparison to the CG.The results of the current study support the work of Myer et al.  who usedFollowing maturation it has been reported that post-pubertal female athletes land with greater knee extension than pre-pubertal female athletes (Yu et al., Steele and Milburn  reportedKnee internal rotation appears to be present in ACL injury situations and usually occurs in combination with valgus knee motion (Koga et al., The effect of the training program on frontal plane knee abduction motion during landing in the DVJ task demonstrated a large and significant increase in the distance between the two lateral knee markers in the trained participants indicating decreased frontal plane knee abduction motion following the 6-week study period. In contrast, the CG showed a decrease in the distance between the two lateral knee markers during landing. The observed increase in the distance between the right and left lateral knee markers designates that the trained participants were able to decrease their knee abduction motion in the frontal plane after completing the 6 week NMT intervention. This may be due to improved landing mechanics as a result of the NMT intervention. It is widely agreed in the literature that female athletes land with increased knee abduction motion in comparison to males (Ford et al., Implementation of NMT to reduce ACL injury incidence has been shown to be more effective for athletes under the age of 18 (Yoo et al., It should be noted that there are potential limitations with the current study. The inability to control the volume and frequency of netball training between each group may have impacted the results of the study. One of the main goals of the present study was to add resistance training to the netball practice of youth athletes in accordance with best practice recommendations for youth resistance training (Faigenbaum et al., Netball has a reputation for a high incidence of knee injuries, with most injuries occurring during abrupt landings from a jump combined with incorrect landing technique. Therefore, it appears important that implementation of NMT programs that teach youth netball athletes proper landing technique and to safely absorb landing forces are important in the prevention of ACL injuries. The results of this study provide evidence that NMT programs encompassing progressive resistance and plyometric training decrease associated risk factors for non-contact ACL injuryincluding poor knee-joint biomechanics and increased landing forces in youth female netball athletes. Much of the benefit of the program appeared to be through improved knee flexion-extension ROM, increased knee external rotation angle, decreased frontal plane knee abduction motion, and decreased landing forces. It is important to note that the current study used athletes who had no history of strength training prior to participating in the present study. As such for ongoing strength and performance adaptations to occur this program should be contextualized as part of a long-term athlete development program which when appropriately applied results in long term physical adaptations that are above and beyond that of growth and maturation.AH: worked on study design, data collection, data analysis, and manuscript preparation. EH: assisted with training intervention design, delivery of training, data collection, and manuscript preparation. CJ: assisted with study design, worked on all biomechanical analyses, and assisted with manuscript preparation. RL: assisted with study design, data analysis and manuscript preparation. GH: assisted with study design, data collection, data analysis, and manuscript preparation.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.


Five older participants with major depression and 13 controls underwent PET and multimodal 3T magnetic resonance imaging (MRI), with blood taken to measure C-reactive protein (CRP). We found significantly higher CRP levels in those with late-life depression and raised [11C]PK11195 binding compared with controls in brain regions associated with depression, including subgenual anterior cingulate cortex, and significant hippocampal subfield atrophy in cornu ammonis 1 and subiculum. Our findings suggest neuroinflammation requires further investigation in late-life depression, both as a possible aetiological factor and a potential therapeutic target.We studied neuroinflammation in individuals with late-life depression, as a risk factor for dementia, using [ We have previously shown an increase in inflammatory cytokines in the blood in older individuals with depression.2 In the current study we aimed to show whether we could demonstrate an increase in central (brain) inflammation in vivo using [11C]PK11195 positron emission tomography (PET) imaging. [11C]PK11195 is a radioligand that selectively binds to the translocator protein (TSPO), a receptor expressed on activated microglia. Increased binding has been found in stroke, traumatic brain injury and some neurodegenerative diseases, such as Alzheimer's disease.3 We also investigated vascular and structural changes in late-life depression using multimodal magnetic resonance imaging (MRI).Late-life depression is known to be associated with specific clinical features, such as cognitive impairments, it typically has a poor outcome and is a risk factor for dementia. Vascular factors have been implicated in its aetiology,4 and 13 controls (59–81 years) from the NIHR Clinical Research Network. Participants had full clinical and cognitive assessment and venepuncture for C-reactive protein (CRP) measurement. All participants provided written informed consent. Ethics approval for the study protocol