Multiple Recognition Motifs in Nucleoporin Nup159 Provide a Stable and Rigid Nup159-Dyn2 Assembly

Dyn2 is the yeast ortholog of the molecular hub LC8, which binds disordered proteins and promotes their self-association and higher order assembly. Dyn2 is proposed to dimerize and stabilize the Nup82-Nsp1-Nup159 complex of the nuclear pore assembly through its interaction with nucleoporin Nup159. Nup159 has six LC8 recognition motifs separated by short linkers. NMR experiments reported here show that the Dyn2 binding domain of Nup159 is intrinsically disordered and that binding of one equivalent of Dyn2 dimer aligns two Nup159 chains along the full Dyn2 binding domain to form a bivalent scaffold that promotes binding of other Dyn2 dimers. Isothermal titration calorimetry of Dyn2 binding to Nup constructs of increasing lengths determine that the third LC8 recognition motifs does not bind Dyn2. A new approach to identifying active LC8 recognition motifs based on NMR-detected β-sheet propensities is presented. Isothermal titration calorimetry experiments also show that, due to unfavorable entropy changes, a Nup-Dyn2 complex with three Dyn2 dimers is more stable than the wild-type complex with five Dyn2 dimers. The calorimetric results argue that, from a thermodynamics perspective, only three Dyn2 dimers are needed for optimal stability and suggest that the evolutionary adaptation of multiple tandem LC8 recognition motifs imparts to the complex other properties such as rigidity and a kink in the rod-like structure. These findings extend the repertoire of functions of intrinsically disordered protein to fine-tuning and versatile assembly of higher order macromolecular complexes.     

The Ba2+ sensitivity of the Na+-induced Ca2+ efflux in heart mitochondria: the site of inhibitory action

The Na+-induced Ca2+ release from rat heart mitochondria was measured in the presence of Ruthenium red. Ba2+ effectively inhibited the Na+-induced Ca2+ release. At 10 mM Na+ 50% inhibition was reached by 1.51 +/- 0.48 (S.D., n = 8) microM Ba2+ in the presence of 0.1 mg/ml albumin and by 0.87 +/- 0.25 (S.D., n = 3) microM Ba2+ without albumin. In order to inhibit, it was not required that Ba2+ ions enter the matrix. 140Ba2+ was not accumulated in the mitochondrial matrix space; further, in contrast to liver mitochondria, Ba2+ inhibition was immediate. The Na+-induced Ca2+ release was inhibited by Ba2+ non-competitively, with respect of the extramitochondrial Na+. The double inhibitor titration of the Na+-Ca2+ exchanger with Ba2+ in the presence and absence of extramitochondrial Ca2+ revealed that the exchanger possesses a common binding site for extramitochondrial Ca2+ and Ba2+, presumably the regulatory binding site of the Na+-Ca2+ exchanger, which was described by Hayat and Crompton (Biochem. J. 202 (1982) 509-518). All these observations indicate that Ba2+ acts at the cytoplasmic surface of the inner mitochondrial membrane. The inhibitory properties of Ba2+ on the Na+-dependent Ca2+ release in heart mitochondria are basically different from those found on Na+-independent Ca2+ release in liver mitochondria (Lukács, G.L. and Fonyó, A. (1985) Biochim. Biophys. Acta 809, 160-166).     

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells,Growth, Photosynthetic Capacity,and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.     

Regret on Choice of Colorectal Cancer Screening Modality Was Associated with Poorer Screening Compliance: A 4-Year Prospective Cohort Study

Purpose

Very few studies examined the issue of regret on choosing colorectal cancer (CRC) screening tests. We evaluated the determinants of regret and tested the hypothesis that regret over screening choices was associated with poorer screening compliance.

Methods

A bowel cancer screening centre invited all Hong Kong citizens aged 50-70 years who were asymptomatic of CRC to participate in free-of-charge screening programmes. Upon attendance they attended health seminars on CRC and its screening, and were offered an option to choose yearly faecal immunochemical test (FIT) for up to four years vs. one direct colonoscopy. They were not allowed to switch the screening option after decision. A self-administered, four-item validated survey was used to assess whether they regretted over their choice (> 2 = regretful from a scale of 0 [no regret]-5 [extreme regret]). A binary logistic regression model evaluated if initial regret over their choice was associated with poorer programme compliance.

