Development of cyan fluorescent protein (CFP) reporter system in green alga Chlamydomonas reinhardtii and macroalgae Pyropia sp.

Various fluorescent proteins have been developed for in vivo reporter systems in diverse prokaryotes and eukaryotes. However, few in vivo imaging systems have been reported for the model algae Chlamydomonas reinhardtii or Pyropia sp. In this study, an effective imaging system using cyan fluorescent protein (CFP) was developed for the green alga C. reinhardtii, and its application was also successful in the red macroalgae Pyropia tenera and P. yezoensis. For optimization of CFP expression in C. reinhardtii and Pyropia sp., we modified codon usage in the CFP gene (CFP), generating PtCrCFP (Pyropia tenera/Chlamydomonas reinhardtii CFP). PtCrCFP was successfully expressed in PtCrCFP-expressing UVM11 transgenic lines, and high accumulation levels of PtCrCFP were found by western blotting. Consistent with these results, PtCrCFP fluorescence was clearly detected with a low level of chlorophyll background fluorescence in PtCrCFP-expressing UVM11 transgenic lines. In Pyropia sp. gametophytic cells, transient expression of PtCrCFP fluorescence was distinctly visualized. PtCrCFP fluorescence was also observed during the regeneration of monospores and young gametophytes from PtCrCFP-expressing P. yezoensis gametophytic cells. These results suggest that PtCrCFP may be useful as an in vivo reporter in green algae due to the short emission wavelength of CFP, which provides a low level of chlorophyll background fluorescence. This study also presents the possibility of PtCrCFP’s use as a visible selection marker for the generation of transgenic lines in the red algae Pyropia sp. Thus, PtCrCFP as an in vivo visualization tool may offer new opportunities for the functional analysis of genetic studies in both green and red algae.     

How Fast Does a Signal Propagate through Proteins?

As the molecular basis of signal propagation in the cell, proteins are regulated by perturbations, such as mechanical forces or ligand binding. The question arises how fast such a signal propagates through the protein molecular scaffold. As a first step, we have investigated numerically the dynamics of force propagation through a single (Ala) protein following a sudden increase in the stretching forces applied to its end termini. The force propagates along the backbone into the center of the chain on the picosecond scale. Both conformational and tension dynamics are found in good agreement with a coarse-grained theory of force propagation through semiflexible polymers. The speed of force propagation of 50Å ps⁻¹ derived from these simulations is likely to determine an upper speed limit of mechanical signal transfer in allosteric proteins or molecular machines.     

Proteomic Evaluation of Acquired Enamel Pellicle during In Vivo Formation

Acquired enamel pellicle (AEP) is a protein film that forms on the enamel surface of teeth by selective adsorption of proteins and peptides present in the mouth. This protein film forms the interface between enamel and the damage oral biofilm, which modulates the attachment of bacteria found in oral biofilm. The overall goal of this study was to gain insight into the biological formation of the human in vivo AEP. This study hypothesized that AEP is created by the formation of successive protein layers, which consist of initial binding to enamel and subsequent protein-protein interactions. This hypothesis was examined by observing quantitative and qualitative changes in pellicle composition during the first two hours of AEP formation in the oral cavity. Quantitative mass spectrometry approaches were used to generate an AEP protein profile for each time-point studied. Relative proteomic quantification was carried out for the 50 proteins observed in all four time-points. Notably, the abundance of important salivary proteins, such as histatin 1, decrease with increasing of the AEP formation, while other essential proteins such as statherin showed constant relative abundance in all time-points. In summary, this is the first study that investigates the dynamic process to the AEP formation by using proteomic approaches. Our data demonstrated that there are significant qualitative and quantitative proteome changes during the AEP formation, which in turn will likely impact the development of oral biofilms.     

Germline Mutations in the Polyposis-Associated Genes BMPR1A,SMAD4, PTEN,MUTYH and GREM1 Are Not Common in Individuals with Serrated Polyposis Syndrome

Background

Recent reports have observed that individuals with serrated polyps, some of whom meet the clinical diagnostic criteria for Serrated Polyposis Syndrome (SPS), are among those who carry germline mutations in genes associated with polyposis syndromes including; (1) genes known to underlie hamartomatous polyposes (SMAD4, BMPR1A, and PTEN), (2) MUTYH-associated polyposis and (3) GREM1 in Hereditary Mixed Polyposis Syndrome (HMPS). The aim of this study was to characterise individuals fulfilling the current WHO criteria for SPS for germline mutations in these polyposis-associated genes.

