Modified ribosome profiling reveals high abundance of ribosome protected mRNA fragments derived from 3′ untranslated regions

Ribosome profiling identifies ribosome positions on translated mRNAs. A prominent feature of published datasets is the near complete absence of ribosomes in 3′ untranslated regions (3′UTR) although substantial ribosome density can be observed on non-coding RNAs. Here we perform ribosome profiling in cultured Drosophila and human cells and show that different features of translation are revealed depending on the nuclease and the digestion conditions used. Most importantly, we observe high abundance of ribosome protected fragments in 3′UTRs of thousands of genes without manipulation of translation termination. Affinity purification of ribosomes indicates that the 3′UTR reads originate from ribosome protected fragments. Association of ribosomes with the 3′UTR may be due to ribosome migration through the stop codon or 3′UTR mRNA binding to ribosomes on the coding sequence. This association depends primarily on the relative length of the 3′UTR and may be related to translational regulation or ribosome recycling, for which the efficiency is known to inversely correlate with 3′UTR length. Together our results indicate that ribosome profiling is highly dependent on digestion conditions and that ribosomes commonly associate with the 3′UTR, which may have a role in translational regulation.     

Risk Stratification by Self-Measured Home Blood Pressure across Categories of Conventional Blood Pressure: A Participant-Level Meta-Analysis

Background

The Global Burden of Diseases Study 2010 reported that hypertension is worldwide the leading risk factor for cardiovascular disease, causing 9.4 million deaths annually. We examined to what extent self-measurement of home blood pressure (HBP) refines risk stratification across increasing categories of conventional blood pressure (CBP).

Methods and Findings

This meta-analysis included 5,008 individuals randomly recruited from five populations (56.6% women; mean age, 57.1 y). All were not treated with antihypertensive drugs. In multivariable analyses, hazard ratios (HRs) associated with 10-mm Hg increases in systolic HBP were computed across CBP categories, using the following systolic/diastolic CBP thresholds (in mm Hg): optimal, <120/<80; normal, 120–129/80–84; high-normal, 130–139/85–89; mild hypertension, 140–159/90–99; and severe hypertension, ≥160/≥100.Over 8.3 y, 522 participants died, and 414, 225, and 194 had cardiovascular, cardiac, and cerebrovascular events, respectively. In participants with optimal or normal CBP, HRs for a composite cardiovascular end point associated with a 10-mm Hg higher systolic HBP were 1.28 (1.01–1.62) and 1.22 (1.00–1.49), respectively. At high-normal CBP and in mild hypertension, the HRs were 1.24 (1.03–1.49) and 1.20 (1.06–1.37), respectively, for all cardiovascular events and 1.33 (1.07–1.65) and 1.30 (1.09–1.56), respectively, for stroke. In severe hypertension, the HRs were not significant (p≥0.20). Among people with optimal, normal, and high-normal CBP, 67 (5.0%), 187 (18.4%), and 315 (30.3%), respectively, had masked hypertension (HBP≥130 mm Hg systolic or ≥85 mm Hg diastolic). Compared to true optimal CBP, masked hypertension was associated with a 2.3-fold (1.5–3.5) higher cardiovascular risk. A limitation was few data from low- and middle-income countries.

Conclusions

HBP substantially refines risk stratification at CBP levels assumed to carry no or only mildly increased risk, in particular in the presence of masked hypertension. Randomized trials could help determine the best use of CBP vs. HBP in guiding BP management. Our study identified a novel indication for HBP, which, in view of its low cost and the increased availability of electronic communication, might be globally applicable, even in remote areas or in low-resource settings. Please see later in the article for the Editors'' Summary     

The Effects of Peatland Restoration on Water‐Table Depth,Elemental Concentrations,and Vegetation: 10 Years of Changes

We studied the effects of restoration on water‐table depth (WTD), element concentrations of peat and vegetation composition of peatlands drained for forestry in southern Finland. The restoration aimed to return the trajectory of vegetation succession toward that of undisturbed systems through the blockage of ditches and the removal of trees. Permanent plots established on a bog and a fen were sampled 1 year before, and 1, 2, 3, and 10 years after the restoration. The restoration resulted in a long‐term rise of the water‐table in both peatlands. Ten years after restoration, the mineral element concentrations (Ca, K, Mg, Mn, and P) of peat corresponded to those reported from comparable pristine peatlands. In particular, the increase of K and Mn concentrations at both sites suggests the recovery of ecosystem functionality in terms of nutrient cycling between peat and plants. The restoration resulted in the succession of plant communities toward the targeted peatland vegetation of wetter condition at both sites. This was evident from the decreased abundance of species benefiting from drainage and the corresponding increase of peatland species. However, many species typical of pristine peatlands were missing 10 years after restoration. We conclude that the restoration led to a reversal of the effects of drainage in vegetation and studied habitat conditions. However, due to the slow recovery of peatland ecosystems and the possibility that certain failures in the restoration measures may become apparent only after extended time periods, long‐term monitoring is needed to determine whether the goals of restoration will be met.     

