We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2'-O, 4'-C-methylene bridge. We have investigated a modified 9-mer single stranded oligonucleotide as well as three 9- and 10-mer modified oligonucleotides hybridized to unmodified DNA. The single-stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson-Crick base-pairing with all the nucleotides in anti-conformation. By use of selective DQF-COSY spectra we determined the ratio between the N-type (C3'-endo) and S-type (C2'-endo) sugar conformations of the nucleotides. In contrast to the corresponding single-stranded DNA (ssDNA), we found that the sugar conformations of the single-stranded LNA oligonucleotide (ssLNA) cannot be described by a major S-type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N-type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N-type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S-type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N-type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases.

LNA is a bicyclic nucleic acid analogue that contains one or more 2'-O,4'-C methylene linkage(s), which effectively locks the furanose ring in a C3'-endo conformation. We report here the NMR solution structure of a nonamer LNA:RNA hybrid and a structural characterization of a nonamer LNA:DNA hybrid, where the LNA strands are composed entirely of LNA nucleotides. This is the first structural characterization of fully modified LNA oligonucleotides. The high-resolution structure reveals that the LNA:RNA hybrid adopts an almost canonical A-type duplex morphology. The helix axis is almost straight and the duplex geometry is regular. This shows that fully modified LNA oligomers can hybridize with complementary RNA and form duplexes within the Watson-Crick framework. The LNA:DNA hybrid structurally resembles an RNA:DNA hybrid as shown by determination of deoxyribose sugar puckers and analysis of NOESY NMR spectra.

Background— For the majority of cases, the cause of spontaneous aortic dissection and rupture is unknown. An inherited risk is associated with Marfan syndrome, Ehlers-Danlos syndrome type IV, and loci mapped to diverse autosomal chromosomes. Analysis of pedigrees however has indicated that it may be also inherited as an X-linked trait. The biglycan gene, found on chromosome X in humans and mice, encodes a small leucine-rich proteoglycan involved in the integrity of the extracellular matrix. A vascular phenotype has never been described in mice deficient in the gene for small leucine-rich proteoglycans. In the breeding of BALB/cA mice homozygous for a null mutation of the biglycan gene, we observed that 50% of biglycan-deficient male mice died suddenly within the first 3 months of life. Methods and Results— Necropsies revealed a major hemorrhage in the thoracic or abdominal cavity, and histology showed aortic rupture that involved an intimal and medial tear as well as dissection between the media and adventitia. By transmission electron microscopy and biomechanical testing, the aortas of biglycan-deficient mice showed structural abnormalities of collagen fibrils and reduced tensile strength. Similar collagen fibril changes were observed in male as well as in female biglycan-deficient mice, which implies a role of additional determinants such as gender-related response to stress in the development of this vascular catastrophe only in male mice. Conclusions— The spontaneous death of biglycan-deficient male mice from aortic rupture implicates biglycan as essential for the structural and functional integrity of the aortic wall and suggests a potential role of biglycan gene defects in the pathogenesis of aortic dissection and rupture in humans.

We have used two-dimensional (1)H NMR spectroscopy at 750 MHz to determine a high-resolution solution structure of an oligonucleotide containing restricted nucleotides with a 2'-O, 4'-C-methylene bridge (LNA) hybridized to the complementary DNA strand. The LNA:DNA duplex examined contained four thymidine LNA modifications (T(L), d(C1T(L)2G3C4T(L)5T(L)6C7T(L)8G9C10):d( G11C12A13G14A15A16G17C 18A19G20). A total relaxation matrix approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Forty final structures were generated for the duplex from A-form and B-form DNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the 40 structures of the complex was 0.6 A. The sugar puckerings are averaged values of a dynamic interchange between N- and S-type conformation except in case of the locked nucleotides that were found to be fixed in the C3'-endo conformation. Among the other nucleotides in the modified strand, the furanose ring of C7 and G9 is predominantly in the N-type conformation whereas that of G3 is in a mixed conformation. The furanose rings of the nucleotides in the unmodified complementary strand are almost exclusively in the S-type conformation. Due to these different conformations of the sugars in the two strands, there is a structural strain between the A-type modified strand and the B-type unmodified complementary strand. This strain is relaxed by decreasing the value of rise and compensating with tip, buckle, and propeller twist. The values of twist vary along the strand but for a majority of the base pairs a value even lower than that of A-DNA is observed. The average twist over the sequence is 32+/-1 degrees. On the basis of the structure, we conclude that the high stability of LNA:DNA duplexes is caused by a local change of the phosphate backbone geometry that favors a higher degree of stacking.

