TC10 Is Regulated by Caveolin in 3T3-L1 Adipocytes

Background

TC10 is a small GTPase found in lipid raft microdomains of adipocytes. The protein undergoes activation in response to insulin, and plays a key role in the regulation of glucose uptake by the hormone.

Methodology/Principal Findings

TC10 requires high concentrations of magnesium in order to stabilize guanine nucleotide binding. Kinetic analysis of this process revealed that magnesium acutely decreased the nucleotide release and exchange rates of TC10, suggesting that the G protein may behave as a rapidly exchanging, and therefore active protein in vivo. However, in adipocytes, the activity of TC10 is not constitutive, indicating that mechanisms must exist to maintain the G protein in a low activity state in untreated cells. Thus, we searched for proteins that might bind to and stabilize TC10 in the inactive state. We found that Caveolin interacts with TC10 only when GDP-bound and stabilizes GDP binding. Moreover, knockdown of Caveolin 1 in 3T3-L1 adipocytes increased the basal activity state of TC10.

Conclusions/Significance

Together these data suggest that TC10 is intrinsically active in vivo, but is maintained in the inactive state by binding to Caveolin 1 in 3T3-L1 adipocytes under basal conditions, permitting its activation by insulin.     

In Vivo Early Intervention and the Therapeutic Effects of 20(S)-Ginsenoside Rg3 on Hypertrophic Scar Formation

Background

Intra-lesional injections of corticosteroids, interferon, and chemotherapeutic drugs are currently the most popular treatments of hypertrophic scar formation. However, these drugs can only be used after HS is formed, and not during the inflammatory phase of wound healing, which regulates the HS forming process.

Objective

To investigate a new, effective, combining therapeutic and safe drug for early intervention and treatment for hypertrophic scars.

Methods

Cell viability assay and flow cytometric analysis were studied in vitro. Animal studies were done to investigate the combining therapeutic effects of 20(S)-ginsenoside Rg3 (Rg3) on the inflammatory phase of wound healing and HS formation.

Results

In vitro studies showed that Rg3 can inhibit HS fibroblasts proliferation and induce HSF apoptosis in a concentration-dependent manner. In vivo studies demonstrated that Rg3 can limit the exaggerated inflammation, and do not delay the wound healing process, which indicates that Rg3 could be used as an early intervention to reduce HS formation. Topical injection of 4 mg/mL Rg3 can reduce HS formation by 34%. Histological and molecular studies revealed that Rg3 injection inhibits fibroblasts proliferation thus reduced the accumulation of collagen fibers, and down-regulates VEGF expression in the HS tissue.

Conclusion

Rg3 can be employed as an early intervention and a combining therapeutic drug to reduce inflammation and HS formation as well.     

Characterization of ftsZ Mutations that Render Bacillus subtilis Resistant to MinC

Background

Cell division in Bacillus subtilis occurs precisely at midcell. Positional control of cell division is exerted by two mechanisms: nucleoid occlusion, through Noc, which prevents division through nucleoids, and the Min system, where the combined action of the MinC, D and J proteins prevents formation of the FtsZ ring at cell poles or recently completed division sites.

Methodology/Principal Findings

We used a genetic screen to identify mutations in ftsZ that confer resistance to the lethal overexpression of the MinC/MinD division inhibitor. The FtsZ mutants were purified and found to polymerize to a similar or lesser extent as wild type FtsZ, and all mutants displayed reduced GTP hydrolysis activity indicative of a reduced polymerization turnover. We found that even though the mutations conferred in vivo resistance to MinC/D, the purified FtsZ mutants did not display strong resistance to MinC in vitro.

Conclusions/Significance

Our results show that in B. subtilis, overproduction of MinC can be countered by mutations that alter FtsZ polymerization dynamics. Even though it would be very likely that the FtsZ mutants found depend on other Z-ring stabilizing proteins such as ZapA, FtsA or SepF, we found this not to be the case. This indicates that the cell division process in B. subtilis is extremely robust.     

Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2) from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis

Background

Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence.

Findings

By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin.

Conclusions

Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0012-0) contains supplementary material, which is available to authorized users.     