Impact of protein binding on the analytical detectability and anticancer activity of thymoquinone

Thymoquinone (TQ), an active component of Nigella sativa L., is known to have anti-cancer and anti-inflammatory effects; however, no studies on its analytical detection in serum and its protein binding have been published. Using high performance liquid chromatography analysis, we show that the average recovery of TQ from serum is 2.5% at 10 μg/ml of TQ and 72% at 100 μg/ml. The low recovery of TQ from serum is due to its extensive binding to plasma proteins, as more than 99% of TQ was bound within 30 min of incubation. The binding of TQ to the major plasma proteins, bovine serum albumin (BSA) and alpha −1 acid glycoprotein (AGP), was studied and found to be 94.5 ± 1.7% for BSA and 99.1 ± 0.1% for AGP. Mass spectrometric analysis revealed that TQ was bound covalently to BSA, specifically on Cyst-34. Using WST-1 proliferation assay, we showed that BSA plays a protective role against TQ-induced cell death; pre-incubation with BSA prevented TQ from exerting its anti-proliferative effects against DLD-1 and HCT-116 human colon cancer cells. On the other hand, binding of TQ to AGP did not alter its anti-proliferative activity against both cell lines. When TQ was pre-incubated with AGP prior to the addition of BSA, the activity of TQ against DLD-1 was maintained, suggesting that AGP prevented the binding of TQ to BSA. This is the first time the covalent binding and inhibitory effect of BSA on TQ is documented. These data offer new grounds for TQ future pharmacokinetic analysis in vivo.     

Protein diffusion in mammalian cell cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.     

Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) Kinases Promote Human T Helper 1 Cell Differentiation

The differentiation of human primary T helper 1 (Th1) cells from naïve precursor cells is regulated by a complex, interrelated signaling network. The identification of factors regulating the early steps of Th1 cell polarization can provide important insight in the development of therapeutics for many inflammatory and autoimmune diseases. The serine/threonine-specific proviral integration site for Moloney murine leukemia virus (PIM) kinases PIM1 and PIM2 have been implicated in the cytokine-dependent proliferation and survival of lymphocytes. We have established that the third member of this family, PIM3, is also expressed in human primary Th cells and identified a new function for the entire PIM kinase family in T lymphocytes. Although PIM kinases are expressed more in Th1 than Th2 cells, we demonstrate here that these kinases positively influence Th1 cell differentiation. Our RNA interference results from human primary Th cells also suggest that PIM kinases promote the production of IFNγ, the hallmark cytokine produced by Th1 cells. Consistent with this, they also seem to be important for the up-regulation of the critical Th1-driving factor, T box expressed in T cells (T-BET), and the IL-12/STAT4 signaling pathway during the early Th1 differentiation process. In summary, we have identified PIM kinases as new regulators of human primary Th1 cell differentiation, thus providing new insights into the mechanisms controlling the selective development of human Th cell subsets.     

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved β-loop of the helicase domain.     

Predicting AD Conversion: Comparison between Prodromal AD Guidelines and Computer Assisted PredictAD Tool

Purpose

To compare the accuracies of predicting AD conversion by using a decision support system (PredictAD tool) and current research criteria of prodromal AD as identified by combinations of episodic memory impairment of hippocampal type and visual assessment of medial temporal lobe atrophy (MTA) on MRI and CSF biomarkers.

Methods

Altogether 391 MCI cases (158 AD converters) were selected from the ADNI cohort. All the cases had baseline cognitive tests, MRI and/or CSF levels of Aβ1–42 and Tau. Using baseline data, the status of MCI patients (AD or MCI) three years later was predicted using current diagnostic research guidelines and the PredictAD software tool designed for supporting clinical diagnostics. The data used were 1) clinical criteria for episodic memory loss of the hippocampal type, 2) visual MTA, 3) positive CSF markers, 4) their combinations, and 5) when the PredictAD tool was applied, automatically computed MRI measures were used instead of the visual MTA results. The accuracies of diagnosis were evaluated with the diagnosis made 3 years later.

Results

The PredictAD tool achieved the overall accuracy of 72% (sensitivity 73%, specificity 71%) in predicting the AD diagnosis. The corresponding number for a clinician’s prediction with the assistance of the PredictAD tool was 71% (sensitivity 75%, specificity 68%). Diagnosis with the PredictAD tool was significantly better than diagnosis by biomarkers alone or the combinations of clinical diagnosis of hippocampal pattern for the memory loss and biomarkers (p≤0.037).

Conclusion

With the assistance of PredictAD tool, the clinician can predict AD conversion more accurately than the current diagnostic criteria.     

Effects of NR1H3 Genetic Variation on the Expression of Liver X Receptor α and the Progression of Alzheimer's Disease

Alzheimer''s disease (AD) has been postulated to involve defects in the clearance of amyloid-β (Aβ). Activation of liver X receptor α (LXRα) increases the expression of apolipoprotein E (ApoE) as well as cholesterol transporters ABCA1 and ABCG1, leading to augmented clearance of Aβ. We have previously shown that the C allele of rs7120118 in the NR1H3 gene encoding LXRα reduces the risk of AD. Here, we wanted to assess whether the rs7120118 variation affects the progression of AD and modulates the expression of NR1H3 and its downstream targets APOE, ABCA1 and ABCG1.We utilized tissue samples from the inferior temporal cortex of 87 subjects, which were subdivided according to Braak staging into mild, moderate and severe AD groups on the basis of AD-related neurofibrillary pathology. APOE ε4 allele increased soluble Aβ42 levels in the tissue samples in a dose-dependent manner, but did not affect the expression status of APOE. In contrast, the CC genotype of rs7120118 was underrepresented in the severe group, although this result did not reach statistical significance. Also, patients with the CC genotype of rs7120118 showed significantly decreased soluble Aβ42 levels as compared to the patients with TT genotype. Although the severity of AD did not affect NR1H3 expression, the mRNA levels of NR1H3 among the patients with CT genotype of rs7120118 were significantly increased as compared to the patients with TT genotype. These results suggest that genetic variation in NR1H3 modulates the expression of LXRα and the levels of soluble Aβ42.