Dengue (DEN) is a mosquito-borne viral disease and represents a serious public health threat and an economical burden throughout the tropics. Dengue clinical manifestations range from mild acute febrile illness to severe DEN hemorrhagic fever/DEN shock syndrome (DHF/DSS). Currently, resuscitation with large volumes of isotonic fluid remains the gold standard of care for DEN patients who develop vascular leakage and shock. Here, we investigated the ability of small volume of hypertonic saline (HTS) suspensions to control vascular permeability in a mouse model of severe DEN associated with vascular leakage. Several HTS treatment regimens were considered and our results indicated that a single bolus of 7.5% NaCl at 4 mL per kg of body weight administered at the onset of detectable vascular leakage rapidly and significantly reduced vascular leak for several days after injection. This transient reduction of vascular leakage correlated with reduced intestine and liver damage with restoration of the hepatic functions, and resulted in delayed death of the infected animals. Mechanistically, we showed that HTS did not directly impact on the viral titers but resulted in lower immune cells counts and decreased systemic levels of soluble mediators involved in vascular permeability. In addition, we demonstrated that neutrophils do not play a critical role in DEN-associated vascular leakage and that the therapeutic effect of HTS is not mediated by its impact on the neutrophil counts. Together our data indicate that HTS treatment can transiently but rapidly reduce dengue-associated vascular leakage, and support the findings of a recent clinical trial which evaluated the efficacy of a hypertonic suspension to impact on vascular permeability in DSS children. Dengue (DEN) is an arthropod-borne viral disease that is endemic in subtropical and tropical countries. With approximately half the world’s population residing in DEN endemic regions and more than 50 million new infections projected to occur annually, DEN certainly poses as a global health threat and economical burden DENV infection in human can be asymptomatic or ranges from mild acute febrile illness associated with the classical DEN fever (DF) to severe DEN hemorrhagic fever/DEN shock syndrome (DHF/DSS). In DHF/DSS patients, capillary leakage develops rapidly over a period of hours at the time of defervescence Currently, resuscitation with isotonic fluid such as Ringer’s lactate (RL) and normal saline (0.9% NaCl) remains the gold standard of care for DEN shock and involves the administration of large volumes of fluid at various rate and over several hours period to restore microvascular perfusion As an alternative strategy to the current fluid resuscitation approach, we investigated whether treatment with a small volume of hypertonic saline (HTS) solution may be beneficial to dengue patients that experience shock. The clinical application of HTS has been described for severe hypovolemia and shock almost two decades ago In this work, we explored the ability of HTS to reduce vascular permeability in a mouse model of severe DEN associated with vascular leakage. Our study demonstrates that a single bolus of HTS rapidly and significantly reduces vascular leakage for several days, and supports the conclusions of a recent clinical trial which evaluated the efficacy of a hypertonic suspension to impact on vascular permeability in DSS children http://nus.edu.sg/iacuc/). NUS has obtained a license (#VR008) from the governing body Agri-Food & Veterinary Authority of Singapore (AVA) to operate an Animal Research Facility. The animal experiments described in this work were approved by the IACUC from National University of Singapore under protocol number 009/09. Non-terminal procedures were performed under anesthesia, and all efforts were made to minimize suffering.All the animal experiments were carried out under the guidelines of the National Advisory Committee for Laboratory Animal Research (NACLAR) in the AAALAC-accredited NUS animal facilities . BHK-21 monolayers were infected with 100 µl of ten-fold serial dilutions of virus suspension [10−1 to 10−8 in RPMI 1640 (GIBCO)]. After 1 hr incubation at 37°C and 5% CO2, 1 ml of 1% (w/v) carboxymethyl cellulose in RPMI supplemented with 2% FCS was added. After 4 days incubation, the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet dissolved in 37% formaldehyde. After thorough rinsing with water, the plates were dried and the plaques were scored visually and expressed as the number of plaque forming units (pfu). Triplicates were run for each dilution sample. The limit of detection was set at 10 pfu per ml or per g of tissue.Plaque assay was carried out in BHK-21 cells as described previously 4 pfu of D2Y98P-PP1 via the subcutaneous (sc) route (0.1 ml in sterile PBS) as described previously AG129 breeders [129/Sv mice deficient in both alpha/beta (IFN-α/β) and gamma (IFN-γ) interferon receptors] were obtained from B&K Universal (UK). They were housed under specific pathogen-free conditions in individual ventilated cages. Eight to nine week-old mice were administered with 10Mice were intravenously administered at a dose of 4 ml/kg of body weight with either one or three consecutive doses of sterile 5% or 7.5% NaCl suspensions at the indicated time points post-infection.4) were assayed by plaque assay as described above. Triplicates were run for each dilution sample. Data are expressed as log10 [mean ± SD] in pfu per gram of wet tissue with a limit of sensitivity set at 1.0 log10 pfu/g of tissue. Five mice per time point per group were assessed. Results are representative of at least two independent experiments.The levels of infectious virus in the plasma and tissues from infected mice were assessed as described previously 620nm per g of tissue wet weight compared to the uninfected control.Vascular leakage was determined using the Evans Blue extravasation assay as described previously Mice were euthanized, and tissues were harvested and immediately fixed in 10% formalin in PBS. Fixed tissues were paraffin embedded, sectioned and stained with Hematoxylin and Eosin (H&E).analyzer Cell Dyn –3700 (Abbott). Serum alanine (ALT) and aspartate (AST) aminotransferases, albumin total protein, sodium, chloride and creatinine levels were quantified using chemistry analyzer COBAS C111 (ROCHE).Mouse blood samples were collected in K2EDTA and serum tubes (Biomed Diagnostics). Whole blood was immediately analysed for cell counts using automated hematology Serum levels of various cytokines and other soluble factors were measured using individual detection kits (R&D), according to the manufacturer’s instructions.Mouse blood was subjected to red blood cells lysis and resuspended in MACS buffer for antibodies staining. Anti-Ly6G (BD Biosciences) antibody was used to identify the neutrophil population. Neutrophil activation was assessed using rat anti-mouse CD18 detected with anti-rat-APC and PerCPCy5.5-conjugated anti-CD62L (BD Biosciences). For detection of VEGF-A and TNF-α production, surface antigen staining was performed prior to intracellular staining for VEGF-A or TNF-α using the BD Fixed/Permeabilization Kit®, and rabbit purified anti-VEGF-A (Santa Cruz) antibody revealed with APC-Cy7-conjugated anti-rabbit IgG (Santa Cruz) or FITC-conjugated TNF-α antibody (Biolegend). FACS analysis was performed using a CyAn ADP Analyzer (Beckman Coulter) and data were analyzed with Flowjo software (Treestar).Groups (n = 5) of DENV-infected AG129 mice were injected twice intraperitoneally at day 5 and 6 p.i., with anti-neutrophil antibody NIMP-R14 at 250 µg per mouse t test and p values <0.05 were considered significant.All statistical analysis was done with GraphPad Prism Version 5.0 . Survival rates were plotted and analyzed according to the Kaplan Meier’s method. Other data were analyzed using two-tailed Student’s Adult AG129 mice were sc. infected with the D2Y98P-PP1 strain as described previously Systemic vascular leakage was assessed 3 hours post-HTS treatment by Evan’s blue extravasation as described previously Altogether, the data indicated that a single-dose iv. administration of 7.5% NaCl at day 6 p.i. is able to reduce DEN-associated vascular leakage without organ (kidney and spleen in particular) damage or dysfunction.To investigate whether a lower salt concentration would result in similar effects, DENV-infected mice were administered iv. with a single bolus of 5% NaCl at day 6 p.i. and the survival and clinical score were determined and compared to those observed with DENV-infected mice treated with 7.5% NaCl. The results indicated that 5% NaCl treatment significantly delayed the death of the animals but not as effectively as 7.5% NaCl treatment . ConsistDay 6 p.i. corresponds to the onset of detectable vascular leakage in the DENV-infected mice To further characterize the effects of a single bolus of HTS given at day 6 p.i., the viral titers were determined in HTS-treated mice and compared to PBS-treated or untreated mice. The data indicated that HTS treatment generally did not affect the kinetic of the virus dissemination and replication . NoticeaHistology analysis of key organs revealed that hepatic necrosis characterized by pyknotic nuclei and cytoplasmic vacuolation of hepatocytes was much less pronounced in the HTS-treated animals compared to PBS-treated mice . LikewisAnalysis of the blood parameters revealed that the white blood cells (WBC), neutrophils (NEU) and lymphocytes (LYM) counts were generally lower in the HTS-treated infected mice than those measured in the PBS-treated infected animals, although the counts remained higher than those measured in the uninfected controls . PlateleThe serum levels of IL-6, TNF-α, metalloprotease 9 (MMP9), C5a complement protein, vascular endothelial growth factor A (VEGFA) and its receptor VEGFR2 were measured in the PBS- or HTS-treated DENV infected mice. The results showed that the systemic concentration of IL-6 and TNF-α was significantly lower in the HTS-treated group at day 7 p.i., i.e one day post-treatment compared to those that received PBS . Serum lPrevious literature reported that HTS reduces the expression of neutrophil adhesion molecules CD11b and CD62L, thereby impairing their activation status which may reduce inflammation and organ damage To further investigate the role of neutrophils during DENV infection, DENV-infected animals were treated with NIMP-R14 antibody that selectively depletes the neutrophil population Together, these observations thus indicate that neutrophils contribute to the serum levels of IL-6, TNF-α and VEGFA but are not directly responsible for DEN-associated vascular leakage. The data also suggest that the lower neutrophil counts measured in HTS-treated animals do not account for the reduction of vascular leakage.A number of hypertonic resuscitation studies have been reported and together with animal studies have demonstrated that HTS infusion in small volumes (3 to 6 mL/kg) rapidly improves cardiovascular and metabolic function through a combination of plasma volume expansion, systemic vasodilation, and improved myocardial performance Some safety concerns have also been raised over electrolyte imbalance upon HTS administration. Hypernatremia, hyperosmolality, hypokalemia, and hyperchloremia may indeed result in disorientation, confusion and seizure, and can increase the risk of renal failure, pulmonary edema and congestive heart failure A single bolus of 7.5% NaCl administered at day 6 post-infection however reduced vascular leakage only transiently (up to 3 days post-administration). In an attempt to sustain the vascular leak reduction over a longer period of time, 3 consecutive daily administrations were given (day 6–8). This treatment was found more effective than a single bolus at reducing vascular leakage and at delaying the animals’ death. However, signs of kidney impairment indicated that such regimen is not advisable. We have also explored the injection of a second bolus of HTS 6 days after the first bolus (day 12 pi.) when vascular leakage is back to levels comparable to the untreated infected animals. This second bolus allowed reducing vascular leakage and delayed further the animals’ death compared to the animal group treated only once at day 6 pi.