The potential roles of strigolactones and brassinosteroids in the autoregulation of nodulation pathway

Background and Aims

The number of nodules formed on a legume root system is under the strict genetic control of the autoregulation of nodulation (AON) pathway. Plant hormones are thought to play a role in AON; however, the involvement of two hormones recently described as having a largely positive role in nodulation, strigolactones and brassinosteroids, has not been examined in the AON process.

Methods

A genetic approach was used to examine if strigolactones or brassinosteroids interact with the AON system in pea (Pisum sativum). Double mutants between shoot-acting (Psclv2, Psnark) and root-acting (Psrdn1) mutants of the AON pathway and strigolactone-deficient (Psccd8) or brassinosteroid-deficient (lk) mutants were generated and assessed for various aspects of nodulation. Strigolactone production by AON mutant roots was also investigated.

Key Results

Supernodulation of the roots was observed in both brassinosteroid- and strigolactone-deficient AON double-mutant plants. This is despite the fact that the shoots of these plants displayed classic strigolactone-deficient (increased shoot branching) or brassinosteroid-deficient (extreme dwarf) phenotypes. No consistent effect of disruption of the AON pathway on strigolactone production was found, but root-acting Psrdn1 mutants did produce significantly more strigolactones.

Conclusions

No evidence was found that strigolactones or brassinosteroids act downstream of the AON genes examined. While in pea the AON mutants are epistatic to brassinosteroid and strigolactone synthesis genes, we argue that these hormones are likely to act independently of the AON system, having a role in the promotion of nodule formation.     

Mice Lacking the SLAM Family Member CD84 Display Unaltered Platelet Function in Hemostasis and Thrombosis

Background

Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation—platelet adhesion and aggregation—have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.

Objective

The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.

Methods and Results

We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84^−/− platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84^−/− mice. In vivo, Cd84^−/− mice exhibited unaltered hemostatic function and arterial thrombus formation.

Conclusion

These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.     

CXCL8 Chemokines in Teleost Fish: Two Lineages with Distinct Expression Profiles during Early Phases of Inflammation

Background

During the inflammatory process, chemokine CXCL8 plays a pivotal role in recruitment of human neutrophilic granulocytes. A diversity of sequences similar to CXCL8 was reported in fish, but their evolutionary relationships and functional homology with their human homolog remain unclear.

Principal Findings

We screened fish genomes to seek for sequences related to CXCL8. A first lineage was retrieved in all teleosts, while a second CXCL8 lineage was found in zebrafish and carp only. An early inflammatory function for both lineages was indicated by several lines of evidence. The induction of carp CXCL8s, CXCb, and CXC receptor-1 and -2 was analyzed after in vitro stimulation of leukocyte subpopulations and in two in vivo inflammation models. Recombinant proteins of carp CXCL8 proteins were produced and showed significant chemotactic activity for carp leukocytes.

Conclusions

While both carp CXCL8s appear to be functional homologs of mammalian CXCL8, their different induction requirements and kinetics evoke a gene-specific sub-functionalization.     

Increased Migration of Human Mesenchymal Stromal Cells by Autocrine Motility Factor (AMF) Resulted in Enhanced Recruitment towards Hepatocellular Carcinoma

Background and Aims

Several reports described the migration of human mesenchymal stromal cells (MSCs) towards tumor-released factors. Autocrine motility factor (AMF) is produced by several tumors including hepatocellular carcinoma (HCC). The aim of this study was to analyze AMF involvement on MSC migration towards human HCC.

Methods

Production of AMF by HCC tumors was evaluated by western analysis. The effects of AMF on MSCs from different sources (bone marrow, adipose tissue and perivascular cells from umbilical cord) were analyzed using in vitro migration assay; metalloproteinase 2 (MMP2) activity and expression of critical genes were studied by zymography and qRT-PCR, respectively. To assess AMF involvement on the in vivo MSC migration, noninvasive fluorescence imaging was performed. To test the effect of AMF-primed MSCs on tumor development, in vitro proliferation and spheroids growth and in vivo tumor volume were evaluated.

