A Temporal Threshold for Formaldehyde Crosslinking and Fixation

Background

Formaldehyde crosslinking is in widespread use as a biological fixative for microscopy and molecular biology. An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the minimum length of time required for an interaction to become fixed has not been determined.

Methodology

Using a unique series of mutations in the DNA binding protein MeCP2, we show that in vivo interactions lasting less than 5 seconds are invisible in the microscope after formaldehyde fixation, though they are obvious in live cells. The stark contrast between live cell and fixed cell images illustrates hitherto unsuspected limitations to the fixation process. We show that chromatin immunoprecipitation, a technique in widespread use that depends on formaldehyde crosslinking, also fails to capture these transient interactions.

Conclusions/Significance

Our findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research.     

Histone Demethylase Retinoblastoma Binding Protein 2 is Overexpressed in Hepatocellular Carcinoma and Negatively Regulated by hsa-miR-212

Background

The H3K4 demethylase retinoblastoma binding protein 2 (RBP2) is involved in the pathogenesis of gastric cancer, but its role and regulation in hepatocellular carcinoma (HCC) is unknown. We determined the function of RBP2 and its regulation in HCC in vitro and in human tissues.

Methods

We analyzed gene expression in 20 specimens each of human HCC and normal liver tissue by quantitative real-time PCR and immunohistochemistry. Proliferation was analyzed by foci formation and senescence by β-galactosidase staining. Promoter activity was detected by luciferase reporter assay.

Results

The expression of RBP2 was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, Homo sapiens miR-212 (hsa-miR-212), showed an opposite pattern. SiRNA knockdown of RBP2 significantly upregulated cyclin-dependent kinase inhibitors (CDKIs), with suppression of HCC cell proliferation and induction of senescence. Overexpression of hsa-miR-212 suppressed RBP2 expression, with inhibited cell proliferation and induced cellular senescence, which coincided with upregulated CDKIs; with low hsa-miR-212 expression, CDKIs were downregulated in HCC tissue. Inhibition of hsa-miR-212 expression upregulated RBP2 expression. Luciferase reporter assay detected the direct binding of hsa-miR-212 to the RBP2 3′ UTR.

Conclusions

RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212–RBP2–CDKI pathway may be important in the pathogenesis of HCC.     

CLPM: A Cross-Linked Peptide Mapping Algorithm for Mass Spectrometric Analysis

Background

Protein-protein, protein-DNA and protein-RNA interactions are of central importance in biological systems. Quadrapole Time-of-flight (Q-TOF) mass spectrometry is a sensitive, promising tool for studying these interactions. Combining this technique with chemical crosslinking, it is possible to identify the sites of interactions within these complexes. Due to the complexities of the mass spectrometric data of crosslinked proteins, new software is required to analyze the resulting products of these studies.

Result

We designed a Cross-Linked Peptide Mapping (CLPM) algorithm which takes advantage of all of the information available in the experiment including the amino acid sequence from each protein, the identity of the crosslinker, the identity of the digesting enzyme, the level of missed cleavage, and possible chemical modifications. The algorithm does in silico digestion and crosslinking, calculates all possible mass values and matches the theoretical data to the actual experimental data provided by the mass spectrometry analysis to identify the crosslinked peptides.

Conclusion

Identifying peptides by their masses can be an efficient starting point for direct sequence confirmation. The CLPM algorithm provides a powerful tool in identifying these potential interaction sites in combination with chemical crosslinking and mass spectrometry. Through this cost-effective approach, subsequent efforts can quickly focus attention on investigating these specific interaction sites.

     

Elevated Serum Triglyceride and Retinol-Binding Protein 4 Levels Associated with Fructose-Sweetened Beverages in Adolescents

Background

The metabolic effect of fructose in sugar-sweetened beverages (SSB) has been linked to de novo lipogenesis and uric acid (UA) production.

Objectives

This study investigated the biological effects of SSB consumption on serum lipid profiles and retinol-binding protein 4 (RBP4) among Taiwanese adolescents.

Methods

We evaluated the anthropometric parameters and biochemical outcomes of 200 representative adolescents (98 boys and 102 girls) who were randomly selected from a large-scale cross-sectional study. Data were analyzed using multiple regression models adjusted for covariates.