Although RANK-L is essential for osteoclast formation, factors such as transforming growth factor-␤ (TGF-␤) are potent modulators of osteoclastogenic stimuli. To systematically investigate the role of TGF-␤ in human osteoclastogenesis, monocytes were isolated from peripheral blood by three distinct approaches, resulting in either a lymphocyte-rich, a lymphocyte-poor, or a pure osteoclast precursor (CD14-positive) cell population. In each of these osteoclast precursor populations, the effect of TGF-␤ on proliferation, TRAP activity, and bone resorption was investigated with respect to time and length of exposure. When using the highly pure CD14 osteoclast precursor cell population, the effect of TGF-␤ was strongly dependent on the stage of osteoclast maturation. When monocytes were exposed to TGF-␤ during the initial culture period (days 1-7), TRAP activity and bone resorption were increased by 40%, whereas the cell number was reduced by 25%. A similar decrease in cell number was observed when TGF-␤ was present during the entire culture period (days 1-21), but in direct contrast, TRAP activity, cell fusion, cathepsin K, and matrix metalloproteinase (MMP)-9 expression as well as bone resorption were almost completely abrogated. Moreover, we found that latent TGF-␤ was strongly activated by incubation with MMP-9 and suggest this to be a highly relevant mechanism for regulating osteoclast activity. To further investigate the molecular mechanism responsible for the divergent effects of continuous versus discontinuous exposure to TGF-␤, we examined RANK expression and p38 MAPK activation. We found the TGF-␤ strongly induced p38 MAPK in monocytes, but not in mature osteoclasts, and that continuous exposure of TGF-␤ to monocytes down-regulated RANK expression. The current results suggest that TGF-␤ promotes human osteoclastogenesis in monocytes through stimulation of the p38 MAPK, whereas continuous exposure to TGF-␤ abrogates osteoclastogenesis through down-regulation of RANK expression and therefore attenuation of RANK-RANK-L signaling.

One hundred and forty-five patients suffering from diseases requiring long-term treatment with high doses of corticosteroids (30 mg/day or greater of prednisolone) were recruited to the study. Patients had to be steroid naive on entry to the study (not more than 15 days of treatment with a corticosteroid within the previous 24 months). Patients were randomized to receive either 1 microgram/day alfacalcidol or placebo capsules for 12 months. Bone mineral density (BMD) of the lumbar spine was assessed by dual-photon absorptiometry on entry and after 3, 6 and 12 months' treatment. Safety was monitored by the recording of all adverse events reported by patients and the regular screening of blood samples for hematology and serum biochemistry. Of the 145 patients, 74 were randomized to alfacalcidol and 71 to placebo. The treatment groups were well matched at baseline with no significant differences in demographic, clinical or biochemical parameters. The mean equivalent dose of prednisolone at baseline was 46.6 mg/day and 46.3 mg/day for the alfacalcidol and placebo group respectively. From the 145 patients randomized to treatment, 71 (38 who received alfacalcidol and 33 who received placebo) provided BMD data both at baseline and at 3, 6 and 12 months. The percentage change in BMD after 6 months' treatment was -2.11% in the alfacalcidol group and -4.00% in the placebo group (p = 0.39). After 12 months the percentage change in BMD was +0.39% (CI: -4.28 to 4.81) in the alfacalcidol group and -5.67% (CI: -8.13 to -3.21) in the placebo group, this difference (6.06%, CI: 0.88 to 11.24) being statistically significant (p = 0.02). An intention to treat analysis also showed a significant difference between the two treatment groups in alfacalcidol's favor (3.81%, p = 0.01; CI: 0.92 to 6.70). There was no significant difference between the two treatment groups in the corticosteroid dose at any time point during the study. Serum calcium was measured throughout and there were no significant differences between the two treatment groups at any visit. This study suggests that alfacalcidol can prevent corticosteroid-induced bone loss from the lumbar spine. Long-term use of alfacalcidol was not associated with any significant adverse effects in this diverse group of patients.

Topic: Diagnostic performance of deep learning-based algorithms in screening patients with diabetes for diabetic retinopathy (DR). The algorithms were compared to the current gold standard of classification by human specialists. Clinical relevance: As DR is a common cause of visual impairment, screening is indicated to avoid irreversible vision loss. Automated DR-classification using deep learning may be a suitable new screening tool that could improve diagnostic performance and reduce manpower. Methods: For this systematic review, we aimed to identify studies that incorporated the use of deep learning in classifying full-scale DR in retinal fundus images of patients with diabetes. The studies had to provide a DR-grading scale, a human grader as a reference standard and a deep learning performance score. A systematic search on April 5, 2018 through Medline and Embase yielded 304 publications. To identify potentially missed publications, the reference lists of the final included studies were manually screened, yielding no additional publications. The Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool was used for risk of bias and applicability assessment. Results: Based on objective selection, we included 11 diagnostic accuracy studies that validated the performance of their deep learning method using either a new group of patients or retrospective datasets. Eight studies reported sensitivity and specificity of 80.28%-100.0% and 84.0%-99.0%, respectively. Two studies report accuracies of 78.7% and 81.0%. One study provides an area under the receiver operating curve (AUC) of 0.955. In addition to diagnostic performance, one study also reported on patient satisfaction, showing that 78% of patients preferred an automated deep learning model over manual human grading. Conclusion: Advantages of implementing deep learning-based algorithms in DR-screening include reduction in manpower, cost of screening and issues relating to intra-and intergrader variability. However, limitations which may hinder such an implementation particularly revolves around ethical concerns regarding lack of trust in the diagnostic accuracy of computers. Considering both strengths and limitations as well as the high performance of deep learningbased algorithms, automated DR classification using deep learning could be feasible in a real-world screening scenario.

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