Results

AMF produced by HCC was found to induce migration of different MSCs in vitro and to enhance their MMP2 activity. Stimulation of MSCs with recombinant AMF (rAMF) also induced the in vitro adhesion to endothelial cells in coincidence with changes in the expression levels of MMP3, AMF receptor, caveolin-1, and -2 and GDI-2. Importantly, stimulation of MSCs with rAMF increased the in vivo migration of MSCs towards experimental HCC tumors. AMF-priming of MSCs did not induce a pro-tumorigenic effect on HCC cells neither in vivo nor in vitro.

Conclusion

AMF plays a role in MSC recruitment towards HCC. However, its ability to increase MSC migration to HCC for therapeutic purposes merits further evaluation.     

Anti-Angiogenic Effects of a Mutant Endostatin: A New Prospect for Treating Retinal and Choroidal Neovascularization

Purpose

Pathological fundus angiogenesis is a major cause of vision loss in retina diseases. Endostatin, a C-terminal fragment of collagen XVIII, is an endogenous anti-angiogenic protein. The present study aimed to investigate the in vitro and in vivo anti-angiogenic properties of two proteins: an N-terminal H1D/H3D mutant endostatin (M-ES) and a polyethylene glycol propionaldehyde (PEG) covalent M-ES (PEG-M-ES).

Methods

M-ES and PEG-M-ES properties were characterized in vitro using a zinc ion binding assay and a stability test. Activity assays, including migration, proliferation, and tube formation assays, were performed with human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs). Mouse oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV) models were used to evaluate in vivo anti-angiogenic effects. In addition, a rabbit model was used to study the retinal pharmacokinetic profile following an intravitreal injection.

Results

The results indicated that the H1D/H3D mutations of endostatin reduced the zinc binding capacity of M-ES and facilitated PEG covalent binding. PEG-M-ES was more stable and persisted longer in the retina compared with M-ES. The in vitro studies demonstrated that M-ES and PEG-M-ES inhibited HRMEC and HUVEC proliferation, migration, and tube formation more efficiently than ES. In vivo, a single intravitreal injection of M-ES and PEG-M-ES significantly decreased neovascularization in both the OIR and CNV animal models.

Conclusion

The present study demonstrated for the first time that PEG-M-ES exhibits a long-term inhibitory effect on neovascularization in vitro and in vivo. These data suggest that PEG-M-ES may represent an innovative therapeutic strategy to prevent fundus neovascularization.     

C. albicans colonization of human mucosal surfaces

Background

Candida albicans is a low level commensal organism in normal human populations with the continuous potential to expand and cause a spectrum of clinical conditions.

Methodology/Principal Findings

Using ex vivo human organ cultures and populations of primary human cells, we have developed several related experimental systems to examine early-stage interactions between C. albicans and mucosal surfaces. Experiments have been conducted both with exogenously added C. albicans and with overtly normal human mucosal surfaces supporting pre-existing infections with natural isolates of Candida. Under different culture conditions, we have demonstrated the formation of C. albicans colonies on human target cells and filament formation, equivalent to tissue invasion.

Conclusions/Significance

These organ culture systems provide a valuable new resource to examine the molecular and cellular basis for Candida colonization of human mucosal surfaces.     

Use of Autologous Human mesenchymal Stromal Cell/Fibrin Clot Constructs in Upper Limb Non-Unions: Long-Term Assessment

Background

Tissue engineering appears to be an attractive alternative to the traditional approach in the treatment of fracture non-unions. Mesenchymal stromal cells (MSCs) are considered an appealing cell source for clinical intervention. However, ex vivo cell expansion and differentiation towards the osteogenic lineage, together with the design of a suitable scaffold have yet to be optimized. Major concerns exist about the safety of MSC-based therapies, including possible abnormal overgrowth and potential cancer evolution.

Aims

We examined the long-term efficacy and safety of ex vivo expanded bone marrow MSCs, embedded in autologous fibrin clots, for the healing of atrophic pseudarthrosis of the upper limb. Our research work relied on three main issues: use of an entirely autologous context (cells, serum for ex vivo cell culture, scaffold components), reduced ex vivo cell expansion, and short-term MSC osteoinduction before implantation.

