Comparison of techniques to control the aggressive environmental invasive species Galenia pubescens in a degraded grassland reserve, Victoria, Australia.

Across many southern regions of Australia, native grasslands have become seriously threatened by human activity, with only a fraction of the original areas remaining undisturbed. In particular, the introduction and establishment of exotic invasive weeds has caused significant degradation to the ecosystems in these areas by contributing to a decrease in native plant density and diversity, and this has ultimately led to major changes to the ecosystem structure and function. One such example is Galenia pubescens. Our objective of this study was to assess the effectiveness of four different attempts to control G. pubescens: herbicide control with glyphosate; organic herbicide control with pine oil; the application of mulch; and the addition of seeds of native species to the seedbank. Results shows that any one single control strategy is insufficient to control G. pubescens, and, in addition, it has shown that regeneration of native vegetation is limited unless direct seeding is applied. There was a strong indication that a combined strategy employing more than two of the aforementioned techniques is likely to be the most effective approach, at least in the short term. Underscoring the complexity of this task, our analysis on foliage cover of G. pubescens shows that the interaction of pine oil and glyphosate treatments appeared to be very effective after six months, but were not so effective after 18 months. By contrast, seeding with native seeds was not particularly effective at six months, but its longer-term contribution appears to be effective at 18 months. Further, our results obtained from the seedbank abundance study indicate that time alone was not a significant factor in restoration of the grasslands (p = 0.165); however there were interactions with time, shown by time*glyphosate (p = 0.008) and time*seeding (p = 0.016). Both interactions indicated that the applications of glyphosate and seeding were more beneficial after 18 months compared to six months. However, full regeneration of invaded native grasslands may not be possible unless further restoration programs are re-implemented after the first cycle of G. pubescens' treatments have been completed.

Intra-luminal gene therapy in the porcine artery using a recombinant adeno-associated virus 9.

The ability to improve or restore blood flow and promote healing in ischemic tissue has many potential clinical applications. Augmentation by direct delivery of growth factors may further enhance results, but requires a method for sustained delivery. In this study, we have tested the ability of adeno-associated virus 9 (AAV9) delivered within the lumen of a porcine artery to transfect the vessel and produce a desired product. The marker chosen was green fluorescent protein (GFP) (Ke et al., 2011). In 4 farm pigs the cranial tibial artery was surgically exposed. The vessel was temporarily clamped proximally, and divided distally. A cannula was placed intraluminally, and the arterial segment was injected with 1×10E13 particles of AAV9.CB7.CI.GFP·WPRE.rBG. At 14days the transfected cranial tibial artery as well as the liver, spleen and kidneys were harvested. ELISA and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were used to analyze the artery for GFP production. Significant GFP expression was seen in all transfected cranial tibial vessels, as determined by both GFP protein production (ELISA) and mRNA (RT-qPCR). No GFP was identified in liver, spleen or kidney, nor in the no-GFP control animal artery. Adeno-associated virus 9 is an appropriate vector for gene therapy experiments in the porcine artery model. This vector, and the intraluminal deliver method described result in robust gene expression at 2weeks without evident systemic spill of the virus. The ability to limit delivery of the gene to an isolated segment of vessel is desirable for future research applications.

CXCR4 and CXCR7 Mediate TFF3-Induced Cell Migration Independently From the ERK1/2 Signaling Pathway.