was obtained from the National Research Ethics Service – East of England Committee.Within the Neuroimaging of Inflammation in Memory and Other Disorders (NIMROD) study, we recruited five participants with depression aged 65–78 years (depression group) from secondary care National Health Service (NHS) psychiatry services, who had met DSM-IV criteria for major depression T1 weighted structural  = 2300 ms, echo time (TE) = 2.98 ms, flip angle 9), T2 FLAIR  and high-resolution hippocampal T2 coronal . Within 4 months of MRI scans, PET imaging was performed on a GE Advance scanner  for 75 min following bolus intravenous injection of [11C]PK11195 (500 MBq), with a pre-injection 15 min 68Ge/68Ga transmission scan used for attenuation correction.Participants underwent multimodal MRI on a 3T Siemens Verio scanner including ND) was determined from [11C]PK11195 PET data with the guidance of T1 weighted MRI.5 Regional white matter hyperintensity (WMH) volumes were segmented and quantified using T2 FLAIR images. T2 hippocampal coronal scans were manually segmented for cornu ammonis 1 (CA1), CA2, CA3/dentate gyrus, and measured for subiculum and entorhinal thickness. To estimate TSPO binding site density, non-displaceable binding potential  Results are reported without correction for multiple comparisons, noting that the primary outcome measure related to our principal hypothesis was [11C]PK11195 BPND in limbic cortical regions associated with depression and dementia.As a result of the relatively small sample size in the depression group, we did not assume Gaussian data distributions. Group-level statistical comparisons of regional PK11195 BPND in the aforementioned brain regions, confirming the group-level statistical inference.Although largely recovered from their depression at time of imaging, at the group level, participants with depression had significantly higher PK11195 BPND showed the greatest effect size compared with other modalities, with a 300% increase from controls , suggesting a strong biomarker potential for late-life depression.We found evidence of both central and peripheral inflammation in older individuals with depression, including changes in the anterior cingulate and medial temporal lobe, which play a key role in the regulation of mood and cognitive functioning.11 In addition, the hippocampus is a key component in the hypothalamic–pituitary–adrenal (HPA) axis. Increases in cytokine levels can lead to increases in oxidative stress and glucocorticoid as well as decreases in serotonin and other neurotransmitters in HPA resulting in impaired mood and cognition.13There was no major cognitive impairment in our cohort, although the depression group showed significant atrophy in the hippocampus and subiculum, which have been shown to correlate with greater risk of cognitive impairment and Alzheimer's disease.11C]PK11195 was in keeping with our principal hypothesis and was supported by both a primary group-level test and secondary individual statistical tests. Cross-sectional studies provide limited information about whether neuroinflammation was the cause or consequence of neuronal damage in affected brain areas, so future longitudinal studies are needed. In conclusion, we suggest that neuroinflammation may be an important mechanism in late-life depression and merits further investigation as a potential target for novel therapeutics in a condition that responds poorly to conventional antidepressant therapy.Our results were not corrected for multiple comparisons, and further replication is required in a larger cohort. However, the large effect size of [


Allelic variation at a microsatellite locus in the 5′ regulatory region of these genes is associated with fitness in the bank vole Myodes glareolus. Given the low frequency of long and short alleles at these microsatellite loci in wild bank voles, we used breeding trials to determine whether selection acts against long and short alleles. Female bank voles with intermediate length avpr1a alleles had the highest probability of breeding, while male voles whose avpr1a alleles were very different in length had reduced probability of breeding. Moreover, there was a significant interaction between male and female oxtr genotypes, where potential breeding pairs with dissimilar length alleles had reduced probability of breeding. These data show how genetic variation at microsatellite loci associated with avpr1a and oxtr is associated with fitness, and highlight complex patterns of selection at these loci. More widely, these data show how stabilizing selection might act on allele length frequency distributions at gene-associated microsatellite loci.The loci arginine vasopressin receptor 1a ( Behav density ,5,6 stimeference –9. Assocd humans –15.avpr1a enjoy greater reproductive success in field experiments [avpr1a microsatellite alleles produce more offspring [M. glareolus, allele length is associated with fitness at the microsatellite locus located in the 5′ regulatory region of avpr1a; males with longer avpr1a alleles sired more offspring than did males with shorter alleles, but shorter alleles were associated with increased reproductive success in female bank voles [avpr1a, there is no macroevolutionary conservation of a microsatellite locus in the 5′ regulatory region of oxytocin receptor (oxtr). Nonetheless, allelic variation at a microsatellite locus located upstream of the oxtr transcription start site in the bank vole is associated with fitness, for example, with shorter oxtr alleles increasing male reproductive success [Evidence for selection on microsatellite allele length is derived from studies of microtine voles. Prairie vole males with short or long microsatellite alleles at eriments  and in weriments , respectffspring . In banknk voles . By cont success . One cor−2 and 10−6 mutations per locus per generation) [avpr1a and oxtr  ,21, withavpr1a) and oxytocin receptor (oxtr) loci. We concentrate on these loci as they (i) show clear fitness effects in microtine voles and (ii) have evolutionarily conserved roles in vertebrate social and sexual behaviour [To test the hypothesis that selection acts against long and short alleles, we quantified breeding success in bank voles with different microsatellite genotypes at arginine vasopressin receptor 1a (ehaviour . We find2.