Results

From 4,341 screening participants who have chosen FIT or colonoscopy, 120 (2.8%) regretted over their decision and 1,029 (23.7%) were non-compliant with the screening programme. Younger subjects and people who felt pressure when making their decision were associated with regret. People who regretted their decision were 2.189 (95% C.I. 1.361-3.521, p = 0.001) times more likely to be non-compliant with the programme.

Conclusions

This study is the first to show that regret over the initial CRC screening choice was associated with later non-compliance. Screening participants who expressed regret over their choice should receive additional reminders to improve their programmatic compliance.     

Hairpins participating in folding of human telomeric sequence quadruplexes studied by standard and T-REMD simulations

DNA G-hairpins are potential key structures participating in folding of human telomeric guanine quadruplexes (GQ). We examined their properties by standard MD simulations starting from the folded state and long T-REMD starting from the unfolded state, accumulating ∼130 μs of atomistic simulations. Antiparallel G-hairpins should spontaneously form in all stages of the folding to support lateral and diagonal loops, with sub-μs scale rearrangements between them. We found no clear predisposition for direct folding into specific GQ topologies with specific syn/anti patterns. Our key prediction stemming from the T-REMD is that an ideal unfolded ensemble of the full GQ sequence populates all 4096 syn/anti combinations of its four G-stretches. The simulations can propose idealized folding pathways but we explain that such few-state pathways may be misleading. In the context of the available experimental data, the simulations strongly suggest that the GQ folding could be best understood by the kinetic partitioning mechanism with a set of deep competing minima on the folding landscape, with only a small fraction of molecules directly folding to the native fold. The landscape should further include non-specific collapse processes where the molecules move via diffusion and consecutive random rare transitions, which could, e.g. structure the propeller loops.     

Localisation of the Notch family in the human endometrium of fertile and infertile women

To investigate the spatial and temporal immunolocalisation and staining intensity of the Notch signalling family in endometrium of fertile and infertile women, endometrial biopsies were collected by curettage from 25 fertile women across the menstrual cycle and 10 infertile women in the mid secretory phase of menstrual cycle. Immunohisotchemistry was completed for NOTCH1, -2, -3, -4, cleaved Notch, DLL1, -3, -4, JAGGED1, -2, HES and NUMB and immunostaining intensity measured in both the endometrial glandular and luminal epithelium. NOTCH1 and the ligands DLL1 and JAGGED1 were key proteins displaying increased staining intensity during the receptive phase of the menstrual cycle and dysregulated in infertile endometrium. Conversely, NUMB a negative regulator of Notch signalling was decreased in the mid secretory phase of the menstrual cycle in fertile women and increased with infertility.     

The Use of Anchored Agonists of Phagocytic Receptors for Cancer Immunotherapy: B16-F10 Murine Melanoma Model

The application of the phagocytic receptor agonists in cancer immunotherapy was studied. Agonists (laminarin, molecules with terminal mannose, N-Formyl-methioninyl-leucyl-phenylalanine) were firmly anchored to the tumor cell surface. When particular agonists of phagocytic receptors were used together with LPS (Toll-like receptor agonist), high synergy causing tumour shrinkage and a temporary or permanent disappearance was observed. Methods of anchoring phagocytic receptor agonists (charge interactions, anchoring based on hydrophobic chains, covalent bonds) and various regimes of phagocytic agonist/LPS mixture applications were tested to achieve maximum therapeutic effect. Combinations of mannan/LPS and f-MLF/LPS (hydrophobic anchors) in appropriate (pulse) regimes resulted in an 80% and 60% recovery for mice, respectively. We propose that substantial synergy between agonists of phagocytic and Toll-like receptors (TLR) is based on two events. The TLR ligand induces early and massive inflammatory infiltration of tumors. The effect of this cell infiltrate is directed towards tumor cells, bearing agonists of phagocytic receptors on their surface. The result of these processes was effective killing of tumor cells. This novel approach represents exploitation of innate immunity mechanisms for treating cancer.     