PURPOSE: Trefoil factor family (TFF) peptides, and in particular TFF3, are characteristic secretory products of mucous epithelia that promote antiapoptosis, epithelial migration, restitution, and wound healing. For a long time, a receptor for TFF3 had not yet been identified. However, the chemokine receptor CXCR4 has been described as a low affinity receptor for TFF2. Additionally, CXCR7, which is able to heterodimerize with CXCR4, has also been discussed as a potential TFF2 receptor. Since there are distinct structural similarities between the three known TFF peptides, this study evaluated whether CXCR4 and CXCR7 may also act as putative TFF3 receptors. METHODS: We evaluated the expression of both CXCR4 and CXCR7 in samples of human ocular surface tissues and cell lines, using RT-PCR, immunohistochemistry, and Western blot analysis. Furthermore, we studied possible binding interactions between TFF3 and the receptor proteins in an x-ray structure-based modeling system. Functional studies of TFF3-CXCR4/CXCR7 interaction were accomplished by cell culture-based migration assays, flow cytometry, and evaluation of activation of the mitogen-activated protein (MAP) kinase signaling cascade. RESULTS: We detected both receptors at mRNA and protein level in all analyzed ocular surface tissues, and in lesser amount in ocular surface cell lines. X-ray structure-based modeling revealed CXCR4 and CXCR7 dimers as possible binding partners to TFF3. Cell culture-based assays revealed enhanced cell migration under TFF3 stimulation in a conjunctival epithelial cell line, which was completely suppressed by blocking CXCR4 and/or CXCR7. Flow cytometry showed increased proliferation rates after TFF3 treatment, while blocking both receptors had no effect on this increase. Trefoil factor family 3 also activated the MAP kinase signaling cascade independently from receptor activity. CONCLUSIONS: Dimers CXCR4 and CXCR7 are involved in TFF3-dependent activation of cell migration, but not cell proliferation. The ERK1/2 pathway is activated in the process, but not influenced by CXCR4 or CXCR7. These results implicate a dependence of TFF3 activity as to cell migration on the chemokine receptors CXCR4 and CXCR7 at the ocular surface.

Isolation and Investigation of Presumptive Murine Lacrimal Gland Stem Cells.

PURPOSE: Aqueous tear deficiency due to lacrimal gland insufficiency is one of the major causes of dry eye disease. In severe cases, such as Sjogren's syndrome, Stevens-Johnson syndrome, or ocular cicatricial pemphigoid, therapy with artificial tears is often insufficient to relieve severe discomfort, prevent progressive ocular surface disease, or enable visual rehabilitation by corneal transplantation. Cell or organ generation from stem cells, resulting in tear-like secretion, presents an option as a suitable alternative treatment. To obtain deeper insights into lacrimal gland stem cells we analyzed murine lacrimal glands for markers of pluripotency, self-renewal, and differentiation. METHODS: A special, patented technique with mechanical and enzymatic digestion was used to generate high numbers of cells in vitro from murine lacrimal glands. These presumptive "murine lacrimal gland stem cells" ("mLGSCs") can be propagated as monolayer cultures over multiple passages. By means of RT-PCR, Western blot, and immunohistochemistry, markers of pluripotency and differentiation were demonstrated. Hanging drop culture was used to build organoid bodies from mLGSCs to investigate their spontaneous differentiation in three-dimensional culture with histology, immunohistochemistry, and transmission electron microscopy methods. RESULTS: Isolated mLGSCs were cultured over more than 65 passages. Murine lacrimal gland stem cells expressed markers of pluripotency such as Nanog, Sox2, Kruppel-like factor 4 (Klf4), as well as early-lineage markers of all three germ layers. Three-dimensional culture of these cells revealed their ability to differentiate into various cell types. CONCLUSIONS: Our results suggest that mLGSCs were isolated and cultured successfully. These cells have the ability to differentiate into all three germ layers. The results provide further insights into lacrimal gland stem cell physiology for engineering of a lacrimal gland construct to treat severe cases of tear deficiency in the future.

Comparing Humoral and Cellular Immune Response Against HBV Vaccine in Kidney Transplant Patients.

