Building a path in cell biology

Setting up a new lab is an exciting but challenging prospect. We discuss our experiences in finding a path to tackle some of the key current questions in cell biology and the hurdles that we have encountered along the way.We were both fortunate to receive the ASCB Early Career Life Scientist Award this year and were offered this opportunity to discuss our science and experiences. As we are at very similar stages in our careers (Iain has had a lab for 5 years, and Gia has had a lab for 6 years), we decided to compare our experiences starting our labs and conducting science and to discuss the decisions that we have made along the way.     

Evaluation of a novel monoclonal-based antigen-in-stool enzyme immunoassay (Premier Platinum HpSA PLUS) for diagnosis of Helicobacter pylori infection in Vietnamese children

Background: Helicobacter pylori infection is difficult to diagnose in children, especially in developing countries where noninvasive methods such as urea breath test are often not available. We evaluate the sensitivity and specificity of a new monoclonal antibody-based antigen-in-stool enzyme immunoassay (Premier Platinum HpSA PLUS) for diagnosis of H. pylori infection in Vietnamese children.
Materials and Methods: Sensitivity of the antigen-in-stool test was evaluated in 232 children, 3–15 years of age, who were positive for H. pylori infection by culture from biopsies. For evaluation of the specificity 98 children of similar age with nongastrointestinal conditions and who were negative for H. pylori infection by serologic assays were included with blood and stool samples.
Results: Of the 232 culture-positive children, 224 were also positive by Premier Platinum HpSA PLUS. Of the 98 control children, 93 were H. pylori negative also in the stool test. The sensitivity of Premier Platinum HpSA PLUS was thus 96.6% (95% CI 93.3–98.5) and the specificity was 94.9% (95% CI 88.5–98.3).
Conclusions: The findings have demonstrated Premium Platinum HpSA PLUS to be a reliable method for detection of H. pylori infection also in children in our area.     

3-[6-(2-Dimethylamino-1-imidazol-1-yl-butyl)-naphthalen-2-yloxy]-2,2-dimethyl-propionic acid as a highly potent and selective retinoic acid metabolic blocking agent

3-[6-(2-Dimethylamino-1-imidazol-1-yl-butyl)-naphthalen-2-yloxy]-2,2-dimethyl-propionic acid and analogs were designed and synthesized as highly potent and selective CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBAs), with demonstrated in vivo efficacy to increase the half-life of exogenous atRA.     

BRAF Mutation is Associated with an Improved Survival in Glioma—a Systematic Review and Meta-analysis

Newly emerged molecular markers in gliomas provide prognostic values beyond the capabilities of histologic classification. BRAF mutation, especially BRAF V600E, is common in a subset of gliomas and may represent a potential prognostic marker. The aim of our study is to investigate the potential use of BRAF mutations on prognosis of glioma patients. Four electronic databases were searched for potential articles, including PubMed, Scopus, ISI Web of Science, and Virtual Health Library (VHL). Data of hazard ratio (HR) for overall survival (OS) and progression-free survival (PFS) were directly obtained from original papers or indirectly estimated from Kaplan Meier curve (KMC). A random effect model weighted by inverse variance method was used to calculate the pooled HR. From 705 articles, we finally included 11 articles with 1308 glioma patients for the final analysis. The overall estimates showed that BRAF V600E was associated with an improved overall survival (OS) in glioma patients (HR = 0.60; 95% CI = 0.44–0.80). Results for progression-free survival (PFS), however, were not statistically significant (HR = 1.39; 95% CI = 0.82–2.34). In subgroup analyses, BRAF V600E showed its effect in improving survival in pediatric and young adult gliomas (under 35 years) but did not have prognostic value in old adult. Additionally, BRAF V600E was only associated with a favorable prognosis in lower grade glioma. Our meta-analysis provides evidence that BRAF mutation has a favorable prognostic impact in gliomas and its prognostic value might be dependent on patient age and tumor grade. This mutation can be used as a prognostic factor in glioma but additional studies are required to clarify its prognostic value taking into account other confounding factors.     

Myomesin is a molecular spring with adaptable elasticity

The M-band is a transverse structure in the center of the sarcomere, which is thought to stabilize the thick filament lattice. It was shown recently that the constitutive vertebrate M-band component myomesin can form antiparallel dimers, which might cross-link the neighboring thick filaments. Myomesin consists mainly of immunoglobulin-like (Ig) and fibronectin type III (Fn) domains, while several muscle types express the EH-myomesin splice isoform, generated by the inclusion of the unique EH-segment of about 100 amino acid residues (aa) in the center of the molecule. Here we use atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy for the biophysical characterization of myomesin. The AFM identifies the "mechanical fingerprints" of the modules constituting the myomesin molecule. Stretching of homomeric polyproteins, constructed of Ig and Fn domains of human myomesin, produces a typical saw-tooth pattern in the force-extension curve. The domains readily refold after relaxation. In contrast, stretching of a heterogeneous polyprotein, containing several repeats of the My6-EH fragment reveals a long initial plateau corresponding to the sum of EH-segment contour lengths, followed by several My6 unfolding peaks. According to this, the EH-segment is characterized as an entropic chain with a persistence length of about 0.3nm. In TEM pictures, the EH-domain appears as a gap in the molecule, indicating a random coil conformation similar to the PEVK region of titin. CD spectroscopy measurements support this result, demonstrating a mostly non-folded conformation for the EH-segment. We suggest that similarly to titin, myomesin is a molecular spring, whose elasticity is modulated by alternative splicing. The Ig and Fn domains might function as reversible "shock absorbers" by sequential unfolding in the case of extremely high or long sustained stretching forces. These complex visco-elastic properties of myomesin might be crucial for the stability of the sarcomere.     

