Association analysis of the vitamin D receptor gene, the type I collagen gene COL1A1, and the estrogen receptor gene in idiopathic osteoarthritis

OBJECTIVE: Evidence has accumulated supporting a role for genes in the etiology of osteoarthritis (OA). Several candidates have been targeted as potential susceptibility loci including genes that are involved in the regulation of bone density. Genetic association analysis has suggested a role for the vitamin D receptor gene (VDR) and the estrogen receptor gene (ER) in susceptibility. Such findings must be tested in additional independent cohorts. We tested for association of these 2 genes, plus a third gene implicated in bone density, COL1A1, with idiopathic OA. METHODS: A case-control cohort of 371 affected probands and 369 unaffected spouses was used. Association was tested using 4 intragenic single nucleotide polymorphisms (SNP), one each for the VDR and COL1A1 genes, and 2 for the ER gene. The VDR and ER SNP are the same SNP that have been associated with OA. All 4 SNP affect restriction enzyme sites and were genotyped using polymerase chain reaction and enzyme digestion. Allele and genotype distributions for each SNP were compared between cases and controls and analyzed using Fisher's exact test. RESULTS: There was no evidence of association of the VDR or the ER gene SNP to OA. There was weak evidence of association of the COL1A1 SNP in female cases (p = 0.017), reflected by a difference in the distribution of genotypes at this SNP between female cases and controls (p = 0.027). However, when corrected for multiple testing, these results were not significant. CONCLUSION: If the VDR, ER, or COL1A1 genes do encode predisposition to OA then the 4 SNP tested are not associated with major susceptibility alleles at these 3 loci.     

Real-time observation of Cas9 postcatalytic domain motions

CRISPR-Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease, which has become the most popular genome editing tool. Coordinated domain motions of Cas9 prior to DNA cleavage have been extensively characterized but our understanding of Cas9 conformations postcatalysis is limited. Because Cas9 can remain stably bound to the cleaved DNA for hours, its postcatalytic conformation may influence genome editing mechanisms. Here, we use single-molecule fluorescence resonance energy transfer to characterize the HNH domain motions of Cas9 that are coupled with cleavage activity of the target strand (TS) or nontarget strand (NTS) of DNA substrate. We reveal an NTS-cleavage-competent conformation following the HNH domain conformational activation. The 3′ flap generated by NTS cleavage can be rapidly digested by a 3′ to 5′ single-stranded DNA-specific exonuclease, indicating Cas9 exposes the 3′ flap for potential interaction with the DNA repair machinery. We find evidence that the HNH domain is highly flexible post-TS cleavage, explaining a recent observation that the HNH domain was not visible in a postcatalytic cryo-EM structure. Our results illuminate previously unappreciated regulatory roles of DNA cleavage activity on Cas9’s conformation and suggest possible biotechnological applications.

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system targets foreign nucleic acids for destruction in bacteria and archaea (1). Among different types of the system, CRISPR-Cas9 from Streptococcus pyogenes has been widely used for genome editing in plant and animal cells (2–5). DNA cleavage by the CRISPR-Cas9 system involves multiple steps (6). The Cas9 enzyme associates with a guide RNA, consisting of a programmable CRISPR RNA (crRNA) and a transactivating RNA (tracrRNA), to form a Cas9 ribonucleoprotein complex (RNP) (7). The DNA substrate of Cas9 RNP contains a protospacer region complementary to the spacer sequence of crRNA, and a protospacer adjacent motif (PAM) (NGG for S. pyogenes Cas9) flanking the protospacer (7). After Cas9 RNP binding, the DNA is directionally unwound from the PAM-proximal region to the PAM-distal region (8–10), and the unwound target strand (TS) is hybridized to the spacer sequence of crRNA. The TS and nontarget strand (NTS) of the DNA are cleaved by the HNH domain (residue 780 to 906) and the RuvC domain (residues 1 to 56, 718 to 765, and 926 to 1,099), respectively (7, 11). Engineering of the active site of the HNH domain or RuvC domain creates NTS nickase (Cas9_dHNH, Cas9 with H840A mutation which cleaves NTS only) or TS nickase (Cas9_dRuvC, Cas9 with D10A mutation which cleaves TS only) (7). The TS and NTS Cas9 nickases have also been used in many genome editing applications to achieve higher editing specificity or avoid generating double-stranded breaks (12–14). Cas9 RNP remains stably bound to the cleaved DNA for hours in vitro (8, 9, 15, 16), likely hindering DNA repair processes in cells (15). Therefore, a characterization of the postcatalysis state of Cas9 and its nickase variants has the potential to provide insights into genome editing mechanisms.Fluorescence resonance energy transfer (FRET) and structural studies have demonstrated the key roles of the HNH domain conformation in Cas9-mediated DNA cleavage (17–24). The HNH domain undergoes large conformational changes from the “undocked” inactive conformations to the “docked” active conformation with respect to its TS substrate upon on-target DNA binding, which represents a conformational activation of the HNH domain preceding DNA cleavage (18, 19). The HNH activation also involves conformational changes of the REC2 domain (residue 167 to 307) and REC3 domain (residue 497 to 713) (25). The HNH domain was not visible in a recent cryo-EM structure of the “product” state Cas9-RNA-DNA complex, suggesting the HNH domain is flexible after DNA cleavage (26). However, the previous FRET study showed that the docked conformation persists after DNA cleavage and the product state HNH domain motions were not observed (except for a special case in which the 3′ flap of the NTS was completely removed after DNA cleavage) (18), possibly because the labeling positions for the FRET donor and acceptor pair were not sensitive to the conformational differences between the docked state and the product state of the HNH domain. In this study, we created new Cas9 FRET constructs with increased sensitivity to small distance changes between the FRET pair and observed postcatalytic HNH domain motions using single-molecule FRET.     