Host protection upon vaccination usually results from the complex interplay of humoral and cellular components of the immune system. Exploring hepatitis B surface antigen (HBsAg)-specific T cell responses and their correlation with humoral responses under immunosuppression, we analyzed 51 renal transplant recipients, differing in HBV vaccine-specific antibody titers (non [NRs]-, low [LRs]-, and high responders [HRs]) and in 22 healthy controls (HCs) in a cross-sectional study. HBsAg-specific T cells were analyzed by flow cytometry according to expression of activation markers CD40L and/or CD69, and the cytokines IFNγ, IL-2, TNFα, and IL-17. No significant differences in responder rate and magnitude of HBsAg-specific T cell responses were found between HCs and HRs. Interestingly, HBsAg-specific Th-cells were also observed in 50% of humoral NRs. Frequencies of HBsAg-specific CD40L+ Th-cells were significantly higher in HRs compared to LRs (p = 0.009) and in LRs in comparison to NRs (p = 0.043). All but NRs showed a predominance of multi-potent HBsAg-specific TNFα+IL-2+ Th-cells. As expected, HBsAg-specific CD8(+) T cells were rarely found. In conclusion, mounting of hepatitis B vaccine-specific T cell responses is possible in kidney transplant recipients despite immunosuppression. Detection of HBV-specific Th-cells in a significant proportion of humoral NRs contributes to the current discussion on conferring immune protection by cellular memory in such patients.

Phosphorylation, nitrosation and plasminogen K3 modulation make VDAC-1 lucid as part of the extrinsic apoptotic pathway-Resulting thesis: Native VDAC-1 indispensible for finalisation of its 3D structure.

Native and recombinant VDAC preparations differ in their acetylation, phosphorylation and nitrosation state; additionally, proteineous modulators are missing in the latter. They thus vary in channel characteristics, as can be taken from comparative black lipid bilayer experiments. Furthermore, the multi-compartment expression makes expect even differing native VDAC-1 molecules. Recent structural work on mammalian VDAC-1 has only used recombinant material, refolded from Escherichia coli inclusion bodies. While this approach established the basic three-dimensional structure of VDAC-1, a ß-barrel set up by nineteen ß-pleated sheets, dissent is on positioning and movements of its free N-terminal helical peptide stretch preceding ß-pleated sheet-1. A synopsis of data concerning posttranslational modifications, cyto-topology and physiology of native VDAC-1, from my point of view, suggests that the finalisation of its three-dimensional structure will need native channel preparations to be studied. Concerning relevance, recent evidence on the regulation of cell membrane-integrated VDAC-1 by posttranslational modifications and proteineous modulators, taken together with experimental demonstrations that VDAC-1 is involved in cell volume regulation, it thus may be part of the extrinsic apoptotic pathway can hopefully help to understand some relevant medical syndromes, e.g. cystic fibrosis, Alzheimer's disease, autism and malaria.

Morphological alterations within the peripheral fixation of the iris dilator muscle in eyes with pigmentary glaucoma.

PURPOSE: To analyze the peripheral fixation of the iris dilator muscle in normal eyes and in eyes with pigmentary glaucoma (PG). METHODS: Using 63 control eyes (age 18 months-99 years), the peripheral iris dilator was investigated by light microscopy, immunohistochemistry, and electron microscopy. Development was studied using 18 differently aged fetal eyes stained immunohistochemically against α-smooth muscle (SM) actin. The peripheral iris dilator muscle in PG was analyzed using semithin and ultrathin sections of six glutaraldehyde-fixed eyes from three donors aged 38, 62, and 74 years. RESULTS: In normal eyes, the peripheral end of the iris dilator muscle is arranged in a sphincter-like manner. Arcade-shaped tendinous connections associated with myofibroblasts (iridial strands) anchor the iris dilator within the elastic-fibromuscular ciliary meshwork that also serves as fixation area for the elastic tendons of the inner ciliary muscle portions. The iridial strands are innervated and can adapt their length during accommodation. The PG eyes show incomplete circular bundles and iridial strands that are mainly anchored to the iris stroma and the flexible uveal parts of the trabecular meshwork. CONCLUSIONS: The normal anchorage of the peripheral iris dilator and its presumably neuronally regulated length adaptation stabilize the peripheral iris during accommodation. Insufficient fixation in PG could promote posterior bowing of the iris with rubbing against the zonular fibers and pigment liberation from the iris pigmented epithelium.

Search for solar axions by the CERN axion solar telescope with 3He buffer gas: closing the hot dark matter gap.