Methods and Findings

Bone marrow MSCs isolated from 8 patients were expanded ex vivo until passage 1 and short-term osteo-differentiated in autologous-based culture conditions. Tissue-engineered constructs designed to embed MSCs in autologous fibrin clots were locally implanted with bone grafts, calibrating their number on the extension of bone damage. Radiographic healing was evaluated with short- and long-term follow-ups (range averages: 6.7 and 76.0 months, respectively). All patients recovered limb function, with no evidence of tissue overgrowth or tumor formation.

Conclusions

Our study indicates that highly autologous treatment can be effective and safe in the long-term healing of bone non-unions. This tissue engineering approach resulted in successful clinical and functional outcomes for all patients.     

Predicted changes in vegetation structure affect the susceptibility to invasion of bryophyte-dominated subarctic heath

Background and Aims

A meta-analysis of global change experiments in arctic tundra sites suggests that plant productivity and the cover of shrubs, grasses and dead plant material (i.e. litter) will increase and the cover of bryophytes will decrease in response to higher air temperatures. However, little is known about which effects these changes in vegetation structure will have on seedling recruitment of species and invasibility of arctic ecosystems.

Methods

A field experiment was done in a bryophyte-dominated, species-rich subarctic heath by manipulating the cover of bryophytes and litter in a factorial design. Three phases of seedling recruitment (seedling emergence, summer seedling survival, first-year recruitment) of the grass Anthoxanthum alpinum and the shrub Betula nana were analysed after they were sown into the experimental plots.

Key Results

Bryophyte and litter removal significantly increased seedling emergence of both species but the effects of manipulations of vegetation structure varied strongly for the later phases of recruitment. Summer survival and first-year recruitment were significantly higher in Anthoxanthum. Although bryophyte removal generally increased summer survival and recruitment, seedlings of Betula showed high mortality in early August on plots where bryophytes had been removed.

Conclusions

Large species-specific variation and significant effects of experimental manipulations on seedling recruitment suggest that changes in vegetation structure as a consequence of global warming will affect the abundance of grasses and shrubs, the species composition and the susceptibility to invasion of subarctic heath vegetation.     

Spatial Distribution of Factor Xa,Thrombin, and Fibrin(ogen) on Thrombi at Venous Shear

Background

The generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro)thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.

Methodology/Principal Findings

Fluorescence microscopy was used to study the accumulation of platelets, FXa, (pro)thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca²⁺ signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro)thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl₃. Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro)thrombin and fibrin(ogen).

Conclusions/Significance

FXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).     

Habitat of In Vivo Transformation Influences the Levels of Free Radical Scavengers in Clinostomum complanatum: Implications for Free Radical Scavenger Based Vaccines against Trematode Infections

Background

Since free radical scavengers of parasite origin like glutathione-S-transferase and superoxide dismutase are being explored as prospective vaccine targets, availability of these molecules within the parasite infecting different hosts as well as different sites of infection is of considerable importance. Using Clinostomum complanatum, as a model helminth parasite, we analysed the effects of habitat of in vivo transformation on free radical scavengers of this trematode parasite.

Methods

Using three different animal models for in vivo transformation and markedly different sites of infection, progenetic metacercaria of C. complanatum were transformed to adult ovigerous worms. Whole worm homogenates were used to estimate the levels of lipid peroxidation, a marker of oxidative stress and free radical scavengers.

Results

Site of in vivo transformation was found to drastically affect the levels of free radical scavengers in this model trematode parasite. It was observed that oxygen availability at the site of infection probably influences levels of free radical scavengers in trematode parasites.

Conclusion

This is the first report showing that habitat of in vivo transformation affects levels of free radical scavengers in trematode parasites. Since free radical scavengers are prospective vaccine targets and parasite infection at ectopic sites is common, we propose that infections at different sites, may respond differently to free radical scavenger based vaccines.     

Confocal Laser Endomicroscopy for In Vivo Diagnosis of Clostridium difficile Associated Colitis — A Pilot Study

Background

Clostridium difficile infection (CDI) is one of the most dreaded causes of hospital-acquired diarrhea. Main objective was to investigate whether confocal laser endomicroscopy (CLE) has the capability for in vivo diagnosis of C. difficile associated histological changes. Second objective was to prove the presence of intramucosal bacteria using CLE.