Methods

A total of 65 individuals with SPS (fulfilling WHO criteria 1 or 3), were recruited to the Genetics of Serrated Neoplasia study between 2000 and 2012, through multiple Genetics or Family Cancer Clinics within Australia, or from the New Zealand Familial Gastrointestinal Cancer Service. Individuals with SPS were tested for coding mutations and large deletions in the PTEN, SMAD4, and BMPR1A genes, for the MUTYH variants in exons 7 (Y179C) and 13 (G396D), and for the duplication upstream of GREM1.

Results

We found no variants that were likely to be deleterious germline mutations in the SPS cases in the PTEN, SMAD4, and BMPR1A genes. A novel variant in intron 2 (c.164+223T>C) of PTEN was identified in one individual and was predicted by in silico analysis to have no functional consequences. One further individual with SPS was found to be mono-allelic for the MUTYH G396D mutation. No individuals carried the recently reported duplication within GREM1.

Conclusions

Genes involved in the gastrointestinal hamartomatous polyposis, Hereditary Mixed Polyposis Syndrome and MUTYH-associated polyposis syndromes are not commonly altered in individuals with SPS.     

Identification of GPCR-Interacting Cytosolic Proteins Using HDL Particles and Mass Spectrometry-Based Proteomic Approach

G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the β₂-adrenergic receptor (β₂AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified β₂AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated β₂AR pull-down, 242 proteins in the inverse agonist-activated β₂AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the β₂AR-interacting proteins isolated was confirmed by Western blot; three known β₂AR-interacting proteins (Gsα, NHERF-2, and Grb2) and 3 newly identified known β₂AR-interacting proteins (AMPKα, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of β₂AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis.     

PKCδ as a Regulator for TGFβ1-Induced α-SMA Production in a Murine Nonalcoholic Steatohepatitis Model

The precise mechanism of TGFβ1 signaling in the progression of non-alcoholic steatohepatitis (NASH) has remained unclear. In particular, a potential regulatory mechanism by which PKCδ affects profibrogenic gene expression had never been explored. In this study, therefore, the role of PKCδ in TGFβ1 mediated α-SMA expression was investigated using NASH model mice. In preparation of the NASH model, male C57BL6/J mice were fed a methionine-choline-deficient (MCD) diet for 3 weeks, after which time they were intraperitoneally injected with lipopolysaccharide (LPS). In addition, Tlr4^Lps-d (CH3/HeJ) mice were used to demonstrate the TGFβ1 signaling’s dependency on TLR4 induction. Liver histology and hepatic hepatitis markers were investigated, and hepatic gene expression levels were determined by real-time PCR. Acute liver injury by LPS injection specifically elevated not only α-SMA expression but also phospho-PKCδ in this model. In contrast, Tlr4^Lps-d (CH3/HeJ) and blockade of TGFβ1 receptor by SB431542 resulted in a significant reduction of PKCδ activation and α-SMA expression. Moreover, the TGFβ1-induced α-SMA production was significantly reduced by a specific PKCδ inhibitor. These findings suggested that PKCδ plays a critical role in TGFβ1-induced α-SMA production in a NASH model. Thus, this was the first demonstration of the involvement of PKCδ in the regulation of α-SMA expression in NASH liver tissues, and the impaired induction of PKCδ phosphorylation by LPS in a steatohepatitis condition. Interestingly, treatment by PKCδ inhibitor caused dramatic reduction of myofibroblast activation, indicating that PKCδ represents a promising target for treating NASH.     

