Phospholipase C Gamma 2 Is Critical for Development of a Murine Model of Inflammatory Arthritis by Affecting Actin Dynamics in Dendritic Cells

Background

Dendritic cells (DCs) are highly specialized cells, which capture antigen in peripheral tissues and migrate to lymph nodes, where they dynamically interact with and activate T cells. Both migration and formation of DC-T cell contacts depend on cytoskeleton plasticity. However, the molecular bases governing these events have not been completely defined.

Methodology/Principal Findings

Utilizing a T cell-dependent model of arthritis, we find that PLCγ2−/− mice are protected from local inflammation and bone erosion. PLCγ2 controls actin remodeling in dendritic cells, thereby affecting their capacity to prime T cells. DCs from PLCγ2−/− mice mature normally, however they lack podosomes, typical actin structures of motile cells. Absence of PLCγ2 impacts both DC trafficking to the lymph nodes and migration towards CCL21. The interaction with T cells is also affected by PLCγ2 deficiency. Mechanistically, PLCγ2 is activated by CCL21 and modulates Rac activation. Rac1/2−/− DCs also lack podosomes and do not respond to CCL21. Finally, antigen pulsed PLCγ2−/− DCs fail to promote T cell activation and induce inflammation in vivo when injected into WT mice. Conversely, injection of WT DCs into PLCγ2−/− mice rescues the inflammatory response but not focal osteolysis, confirming the importance of PLCγ2 both in immune and bone systems.

Conclusions/Significance

This study demonstrates a critical role for PLCγ2 in eliciting inflammatory responses by regulating actin dynamics in DCs and positions the PLCγ2 pathway as a common orchestrator of bone and immune cell functions during arthritis.     

Comorbidities in Children and Adolescents with AIDS Acquired by HIV Vertical Transmission in Vitória,Brazil

Background

Studying diseases associated with AIDS is essential for establishing intervention strategies because comorbidities can lead to death. The objectives were to describe the frequency of comorbidities and verify their distribution according to demographic, epidemiological and clinical data as well as to classify diseases in children and adolescents with AIDS in Vitória, Brazil.

Methods

A retrospective cohort study was conducted among children with AIDS, as defined according to the criteria established by the Ministry of Health, who acquired HIV via vertical transmission, were aged 0 to 18 years, and were monitored at a referral hospital from January 2001 to December 2011.

Results

A total of 177 patients were included, of whom 97 were female (55%). There were 60 patients (34%) <1 year old, 67 patients (38%) between the ages of 1 and 5, and 50 patients (28%) ≥6 years of age included at the time of admission to the Infectious Diseases Ward. Regarding clinical-immunological classification, 146 patients (82.5%) showed moderate/severe forms of the disease at the time of admission into the Ward, and 26 patients (14.7%) died during the study. The most common clinical signs were hepatomegaly (81.62%), splenomegaly (63.8%), lymphadenopathy (68.4%) and persistent fever (32.8%). The most common comorbidities were anaemia (67.2%), pneumonia/septicaemia/acute bacterial meningitis (ABM) (64.2%), acute otitis media (AOM)/recurrent sinusitis (55.4%), recurrent severe bacterial infections (47.4%) and dermatitis (43.1%). An association between severe clinical-immunological classification and admission to the Ward for children aged less than one year old was found for several comorbidities (p<0.001).

Conclusion

Delayed diagnosis was observed because the majority of patients were admitted to the Infectious Diseases Ward at ≥1 year of age and were already presenting with serious diseases. The general paediatrician should be alert to this possibility to make an early diagnosis in children infected with HIV.     