(data not shown). However, the safety aspect of this 6-days apart double dose regimen remains to be investigated.Fluid management of dengue patients consists of the intravenous administration of isotonic fluids (RL and others) at various rates and over a several hours period. Such intervention procedure is not possible to perform in a mouse model. We have thus not been able to compare in our experimental set up HTS treatment with the standard care approach. In our study, the PBS control allows demonstrate that the effects observed are due to the hypertonic nature of the HTS suspension rather than the volume of liquid injected. In addition to a rapid correction of fluid loss, the advantage of HTS versus isotonic fluid is the administration of a much smaller volume which avoids the risk of excessive fluid accumulation in tissues. Therefore, although transient in our mouse model, HTS treatment may represent a faster therapeutic intervention compared to isotonic fluid for a rapid correction of fluid loss, in particular in severe shock cases. Combining a single bolus  of HTS for a rapid correction of fluid loss followed by isotonic fluid administration for maintenance may be worth considering further.The actual cause of death in the DENV-infected AG129 mice (HTS treated or untreated) has remained unclear. Our study showed that a single bolus of HTS delayed the animals’ death but led to increased viral titers in the CNS. It may thus be possible that mice die as a result of CNS damage. However, as mentioned above, we observed that a second bolus of HTS given at day 12 post-infection reduced vascular leakage and further delayed the animals’ death with a median of 6 days difference between mice treated once (day 6 pi) and mice treated twice (day 6 and 12 pi.) (data not shown). Furthermore, in this two doses-treated group, we found a clear correlation between moribund state and increased vascular leakage whereby moribund animals presented higher vascular leak compared to non-moribund animals. These observations thus support that instead of CNS involvement due to increased viral loads, vascular leakage is the primary cause of death in the infected animals. Meningitis and CNS involvement have rarely been reported in DEN patients who are mainly immunocompetent, and this feature is one of the weaknesses of the AG129 mouse model. A recent study has indeed shown that absence of the type I interferon pathway in these mice is responsible for the viral spread to the CNS The current paradigm of the mechanisms responsible for DEN associated vascular leakage involves a variety of soluble mediators known to affect vascular permeability, including pro-inflammatory cytokines and chemokines as well as non-inflammatory mediators In conclusion, our study demonstrates that administration of a hypertonic solution effectively but transiently reduces vascular leakage in a mouse model of severe dengue infection. Our findings are consistent with the conclusions of a recent clinical trial conducted in Indonesia in DSS children


The motor cortex represents muscle and joint control and projects to spinal cord interneurons and–in many primates, including humans–motoneurons, via the corticospinal tract (CST). To examine these spinal CST anatomical mechanisms, we determined if motor cortex sites controlling individual forelimb joints project differentially to distinct cervical spinal cord territories, defined regionally and by the locations of putative last-order interneurons that were transneuronally labeled by intramuscular injection of pseudorabies virus. Motor cortex joint-specific sites were identified using intracortical-microstimulation. CST segmental termination fields from joint-specific sites, determined using anterograde tracers, comprised a high density core of terminations that was consistent between animals and a surrounding lower density projection that was more variable. Core terminations from shoulder, elbow, and wrist control sites overlapped in the medial dorsal horn and intermediate zone at C5/C6 but were separated at C7/C8. Shoulder sites preferentially terminated dorsally, in the dorsal horn; wrist/digit sites, more ventrally in the intermediate zone; and elbow sites, medially in the dorsal horn and intermediate zone. Pseudorabies virus injected in shoulder, elbow, or wrist muscles labeled overlapping populations of predominantly muscle-specific putative premotor interneurons, at a survival time for disynaptic transfer from muscle. At C5/C6, CST core projections from all joint zones were located medial to regions of densely labeled last-order interneurons, irrespective of injected muscle. At C7/C8 wrist CST core projections overlapped the densest interneuron territory, which was located in the lateral intermediate zone. In contrast, elbow CST core projections were located medial to the densest interneuron territories, and shoulder CST core projections were located dorsally and only partially overlapped the densest interneuron territory. Our findings show a surprising fractionation of CST terminations in the caudal cervical enlargement that may be organized to engage different spinal premotor circuits for distal and proximal joint control. The corticospinal tract (CST) provides a direct path for the motor cortex to spinal motor circuits. From the earliest studies, we learned of the predominance of the motor cortex muscle and joint representation; the familiar homunculus in humans The CST projects to the spinal cord to contact motoneurons in select species and interneurons, in all species In this study, we focused on the anatomical organization of motor cortex joint control at the spinal level. As sites in motor cortex preferentially represent different forelimb joints, it follows that during tasks that recruit the CST, the cortical representations of different forelimb joints We focused on the forelimb motor cortex representation and the cervical enlargement segments that are the substrates for forelimb control. We studied this in the mouse because we could identify putative spinal premotor interneurons using retrograde transneuronal transport of pseudorabies virus (PRV) in mature mice and because a functional neuroanatomical study in that species could inform molecular genetic studies of cortical projection and spinal interneurons Experiments were conducted on adult male and female C57/BL6 mice. All procedures were approved by the Institutional Animal Care and Use Committees of City College of the City University of New York, New York State Psychiatric Institute, and Columbia University.For ICMS motor mapping, anesthesia was induced with a ketamine/xylazine mixture  and maintained using IP ketamine injections to render the animal unresponsive to paw pinch while maintaining muscle tone. Animals were placed in a stereotaxic frame. Body temperature was maintained at 39°C by a heating pad. A craniotomy was made over the forelimb area of M1. We used tungsten microelectrodes . Electrode penetrations were made perpendicular to the pial surface and approximately 0.3 mm apart. In all animals, the region sampled was the same, from 1 to 1.9 mm lateral to bregma and up to 2.1 mm rostral to bregma. Motor effects produced by microstimulation occurred at the lowest stimulus current when the electrode was at the depth that we recorded multiunit activity with the largest amplitude spikes ; this was presumably laver V.Stimuli  were delivered using an isolated pulse stimulator in constant current stimulation mode (A–M Systems). The threshold was defined as the lowest current that consistently produced a motor effect on >50% of trials. For a given site, we started at a low current and first determined the threshold for evoking a contralateral response. The thresholds were then examined in reverse through the loss of the responses with decreasing currents. We randomized placement of the electrode to prevent biasing our results by anesthesia level or other state-dependent changes. Stimulation currents were up to 100 µA. For each penetration, the type of motor effect produced by a threshold stimulus was determined on the basis of the evoked phasic kinematic change; adjacent joints were stabilized. Limb posture was the same for all experiments; with the shoulder and elbow extended, and the wrist plantar flexed.We recorded electromyographic (EMG) responses from forelimb muscles using percutaneous Ni-chrome wire electrodes and a differential AC amplifier with low and high pass filtration (A–M Systems). EMG recording wires were deinsulated at the tip (1 mm), a small hook was formed by bending the wire over the needle tip, and inserted into muscle using a 26-gauge hypodermic needle. Because of the hook at the end of the EMG lead, when the needle was withdrawn the wire remained securely embedded within the muscle. We recorded differentially, with two wire electrodes within each muscle. EMG signals were acquired using an analog-to-digital converter  at 20 kHz per channel and processed using the program AxoGraph for the Apple Macintosh computer. For analysis and display, EMGs were first rectified and then averaged.Immediately after ICMS, in selected experiments we injected biotinylated dextran amine , Lucifer yellow dextran amine  or dextran alexa fluor  into specific sites of the forelimb area of motor cortex to anterogradely label joint-specific CST projections. Injections were made using glass micropipettes (15–20 µm tip diameter). The current for iontophoresis was set to 7 µA for 5 min in alternating mode . The average injection site diameter was 279±11µM (n = 3 mice). For each animal, we injected BDA with either LY-DA or DAF at sites separated by at least 400 µm. After a minimum and maximum post surgery survival of 14 and 21 d respectively, mice were given an anesthetic overdose and perfused through the heart with heparinized (0.1%) saline followed by 4% paraformaldehyde. The brain and spinal cord were removed, post-fixed in the same fixative at room temperature for 2 hr, and transferred to 20% sucrose in 0.1 M PB at 4°C overnight. Transverse sections of the cervical enlargement  were cut at 40 µm and processed for tracer histochemistry. For visualization of BDA, sections were incubated with ExtrAvidin cyanine 3  overnight at 4°C. For visualization of LY-DA and DAF, sections were incubated at 4°C overnight in PBS containing rabbit anti-LY-DA antibody  or rabbit anti-alexa fluor antibody  in blocking buffer . After rinsing, sections were incubated for 2 hr at room temperature (RT) in blocking buffer containing 0.2% anti-rabbit secondary antibody conjugated to FITC .We developed a quantitative method for determining the topographic distribution of label within the gray matter in the cervical enlargement. For quantitative assessment of CST labeling we measured the amount of label within 4 regions of interest (ROI) in the contralateral gray matter: portion of laminae 3–4, located at the level of the “notch” on the lateral dorsal horn; laminae 4–5, to capture the region of dense labeling in between adjoining ROIs; lamina 6, at the base of the dorsal column; and a portion of laminae 7–9, just ventral to the ventral commissure.Cholera toxin b subunit (CTb) was used to retrogradely label the motor pools of selected forelimb muscles and to anterogradely  label muscle afferents as a means to help localize CST terminations. CTb preferentially labels myelinated afferents 8 or 4.02×108 pfu/ml) 8 pfu/ml) Like CTb, injections of pseudorabies virus (PRV) were also made unilaterally into the deltoid, biceps, the wrist extensor compartment (4×2.5 µL). We used two types of the Bartha strains–152, expressing green fluorescent protein . After rinsing sections in 1×PBS, sections were then incubated for 1 hour at RT in blocking buffer . The sections were next incubated at 4°C overnight in PBS containing primary anti- GFP antibody (1∶1000) After rinsing with 1×PBS, sections were incubated for 2 hr at RT with FITC-conjugated donkey anti-rabbit antibody . Sections were then rinsed again in 1×PBS and incubated at 4°C overnight in PBS containing primary anti-RFP antibody (1∶1000). After rinsing with 1×PBS, sections were incubated for 2 hr at RT with donkey anti-goat conjugated to Cy3 (1∶800). All sections were mounted on gelatin-coated slides, air-dried and coverslipped with Vectashield (Vector Laboratories). For PRV-ChAT double immunostaining, the same double-staining process as above was repeated using anti-GFP to label PRV-infected interneurons. Sections were then immunostained for ChAT using goat anti-ChAT  followed by donkey anti-goat conjugated to Cy3 (1∶800).To quantify PRV labeling, we randomly selected and processed (as above) sections through the cervical enlargement of the spinal cord. Using Neurolucida (Microbrightfield), we counted all labeled neurons on at least 4 sections per animal for