The CERN Axion Solar Telescope has finished its search for solar axions with (3)He buffer gas, covering the search range 0.64 eV ≲ ma ≲ 1.17 eV. This closes the gap to the cosmological hot dark matter limit and actually overlaps with it. From the absence of excess x rays when the magnet was pointing to the Sun we set a typical upper limit on the axion-photon coupling of gaγ ≲ 3.3 × 10(-10) GeV(-1) at 95% C.L., with the exact value depending on the pressure setting. Future direct solar axion searches will focus on increasing the sensitivity to smaller values of gaγ, for example by the currently discussed next generation helioscope International AXion Observatory.

Opening up of plasmalemma type-1 VDAC to form apoptotic "find me signal" pathways is essential in early apoptosis - evidence from the pathogenesis of cystic fibrosis resulting from failure of apoptotic cell clearance followed by sterile inflammation.

Cell membrane-standing type-1 VDAC is involved in cell volume regulation and thus apoptosis. The channel has been shown to figure as a pathway for osmolytes of varying classes, ATP included. An early event in apoptotic cell death is the release of "find me signals" by cells that enter the apoptotic process. ATP is one of those signals. Apoptotic cells this way attract phagocytes for an immunologically silent cell clearance. Thus, whenever apoptosis fails by a blockade of plasmalemma type-1 VDAC processes of sterile inflammation must be assumed for cell elimination. This is evident from a close look on the pathogenetic process of cystic fibrosis (CF). However, in normal airway epithelia two different anion channels cooperate to guarantee an appropriate volume of airway surface liquid (ASL) necessary for surface clearing: the cystic fibrosis conductance regulator (CFTR) and the outwardly rectifying chloride channel (ORCC) complex also called "alternate chloride channel" and under the control of the CFTR. There are arguments, that type-1 VDAC forms the channel part of the ORCC complex, and it has been shown that CFTR and type-1 VDAC co-localize in the apical membranes of human surface respiratory epithelium. In cystic fibrosis, the central cAMP-dependent regulation of ion and water transport via functional CFTR is lost. Here, CFTR molecules do not reach the apical membranes of airway epithelia anymore or work in an insufficient way, respectively. In addition, type-1 VDAC is no longer available to work as a "find me signal" pathway. In consequence, clearing away of apoptotic cells is blocked. There are experimental data on the channel characteristics of type-1 VDAC under the anion channel blocker DIDS (4,4-diisothiocyanato-stilbenedisulphonic acid) that argue in favor of this hypothesis. Together, type-1 VDAC should be kept as a "find me signal" pathway, which may give way to several classes of such signals.

Parents who refuse to vaccinate their infants: paediatricians' feelings and treatment intentions.

AIM: The aim of this study was to evaluate paediatricians' attitudes and emotions towards parents who refuse to vaccinate their infants and to assess their reactions, suggestions and practices. DESIGN: The study group consisted of 376 paediatricians in Israel, who completed the emailed research questionnaire anonymously. RESULTS: Although the vast majority of paediatricians agreed that vaccination was in the baby's best interest (92.2%), only a small percentage (3.5%) felt that there should be some scientific justification behind a parent's refusal. The majority (70.7%) of those surveyed expressed negative feelings towards refusing parents. Despite this, more than a third (36.9%) agreed that parents have the right to decide (28.9% disagreed) and a third (36.8%) agreed that vaccinations should be officially enforced (35.8% disagreed). Only a very small percentage of the paediatricians (1.8%) said they would object to treating infants who had not been vaccinated. CONCLUSION: Paediatricians face a conflict between two opposing values: the importance of immunization versus the parents' rights to decide what is best for their own child. Therefore, they are in favour of gentle persuasion or official enforcement. We believe that experts in modern communication could help paediatricians to convey the positive benefits of vaccination to parents.

Insulin-like factor 3 promotes wound healing at the ocular surface.