Results

Increased SSB consumption was associated with increased waist and hip circumferences, body mass index (BMI) values and serum UA, triglyceride (TG) and RBP4 levels. Adolescents who consumed >500 ml/day of beverages half-to-heavily sweetened with high-fructose corn syrup (HFCS) exhibited TG and RBP4 levels 22.7 mg/dl and 13.92 ng/ml higher than non-drinkers, respectively. HFCS drinkers with hyperuricemia had higher TG levels than HFCS drinkers with normal UA levels (98.6 vs. 81.6 mg/dl). The intake of HFCS-rich SSBs and high value of BMI (≥24) interactively reinforced RBP4 levels among overweight/obese adolescents. Circulating RBP4 levels were significantly correlated with weight-related outcomes and TG and UA concentration among HFCS drinkers (r = 0.253 to 0.404), but not among non-drinkers.

Conclusions

High-quantity HFCS-rich beverage consumption is associated with higher TG and RBP4 levels. Hyperuricemia is likely to intensify the influence of HFCS-rich SSB intake on elevated TG levels, and in overweight and obese adolescents, high BMI may modify the action of fructose on higher circulating levels of RBP4.     

Dopaminergic Neuronal Imaging in Genetic Parkinson's Disease: Insights into Pathogenesis

Objectives

To compare the dopaminergic neuronal imaging features of different subtypes of genetic Parkinson''s Disease.

Methods

A retrospective study of genetic Parkinson''s diseases cases in which DaTSCAN (123I-FP-CIT) had been performed. Specific non-displaceable binding was calculated for bilateral caudate and putamen for each case. The right:left asymmetry index and striatal asymmetry index was calculated.

Results

Scans were available from 37 cases of monogenetic Parkinson''s disease (7 glucocerebrosidase (GBA) mutations, 8 alpha-synuclein, 3 LRRK2, 7 PINK1, 12 Parkin). The asymmetry of radioligand uptake for Parkinson''s disease with GBA or LRRK2 mutations was greater than that for Parkinson''s disease with alpha synuclein, PINK1 or Parkin mutations.

Conclusions

The asymmetry of radioligand uptake in Parkinsons disease associated with GBA or LRRK2 mutations suggests that interactions with additional genetic or environmental factors may be associated with dopaminergic neuronal loss.     

Spirometry Reference Equations for Central European Populations from School Age to Old Age

Background

Spirometry reference values are important for the interpretation of spirometry results. Reference values should be updated regularly, derived from a population as similar to the population for which they are to be used and span across all ages. Such spirometry reference equations are currently lacking for central European populations.

Objective

To develop spirometry reference equations for central European populations between 8 and 90 years of age.

Materials

We used data collected between January 1993 and December 2010 from a central European population. The data was modelled using “Generalized Additive Models for Location, Scale and Shape” (GAMLSS).

Results

The spirometry reference equations were derived from 118''891 individuals consisting of 60''624 (51%) females and 58''267 (49%) males. Altogether, there were 18''211 (15.3%) children under the age of 18 years.

Conclusion

We developed spirometry reference equations for a central European population between 8 and 90 years of age that can be implemented in a wide range of clinical settings.     

US News and World Report Cancer Hospital Rankings: Do They Reflect Measures of Research Productivity?

Context

Prior research has faulted the US News and World Report hospital specialty rankings for excessive reliance on reputation, a subjective measure of a hospital''s performance.

Objective

To determine whether and to what extent reputation correlates with objective measures of research productivity among cancer hospitals.

Design

A retrospective observational study.

Setting

Automated search of NIH Reporter, BioEntrez, BioMedline and Clinicaltrials.gov databases.

Participants

The 50 highest ranked cancer hospitals in 2013''s US News and World Report Rankings.

Exposure

We ascertained the number of NCI funded grants, and the cumulative funds received by each cancer center. Additionally, we identified the number of phase I, phase II, and phase III studies published and indexed in MEDLINE, and registered at clinicaltrials.gov. All counts were over the preceding 5 years. For published articles, we summed the impact factor of the journals in which they appeared. Trials were attributed to centers on the basis of the affiliation of the lead author or study principal investigator.

Main Outcome

Correlation coefficients from simple and multiple linear regressions for measures of research productivity and a center''s reputation.

Results

All measures of research productivity demonstrated robust correlation with reputation (mean r-squared  = 0.65, median r-squared = 0.68, minimum r-squared = .41, maximum r-squared = 0.80). A multivariable model showed that 93% of the variation in reputation is explained by objective measures.