combined C5/C6 levels and C7/C8 levels. The images of the counted sections were processed in Matlab to produce regional density maps (see above). All sections sampled were of similar sizes therefore removing the need to re-size the images as was the case when determining CST topography.To determine whether CST axons contacted spinal cord interneurons directly and to image PRV-ChAT double-labeled interneurons, we used laser-scanning confocal microscopy  using two different fluorescent markers: FITC  and Cy3/rhodamine . To adjust color balance, contrast, and brightness in the confocal images, ImageJ (NIH) and Adobe Photoshop (Adobe Systems) were used. When comparing images, all capture and adjustment parameters were kept identical.Standard statistical tests including student’s t-test and analysis of variance (ANOVA) were conducted using Microsoft Excel (Microsoft), Prism 4 (Graph Pad), and Statview. We used the Fisher’s PLSD post hoc test for repeated measures ANOVA.We investigated the functional organization and spinal connectivity of the forelimb area of the motor cortex in adult mice, which includes the caudal and rostral forelimb areas ICMS evoked movement about the shoulder, elbow, wrist, and digit joints (n = 35 mice), as other studies have shown in the mouse Within the territory explored, we examined a total of 304 sites across all animals and forelimb responses were evoked at 273 of these sites . In addiFor the forelimb, responses evoked at the elbow were most common, followed by the wrist, shoulder, and digit. The maps in The average threshold for the shoulder sites (15±2 µA) was significantly lower  than the elbow, wrist and digit, which were not different from each other . For a minority of responses (12.8%), which were without any motor cortex regional localization, it was not possible to distinguish among the multiple joint movements at threshold and these were termed multi-joint responses. The most common multi-joint responses involved the elbow with either of the other joints (68.6%). Overall, the average threshold of multi-joint responses was not significantly different from single joint responses .We constructed a composite map (F) and a map of the probability of evoking a particular response from the composite map (G). The saturation of the color representing that joint is proportional to the probability for evoking the dominant response. At each of the 32 sites sampled, one joint was most likely to be evoked. Several shoulder- and wrist-dominant sites co-represented an additional joint. The elbow was the only joint that was solely represented at some coordinates . Although our findings indicate an underlying consistent somatotopy, they stress that mouse forelimb motor cortex is organized into regions where a given joint can be represented at multiple sites, as others have reported for monkey To identify contralateral spinal termination patterns from motor cortex sites evoking movements of each of the forelimb joints, we injected anterograde tracers  at sites whose principal joint actions were identified using ICMS , differences in the CST termination fields for motor cortex shoulder, elbow, and wrist joint zones were not apparent, as shown on the averaged color coded regional density plot (or “heat map”). The light gray lines enclose the region of densest labeling in each animal . The black line marks the average dense region based on the average heatmap. In all animals, the core of densest labeling for each joint was located in the medial dorsal horn-intermediate zone. These data are summarized in B4 , which shows overlap of both the dense zones  and the sparsely labeled regions .By contrast, there were clear differences in joint termination fields at the C7/C8 level (C1–3). CST shoulder site core terminations for all animals were restricted to laminae 4–5, as shown on the averaged color coded regional density plot (C1). M1 elbow sites (C2) terminated primarily in the medial portion of the deep laminae of dorsal horn and intermediate zone, ventromedial to the shoulder region. We noted for the elbow that more rostral sites in motor cortex had slightly more axons terminating ventrally in the spinal intermediate zone. The average antero-posterior bregma coordinate for this group (n = 4) was 1.5±0.2 and the remaining sites (n = 7), 0.8 mm ±0.1 mm . Core CST label from motor cortex wrist sites (C3) was preferentially located within the deeper dorsal horn laminae and the central intermediate zone (laminae 5–7) lateral to the elbow and ventrolateral to the shoulder regions. The region of sparser wrist labeling had the most ventral pattern of all joints. In some experiments we identified sparse projections from wrist sites into the region of the dorsolateral motor pools. In a subset of animals (n = 7), two different tracers labeled different motor cortex joint zones effectively. In these side-by-side comparisons, there were differences in the CST termination patterns for the two different joint zones. We conducted a laminar analysis to determine if these topographic differences in joint-specific projection patterns were significant across animals. We analyzed CST label density in 4 regions of interest ROI, 2, inset.The topographic and ROI analyses described above point to potential important laminar termination differences for the motor cortex joint zones. To examine this further we plotted average label density for each joint zone across the 4 dorsoventral ROIs 1, B1. MoThe complementary view to the differential laminar projections of the motor cortex joint zones is that each spinal laminar region collects CST inputs preferentially from one or another M1 joint zone: laminae 3/4 receive mostly shoulder input; laminae 4/5 elbow and wrist, but with wrist lateral to elbow; and laminae 6 and 7–9 receiving mostly wrist input. The stacked bar graphs  interneurons. Our starting hypothesis was that the different motor cortex joint-specific zones each target the spinal region containing the last-order interneurons that synapse on motoneurons innervating muscles acting on the represented joint. To identify last-order interneurons, we used retrograde transneuronal transport of pseudorabies virus (PRV) following intramuscular viral injection A post-injection survival period of 36 hrs did not result in viral labeling of any cervical neurons. Monosynaptic transfer across the neuromuscular junction, from muscle to the motoneurons, began to occur by 48 hr after the viral injections. At this survival time, only motoneurons were labeled. At 56 hr survival we observed very sparse labeling of interneurons, in addition to motoneuronal labeling. Sixty-four hours was the first survival time with substantial interneuronal labeling and, importantly, no label in motor cortex. This is consistent with the finding that monosynaptic connections between CST and motoneurons are sparse To determine shoulder, elbow, and wrist premotor interneurons, we injected PRV into the deltoid (n = 5), biceps (n = 4), and wrist extensors (n = 4) muscle compartments. Bartha strains 152 and 614 (expressing green and red fluorescent protein respectively) were injected into muscle pairs . We sampled a total of 57 sections for cervical levels C5/C6 and 46 sections for cervical levels C7 and C8 . PRV-labeled interneurons associated with muscles at each joint were distributed throughout the ipsilateral gray matter and ventromedial contralateral gray matter ; B1, B2.For C5/C6, the densest CST termination zones  the dense joint-specific projections spared the territories of putative premotor interneurons. Importantly, this rostrocaudal difference is not explained by gross differences in the location of motor pools. As we show, and as recently reported Our findings suggest two populations of CST termination, one dense (“core”), which differentially targeted particular segmental territories at C7/C8, and a second that is sparse and diffuse. Whereas we show that CST axons can contact premotor interneurons, our interpretation stresses that it is the population of densest CST terminations that provides the most effective disynaptic access to motoneurons. While a single CST axon branch may make only a few contacts with a premotor interneuron, when this branch is located within the densest CST termination field it could affect the spinal neuron’s excitability together with the large cohort of other CST axons projecting to the same territory. It is unlikely that the sparse CST projections to premotor interneuron fields will be as effective as those within the dense focus because they function in relative isolation. The function of CST connections in the sparse fields may depend on convergent signaling from multiple cortical sites, other descending pathways, or somatic afferent inputs; they may serve a more modulatory function. The core/dense projection is better suited for feed-forward drive.Co-registration of the cortical motor map and injection sites reveals the basic motor cortex representations of joint and, as we now show, segmental termination patterns that could provide differential access to segmental interneuron populations. However, the cortical joint map is not somatotopically fixed across animals. The probability of evoking a particular joint movement at a given location was high, but not 100%. Furthermore, at most sites, another joint could be recruited at a higher current. Multijoint and more integrative effects may depend on stronger drive to the sparse segmental territories, which might come about with longer duration stimuli in mapping studies The joint-specific CST projections to C7/C8 show a remarkable amount of specificity that could lead to fractionation of connections with particular last-order and higher-order spinal interneurons Cortical distal limb control may tap preferentially into a network in the intermediate zone that has direct access to motoneurons through last-order interneurons. Cortical wrist zones, but not shoulder or elbow zones, preferentially project to the territory of spinal dI3 interneurons in the cervical cord Surprisingly, motor cortex elbow sites had their densest projections targeted to a restricted medial cervical region. This is a conserved CST termination zone, present also in humans examined using degeneration techniques Although our focus is on spinal mechanisms, our findings help inform the organization of motor cortex in the context of forelimb control. The forelimb motor map in mouse motor cortex, like other species, contains a complete body joint/muscle map Our findings provide two new perspectives on the anatomical mechanisms underlying CST control of limb muscles and joints. First, it was not known, nor even suspected, that joint-specific sites in motor cortex would have CST projections with differential segmental terminations. This is a new finding that begins to provide an anatomical explanation for the cortical joint/limb segment map. Importantly, the finding also provides a plausible new model for “upper motor neuron” control by conferring access to particular spinal interneuron circuits for joint control, especially premotor interneurons for distal joints. Second, differential segmental CST termination patterns reveal an unsuspected organization in the spinal cord. The patterns are neither an imprint of premotor interneurons, nor a simple means for integrating cortical control signals with muscle-specific proprioceptive afferents. Rather, it likely reflects the complex logic of the underlying circuits in those particular regions of the cervical cord. Whereas this logic that has yet to be elucidated, two circuits come to mind. One mediates “motor primitives,” limb movements with end-point control elicited by focal activation of lumbar spinal circuits There is a growing inventory of genetically-identified spinal interneuron subtypes in the mouse Figure S1Spinal terminations of wrist and shoulder zones of the same animal. BDA was injected into a wrist zone (A) and Lucifer yellow dextran amine, into a shoulder zone (B) in motor cortex. The locations of the injection sites are shown in the inset . Scale, 500 µm.(TIF)Click here for additional data file.Figure S2Biceps motor pool at C7/C8 labeled by retrograde transport of CTb and PRV in the same animal. A. CTb was injected into biceps to mark the locations of biceps motoneurons (n = 8 sections). The motor pool is located ventrolaterally in lamina 10. B. PRV was injected into biceps several days later. Note that biceps motoneurons are also located within ventrolateral lamina 10.(TIF)Click here for additional data file.