Tear fluid is known to contain many different hormones with relevance for ocular surface homeostasis. We studied the presence and functional role of insulin-like factor 3 (INSL3) and its cognate receptor RXFP2 (relaxin/insulin-like family peptide receptor 2) at the ocular surface and in tears. Expression of human INSL3 and RXFP2 was determined in tissues of the ocular surface and lacrimal apparatus; in human corneal (HCE), conjunctival (HCjE), and sebaceous (SC) epithelial cell lines; and in human tears by RT-PCR and ELISA. We investigated effects of human recombinant INSL3 (hrINSL3) on cell proliferation and cell migration and the influence of hrINSL3 on the expression of MMP2, -9, and -13 and TIMP1 and -2 was quantified by real-time PCR and ELISA in HCE, HCjE, and SC cells. We used a C57BL/6 mouse corneal defect model to elucidate the effect of topical application of hrINSL3 on corneal wound healing. INSL3 and RXFP2 transcripts and INSL3 protein were detected in all tissues and cell lines investigated. Significantly higher concentrations of INSL3 were detected in tears from male vs. female volunteers. Stimulation of HCE, HCjE, and SC with hrINSL3 significantly increased cell proliferation in HCjE and SC and migration of HCjE. Treatment with hrINSL3 for 24 hours regulated MMP2, TIMP1, and TIMP2 expression. The local application of hrINSL3 onto denuded corneal surface resulted in significantly accelerated corneal wound healing in mice. These findings suggest a novel and gender-specific role for INSL3 and cognate receptor RXFP2 signaling in ocular surface homeostasis and determined a novel role for hrINSL3 in corneal wound healing.

New findings concerning vertebrate porin II--on the relevance of glycine motifs of type-1 VDAC.

New findings concerning vertebrate porin part I was published in 1997, then summarizing early data and reflections regarding the molecular structure of vertebrate voltage-dependent anion-selective channels, VDAC/eukaryotic porin, and the extra-mitochondrial expression pattern of human type-1 VDAC. Meanwhile, endeavors of different laboratories confirmed and widened this beginning by encircling the function of the channels. Regarding the function of mitochondrial outer membrane-standing VDACs the channels are established parts of the intrinsic apoptotic pathway and thus therapeutic targets in studies on several diseases: cancer, Alzheimer's disease, Down Syndrome, Parkinson's disease, Amyotrophic Lateral Sclerosis, cystic fibrosis and malaria. Regarding cell membrane-integrated type-1 VDAC it has been documented by different approaches that this porin channel is engaged in cell volume regulation, trans-membrane electron transport and apoptosis. Furthermore, new data insinuate a bridging of extrinsic and intrinsic apoptotic pathways, putatively gaining relevance in Alzheimer research. Mammalian type-1 VDAC, a ß-barrel, is basically built up by nineteen ß-sheets connected by peptide stretches of varying lengths. The molecule also comprises an N-terminal stretch of some twenty amino acids which, according to biochemical data, traverses the channel lumen towards the cytosolic surface of outer mitochondrial membranes or the plasma lemma, respectively and works as voltage sensor in channel gating. In artificial lipid bilayers VDACs figure as anion or cation-channels, as VDACs are permeable to both cations and anions, with voltage shifts changing the relative permeability. Type-1 VDAC carries several motifs where glycine residues are in critical positions. Motifs of this type, on the on hand, are established nucleotide binding sites. On the other hand, the GxxxG motifs are also discussed as relevant peptide dimerization/aggregation/membrane perturbation motifs. Finally, GxxxG motifs bind cholesterol. Type-1 VDAC shows one such GxxxG motif at the proximal end of its N-terminal voltage sensor while amyloid Aß peptides include three of them in series. Noteworthy, two additional may be modified versions, GxxxGxG and GxxGxxxG, are found on ß-sheet 19 or 9, respectively. Recent data have allowed speculating that amyloid Aß induces apoptosis via opening type-1 VDAC in cell membranes of hypo-metabolic neurons, a process most likely running over life time--as leaves fall from trees in the tropics--and ending in Alzheimer's disease whenever critical brain regions are affected. The expression of GxxxG motifs on either reactant under consideration is in line with this model of Alzheimer's disease pathogenesis, which clearly differs from the amyloid Aß cascade theory, and which can, furthermore, be understood as a basic model for apoptosis induction. However, to assume randomly distributed interactions of body wide found amyloid Aß peptides with the N-terminal voltage sensors of ubiquitously expressed cell membrane-standing human type-1 VDAC opens up a new view on Alzheimer's disease, which might even include a clue on systemic aspects of the disease. While elaborating this concept, my focus was at first only on the GxxxG motif at the proximal end of the N-terminal voltage sensor of type-1 VDAC. Here, I include a corresponding sequence stretch on the channel's ß-sheet 19, too.

