Versatile Solid Supports for Oligonucleotide Synthesis that Incorporate Urea Bridge

The universal solid support, USIII, representing a new and improved version of commercial USII, as well as 2 ′-deoxynucleoside and 2 ′-deoxy-2 ′-fluoronucleoside bound supports, incorporating a labile phenoxyacetyl fragment, was synthesized by an aminomethyl polystyrene carbamoylation with corresponding azides in the presence of aqueous triethylammonium bicarbonate. All three solid phases incorporate a stable urea tether, thus bridging the polymer and functional linker. These new matrices proved to be potent solid phases for the synthesis of DNA, RNA, or modified oligonucleotides as well as randomized mixed 2 ′-ribo/2 ′-deoxy-2 ′-fluoro-RNA libraries and/or DNA libraries, randomized with trinucleotides (codons).     

The Solution Synthesis of Antisense Oligonucleotide‐Peptide Conjugates Directly Linked via Phosphoramide Bond by Using a Fragment Coupling Approach

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell‐penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e., phosphorylation of a peptide amino group by an oligonucleotide terminal phosphate 1‐hydroxybenzotriazole ester in aqueous media or condensation of phosphate and amino groups in presence of triphenylphosphine, 2,2′‐dithiopyridine and 4‐dimethylaminopyridine in organic media. Several oligonucleotides, including a 18‐mer antisense oligodeoxyribonucleotide complementary to an internal coding region of the reporter gene of the green fluorescent protein (GFP) were prepared. Peptides derived from the third helix of the homeodomain of Antennapedia, the influenza envelope hemagglutinin subunit as well as melittin and polymyxin B were used for the conjugates' synthesis. The peptides with various amino acid composition were chosen to confirm that these coupling methods are of a general use.     

E. COLI RNASE HI AND THE PHOSPHONATE-DNA/RNA HYBRID: MOLECULAR DYNAMICS SIMULATIONS

A model for the complex between E. coli RNase HI and the DNA/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3′–O–CH₂–P–O–5′ or 3′–O–P–CH₂–O–5′) on the ability of the modified-DNA/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to the 3′-end of the DNA strand to interfere with the DNA binding site of RNase H. Altogether, six trajectories were produced (length 1.5). Mutual hydrogen bonds connecting both strands of the nucleic acids hybrid, DNA with RNase H, RNA with RNase H, and the scissile bond with the Mg⁺⁺ · 4H₂O chelate complex (bound in the active site) were analyzed in detail. Many residues were involved in binding of the DNA (Arg88, Asn84, Trp85, Trp104, Tyr73, Lys99, Asn100, Thr43, and Asn16) and RNA (Gln76, Gln72, Tyr73, Lys122, Glu48, Asn44, and Cys13) strand to the substrate-binding site of the RNase H enzyme. The most remarkable disturbance of the hydrogen bonding net was observed for structures with modified internucleotide linkages positioned in a way to interact with the Trp104, Tyr73, Lys99, and Asn100 residues (situated in the middle of the DNA binding site, where a cluster of Trp residues forms a rigid core of the protein structure).     

Synthesis and Application of Negatively Charged PNA Analogues

Negatively charged DNA mimics containing phosphonate analogues of peptide nucleic acids were designed, and their physicochemical and biological properties were evaluated in the comparison with natural oligonucleotides, classical peptide nucleic acids, and morpholino phosphorodiamidate oligonucleotide analogues. The results obtained revealed a high potential of phosphonate-containing PNA derivatives for a number of biological applications, such as diagnostic, nucleic acids analysis, and inhibition of gene expression.     

Studies on the Pathophysiology and Genetic Basis of Migraine

Migraine is a neurological disorder that affects the central nervous system causing painful attacks of headache. A genetic vulnerability and exposure to environmental triggers can influence the migraine phenotype. Migraine interferes in many facets of people’s daily life including employment commitments and their ability to look after their families resulting in a reduced quality of life. Identification of the biological processes that underlie this relatively common affliction has been difficult because migraine does not have any clearly identifiable pathology or structural lesion detectable by current medical technology. Theories to explain the symptoms of migraine have focused on the physiological mechanisms involved in the various phases of headache and include the vascular and neurogenic theories. In relation to migraine pathophysiology the trigeminovascular system and cortical spreading depression have also been implicated with supporting evidence from imaging studies and animal models. The objective of current research is to better understand the pathways and mechanisms involved in causing pain and headache to be able to target interventions. The genetic component of migraine has been teased apart using linkage studies and both candidate gene and genome-wide association studies, in family and case-control cohorts. Genomic regions that increase individual risk to migraine have been identified in neurological, vascular and hormonal pathways. This review discusses knowledge of the pathophysiology and genetic basis of migraine with the latest scientific evidence from genetic studies.     