(a)avpr1a of the bank vole consists of (CA) and (GA) motifs, and is located approximately 920 bp upstream of the avpr1a exon 1 [avpr1a exon 1 in the prairie vole , mouse , Norway rat  and in eight species of deer mice , respectively. The microsatellite located in the 5′ regulatory region of oxtr in the bank vole genome is predominantly (CT) and (GA) motifs located upstream of the putative transcription start site [M. musculus, a microsatellite is located immediately (approx. 10 bp) upstream of oxtr transcript variant X1 (XM_006505723) or 1448 bp upstream of the oxtr transcription start site (NM_001081147).The microsatellite within the 5′ regulatory region of a exon 1 ; this miart site . In M. m(b)M. glareolus is a small rodent that inhabits forests and fields in the Palearctic, extending its distribution from Europe into western Siberia. Female bank voles defend breeding territories and care for their young, while males do not provision their young; both sexes mate multiply [The bank vole multiply .avpr1a and oxtr microsatellite loci, and (ii) examine the influence of microsatellite allele length on breeding success, we caught 325 wild bank voles from central Finland . Animals were caught at 20 trapping locations distributed over approximately 100 km2, with any two trapping locations up to 4.3 km apart. Each location contained four Ugglan Special multiple-capture live traps  located at the corners of a 15 × 15 m2 (see [To (i) quantify natural levels of polymorphisms in the  m2  with sawdust and hay for bedding, with food  and water ad libitum, at 22°C and on a 16 : 8 h L/D photoperiod (dark period is between 22.00 and 06.00). All animals were implanted with electronic identification microchips (Trovan Unique).avpr1a and oxtr genotypes (see Genotyping) in the same cage for two weeks, after which time the animals were separated and the female was monitored daily for birth. Some of the offspring of successful matings were used in the breeding trials. We kept a pedigree to ensure that potential breeding pairs were not close relatives .Fitness associated with microsatellite allele length is quantified as ‘breeding success’ (production of some or no offspring) using breeding trials: essentially, failure to produce any offspring after an opportunity to mate is zero fitness. To quantify how microsatellite allele length affects breeding success, we paired a male and a female bank vole with known (c)avpr1a and oxtr microsatellite loci are provided in [eneMapper v. 3.7 (Applied Biosystems).A small sample of ear tissue (field-caught adults) or the tip of tail (laboratory-reared young) was taken from as a source of DNA. DNA was extracted using the Qiagen DNeasy Tissue kit and a Kingfisher magnetic particle processor (Thermo Fisher Scientific). Primer sequences and thermal cycling conditions to amplify vided in . PCR pro(d)avpr1a and oxtr, as microsatellite allele length had little effect on the litter size of the breeding voles . Models (described below) were run separately for avpr1a and oxtr as they are independent loci [Our aim was to determine whether the microsatellite allele length of an individual affected its breeding success. We examined the probability that animals produced offspring  after an opportunity to mate in relation to microsatellite allele length at ent loci .n breeding trials per individual  using generalized linear modelling (GLM) with binomial error distribution and logit link function. As many individuals were paired with several partners, the partner's genotype was not considered in this first analysis and the sexes were examined separately. Potential explanatory variables included in the full global model were: (i) MAL—mean (over the diploid genotype) allele length of an individual  and (ii) its polynomial term , and (iii) DAL—the difference in allele length between the two alleles within an individual, and (iv) the origin of the individual (i.e. wild-caught or laboratory-born). In addition, (v) all two-way interactions were included in the full model. The full model showed some overdispersion (dispersion parameter 1.1–1.7) that was taken into account using quasi-binomial models [Our first analysis considered whether an individual's allele length affected breeding success. We estimated the breeding probability as the proportion of successful matings  out of l models . Model sl models . The mosl models  and usedl models  for R v.l models .Our second analysis quantified whether breeding success was associated with the interaction between the genotypes of pairs: whether the combination of microsatellite alleles affected reproduction. For these analyses, we considered only data for the pairs of individuals that had successfully bred a least once in the laboratory. We estimated the breeding probability (binary outcome) using the generalized linear mixed modelling (GLMM) approach implemented by the glmer function in lme4 , with a 3.