SMG7 is a 14-3-3-like adaptor in the nonsense-mediated mRNA decay pathway

In metazoa, regulation of the phosphorylation state of UPF1 is crucial for nonsense-mediated mRNA decay (NMD), a process by which aberrant mRNAs containing nonsense mutations are degraded. UPF1 is targeted for dephosphorylation by three related proteins, SMG5, SMG6, and SMG7. We report here the crystal structure of the N-terminal domain of SMG7. The structure reveals that SMG7 contains a 14-3-3-like domain. Residues that bind phosphoserine-containing peptides in 14-3-3 are conserved at the equivalent positions in SMG7. Mutation of these residues impairs UPF1 binding to SMG7 in vitro and UPF1 recruitment to cytoplasmic mRNA decay foci in vivo, suggesting that SMG7 acts as an adaptor in targeting mRNAs associated with phosphorylated UPF1 for degradation. The 14-3-3 site of SMG7 is conserved in SMG5 and SMG6. These data also imply that the homologous human Est1 might have a 14-3-3 function at telomeres, and that phosphorylation events may be important for telomerase regulation.     

Application of the Singular Spectrum Analysis Technique to Study the Recent Hiatus on the Global Surface Temperature Record

Global surface temperature has been increasing since the beginning of the 20^th century but with a highly variable warming rate, and the alternation of rapid warming periods with ‘hiatus’ decades is a constant throughout the series. The superimposition of a secular warming trend with natural multidecadal variability is the most accepted explanation for such a pattern. Since the start of the 21^st century, the surface global mean temperature has not risen at the same rate as the top-of-atmosphere radiative energy input or greenhouse gas emissions, provoking scientific and social interest in determining the causes of this apparent discrepancy. Multidecadal natural variability is the most commonly proposed cause for the present hiatus period. Here, we analyze the HadCRUT4 surface temperature database with spectral techniques to separate a multidecadal oscillation (MDV) from a secular trend (ST). Both signals combined account for nearly 88% of the total variability of the temperature series showing the main acceleration/deceleration periods already described elsewhere. Three stalling periods with very little warming could be found within the series, from 1878 to 1907, from 1945 to 1969 and from 2001 to the end of the series, all of them coincided with a cooling phase of the MDV. Henceforth, MDV seems to be the main cause of the different hiatus periods shown by the global surface temperature records. However, and contrary to the two previous events, during the current hiatus period, the ST shows a strong fluctuation on the warming rate, with a large acceleration (0.0085°C year⁻¹ to 0.017°C year⁻¹) during 1992–2001 and a sharp deceleration (0.017°C year⁻¹ to 0.003°C year⁻¹) from 2002 onwards. This is the first time in the observational record that the ST shows such variability, so determining the causes and consequences of this change of behavior needs to be addressed by the scientific community.     

Synthesis of sulfonamide-containing N-hydroxyindole-2-carboxylates as inhibitors of human lactate dehydrogenase-isoform 5

N-Hydroxyindole-2-carboxylates possessing sulfonamide-substituents at either position 5 or 6 were designed and synthesized. The inhibitory activities of these compounds against isoforms 1 and 5 of human lactate dehydrogenase were analysed, and K_i values of the most efficient inhibitors were determined by standard enzyme kinetic studies. Some of these compounds displayed state-of-the-art inhibitory potencies against isoform 5 (K_i values as low as 5.6 μM) and behaved as competitive inhibitors versus both the substrate and the cofactor.     