Methods

80 patients were prospectively included, 10 patients were diagnosed with CDI based on toxigenic culture. To validate the presence of intramucosal bacteria ex vivo, CLE was performed in pure C. difficile culture; additionally fluorescence in situ hybridization (FISH) was performed. Finally, CLE with fluorescence labelled oligonucleotide probe specific for C. difficile was performed ex vivo in order to prove the presence of bacteria.

Results

CLE identified CDI-associated histological changes in vivo (sensitivity and accuracy of 88.9% and 96.3%). In addition, intramucosal bacteria were visualized. The presence of these bacteria could be proven by CLE with labeled, specific molecular C. difficile probe and FISH-technique. Based on comparison between CLE and FISH analyses, sensitivity and specificity for the presence of intramucosal bacteria were 100%.

Conclusion

CLE has the potential for in vivo diagnosis of CDI associated colitis. In addition, CLE allowed the detection of intramucosal bacteria in vivo.     

In vivo outer hair cell length changes expose the active process in the cochlea

Background

Mammalian hearing is refined by amplification of the sound-evoked vibration of the cochlear partition. This amplification is at least partly due to forces produced by protein motors residing in the cylindrical body of the outer hair cell. To transmit power to the cochlear partition, it is required that the outer hair cells dynamically change their length, in addition to generating force. These length changes, which have not previously been measured in vivo, must be correctly timed with the acoustic stimulus to produce amplification.

Methodology/Principal Findings

Using in vivo optical coherence tomography, we demonstrate that outer hair cells in living guinea pigs have length changes with unexpected timing and magnitudes that depend on the stimulus level in the sensitive cochlea.

Conclusions/Significance

The level-dependent length change is a necessary condition for directly validating that power is expended by the active process presumed to underlie normal hearing.     

Wing Defects in Drosophila xenicid Mutant Clones Are Caused by C-Terminal Deletion of Additional Sex Combs (Asx)

Background

The coordinated action of genes that control patterning, cell fate determination, cell size, and cell adhesion is required for proper wing formation in Drosophila. Defects in any of these basic processes can lead to wing aberrations, including blisters. The xenicid mutation was originally identified in a screen designed to uncover regulators of adhesion between wing surfaces [1].

Principal Findings

Here, we demonstrate that expression of the βPS integrin or the patterning protein Engrailed are not affected in developing wing imaginal discs in xenicid mutants. Instead, expression of the homeotic protein Ultrabithorax (Ubx) is strongly increased in xenicid mutant cells.

Conclusion

Our results suggest that upregulation of Ubx transforms cells from a wing blade fate to a haltere fate, and that the presence of haltere cells within the wing blade is the primary defect leading to the adult wing phenotypes observed.     

Comparative Study of Nanosecond Electric Fields In Vitro and In Vivo on Hepatocellular Carcinoma Indicate Macrophage Infiltration Contribute to Tumor Ablation In Vivo

Background and Aim

Recurrence and metastasis are associated with poor prognosis in hepatocellular carcinoma even in the patients who have undergone radical resection. Therefore, effective treatment is urgently needed for improvement of patients'' survival. Previously, we reported that nanosecond pulse electric fields (nsPEFs) can ablate melanoma by induction of apoptosis and inhibition of angiogenesis. This study aims to investigate the in vivo ablation strategy by comparing the dose effect of nanosecond electric fields in vitro and in vivo on hepatocellular carcinoma.

Materials and Methods

Four hepatocellular carcinoma cell lines HepG2, SMMC7721, Hep1-6, and HCCLM3 were pulsed to test the anti-proliferation and anti-migration ability of 100 ns nsPEFs in vitro. The animal model of human subdermal xenograft HCCLM3 cells into BALB/c nude mouse was used to test the anti-tumor growth and macrophage infiltration in vivo.

Results

In vitro assays showed anti-tumor effect of nsPEFs is dose-dependant. But the in vivo study showed the strategy of low dose and multiple treatments is superior to high dose single treatment. The macrophages infiltration significantly increased in the tumors which were treated by multiple low dose nsPEFs.

Conclusion

The low dose multiple nsPEFs application is more efficient than high dose single treatment in inhibiting the tumor volume in vivo, which is quite different from the dose-effect relationship in vitro. Beside the electric field strength, the macrophage involvement must be considered to account for effect variability and toxicology in vivo.