(a)avpr1a, the long (greater than 512 bp) and short (less than 482 bp) alleles occur at low (less than 0.02) frequencies while the intermediate (496–502 bp) length alleles occur at an allele frequency of approximately 0.10 or higher (a); similarly, at oxtr the long (greater than 306 bp) and short (less than 274 bp) alleles occur at lower (approx. 0.02 or less) frequencies than the intermediate length (286–290 bp) alleles  (b).Allele frequency-length distributions at both microsatellite loci show higher frequencies of the intermediate length alleles and lower frequencies of long and short alleles in wild-caught bank voles . For avpr higher a; similaequency) b.(b)(i)Data on individual breeding probability represent breeding trials for 952 bank voles , with 1–13 and 1–15 observations per female and male, respectively: in total, the analyses are based on 1310 breeding trials, of which 410 were successful.avpr1a microsatellite allele genotype is associated with its probability of breeding, with an apparent adverse effect of long and short alleles. Significant effects on female breeding probability were derived from (i) the origin of the individuals  . In males, the best model identified that (i) the individual's origin (p < 0.001) and (ii) the interaction between individual's origin and the difference in the length of avpr1a alleles in a genotype (p = 0.034) had significant associations with breeding probability (b).An individual's = 0.004) . A reduc alleles a. In malbability ; wild-cabability b.Figureoxtr microsatellite length and breeding probability were identified when considering a female's genotype in isolation . In male bank voles, the best model identified significant effects of (i) individual origin (p = 0.001) and (ii) the interaction between the difference between oxtr allele lengths within an individual and the mean allele length oxtr of the individual (p = 0.007) , while in more heterogeneous  males the probability of breeding increased with increasing mean allele length (c).No significant associations between = 0.007) ; in effee length c.(ii)In total, there were 546 breeding trials  between individuals that had ever reproduced in the laboratory; these pairings involved 220 females and 256 males .avpr1a, the probability of breeding by bank voles in relation to their microsatellite length was significantly associated with (i) the origin of the animals , (ii) nonlinear mean allele length of the female's genotype   . Thus, ooxtr, a negative effect of divergent alleles was apparent, and the probability of breeding was associated with (i) the origin of the females (p = 0.003), (ii) the level of dissimilarity in male and female genotypes  (p < 0.001) and (iii) the level of dissimilarity in the length of a female's own alleles (p = 0.011)  associated with lower breeding probability (At = 0.011) . Hence, bability  (see ele4.avpr1a) and oxytocin receptor (oxtr) plays important roles in vertebrate social and sexual behaviour [avpr1a and oxtr, we provide evidence for stabilizing selection against long and short allele lengths. These data show how microsatellite genotypes can impact fitness and allele length distributions at avpr1a and oxtr microsatellite loci.Expression of arginine vasopressin receptor 1a (ehaviour . By exam(a)per se  or (ii) selection favouring genotypes that comprise similar length alleles . How might variation in microsatellite allele length exert tangible fitness consequences? Microsatellite loci located in 5′ regulatory regions can regulate transcription, for example by modifying chromatin structure, overlapping with protein binding sites and/or affecting the spacing of promoter elements [avpr1a microsatellite allele length and gene expression (in specific regions of the brain) has been documented in bank voles [avpr1a in prairie vole brains [avpr1a and oxtr indicates a more general role for microsatellite allele length.Offspring production can be an inherent property of an individual's microsatellite genotype via (i) selection for intermediate length alleles elements –40. Indenk voles , prairienk voles  and in hnk voles ,13. While brains ,41, seleavpr1a and oxtr  caused by a major expansion of the repeat array ,42. Moreand oxtr  necessite genes’ . A negat(b)As outlined previously, hypotheses about the mechanisms that determine microsatellite allele length distributions emphasize mutational processes. One argument against a general effect of selection on microsatellite allele length distributions is a lack of evidence about how subtle differences in length at non-genic microsatellite loci might impact fitness . Here, tAnother feature of our data is the negative association between the difference in length of the alleles within an individual and breeding success. Microsatellite alleles paired with dissimilar length homologues are more likely to mutate, which in turn could drive increased heterozygosity ,47. Sele(c)avpr1a and oxtr. For example, the action of directional selection is consistent with studies of prairie voles where either longer [avpr1a alleles were associated with greater reproductive success. The contrast between the results of Solomon et al. [et al. [avpr1a allele length can differ in male prairie voles. Indeed, in the bank vole, the optimum allele length at the avpr1a and oxtr microsatellite loci depends on an individual's sex and the population density, indicating that balancing selection operates [avpr1a and oxtr microsatellite loci. Several reasons may explain why studies can identify a different mode of selection on avpr1a and oxtr microsatellite loci. First, some analyses of prairie vole reproductive behaviour partitioned avpr1a alleles into short and long categories [avpr1a and oxtr alleles in nature  or w animals . Lonn et [et al.  did not election . These c