Overwintering stress of Vaccinium vitis-idaea in the absence of snow cover

A snow manipulation experiment aimed to assess risks of direct freezing injury, freeze-induced dehydration and winter desiccation in the absence of snow cover on lingonberry (Vaccinium vitis-idaea). Frames with sheet-plastic sides and removable lids were used in this experiment for two purposes: to prevent accumulation of snow in mid-winter and to provide extra heat during early spring. Leaves were analyzed for frost hardiness, tissue water content and osmotic concentrations, and photoinhibition (Fv/Fm) during the period from the 10th of February to the 7th of April. The natural snow accumulation was low indicated by a minor difference in minimum temperatures between the frame treatment and naturally snow-covered plots. The heating effect of the frames started gradually at the end of February along with increasing solar elevation angles, and was highest at the beginning of April. Frost hardiness peaked in March as a consequence of cold periods, but it was practically lost by the beginning of April. Tissue water content decreased gradually at first, becoming greatly decreased later due to the extra heat. In accordance, the tissue osmotic concentrations increased first gradually, followed by a dramatic increase. Photoinhibition increased uniformly with increasing solar radiation, but at the end showed a sharp increment within a few days, obviously also indicating the effect of heating. It was concluded that neither lethal freezing stress nor significant freeze-induced dehydration occurred during the experiment. However, plants that overwintered without snow suffered from severe winter desiccation injuries due to the combination of solar heat and frozen soil. Although the desiccation stress was possibly a lethal factor, it was preceded by long-term and continued photoinhibition. It was concluded that during overwintering, chamaephyte species may suffer from both freezing and winter desiccation in the absence of protecting snow cover. However, during mild winters provided by climatic change scenarios, the risk of winter desiccation will be more probable. In relation to the future climate, it was concluded that winter desiccation and photoinhibition may develop gradually during a snowless winter and would, even if they did not reach a lethal level by themselves, possibly reduce frost hardiness.     

Cross Talk among the Glycoproteins Involved in Herpes Simplex Virus Entry and Fusion: the Interaction between gB and gH/gL Does Not Necessarily Require gD

The gD, gB, and gH/gL glycoprotein quartet constitutes the basic apparatus for herpes simplex virus (HSV) entry into the cell and fusion. gD serves as a receptor binding glycoprotein and trigger of fusion. The conserved gB and gH/gL execute fusion. Central to understanding HSV entry/fusion has become the dissection of how the four glycoproteins engage in cross talk. While the independent interactions of gD with gB and gD with gH/gL have been documented, less is known of the interaction of gB with gH/gL. So far, this interaction has been detected only in the presence of gD by means of a split green fluorescent protein complementation assay. Here, we show that gB interacts with gH/gL in the absence of gD. The gB-gH/gL complex was best detected with a form of gB in which the endocytosis and phosphorylation motif have been deleted; this form of gB persists in the membranes of the exocytic pathway and is not endocytosed. The gB-gH/gL interaction was detected both in whole transfected cells by means of a split yellow fluorescent protein complementation assay and, biochemically, by a pull-down assay. Results with a panel of chimeric forms of gB, in which portions of the glycoprotein bracketed by consecutive cysteines were replaced with the corresponding portions from human herpesvirus 8 gB, favor the view that gB carries multiple sites for interaction with gH/gL, and one of these sites is located in the pleckstrin-like domain 1 carrying the bipartite fusion loop.Entry of herpes simplex virus (HSV) into the cell requires a multipartite apparatus made of a quartet of viral glycoproteins, gD, gB, and the heterodimer gH/gL, and a multistep process that culminates in the fusion of the virion envelope with cell membranes (5, 6, 10, 25, 36, 41). gD serves as the receptor-binding glycoprotein, able to interact with alternative receptors, nectin1, herpesvirus entry mediator (HVEM) and, in some cells, modified heparan sulfate (9, 13, 30, 39). It can also be engineered to accept heterologous ligands able to interact with selected receptors present on tumor cells and thus represents a tool to redirect HSV tropism (21, 28, 29, 42). The heterodimer gH/gL and gB execute fusion and constitute the conserved fusion apparatus across the Herpesviridae family. gB structure in the postfusion conformation shows a trimer with a central coiled coil (19). gH shows elements typical of type 1 fusion glycoproteins, in particular, helices able to interact with membranes, and two heptad repeats potentially able to form a coiled coil (12, 15-18). The discovery that a soluble form of gD enables entry of gD-null virions revealed that gD serves the additional function of triggering fusion and led to the view that the major roles of gD are to sense that virus has reached a receptor-positive cell and to signal to gB and gH/gL that fusion is to be executed (8). Biochemical and structural analyses showed that the C-terminal region of the gD ectodomain, containing the profusion domain required for fusion but not for receptor binding, can undergo major conformational changes (11, 24). Specifically, it binds the gD core and masks or hinders the receptor binding sites, conferring upon the molecule a closed, auto-inhibited conformation (24). Alternatively, it may unfold, conferring upon gD an open conformation. It was proposed that the C terminus of gD unfolds from gD core at receptor binding and recruits gH/gL and gB to a quaternary complex. A key feature of the model was that complexes among the glycoprotein quartet were not preformed, but, rather, they would assemble at the onset of or at fusion execution.Central to understanding HSV entry/fusion has become the dissection of the interactions that occur among the members of the glycoprotein quartet and their significance to the process. A first evidence of a gD-gH/gL interaction was provided in coimmunoprecipitation studies (35). Interactions between gD and gH/gL and between gD and gB were subsequently detected by split green fluorescence protein (GFP) complementation assays, implying that gD can recruit gB and gH/gL independently of one another, a result that argues against a stepwise recruitment of the glycoproteins to gD. In agreement with the proposed model, the interaction between gH/gL and gB was detected in the presence of transfected or soluble gD (1, 2). However, further studies highlighted levels of complexity not foreseen in the initial model. Thus, pull-down analyses showed that the interaction sites in gD with gB and with gH/gL lie in part outside the C-terminal portion of the gD ectodomain, that resting virions contain small amounts of gD in complex with gB and with gH/gL prior to encountering cells, and that de novo gD-gB complexes were not detected at virus entry into the cell (14).A major objective of current studies was to analyze the interaction of gB with gH/gL. We documented the interaction by two independent assays, i.e., by a complementation assay of split yellow fluorescent protein Venus (herein indicated as YFP) (31) in whole cells and, biochemically, by a pull-down assay. The latter was applied recently in our laboratory and is based on the ability of One-Strep-tagged proteins (e.g., gH) to specifically absorb to Strep-Tactin resin and thus retain any protein in complex (14). To preliminarily search for gB regions critical for the interaction with gH/gL, we engineered chimeric forms of HSV-1 and human herpesvirus 8 (HHV-8) gB in which the cysteines were preserved. While none of the chimeras was completely defective in the interaction, the interactions in the chimeras carrying substitutions in the pleckstrin-like domain 1—the domain that carries the bipartite fusion loops—were hampered. Altogether, the results underscore the ability of gB to interact with gH/gL in the absence of gD and favor the view that sites in gB for interaction with gH/gL involve multiple contacts, one of which is located in the domain that carries the fusion loops.     