The NF-κB RelB Protein Is an Oncogenic Driver of Mesenchymal Glioma

High-grade gliomas, such as glioblastomas (GBMs), are very aggressive, invasive brain tumors with low patient survival rates. The recent identification of distinct glioma tumor subtypes offers the potential for understanding disease pathogenesis, responses to treatment and identification of molecular targets for personalized cancer therapies. However, the key alterations that drive tumorigenesis within each subtype are still poorly understood. Although aberrant NF-κB activity has been implicated in glioma, the roles of specific members of this protein family in tumorigenesis and pathogenesis have not been elucidated. In this study, we show that the NF-κB protein RelB is expressed in a particularly aggressive mesenchymal subtype of glioma, and loss of RelB significantly attenuated glioma cell survival, motility and invasion. We find that RelB promotes the expression of mesenchymal genes including YKL-40, a marker of the MES glioma subtype. Additionally, RelB regulates expression of Olig2, a regulator of cancer stem cell proliferation and a candidate marker for the cell of origin in glioma. Furthermore, loss of RelB in glioma cells significantly diminished tumor growth in orthotopic mouse xenografts. The relevance of our studies for human disease was confirmed by analysis of a human GBM genome database, which revealed that high RelB expression strongly correlates with rapid tumor progression and poor patient survival rates. Thus, our findings demonstrate that RelB is an oncogenic driver of mesenchymal glioma tumor growth and invasion, highlighting the therapeutic potential of inhibiting the noncanonical NF-κB (RelB-mediated) pathway to treat these deadly tumors.     

Effect of Sodium Bicarbonate Administration on Mortality in Patients with Lactic Acidosis: A Retrospective Analysis

Background

Lactic acidosis is a common cause of high anion gap metabolic acidosis. Sodium bicarbonate may be considered for an arterial pH <7.15 but paradoxically depresses cardiac performance and exacerbates acidosis by enhancing lactate production. This study aimed to evaluate the cause and mortality rate of lactic acidosis and to investigate the effect of factors, including sodium bicarbonate use, on death.

Methods

We conducted a single center analysis from May 2011 through April 2012. We retrospectively analyzed 103 patients with lactic acidosis among 207 patients with metabolic acidosis. We used SOFA and APACHE II as severity scores to estimate illness severity. Multivariate logistic regression analysis and Cox regression analysis models were used to identify factors that affect mortality.

Results

Of the 103 patients with a mean age of 66.1±11.4 years, eighty-three patients (80.6%) died from sepsis (61.4%), hepatic failure, cardiogenic shock and other causes. The percentage of sodium bicarbonate administration (p = 0.006), catecholamine use, ventilator care and male gender were higher in the non-survival group than the survival group. The non-survival group had significantly higher initial and follow-up lactic acid levels, lower initial albumin, higher SOFA scores and APACHE II scores than the survival group. The mortality rate was significantly higher in patients who received sodium bicarbonate. Sodium bicarbonate administration (p = 0.016) was associated with higher mortality. Independent factors that affected mortality were SOFA score (Exp (B) = 1.72, 95% CI = 1.12–2.63, p = 0.013) and sodium bicarbonate administration (Exp (B) = 6.27, 95% CI = 1.10–35.78, p = 0.039).

Conclusions

Lactic acidosis, which has a high mortality rate, should be evaluated in patients with metabolic acidosis. In addition, sodium bicarbonate should be prescribed with caution in the case of lactic acidosis because sodium bicarbonate administration may affect mortality.     

Two Distinct Functional Patterns of Hepatitis C Virus (HCV)-Specific T Cell Responses in Seronegative,Aviremic Patients

In hepatitis C Virus (HCV) high-risk groups, HCV-specific T cell responses have been detected in seronegative, aviremic persons who have no evidence of HCV infection. Herein, we investigated functional profiles of HCV-specific T-cell responses in seronegative, aviremic patients of a HCV high-risk group. Seventy seven hemodialysis patients with chronic renal disease were analyzed by IFN-γ ELISpot assays, and eight of 71 (11.3%) seronegative, aviremic patients displayed HCV-specific T-cell responses. Their HCV-specific memory T cells were characterized by assessing cytokine polyfunctionality, known to provide antiviral protection. By intracellular staining of IFN-γ, TNF-α, IL-2 and MIP-1β, we identified two distinct populations in the seronegative, aviremic patients: polyfunctional responders and TNF-α-predominant responders. In further analysis, occult HCV infection was excluded as a cause of the HCV-specific T cell response via secondary nested RT-PCR of HCV RNA in peripheral blood mononuclear cell samples. HCV-specific T cells targeted multiple epitopes including non-structural proteins in a single patient, implying that their T cells might have been primed by HCV proteins synthesized within the host. We conclude that HCV-specific memory T cells of seronegative, aviremic patients arise from authentic HCV replication in the host, but not from current occult HCV infection. By functional pattern of HCV-specific T cells, there are two distinct populations in these patients: polyfunctional responders and TNF-α-predominant responders.