Relaxin 2 is functional at the ocular surface and promotes corneal wound healing.

PURPOSE: We aimed to determine if the insulin-like peptide hormone relaxin 2 (RLN2) is expressed at the ocular surface and in tears and if RLN2 influences wound healing at the ocular surface, which is associated with extracellular matrix (ECM) remodeling. METHODS: We analyzed transcript levels of human RLN2 and its cognate relaxin-like receptors RXFP1 and RXFP2 in tissues of the ocular surface, lacrimal apparatus, and human corneal (HCE), conjunctival (HCjE) and sebaceous (SC) cell lines. We analyzed effects of human RLN2 on cell proliferation and migration and quantified mRNA expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in HCE, HCjE, and SC. Using an alkali-induced corneal wounding model, we analyzed the wound healing rate in C57BL/6 mice eyes after topically applied RLN2. RESULTS: The presence of RLN2, RXFP1, and RXFP2 transcripts was detected in lacrimal gland, eyelid, conjunctiva, cornea, primary corneal fibroblasts, nasolacrimal ducts, and all three cell lines. ELISA revealed RLN2 protein in all ocular surface tissues analyzed and in human tears. Stimulation of HCE, HCjE, and SC with RLN2 significantly increased cell proliferation and migration. Relative mRNA expression levels of MMP2, MMP9, TIMP1, and TIMP2 were significantly influenced by RLN2 in all three cell lines at different time points studied. The local application of RLN2 onto denuded corneal surface resulted in significantly elevated corneal wound healing. CONCLUSIONS: Our data support a novel role for the RLN2 ligand-receptor system at the ocular surface and in the lacrimal apparatus as a potential future therapeutic during wound healing at the ocular surface.

On a fully closed state of native human type-1 VDAC enriched in Nonidet P40.

There is indication that human type-1 VDAC/Porin31HL complexes, when purified from highly enriched cell membrane preparations of human B-lymphocytes by classical ion-exchange chromatography in the detergent Nonidet P40, rest in fully closed state, its N-terminus being accessible for mAbs. Cholesterol appears to be involved as a channel modulator. The channel switches to anion-selective or "open state" while being incorporated into black membranes at zero transmembrane potential. In this case, its N-terminus is hidden in the channel lumen. The cation-selective or "closed state" can be induced by transmembrane potentials beyond 30 mV, the N-terminus putatively now being positioned outside the channel lumen. The latter situation might allow one to decide if type-1 VDAC, preincubated with adequate antibodies against its N-terminal part, would enter black membranes in fully closed state or stay in the application medium, respectively, may be complexed to dimers.

Why cancer survivors have a lower risk of Alzheimer disease.

Tying up two recent lines of experimental evidence may help explain this vital issue. On the one hand, data indicate that cancerous transformations of cells include changes in expression level and/or the functionality of multidrug resistance modulators which then disturb chemotherapy. On the other hand, studies have shown that some of the ABC transporters--at the blood-brain barrier--work as effective efflux pumps for amyloid Aß peptides. Amyloid Aß peptides, cut from the amyloid precursor protein of neurons can be assumed to induce brain wide neuronal apoptosis via opening plasma lemma-standing type-1 VDAC/porin channels as shown by in vitro experiments using established neuronal cell lines. However, extrusion of apoptosis inductive Aß by increased ABC transporter activity at the blood-brain barrier from the brain of cancer survivors might abolish this effect. The hypothesis presented can be read as a clue on what in recent literature is referred to as the inverse association of cancer and Alzheimer disease.