Photophysical characterization and photodynamic activity of metallo 5-(4-(trimethylammonium)phenyl)-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin in homogeneous and biomimetic media

The photophysical properties and photodynamic effect of Zn(ii), Pd(ii), Cu(ii) and free-base 5-(4-(trimethylammonium)phenyl)-10,15,20-tris(2,4,6-trimethoxy phenyl)porphyrin (H2P) iodide have been studied in N,N-dimethylformamide (DMF) and in different biomimetic systems. The absorption, fluorescence, triplet state and singlet molecular oxygen production of the metal complexes were all referred to H2P. The photodynamic activity was first analyzed using 9,10-dimethylanthracene and guanosine 5'-monophosphate in N,N-dimethylformamide. The photooxidation processes were also investigated in benzene/benzyl-n-hexadecyldimethyl ammonium chloride/water reverse micelles. Photosensitization efficiency of these porphyrins was H2P approximately ZnP > PdP in homogeneous solution and ZnP > H2P > PdP in micelles, whereas no photooxidation effect was detected using the Cu(ii) complex. Human erythrocytes were used as a biological membrane model. The photohemolytic activity depended on irradiation time, sensitizer and concentration of the agent. When cells were treated with 1 microM sensitizer, the hemolytic activity was H2P > ZnP > CuP. However, it was H2P > ZnP approximately CuP using 5 microM of the respective porphyrin. Although CuP could undergo a type I photoreaction, in all cases the photohemolytic effect considerably diminishes in anoxic conditions, indicating that an oxygen atmosphere is required for the mechanism of cellular membrane damage. The behavior of these amphiphilic metallo porphyrins provides information on the photodynamic activity of these agents in biomimetic microenvironments.