Lipoprotein-associated phospholipase A2 activity in patients with preserved left ventricular ejection fraction

Background: A significant proportion of heart failure (HF) patients have preserved ejection fraction (EF). Considering that inflammation and oxidative stress are involved in HF evolution, we investigated lipoprotein-associated phospholipase A2 (LpPLA2), an enzyme involved in these pathophysiologic processes in relation to EF.

Methods and results: The study included 208 HF patients and 20 healthy controls. HF patients with preserved EF (HFpEF) represented 42.31% of all HF patients. LpPLA2 activity was significantly increased in HF patients when compared with controls and was higher in HFpEF than in HF with reduced EF patients (HFrEF). The incidence of left ventricular hypertrophy was higher in HFpEF than in HFrEF (EF < 50).

Conclusion: Confirming its role as a marker of vascular inflammation, LpPLA2 seems to be a biomarker constantly correlated with HF, regardless of etiology. Elevated plasma values of LpPLA2 in HFpEF are consistent with the exacerbated inflammatory status.     

Cystatin C,a novel urinary biomarker for sensitive detection of acute kidney injury during haemorrhagic fever with renal syndrome

To explore the value of cystatin C for evaluating acute kidney injury (AKI) in haemorrhagic fever with renal syndrome (HFRS), the concentrations of cystatin C in serum and urine samples from HFRS patients were determined. The serum and urinary cystatin C concentrations significantly increased in HFRS patients compared with normal controls (p?<?0.001). In the acute phase of HFRS, urinary cystatin C increased to higher levels than serum creatinine, especially in severe or critical cases in the oliguric stage. Furthermore, higher levels of urinary cystatin C in the acute phase positively correlated with increased severity of the subsequent kidney injury. In conclusion, urinary cystatin C is a more sensitive clinical marker for AKI in HFRS, which may enable us to initiate treatment measures as early as possible.     

Volumetric properties underlying ligand binding in a monomeric hemoglobin: A high-pressure NMR study

The 2/2 hemoglobin of the cyanobacterium Synechococcus sp. PCC 7002, GlbN, coordinates the heme iron with two histidines and exists either with a b heme or with a covalently attached heme. The binding of exogenous ligands displaces the distal histidine and induces a conformational rearrangement involving the reorganization of internal void volumes. The formation of passageways within the resulting conformation is thought to facilitate ligand exchange and play a functional role. Here we monitored the perturbation induced by pressure on the ferric bis-histidine and cyanide-bound states of GlbN using ¹H–¹⁵N HSQC NMR spectroscopy. We inspected the outcome with a statistical analysis of 170 homologous 2/2 hemoglobin sequences. We found that the compression landscape of GlbN, as represented by the variation of an average chemical shift parameter, was highly sensitive to ligand swapping and heme covalent attachment. Stabilization of rare conformers was observed at high pressures and consistent with cavity redistribution upon ligand binding. In all states, the EF loop was found to be exceptionally labile to pressure, suggesting a functional role as a semi-flexible hinge between the adjacent helices. Finally, coevolved clusters presented a common pattern of compensating pressure responses. The high-pressure dissection combined with protein sequence analysis established locations with volumetric signatures relevant to residual communication of 2/2 hemoglobins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

大剂量阿托伐他汀对冠心病合并高尿酸血症PCI术后Scr、hsCRP及NGAL的影响

摘要目的：探讨大剂量阿托伐他汀对冠心病合并高尿酸血症PCI术后scr、hsCRP及NGAL的影响。方法：选取2010年6月至2012年6月间于我院接受诊断和治疗的冠心病合并高尿酸血症患者64例,随机分为观察组与对照组,每组32例。观察组给予大剂量阿托伐他汀治疗,对照组给予常规剂量阿托伐他汀治疗。结果：两组患者治疗前Scr、hsCRP及NGAL水平之间相互比较无显著性差异（P〉0．05）。观察组患者术后24hhsCRP水平为（6．7±1．5）m∥L,72hhsCRP水平回落至（5．7±1．0）mg门L,均明显低于对照组（P〈0．05）。观察组患者术后24hNGAL水平为（59．1±12．3）ng／L,72hNGAL水平回落至（47．8±10_3）ngm,均明显低于对照组（P〈0．05）。观察组心脏不良事件发生率明显低于对照组（P〈O．05）。结论：大剂量阿托伐他汀对于冠心病合并高尿酸血症患者PCI术后有一定的肾脏保护作用,且其安全性良好。     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.