A sustained gene modulatory strategy is necessary for regulating abnormal gene expression in diabetic retinopathy, a long-term complication. We investigated the efficacy of a small interference RNA (siRNA) strategy in mediating the long-term downregulatory effect of fibronectin (FN) overexpression in vivo.Streptozotocin-induced diabetic rats were intravitreally injected with 3 µM of FN-siRNA at six week intervals over a period of 4.5 months. Retinal FN protein expression, vascular basement membrane (BM) thickness, and retinal vascular cell loss were assessed by western blot, electron microscopy, and retinal trypsin digest, respectively.Retinal FN expression and BM thickness were significantly increased in diabetic rat retinas compared to those in non-diabetic control rats . FN-siRNA treatment reduced FN overexpression and BM thickening  and significantly reduced the number of acellular capillaries (35%) and pericyte loss (55%) compared to those of untreated diabetic eyes.These findings suggest that BM thickening is an important target for preventing vascular cell loss in a diabetic retina, and that the siRNA approach could be useful for long-term gene modulation in diabetic retinopathy. Retinal capillary basement membrane (BM) thickening is closely associated with the development of vascular lesions in diabetic retinopathy, a long-term complication of diabetes. The detrimental effects of BM thickening in promoting the development of vascular lesions in diabetic retinopathy are beginning to be established. Studies indicate that BM thickening primarily develops from a process involving hyperglycemia-induced excess synthesis of BM components, such as fibronectin (FN), collagen type IV, and laminin –4. The aThe role of FN overexpression and BM thickening has recently been examined in the retinal and glomerular capillaries of diabetic rats using antisense oligonucleotides . The stuWe previously demonstrated that antisense oligonucleotides could reduce BM thickening by lowering the expression of BM components , and thaIn this study, we selectively targeted FN since previous studies indicated that downregulation of FN expression significantly reduces retinal capillary BM thickening and prevents retinal vascular lesions characteristic of diabetic retinopathy . The siRDifferent concentrations of FN-siRNA  were tested for 1 week to identify an effective dose for reducing FN expression by approximately 30%. The target for achieving 30% downregulation was selected based on our previous studies, which is consistent with the magnitude of the increase caused by diabetes. As such, a 30%–50% decrease in FN expression would bring diabetic levels to a normal range .To determine the duration efficacy of the FN-siRNA strategy in reducing FN expression, retinas of rats intravitreally injected with 3 μM of FN-siRNA were studied at different time points . The retinal proteins from rats sacrificed at these time points were analyzed for their FN protein level, which was then compared to that of uninjected control rats.2 inhalation as described and approved by the Boston University School of Medicine animal care board. No untoward effects were noted from the intravitreal injections in any of the eyes, as there was no redness or opacity as determined through routine eye examinations, as previously tested [In this study, 32 male Sprague Dawley rats were randomly assigned into four groups: normal, diabetic, diabetic injected with FN-siRNA, and diabetic injected with scrambled siRNA. Diabetes was induced using streptozotocin (STZ) as previously described . SiRNA wy tested . One eyeThe 21-mer FN-siRNA (5′-AAC TTC AAA TTA TGA ACA AGA-3′) used in this study was designed on the basis of Tuschl’s rules (AAC19 adjacent nucleotides)  and encoRetinas isolated from enucleated eyes were immediately fixed in 2.5% glutaraldehyde in a 0.1 M cacodylate buffer. The tissues were dehydrated using osmium tetraoxide, ethanol, and propylene oxide . The tissues were then embedded in an Epon-Araldite plastic mixture and baked for 48 h. Ultra-thin sections were serially cut at 60–70 nm using a microtome . The sections were collected and placed on a copper grid and stained with 4% uranyl acetate (EMS) in methanol and viewed under a transmission electron microscope . At least ten random images of retinal capillaries from the outer plexiform and ganglion cell layers from each individual animal were photographed and enlarged to 50,000× magnification for examination. These electron micrographs were analyzed according to the orthogonal intercept method  for BM tRetinal capillary BM thickness was determined from electron micrographs using the Siperstein method . In brie3 (Sigma). After dehydration in ethanol and clearing in xylene, the slides were mounted with Permount. The PAS stained the glycoprotein of the BMs pink, while the cellular nuclei  were stained blue by the hematoxylin. Representative areas of the vascular network were photographed using a Nikon DS-Fi1. The images were analyzed for retinal morphology and in particular for pericyte loss and acellular capillaries.To analyze the retinal vasculature for pericyte loss and acellular retinal capillaries, we used the retinal trypsin digest (RTD) technique as described by Kuwabara and Cogan  with min2) was analyzed for each of the treated and untreated groups to compare the number of acellular capillaries.Approximately 1,200 capillary cells were counted from the midretinal area of each retina following RTD and hematoxylin-PAS staining. At first, the images of retinal capillaries were captured using a Kodak digital camera connected to a computer and then pericyte counts were determined from the images. The specific loss of pericytes could be detected from the empty “shell” left behind after pericyte “dropout.” Capillaries that lacked both pericytes and endothelial cells were considered acellular. A defined area  were examined using confocal microscopy. FN-siRNA localization in the retinal vascular cells of the rat eyes was observed within 2 h post-injection. Based on the double stain from Cy3 and DAPI, fluorescence from the siRNA could be assigned to the nuclei of retinal vascular endothelial cells and pericytes . The lumThe effectiveness of the FN-siRNA strategy was tested using increasing concentrations (0.5–6.0 µM) of siRNA to identify an optimal dose for ~30% downregulation of FN expression. A reduced FN level was observed in rat retinas after intravitreal injection at concentrations ≥1 μM at 1 week post-injection . The 1 µA time course (0–6 weeks) study following intravitreal injection of FN-siRNA showed significant downregulation of FN expression in the retinas. After 1 week, retinal FN expression was reduced by approximately 35%, which persisted to the 6 week time point . ScramblWestern blot analyses indicated a significant increase in FN expression in the retinas of diabetic rats compared to those of non-diabetic control rats , respectively. A significant downregulation in FN expression was observed in the retinas of diabetic rats that were intravitreally injected with FN-siRNA compared to those of uninjected diabetic control eyes or those of diabetic eyes injected with scrambled siRNA  in retinal capillary BM thickness in the diabetic rats compared to those of non-diabetic control rats . Upon intravitreal injection with FN-siRNA, a significant reduction (~75%) in retinal capillary BM thickness was observed in the retinas of diabetic rats compared to those of uninjected control diabetic rats and diabetic rats intravitreally injected with scrambled siRNA  and number of acellular capillaries  observed in diabetic retinas was significantly reduced in retinas of diabetic rats treated with FN-siRNA . The number of acellular capillaries  and the Retinas injected with siRNA and examined 1, 2, 4, and 6 weeks post-injection showed no physical evidence of redness or opacity in the eyes, and retinal capillaries and sections examined in parallel showed no infiltration of macrophages.Fasting blood glucose levels were measured every alternate day and showed hyperglycemic conditions in diabetic animals (307±48 mg/dl) compared to normal animals (122±21 mg/dl). The HbA1c level measured at the three-month interval also confirmed a hyperglycemic state in diabetic animals (11.2±1.6%) versus normal animals  of FN-siRNA, which resulted in a reduction in BM thickness and other beneficial outcomes, including the prevention of pericyte loss and acellular capillaries. This finding was consistent with our previous results demonstrating that reducing overexpression of BM components can decrease BM thickening in retinal capillaries . The preBM is a highly organized structure composed of multiple ECM components linked to each other in an orderly manner. The ordered structure appears to be compromised in the thickened BMs of diabetic retinal capillaries, as evidenced by altered ultrastructures such as a “Swiss cheese-like” appearance. These altered structures are at least in part due to a disturbed ratio among the ECM components – a likely consequence of the abnormal increase in synthesis of the ECM components ,10,16–24One mechanism responsible for triggering apoptotic signaling is the high glucose- or diabetes-induced forkhead box protein 1 (FOXO1) activation . A previPreviously, we identified a FN-siRNA that exhibits a long-term effect in microvascular endothelial cells, in vitro . Time coThe FN siRNA used in this study was specifically targeted to the FN translation initiation site of the FN transcript, with the goal of reducing hyperglycemia-induced FN overexpression by 50%. Our studies indicate that an approximate 50% FN reduction was achieved using the 21-mer FN siRNA. However, it is possible that the efficacy may be increased if longer (25–30 mer) siRNAs  are used