Conclusion

Contrary to prior criticism, the majority of reputation, used in US News and World Rankings, can be explained by objective measures of research productivity among cancer hospitals.     

Variation at GRN 3′-UTR rs5848 Is Not Associated with a Risk of Frontotemporal Lobar Degeneration in Dutch Population

Background

A single nucleotide polymorphism (rs5848) located in the 3′- untranslated region of GRN has recently been associated with a risk of frontotemporal lobar degeneration (FTLD) in North American population particularly in pathologically confirmed cases with neural inclusions immunoreactive for ubiquitin and TAR DNA-binding protein 43 (TDP-43), but negative for tau and alpha-synuclein (FTLD-TDP).

Methodology/Principal Findings

In an effort to replicate these results in a different population, rs5848 was genotyped in 256 FTLD cases and 1695 controls from the Netherlands. Single SNP gender-adjusted logistic regression analysis revealed no significant association between variation at rs5848 and FTLD. Fisher''s exact test, failed to find any significant association between rs5848 and a subset of 23 pathology confirmed FTLD-TDP cases.

Conclusions/Significance

The evidence presented here suggests that variation at rs5848 does not contribute to the etiology of FTLD in the Dutch population.     

Quantitative mass spectrometry evaluation of human retinol binding protein 4 and related variants

Background

Retinol Binding Protein 4 (RBP4) is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA) technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker.

Methods

RBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants.

Results

The linear range of the assay was 7.81–500 ug/mL, and the limit of detection and limit of quantification were 3.36 ug/mL and 6.52 ug/mL, respectively. The intra-assay CVs were determined to be 5.1% and the inter-assay CVs were 9.6%. The percent recovery of the RBP4-MSIA ranged from 95 – 105%. Method comparison of the RBP4 MSIA vs the Immun Diagnostik ELISA yielded a Passing & Bablok fit of MSIA  = 1.05× ELISA – 3.09, while the Cusum linearity p-value was >0.1 and the mean bias determined by the Altman Bland test was 1.2%.

Conclusion

The novel RBP4 MSIA provided a fast, accurate and precise quantitative protein measurement as compared to the standard commercially available ELISA. Moreover, this method also allowed for the detection of RBP4 variants that are present in each sample, which may in the future provide a new dimension in the clinical utility of this biomarker.     

ITAM Signaling by Vav Family Rho Guanine Nucleotide Exchange Factors Regulates Interstitial Transit Rates of Neutrophils In Vivo

Background

In response to infection, neutrophils are quickly recruited from the blood into inflamed tissues. The interstitial migration of neutrophils is crucial for the efficient capture and control of rapidly proliferating microbes before microbial growth can overwhelm the host''s defenses. However, the molecular mechanisms that regulate interstitial migration are incompletely understood.

Methodology/Principal Findings

Here, we use two-photon microscopy (2PM) to study discrete steps of neutrophil responses during subcutaneous infection with bacteria. Our study demonstrates that signals emanating from ITAM-containing receptors mediated by Vav family Rho GEFs control the velocity, but not the directionality, of neutrophil migration towards sites of bacterial infection.

Conclusions/Significance

Here we show that during neutrophil migration towards sites of bacterial infection, signals emanating from ITAM-containing receptors specifically control interstitial neutrophil velocity.     

A Feeding Induced Switch from a Variable to a Homogenous State of the Earthworm Gut Microbiota within a Host Population

Background

The distribution pattern of the earthworm gut microbiota at the host population level is of fundamental importance to understand host-microbiota interactions. Our current understanding of these interactions is very limited. Since feeding represents a main perturbation of the gut microbiota, we determined the effect of a single dose of feed on the microbiota associated with an earthworm population in a simulated microenvironment.

Methodology

Earthworms were sampled 0, 1 and 7 days after feeding. We determined the overall composition of the earthworm-associated microbiota by 16S rRNA gene cloning and sequencing. Based on the 16S rRNA gene data we constructed quantitative PCR''s (Q-PCR) for the seven most dominating bacterial groups.

Principal Findings

Q-PCR revealed low density and highly variable microbiota among the earthworms before feeding, while a high-density homologous microbiota resulted from feeding. We found that the microbiota 1 day after feeding was more equal to the microbiota after 7 days than before feeding. Furthermore, we found that the gut microbiota was very distinct from that of the bedding and the feed.

Significance

The homogenous population response represents fundamental new knowledge about earthworm gut associated bacteria.