Cropping Systems and Cultural Practices Determine the Rhizoctonia Anastomosis Groups Associated with Brassica spp. in Vietnam

Ninety seven Rhizoctonia isolates were collected from different Brassica species with typical Rhizoctonia symptoms in different provinces of Vietnam. The isolates were identified using staining of nuclei and sequencing of the rDNA-ITS barcoding gene. The majority of the isolates were multinucleate R. solani and four isolates were binucleate Rhizoctonia belonging to anastomosis groups (AGs) AG-A and a new subgroup of A-F that we introduce here as AG-Fc on the basis of differences in rDNA-ITS sequence. The most prevalent multinucleate AG was AG 1-IA (45.4% of isolates), followed by AG 1-ID (17.5%), AG 1-IB (13.4%), AG 4-HGI (12.4%), AG 2-2 (5.2%), AG 7 (1.0%) and an unknown AG related to AG 1-IA and AG 1-IE that we introduce here as AG 1-IG (1.0%) on the basis of differences in rDNA-ITS sequence. AG 1-IA and AG 1-ID have not been reported before on Brassica spp. Pathogenicity tests revealed that isolates from all AGs, except AG-A, induced symptoms on detached leaves of several cabbage species. In in vitro tests on white cabbage and Chinese cabbage, both hosts were severely infected by AG 1-IB, AG 2-2, AG 4-HGI, AG 1-IG and AG-Fc isolates, while under greenhouse conditions, only AG 4-HGI, AG 2-2 and AG-Fc isolates could cause severe disease symptoms. The occurrence of the different AGs seems to be correlated with the cropping systems and cultural practices in different sampling areas suggesting that agricultural practices determine the AGs associated with Brassica plants in Vietnam.     

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

The endoplasmic reticulum (ER) is a large, continuous membrane-bound organelle comprised of functionally and structurally distinct domains including the nuclear envelope, peripheral tubular ER, peripheral cisternae, and numerous membrane contact sites at the plasma membrane, mitochondria, Golgi, endosomes, and peroxisomes. These domains are required for multiple cellular processes, including synthesis of proteins and lipids, calcium level regulation, and exchange of macromolecules with various organelles at ER-membrane contact sites. The ER maintains its unique overall structure regardless of dynamics or transfer at ER-organelle contacts. In this review, we describe the numerous factors that contribute to the structure of the ER.The endoplasmic reticulum (ER) is a dynamic organelle responsible for many cellular functions, including the synthesis of proteins and lipids, and regulation of intracellular calcium levels. This review focuses on the distinct and complex morphology of the ER. The structure of the ER is complex because of the numerous distinct domains that exist within one continuous membrane bilayer. These domains are shaped by interactions with the cytoskeleton, by proteins that stabilize membrane shape, and by a homotypic fusion machinery that allows the ER membrane to maintain its continuity and identity. The ER also contains domains that contact the plasma membrane (PM) and other organelles including the Golgi, endosomes, mitochondria, lipid droplets, and peroxisomes. ER contact sites with other organelles and the PM are both abundant and dispersed throughout the cytoplasm, suggesting that they too could influence the overall architecture of the ER. As we will discuss here, ER shape and distribution are regulated by many intrinsic and extrinsic forces.     

The reticulon and DP1/Yop1p proteins form immobile oligomers in the tubular endoplasmic reticulum

We recently identified a class of membrane proteins, the reticulons and DP1/Yop1p, which shape the tubular endoplasmic reticulum (ER) in yeast and mammalian cells. These proteins are highly enriched in the tubular portions of the ER and virtually excluded from other regions. To understand how they promote tubule formation, we characterized their behavior in cellular membranes and addressed how their localization in the ER is determined. Using fluorescence recovery after photobleaching, we found that yeast Rtn1p and Yop1p are less mobile in the membrane than normal ER proteins. Sucrose gradient centrifugation and cross-linking analyses show that they form oligomers. Mutants of yeast Rtn1p, which no longer localize exclusively to the tubular ER or are even totally inactive in inducing ER tubules, are more mobile and oligomerize less extensively. The mammalian reticulons and DP1 are also relatively immobile and can form oligomers. The conserved reticulon homology domain that includes the two membrane-embedded segments is sufficient for the localization of the reticulons to the tubular ER, as well as for their diffusional immobility and oligomerization. Finally, ATP depletion in both yeast and mammalian cells further decreases the mobilities of the reticulons and DP1. We propose that oligomerization of the reticulons and DP1/Yop1p is important for both their localization to the tubular domains of the ER and for their ability to form tubules.     

Sheets, ribbons and tubules - how organelles get their shape

Most membrane-bound organelles have elaborate, dynamic shapes and often include regions with distinct morphologies. These complex structures are relatively conserved throughout evolution, which indicates that they are important for optimal organelle function. Various mechanisms of determining organelle shape have been proposed - proteins that stabilize highly curved membranes, the tethering of organelles to other cellular components and the regulation of membrane fission and fusion might all contribute.