Rhizobial nodulation (Nod) factors activate both nodule morphogenesis and infection thread development during legume nodulation. Nod factors induce two different calcium responses: intra-nuclear calcium oscillations and a calcium influx at the root hair tip. Calcium oscillations activate nodule development; we wanted to test if the calcium influx is associated with infection.Sinorhizobium meliloti nodL and nodF mutations additively reduce infection of Medicago truncatula. Nod-factors made by the nodL mutant lack an acetyl group; mutation of nodF causes the nitrogen (N)-linked C16:2 acyl chain to be replaced by C18:1. We tested whether these Nod-factors differentially induced calcium influx and calcium spiking.nodF and nodL on legume infection. Infection thread development is inhibited by ethylene, which also inhibited Nod-factor-induced calcium influx.The absence of the NodL-determined acetyl group greatly reduced the induction of calcium influx without affecting calcium spiking. The calcium influx was even further reduced if the N-linked C16:2 acyl group was replaced by C18:1. These additive effects on calcium influx correlate with the additive effects of mutations in We conclude that Nod-factor perception differentially activates the two developmental pathways required for nodulation and that activation of the pathway involving the calcium influx is important for efficient infection. These various substitutions are the major determinants of host specificity for a given rhizobial strain -fixing nodules on legumes requires coordinated signalling between rhizobia and the plant. This results in nodule morphogenesis and the development of specialized tube-like infection structures (infection threads), which often initiate in root hair cells; rhizobia grow down infection threads to infect plant cells in the developing nodule   significantly reduced rates of rhizobial infection and reduced, but did not block nodule development vinyl glycine (AVG) and prepared for microscopy as previously described  and nodF/nodL  Nod factors were synthesized using ‘E. coli cell factory’ procedure previously described  was pumped through a C18:1 reverse phase column  and then eluted with 2.5 ml of each of 20%, 40%, 60%, 80% methanol and 4 ml of 100% methanol. The fractions were assayed for root-hair deformation activity  above 0.1 and were considered as candidate cells positive for calcium influx. From close inspection of many traces, we found this approach captured our definition of influx robustly with a strong correlation between the score and our own visual evaluation. Below a score of 0.1 traces were classified as negative.Confidently identifying a calcium influx in root hairs is difficult because it is a single transient event that can be affected by cytoplasmic streaming. To develop an objective analysis of what constitutes a calcium influx, we defined an influx by an above-average, broad variation in the background trend  and madeMedicago truncatula seedlings expressing Yellow Cameleon YC2.1  added to Medicago truncatula seedlings expressing Yellow Cameleon YC2.1  induced calcium influx in 13 of the 15 the cells tested  and also lacks the NodL-determined acetate group . This signal , equivalent to that produced by the S. meliloti nodF,nodL double mutant, (referred to hereafter as the nodF/nodL-Nod factor) did not induce a calcium influx at 10 nM , to WT roots and found that it significantly reduced the number of root-hair cells inducing a calcium influx after the addition of 10 nM WT-Nod factor Fig. b confirmnodL-Nod factor induces normal calcium spiking but is much less potent than WT-Nod factor for the induction of the calcium influx and both these responses require NFP , these two forms of polar growth may have some similar properties. Root-hair tip elongation requires NADPH-oxidase driven production of ROS, activated by small ROP-family GTPases which can bind to and activate NADPH oxidase  fits with a model in which Nod-factor binding normally activates two pathways, one being calcium spiking and the other involving NADPH-oxidase and the calcium influx. It is proposed that this latter pathway could help activate infection by rhizobia  the calcium influx, membrane depolarization and production of ROS are induced by similar concentrations of Nod factor , similar to that reported for chitin binding to the Arabidopsis CERK1 receptor (Liu et al., et al., et al., et al., et al., HCL encoding the LYK3 receptor might play such a role, but the hcl mutant retained the Nod-factor-induced calcium influx so the Nod-factor-induced calcium influx observed in the hcl-1 mutant could be explained by an interaction between NFP and one or more of the other predicted receptors in this family.How could Nod-factor binding to cognate receptors induce both calcium-spiking-driven nodulation development and the proposed NADPH-oxidase-calcium influx-dependent infection pathway? The et al., Medicago truncatula skl mutant is defective in ethylene signalling due to a mutation in the EIN2 gene and infection threads in this mutant are more prolific and grew in an uncontrolled fashion throughout the cortex (Penmetsa & Cook, et al., skl mutant was more sensitive to Nod factor for induction of calcium influx, together with the observation that the ethylene precursor, ACC, inhibited induction of calcium influx shows that ethylene can suppress the calcium influx and in this way may regulate infection. The combined observations of the effect of ethylene and the different Nod-factor structures on the activation of calcium influx and promotion of bacterial infection, all point towards a role for the calcium influx in the initiation and/or proliferation of bacterial infection.Ethylene reduces the sensitivity of legumes to Nod factor based on analyses of calcium spiking and gene induction (Oldroyd


Obesity leads to changes in the gut microbial community which contribute to the metabolic dysregulation in obesity. Dietary fat and fiber affect the caloric density of foods. The impact of dietary fat content and fiber type on the microbial community in the hind gut is unknown. Effect of dietary fat level and fiber type on hindgut microbiota and volatile fatty acid (VFA) profiles was investigated. Expression of metabolic marker genes in the gut, adipose tissue and liver was determined. A 2×2 experiment was conducted in pigs fed at two dietary fat levels (5% or 17.5% swine grease) and two fiber types . High fat diets (HFD) resulted in a higher (P<0.05) total body weight gain, feed efficiency and back fat accumulation than the low fat diet. Feeding of inulin, but not solka floc, attenuated (P<0.05) the HFD-induced higher body weight gain and fat mass accumulation. Inulin feeding tended to lead to higher total VFA production in the cecum and resulted in a higher (P<0.05) expression of acyl coA oxidase (ACO), a marker of peroxisomal β-oxidation. Inulin feeding also resulted in lower expression of sterol regulatory element binding protein 1c (SREBP-1c), a marker of lipid anabolism. Bacteria community structure characterized by DGGE analysis of PCR amplified 16S rRNA gene fragments showed that inulin feeding resulted in greater bacterial population richness than solka floc feeding. Cluster analysis of pairwise Dice similarity comparisons of the DGGE profiles showed grouping by fiber type but not the level of dietary fat. Canonical correspondence analysis (CCA) of PCR- DGGE profiles showed that inulin feeding negatively correlated with back fat thickness. This study suggests a strong interplay between dietary fat level and fiber type in determining susceptibility to obesity. Recent analysis of the human hindgut microbiome suggests a strong association between microbiome composition and obesity susceptibility Dietary fiber, made of carbohydrates and lignin, are resistant to degradation in the upper gut Gut microbes play a key role in the regulation of energy metabolism and fat storage This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Purdue Animal Care and use Committee (PACUC) approved the protocol. Animals were held under a controlled environment at the Purdue Animal Sciences Research and Extension Center swine facility and all efforts were made to minimize discomfort. Thirty two female pigs  were randomly allocated to 4 treatments in a 2×2 factorial arrangement with two dietary fat levels (5% or 17.5% swine grease) and two types of fiber (4% inulin or solka floc). Thus we have four treatments: 1) high fat diet with solka floc (HFD Sol), 2) low fat diet with solka floc (LFD Sol), 3) high fat diet with inulin (HFD Inu), and 4) low fat diet with inulin (LFD Inu). Nutrient composition of experimental diets is presented in Volatile fatty acid (VFA) determination was conducted on metaphosphoric acid derived samples using gas chromatographic methods described by Playne Total bacterial DNA was extracted from ceca samples using the FastDNA® SPIN Kit for Soil  per manufacturer’s instructions with the exception that 100 mg of ceca samples was used instead of soil PCR-DGGE fingerprint profiles were analyzed using BioNumerics software . The presence and absence of bands in profiles were made into a binary matrix for quantitative comparison between different treatments. The number of bands was counted to estimate richness of the most abundant bacterial populations in each sample Based on binary data of the presence or absence of each band in the two fiber types, Fisher’s exact test  was used to analyze the effect of fiber type on each individual band/population. Based on the Fisher’s exact test, the bands that were most significantly affected by fiber type were excised from the DGGE gel, eluted in sterilized PCR water and re-amplified with primers PRBA338F and PRUN518R without the GC clamp. PCR amplicons from bands migrating the same distance extracted from four different inulin-fed pigs were cloned into pGEM-T Easy cloning vector  and sequenced in both forward and reverse directions at the Purdue Genomics Center. Nucleotide sequence was compared to the National Center for Biotechnology Information (NCBI) using BLASTn Bifidobacterium, Bacteroides and total Bacteria abundances in the extracted DNA was performed using the MyiQ real-time PCR detection system  with the SYBR green RT-PCR mix . Bacteria copy numbers were generated from a standard curve prepared from purified plasmid clones of the16S rRNA gene from Bifidobacterium bifidum and Bacteroides vulgatus. Gene copy number was calculated from the concentration of the extracted plasmid DNA clone assuming 1.096×10−12 g/bp. To eliminate potential differential cell lysis effects on DNA extraction, percentages of Bifidobacterium and Bacteroides with respect to total Bacteria were then calculated. Primer sequences for Bifidobacterium, Bacteroides and total Bacteria used for quantitative PCR are presented in Quantitation of Liver, adipose tissue and intestinal tissues were removed and snap-frozen in liquid nitrogen and then stored at −80°C. Total RNA was extracted using QIAzol lysis reagent . RNA extracts were dissolved in nuclease-free water  and quantified using a Nanodrop 1000 instrument . Reverse transcription was performed using M-MLV reverse transcriptase  with 1 µg RNA. Markers of mitochondrial β-oxidation, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and carnitine palmitoyl transferase 1-alpha (CPT1α) and markers of peroxisomal β-oxidation, acyl-CoA oxidase I (ACO) and peroxisome proliferators activate receptor alpha (PPARα), were analyzed. Markers of lipid anabolism, SREBP-1c and (fatty acid synthase) FAS, were also analyzed. Interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were selected as inflammatory markers. The primers used for reverse transcriptase qPCR are listed in In order to compare the changes in bacterial community structure that came from different fat levels and fiber types, two-way analyses of similarity (ANOSIM) The binary matrices of PCR-DGGE bacterial profiles were further analyzed by canonical correspondence analysis (CCA) to determine the possible experimental variables that were correspondent to gut bacterial community structure. CCA is a multivariate method to illustrate the relationships between biological structures of species and environmental variables, which is widely used in community ecology Our analysis combined the performance data with bacteria fingerprints to find potential connections between performance variables and bacterial community structure.Statistical analysis of performance data and gene expression data was conducted using the GLM program in SAS version 9.0 . The Tukey multiple comparison test was used to determine significant mean differences. Data was expressed as mean ± SE. P values less than 0.05 were considered statistically significant