Muscle Fiber Viability,a Novel Method for the Fast Detection of Ischemic Muscle Injury in Rats

Acute lower extremity ischemia is a limb- and life-threatening clinical problem. Rapid detection of the degree of injury is crucial, however at present there are no exact diagnostic tests available to achieve this purpose. Our goal was to examine a novel technique - which has the potential to accurately assess the degree of ischemic muscle injury within a short period of time - in a clinically relevant rodent model. Male Wistar rats were exposed to 4, 6, 8 and 9 hours of bilateral lower limb ischemia induced by the occlusion of the infrarenal aorta. Additional animals underwent 8 and 9 hours of ischemia followed by 2 hours of reperfusion to examine the effects of revascularization. Muscle samples were collected from the left anterior tibial muscle for viability assessment. The degree of muscle damage (muscle fiber viability) was assessed by morphometric evaluation of NADH-tetrazolium reductase reaction on frozen sections. Right hind limbs were perfusion-fixed with paraformaldehyde and glutaraldehyde for light and electron microscopic examinations. Muscle fiber viability decreased progressively over the time of ischemia, with significant differences found between the consecutive times. High correlation was detected between the length of ischemia and the values of muscle fiber viability. After reperfusion, viability showed significant reduction in the 8-hour-ischemia and 2-hour-reperfusion group compared to the 8-hour-ischemia-only group, and decreased further after 9 hours of ischemia and 2 hours of reperfusion. Light- and electron microscopic findings correlated strongly with the values of muscle fiber viability: lesser viability values represented higher degree of ultrastructural injury while similar viability results corresponded to similar morphological injury. Muscle fiber viability was capable of accurately determining the degree of muscle injury in our rat model. Our method might therefore be useful in clinical settings in the diagnostics of acute ischemic muscle injury.     

Secure Surveillance of Antimicrobial Resistant Organism Colonization or Infection in Ontario Long Term Care Homes

Background

There is stigma attached to the identification of residents carrying antimicrobial resistant organisms (ARO) in long term care homes, yet there is a need to collect data about their prevalence for public health surveillance and intervention purposes.

Objective

We conducted a point prevalence study to assess ARO rates in long term care homes in Ontario using a secure data collection system.

Methods

All long term care homes in the province were asked to provide colonization or infection counts for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and extended-spectrum beta-lactamase (ESBL) as recorded in their electronic medical records, and the number of current residents. Data was collected online during the October-November 2011 period using a Paillier cryptosystem that allows computation on encrypted data.

Results

A provably secure data collection system was implemented. Overall, 82% of the homes in the province responded. MRSA was the most frequent ARO identified at 3 cases per 100 residents, followed by ESBL at 0.83 per 100 residents, and VRE at 0.56 per 100 residents. The microbiological findings and their distribution were consistent with available provincial laboratory data reporting test results for AROs in hospitals.

Conclusions

We describe an ARO point prevalence study which demonstrated the feasibility of collecting data from long term care homes securely across the province and providing strong privacy and confidentiality assurances, while obtaining high response rates.     

Increased Low-Frequency Oscillation Amplitude of Sensorimotor Cortex Associated with the Severity of Structural Impairment in Cervical Myelopathy

Decreases in metabolites and increased motor-related, but decreased sensory-related activation of the sensorimotor cortex (SMC) have been observed in patients with cervical myelopathy (CM) using advanced MRI techniques. However, the nature of intrinsic neuronal activity in the SMC, and the relationship between cerebral function and structural damage of the spinal cord in patients with CM are not fully understood. The purpose of this study was to assess intrinsic neuronal activity by calculating the regional amplitude of low frequency fluctuations (ALFF) using resting-state functional MRI (rs-fMRI), and correlations with clinical and imaging indices. Nineteen patients and 19 age- and sex-matched healthy subjects underwent rs-fMRI scans. ALFF measurements were performed in the SMC, a key brain network likely to impaired or reorganized patients with CM. Compared with healthy subjects, increased amplitude of cortical low-frequency oscillations (LFO) was observed in the right precentral gyrus, right postcentral gyrus, and left supplementary motor area. Furthermore, increased z-ALFF values in the right precentral gyrus and right postcentral gyrus correlated with decreased fractional anisotropy values at the C2 level, which indicated increased intrinsic neuronal activity in the SMC corresponding to the structural impairment in the spinal cord of patients with CM. These findings suggest a complex and diverging relationship of cortical functional reorganization and distal spinal anatomical compression in patients with CM and, thus, add important information in understanding how spinal cord integrity may be a factor in the intrinsic covariance of spontaneous low-frequency fluctuations of BOLD signals involved in cortical plasticity.