whereas P values between 0.05 and 0.1 were considered as showing a trend.Pigs fed HFD had a higher final body weight, average daily weight gain, feed conversion ratio, total back fat (TBF) thickness and inner back fat (IBF) thickness  than thoHFD fed pigs had a lower concentration of acetate, propionate and butyrate than the LFD fed pigs . Inulin Each band in the PCR-DGGE profile theoretically represented one species/population of bacteria. The number of the different bands provides an estimation of the number of abundant populations in each sample The UPGMA dendrogram of Dice pairwise similarity indices of PCR-DGGE profiles illustrated separation of the profiles into two distinct clusters that corresponded with the two different fiber types fed to the pigs . The solPrincipal component analysis (PCA) of the PCR-DGGE fingerprint profiles  also indBased on band binary matrix from DGGE profile of bacterial communities, significant differences were observed between inulin and solka floc diets . Although cluster analysis indicated dietary fat level had a lower influence, especially with inulin diets, the differences in bacterial communities between HFD and LFD were still significant . Two-way perMANOVA analysis also produced similar results .Based on canonical correspondence analysis (CCA), dietary fiber, fat level, total back fat, inner back fat and ADG were significantly associated with PCR-DGGE profiles of bacteria community structure . The firCatenibacterium mitsuokai JCM 10609T (AB030224), in the phylum Firmicutes and family Erysipelotrichaceae. For band 6.00, multiple sequence results were 100% identical from two pigs and the sequence matched with 100% identity to Blautia wexlerae WAL 14507T (EF036467), in the phylum Firmicutes and family Lachnospiraceae. Due to the short sequence of these bands only the genus designation is reliable at this point. Sequences for bands 5.76, 6.41 and 7.30 could not be obtained likely because they were relatively weak and in close proximity to other bands.Fisher’s exact tests indicate that a total number of five bacteria bands were significantly different in response to the two fiber types . These fBifidobacterium bifidum and Bacteroides vulgatus, the average gene copy numbers using Bacteria primers were 2.90×106±1.05×105/ng DNA and 3.62×106±1.56×105/ng DNA, respectively, which had the same order of magnitude as previously reported for pigs Bifidobacterium and Bacteroides relative to total Bacteria indicated that HFD feeding resulted in a significantly (P<0.01) higher relative percentage of Bacteroides but not Bifidobacteria, compared with LFD. There was no significant effect of fiber type on these bacteria. However, in HFD fed pigs, inulin resulted in a higher percentage of Bacteroides than those on solka floc diets  in the liver (P = 0.02)  and subcFor lipid anabolism related genes, LFD feeding resulted in higher expression of SREBP-1c in subcutaneous fat (P<0.01) and a trend (P = 0.10) for a higher expression in the liver as well. Compared with solka floc-fed pigs, expression of SREBP-1c was lower in inulin fed pigs (P = 0.03) in the mesenteric fat. Fat level and fiber type did not significantly affect the expression of FAS.In HFD fed pigs, there was a tendency (P = 0.09) for a higher TNFα mRNA expression in the mesenteric fat, and significantly higher (P = 0.02) IL-6 expression in the subcutaneous fat.There was no significant effect of fiber type and fat level on the expression of genes in the intestinal sections . In the Chronic intake of high fat diet is associated with being overweight and obese Gut microbial community plays an important role in host health, especially in the modulation of adiposity Bifidobacterium and Bacteroides are the two microbial genera that have been reported to have significant effect on substrate fermentation in the hind gut and energy homeostasis Bacteroides was relatively higher in inulin-fed pigs with HFD than other treatment, this may account for inulin-suppressing HFD induced fat accumulation. But the relatively low abundance of Bacteroides to total bacteria in this study suggests that the potential contribution of this group to the observed weight gain and adiposity is very small. Alternatively, this low abundance of Bacteroides may reflect a difference in microbial structure between humans and swine. In swine, Bacteroides may constitute a small fraction of the phylum Bacteroidetes community and other members of this phylum may be related to obesity. Bifidobacterium is another bacterial group that has been shown to have probiotic properties in mammals Bifidobacteria is known to degrade inulin to generate fructo-oligosaccharides Bifidobacterium abundance in vivoBifidobacterium, suggesting that other bacteria may also contribute significantly to the observed animal performance. A metagenomic analysis may be needed in the future to determine the identity of bacteria species that mediate the inulin effect.Catenibacterium and Blautia were exclusively present only in the inulin-fed pigs. Catenibacterium is Gram-positive and an obligatory anaerobe that utilizes glucose to produce acetic, lactic, butyric and iso-butyric acids Blautia is also Gram-positive and anaerobic. It can utilize many kinds of carbohydrates to produce acetic, lactic acids and ethanol Catenibacterium and Blautia with inulin fermentation is not well characterized; however, both of these bacteria belong to the phylum Firmicutes. Studies have indicated that bacteria in the phylum Firmicutes are enriched by inulin both in vivoin vitroFirmicutes versus Bacteroidetes in the microflora of obese vs. lean subjects Firmicutes and inulin presence in the diet. Most butyrate-producing bacteria belong to the phylum FirmicutesCatenibacterium in the cecum may have contributed to the increased VFA production with inulin consumption observed in the study. Inulin feeding resulted in increased cecal VFA concentration (propionate and butyrate)   and the Although fiber consumption is linked to reduction in weight gain and fat accumulation Table S1The nutrient composition of the experimental diets.(DOCX)Click here for additional data file.Table S2Bifidobacterium and Bacteroides.PCR primers for amplification of 16S rRNA gene of Bacteria, (DOCX)Click here for additional data file.Table S3Primers for fat oxidation and inflammatory genes for Real-time PCR.(DOCX)Click here for additional data file.Table S4Effect of different fiber types and fat content on gene expression in intestinal sections.(DOCX)Click here for additional data file.


The formed copper nanoparticles showed extended stability over a period of one year. Copper nanoparticles thus prepared were characterized by powder X-ray diffraction measurements (pXRD), transmission electron microscopy (TEM) and quasi elastic light scattering (QELS) techniques. Powder X-ray diffraction (pXRD) analysis revealed relevant Bragg's reflection for crystal structure of copper. Powder X-ray diffraction plots also revealed no oxidized material of copper nanoparticles. TEM showed nearly uniform distribution of the particles in methanol and confirmed by QELS. Typical applications of copper nanoparticles include uses in conductive films, lubrication and nanofluids. Currently efforts are under way in our laboratory for using these nanoparticles as catalysts for a variety of organic reactions.In this work, we present a novel method for the synthesis of copper nanoparticles. We utilize the charge compensatory effect of ionic liquid [bmim]BF Metal nanoparticles play a significant role in diverse fields such as optical, electronic, catalysis, medical, magnetic, information storage and surface enhanced raman scattering (SERS) The use of Ionic Liquids (ILs) in science and its technological applications is not new as they have already emerged as a green alternative to the conventional and environmentally detrimental volatile solvents. They have attracted a great deal of attention due to their high thermal stability, good conductivity, non volatility, non flammability, suitable polarity, wide electrochemical window and recyclability Our study is focused towards the ionic liquids based on imidazolium cations as they are especially favorable for green industrial applications 4.5H2O), ethylene glycol, hydrazine hydrate (NH2NH2.2H2O), sodium tetrafluoroborate and 1-butyl-3-methylimidazoliumbromide were all of analytical grade and used as such. All the aldehydes and solvents were purchased from spectrochem Pvt. Ltd. Mumbai (India) and were used without any additional purification. All reactions were monitored by thin layer chromatography (TLC) on gel F254 plates. 1H-NMR and 13 C-NMR spectra were recorded in CDCl3 and DMSO-d6 on a Jeol JNN ECX- 400P spectrometer; Melting points were recorded on SECOR Laboratories instruments melting point instruments. The infrared spectra were recorded using a model Perkin Elmer spectrum BX2 FT-IR system. Spectra were recorded with Spectrum V 5.3.1 software in the range 4000–400 cm−1. The KBr pellet technique was adopted for recording the spectra.Copper sulphate pentahydrate (CuSO1H NMR studies. (Sodium tetrafluoroborate and 1-butyl-3-methylimidazoliumbromide in equimolar quantities were stirred in dry acetone under anhydrous conditions for 48–72 h. The mixture was filtered off to remove unreacted sodium tetrafluoroborate and the filtrate was further treated with dichloromethane to remove sodium bromide and again the filtrate obtained was again treated with dichloromethane to check for any further precipitation. The solvents were removed under reduced pressure and the resulting colorless ionic liquid was dried in rotavapor at 70°C for 2 h to remove water. The product 1-butyl-3-methylimidazoliumtetrafuoroborate was characterized by studies. 4.5H2O (100 µl) and the reaction was allowed to stir on a magnetic stirrer under nitrogen atmosphere. After five minutes 3.0 M hydrazine hydrate (100 µl) was added as the reducing agent drop wise continued over a period of ten minutes and the system was stirred for another 30 minutes.  was used to image size and morphology of the powder. X-ray diffraction patterns of the powders were recorded using diffractometer .4. We discover that ionic liquid in conjunction with ethylene glycol play a vital role in the stabilization of copper nanoparticles.We report herein a method to synthesize stabilized copper nanoparticles using hydrazine hydrate as a reducing agent in the presence of ethylene glycol and [bmim]BF4, serves as an excellent media for dispersing copper nanoparticles, controlling their size and preventing their aerial oxidation; however agglomeration could not be avoided in the absence of ethylene glycol. Nanoparticles could also be synthesized in ethylene glycol alone. But the synthesized nanoparticles are not stable for extended periods of time. The probable reason is that the synthesized nanoparticles are oxidized. We intend to use the synthesized nanoparticles for catalysts over multiple cycles and store them. Thus our aim is to make nanoparticles that are stable for extended periods of time. Thus, we find that both ethylene glycol and ionic liquid [bmim]BF4 is required for synthesizing stable, monodispersed nanoparticles. Using ethylene glycol alone in the absence of [bmim]BF4 gives unstable nanoparticles; whereas using [bmim]BF4 alone in the absence of ethylene glycol leads to agglomeration of nanoparticles. Furthermore, we determine that using hydrazine hydrate as a reducing agent in the presence of ethylene glycol and hydrophilic ionic liquid [bmim]BF4 could serve effectively to increase the reaction and nucleation rates without employing any source of heating or microwave irradiation.Ionic liquid [bmim]BF4 is hydrophilic, there was the possibility of it absorbing moisture thereby rendering the nanoparticles unusable over long periods of time. Our experiments showed that the nanoparticles were stabilised for at least one year. Ionic liquid [bmim][BF4] has been demonstrated previously to be a moisture and thermally stable compound 4. Our results are in agreement with the previous work; the added stability could also be attributed to the use of an organic solvent like ethylene glycol.Since the ionic liquid [bmim]BFmax near 580 nm.Color changes during course of reduction indicate complex formation between copper sulphate and hydrazine hydrate. As the hydrazine hydrate solution is introduced drop wise, the blue color of copper sulphate solution turns darker and gradually turns intense yellow and eventually brown during the course of the reaction, indicating formation of highly dispersed metallic copper. UV-Vis spectra as shown in Copper nanoparticles separated from the suspension were analyzed for their size distribution and shape by DLS and TEM. Alongside average sizes were calculated from XRD peak data. TEM images in 4 to give ionic liquid [bmim][BF4] and NaBr. The subsequent synthesis of Cu nanoparticles involves reduction of CuSO4.5H2O by hydrazine in the presence of the ionic liquid (Scheme 1). A simple, convenient and significant method for the reductive synthesis of copper nanoparticles in ionic liquid has thus been uncovered here.Use of ionic liquid-ethylene glycol media for the synthesis of copper nanoparticles stabilised over a period of more than one year has been demonstrated. In the first step, there is an ion-exchange reaction where [bmim][Br] reacts with NaBFSupporting Information S1Graphical Abstract.(DOC)Click here for additional data file.


Phreatomerus latipes. Phylogenetic and phylogeographic relationships amongst P. latipes individuals were examined using a multilocus approach comprising allozymes and mtDNA sequence data. From the Lake Eyre region in South Australia we collected data for 476 individuals from 69 springs for the mtDNA gene COI; in addition, allozyme electrophoresis was conducted on 331 individuals from 19 sites for 25 putative loci. Phylogenetic and population genetic analyses showed three major clades in both allozyme and mtDNA data, with a further nine mtDNA sub-clades, largely supported by the allozymes. Generally, each of these sub-clades was concordant with a traditional geographic grouping known as spring complexes. We observed a coalescent time between ∼2–15 million years ago for haplotypes within each of the nine mtDNA sub-clades, whilst an older total time to coalescence (>15 mya) was observed for the three major clades. Overall we observed that multiple layers of phylogeographic history are exemplified by Phreatomerus, suggesting that major climate events and their impact on the landscape have shaped the observed high levels of diversity and endemism. Our results show that this genus reflects a diverse fauna that existed during the early Miocene and appears to have been regionally restricted. Subsequent aridification events have led to substantial contraction of the original habitat, possibly over repeated Pleistocene ice age cycles, with P. latipes populations becoming restricted in the distribution to desert springs.Desert mound springs of the Great Artesian Basin in central Australia maintain an endemic fauna that have historically been considered ubiquitous throughout all of the springs. Recent studies, however, have shown that several endemic invertebrate species are genetically highly structured and contain previously unrecognised species, suggesting that individuals may be geographically ‘stranded in desert islands’. Here we further tested the generality of this hypothesis by conducting genetic analyses of the obligate aquatic phreatoicid isopod  These groundwater-fed, island-like, wetland habitats are surrounded by sparse desert and sit on the edges of Australia’s largest inland catchment, the Lake Eyre Basin . Their cin situ genetic diversification followed by allopatric speciation. Under this view, groups of GAB springs are equivalent to aquatic islands in a surrounding “sea” of desert. A similar hypothesis has been proposed to explain the diverse subterranean invertebrate fauna  and most conspicuous endemics restricted solely to these springs. With no congeners, P. latipes is considered a biogeographic relict species in the true sense of the term. Interestingly, P. latipes is considered to be ‘widespread’ throughout the southern Lake Eyre mound springs P. latipes does not appear capable of dispersal across the desert landscape in the absence of aquatic connections P. latipes may exhibit strong genetic sub-structuring in this region P. latipes among GAB springs may occur along ephemeral river systems. The springs are located on the edges of the large ephemeral river drainage catchment, the Lake Eyre Basin. Water feeds into this giant inland lake from tributaries coming from all directions and it has been a permanent water feature at various times in Australia’s history Relicts, as defined by Habel P. latipes throughout the highly fragmented Lake Eyre mound springs. Our aim was to investigate whether major climatic events during the history of the Australian arid region have impacted on the spatio-temporal history of a single, supposedly widespread species from freshwater springs in the Australian desert. Based on previous studies of spring invertebrates we hypothesised that a) major periods of aridification are likely to have isolated taxa in the GAB mound springs, in particular during the period of aridification following the late Miocene after which there was a return to wet and the subsequent Pliocene aridification and b) the island-like GAB mound springs have promoted a high degree of genetic isolation among spring groups, leading to substantial genetic diversification within P. latipes.Here we used a multilocus genetic approach using mtDNA and allozymes to investigate the phylogenetic and phylogeographic history of None of the aforementioned field studies involved endangered or protected species, all sites were on private land or in National Parks, and we comprehensively obtained permission to access and sample all sites. For collections in National Parks we obtained a permit to ‘undertake scientific research’ (permit number Z25519 to Dr Nicholas Murphy) using appropriate methods and ethics approval from The Government of South Australia, Department for Environment and Heritage signed for the Minister for Environment and Conservation. For collection on private property we obtained general permission from Greg Campbell (Chief Executive Officer) of S. Kidman & Co Ltd and also directly from a number of station managers to carry out our field collections. The station managers were: Randall Crozier for Anna Creek Station, Peter Paisley for Stuart Creek Station, Bobby Hunter for The Peak Station. We also received permission to access culturally sensitive land at Hermit Hills Springs (Reg Dodd). At the privately owned properties we used the same methods as those in the national parks.P. latipes. The species is known to brood ∼10 live young, born as miniature adults, in a pouch carried by the female 2) to small seeps (0.5 m2) both of which may contain endemic invertebrates. This study was undertaken on the Lake Eyre supergroup of GAB mound springs, located throughout the southern and western portion of the Lake Eyre Basin in central Australia  is available for ustralia . These ig groups . These somplexes  based on2) inland lake that infrequently receives water during major rainfall events, and historically contained large amounts of water Despite relying on groundwater flows for spring existence, the ephemeral rivers and streams of the Lake Eyre Basin may provide an aquatic medium for connectivity between fragmented spring habitats. Major river drainages flow from a number of directions into Lake Eyre, a giant  sampling periods.Individuals were collected using fine mesh nets during 2008–9. Nets were rinsed with water and ethanol between springs to prevent contamination and all specimens were stored in 100% ethanol. Not all springs yielded Cytochrome Oxidase subunit 1 (COI) to examine the phylogeographic structure of P. latipes. This mtDNA gene has been used to successfully elucidate phylogeographic and population genetic relationships within crustacean taxa in previous studies (e.g. 5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) 2+, 2.5 mM of each dNTP, 5.0 µM of each primer, 0.1 units of Eppendorf Hotmaster® Taq Polymerase and ∼1 ng of DNA. These PCR products were sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing kit  and the ABI PRISM 3700 DNA analyzer. All sequences were edited with reference to chromatograms using BioEdit version 7.0.1 We used partial DNA sequences of the mtDNA gene, ies e.g. . DNA wasPhreatomerus (Phreatoicidea: Amphisopidae), namely Amphisopus lintoni Nicholls 1924  D) 0) and after (θ1) expansion) and for the continent-island model of demographic expansion  and m = fraction of individuals from a deme exchanging with other demes). The generalized least-squares approach ARLEQUIN v.3.1 COI sequences were analysed using a parsimony approach with TCS v.1.21 To examine the relationships among haplotypes more closely than the phylogenetic approach used above, we estimated haplotype networks within each of the nine sub-clades. MtDNA COI sequences among sub-clades and major clades. The subprogram BEAUti v1.4.7 Phreatomerus data set using BEAST . Each analysis was run for 10,000,000 generations, with sampling every 100 generations, and the burn-in was 25% of the total sampled trees . Each analysis was run multiple times and all estimated dates were found to be consistent among different runs.BEAST v1.4.7 et al. Acyca < Acycb; Got1< Got2).Allozyme electrophoresis of whole animal homogenates was undertaken on cellulose acetate gels (Cellogel™) according to the principles and procedures of Richardson a priori grouping by spring, spring group, or mtDNA profile. Where discrete PCO groups were evident and diagnosable from one another by fixed differences  and/or near-fixed differences  at two or more loci, individuals within each distinct PCO group were subsequently subjected to a further round of PCO to assess whether significant additional heterogeneity was present in deeper dimensions. Horner & Adams We initially used Principal CO-ordinates Analysis (PCO) to assess the genetic affinities among all individuals, independent of any We also undertook a range of standard population genetic analyses, using the procedures and software employed by Horner & Adams A final set of Bayesian analyses involved using the R statistical package ‘Geneland’  Coward ; one haplotype shared between Beresford and Coward (BC1); Lake Eyre South (L1–L2); Billa Kalina (K1); Neales ; Hermit Hills ; Davenport (D1–D6); FRAncis Swamp only haplotypes (FRA1–FRA3); Strangways (S1); one haplotype shared between Francis Swamp + Strangways (FS1); and FREeling (FRE1–FRE5). All haplotype sequences have been submitted to Genbank (JQ612592–JQ612655).Overall 597 bp of the mtDNA gene ustralia . Ninety-COI haplotypes among P. latipes of the mound springs revealed three primary clades, hereafter referred to by their rough geographic position relative to Lake Eyre: C , S (Southern) and N (Northern)  . While corthern) , most ofd) was generally moderate to high for all spring complexes of P. latipes (d = 0.35–0.39). In contrast, nucleotide diversity (polymorphism) represented by the mean number of pairwise differences (π) was consistently low to moderate with the highest nucleotide diversity at Strangways and Neales River (π = 11.5–12.9) even though only two haplotypes were sampled at Strangways.Haplotype diversity  in haplotype networks where a single dominant haplotype had a few single point mutations leading to novel and rare haplotypes . AdditioCOI was observed in the Lake Eyre GAB mound spring system. Negative and significant estimates of sF, D were observed in Freeling, Beresford and Hermit Hills spring complexes (0) and after (θ1) expansion under the Demographic Expansion model (M = 14.0) with a few exceptions .Some evidence for a departure from neutrality and population expansion in omplexes . Selection model  indicateThe BEAST analyses all reached convergence, with effective sample size values well above 100. Here we present the results of the Yule model tree prior analyses in The final allozyme dataset comprised genotypes at 25 putative loci for 331 individuals from 19 sites. An initial PCO on all individuals  identifiStatistical tests for each of the 19 sites found no evidence for genotypic departure from Hardy Weinberg expectations and for linkage disequilibrium. We therefore considered it appropriate to undertake site-based analyses of population genetic structure for those taxa which have been sampled from multiple spring complexes such as clades S and C  the deep, older, relationships amongst three primary phylogenetic clades , where the distribution of historical lineages was examined, b) the intermediate genetic relationships, where nine sub-clades (I-IX) were examined with respect to the influence of a changing landscape on lineage divergence, and c) the potential influence of Lake Eyre drainages on gene flow between P. latipes sub-clades.The GAB mound springs in Australia’s arid zone are proving to be complex and dynamic ecological systems with multiple layers to their history. The endemic fauna that are restricted to these springs are considered to be ecologically and evolutionarily relictual from a time when central Australia comprised a wet and temperate habitat The oldest relationships amongst the mound spring faunas pose a challenge for reconstructing historic biogeography and understanding species distributions because of the complex climatic and geological history of the region. In particular, repeated climatic changes in aridification since the mid-late Miocene are likely to have regularly overwritten some previous features of the landscape, especially for aquatic habitats. Here we identified three major clades, concordant for both mtDNA (mean estimate 18.7 (12.2–25.8 95% HPD)–13.5 (8.0–19.6 95% HPD) million years divergent) and allozyme profiles . These clades  were found to be completely allopatric. Nevertheless, clade C included an intriguing geographic outlier, namely the Freeling spring complex. As the most northern spring complex, Freeling is geographically much closer to the spring complexes within the Umbum Creek drainage, albeit in the separate Neales Creek drainage. This pattern, observed in both mtDNA and allozymes, is inconsistent with a simple hypothesis of multiple species originating from a single common ancestor and subsequent isolation by distance. Instead it suggests a more complex phylogeographic history for clade C and, by inference, clade N.The geological history of the mound springs dates back to the late Pliocene, early Pleistocene P. latipes with contrasting results. In addition to three major relict clades, indicated by both sets of markers, we also observed nine well-supported mtDNA lineages , clade S: VIII Wangianna and IX Hermit Hills, clade N: V, VI and VII Neales River). Each of the mtDNA sub-clades within the three major clades displayed similarity in coalescent dates (8.8 (5.7–12.6 95% HPD)–2.2 (0.2–5.1 95% HPD) mya, excluding Neales sub-clade VI which had a date of 12.3 (7.2–17.7 95% HPD) mya). We also observed a match between geographic locality and genetic structure within the Lake Eyre mound spring system, with each of these clades corresponding largely to the grouping of spring complex. This historical grouping defines spatially close springs  situated within a similar geomorphic setting and relative position to Lake Eyre. However, two exceptions were observed; clade N , which was split into three sub-clades, suggesting substantial sub-structuring within the Neales complex , plus two sympatric sub-clades at Strangways . Despite several modest differences between the allozyme and mtDNA perspectives for some spring complexes, a general concordance was observed between all of the sub-clades and the natural groupings of spring complexes for both allozyme data and mtDNA. We therefore consider our findings to be robust enough to support the hypothesis that the intervening landscape and isolation between spring complexes has played an important role in the evolutionary patterns observed where strong genetic structure exists. In particular, these sub-clades likely reflect the geomorphic proximity and similarity in physico-chemical composition of the area, but overall a climate and landscape mediated contraction and subsequent diversification event during the history of P. latipes in the mound springs is predicted to reflect its present day divergences.Recent work on spring snails in Lake Eyre mound springs has led to a proposal known as the ‘stranded in desert springs’ hypothesis which states that widespread spring endemics have historically become stranded in GAB spring refugia due to Miocene aridification in situ, notably the calcrete aquifers in Western Australia P. latipes coincides with dates listed for aridification, a return to wet and subsequent Pleistocene aridification. However, the sub-clade dates are substantially earlier than those estimated for a) formation of mound springs (∼1 mya) and the stony deserts (4-2 mya) COI substitution rate of 1.76% per million years was used to calibrate the clock tree P. latipes was stranded in wetland habitats in the Lake Eyre region earlier than that estimated for snails and that events spanning the mid Miocene-Pleistocene were probably most influential. Under this scenario each sub-clade in P. latipes is thought to represent a contraction of populations within the nine sub-clades to regional habitats  during mid-Miocene aridification, at which time a contraction of aquatic habitats and a fall in water tables are likely to have occurred P. latipes as the arid zone habitat became more inhospitable, particularly during the 100 ka glacial cycles of the late Pleistocene. Subsequently, these fragmented pockets of relictual aquatic habitat were able to maintain large populations of invertebrates and thus have more recently promoted and maintained genetic differentiation across the arid landscape. While our date estimates are consistent with the beginning of lineage diversification following Miocene aridification of Australia, it must be noted that our molecular clock analyses were limited by the use of a standard rate calibration Protamphisopus wianamattensisA major period of aridification 15–7 mya and a ‘return to wet’ is thought to have occurred around 6 mya in the Australian arid zone et al. P. latipes. With few exceptions, we observed little evidence in either dataset of present day gene flow (i.e. shared haplotypes) especially between major clades, which were completely allopatric, or between sub-clades. The only evidence of contemporary migration across the desert we observed was between the Central spring complexes: Francis Swamp ↔ Strangways complexes  and Beresford ↔ Coward complexes. River mediated gene flow is a likely explanation for these shared haplotypes with individuals able to move between spring complexes during periods of major flooding. Interestingly, population genetic structure remains very strong amongst these populations based on allozymes and mostly allopatric mtDNA sub-clades suggesting that typically such migration events are likely to be rare and either do not contribute substantially to the population’s gene pool or mostly involve males.For desert spring species that lack terrestrial dispersal abilities and are unable to disperse passively via phoresy P. latipes, one operating at a broad regional scale  and the others at the mid scale with nine sub-clades (i.e. I-IX) and at the local population level of rivers. While future work will investigate the latter in more detail, all of these levels are relevant for conservation of biodiversity within the nationally protected mound spring community around Lake Eyre. Our results are particularly pertinent given the general phylogeographic patterns observed here for P. latipes mirror those displayed in co-occurring mound spring endemics such as amphipods et al. Here we have identified three major layers of evolutionary diversity in Taken together, our mtDNA and allozyme data argue strongly that clades C, S and N should each be regarded as separate species. The number of independent and concordant taxonomic characters , levels of genetic divergence encountered (mtDNA divergence estimates >15 mya), and overall geographic patterns encountered are, when combined, consistent with the standard operating criteria employed under most modern species concepts Click here for additional data file.Table S1Phreatomerus latipes including the collection number (GAB#), the number of specimens sequenced from a particular location (n), the number of haplotypes (h) and their names (name and frequency in parentheses) and their respective position in the final phylogenetic analysis from Data table containing locality data for (DOCX)Click here for additional data file.Table S2Estimates of time since most recent common ancestor (time per million years) of haplotypes from individual sub-clade/clades based on five coalescent models using a Bayesian coalescent approach with BEAST (DOCX)Click here for additional data file.Table S3Summary of between-site assessments of heterogeneity for clade S sites. The lower triangle indicates which pairwise comparisons among sites were statistically significant across all loci, calculated using Fisher’s method; * = 0.01<P<0.05; *** = P<0.001; ns = not significant . The upper triangle presents the FST value obtained using Geneland. None of the analyses found any statistical evidence of heterogeneity among the five sites included in “all other sites”.(DOCX)Click here for additional data file.Table S4Summary of between-site assessments of heterogeneity for clade C sites .(DOCX)Click here for additional data file.