Welcome back survey: exploring concerns impacting HIV care engagement and retention

The current study describes the development of a short pre-clinic survey that helped multidiscipline providers to elicit patient perspective on barriers to HIV primary care. The survey was piloted with 318 patients returning to care after being lost to care for at least 12 months. Reasons for breaks in care were dependent on age, gender, and race. Concerns about confidentiality in care were more commonly reported by African-American, Latino, and younger patients, while concerns relating to acceptance of diagnosis and side effects were greater for women, African-American, and Latino participants. Further, Intimate Partner Violence (IPV) and transportation were greater concerns for women and younger patients in the sample.     

Care Needs and Caregivers: Associations and Effects of Living Arrangements on Caregiving to Older Adults in India

As the ageing phenomenon continues in India, we explore the care needs of older adults and identify caregivers for specific care needs across living arrangements. Using the United Nations Population Fund (UNFPA) conducted Building Knowledge Base on Population Ageing in India (BKPAI 2011) data comprising 9850 older adults, we employed statistical methods to analyze the data, find associations and used binary logistic regression to model the adjusted and unadjusted effects of living arrangements on caregiving to older adults for specific care needs. Care-requiring situations considered were acute sickness, sickness requiring hospitalization, chronic morbidity, functional disability represented by ADL and IADL limitations, and locomotor disability. Results indicate that living arrangements of older adults were significantly associated with health, functional status and disability as well as caregiving patterns. Our results suggest that co-residence with children and all others was beneficial to older adults in obtaining care from a family caregiver for their hospitalization and chronic morbidity needs while living with spouse or living with a partner was advantageous for older adults in receiving care for their ADL limitations and during hospitalizations. Mean number of children was also significantly associated with the availability of a caregiver during hospitalization, locomotor disability, chronic morbidity and acute sickness. The study also highlights a little known phenomenon, that there was familial help available to older adults who lived alone. Notably, non-family sources of caregiving were steadily becoming visible (as high as 8–10 % of the caregiving component) especially among older adults living alone.     

SCAI/ACC/HRS institutional and operator requirements for left atrial appendage occlusion

© 2016 Heart Rhythm Society, the American College of Cardiology Foundation, and the Society for Cardiovascular Angiography and Interventions     

Colorectal Cancer Screening: Stool DNA and Other Noninvasive Modalities

Colorectal cancer screening dates to the discovery of pre-cancerous adenomatous tissue. Screening modalities and guidelines directed at prevention and early detection have evolved and resulted in a significant decrease in the prevalence and mortality of colorectal cancer via direct visualization or using specific markers. Despite continued efforts and an overall reduction in deaths attributed to colorectal cancer over the last 25 years, colorectal cancer remains one of the most common causes of malignancy-associated deaths. In attempt to further reduce the prevalence of colorectal cancer and associated deaths, continued improvement in screening quality and adherence remains key. Noninvasive screening modalities are actively being explored. Identification of specific genetic alterations in the adenoma-cancer sequence allow for the study and development of noninvasive screening modalities beyond guaiac-based fecal occult blood testing which target specific alterations or a panel of alterations. The stool DNA test is the first noninvasive screening tool that targets both human hemoglobin and specific genetic alterations. In this review we discuss stool DNA and other commercially available noninvasive colorectal cancer screening modalities in addition to other targets which previously have been or are currently under study.     

Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation.

We have characterized a family of repetitive DNA elements with homology to the MgPa cellular adhesion operon of Mycoplasma genitalium, a bacterium that has the smallest known genome of any free-living organism. One element, 2272 bp in length and flanked by DNA with no homology to MgPa, was completely sequenced. At least four others were partially sequenced. The complete element is a composite of six regions. Five of these regions show sequence similarity with nonadjacent segments of genes of the MgPa operon. The sixth region, located near the center of the element, is an A+T-rich sequence that has only been found in this repeat family. Open reading frames are present within the five individual regions showing sequence homology to MgPa and the adjacent open reading frame 3 (ORF3) gene. However, termination codons are found between adjacent regions of homology to the MgPa operon and in the A+T-rich sequence. Thus, these repetitive elements do not appear to be directly expressible protein coding sequences. The sequence of one region from five different repetitive elements was compared with the homologous region of the MgPa gene from the type strain G37 and four newly isolated M. genitalium strains. Recombination between repetitive elements of strain G37 and the MgPa operon can explain the majority of polymorphisms within our partial sequences of the MgPa genes of the new isolates. Therefore, we propose that the repetitive elements of M. genitalium provide a reservoir of sequence that contributes to antigenic variation in proteins of the MgPa cellular adhesion operon.     

Extent and distribution of in-stent intimal hyperplasia and edge effect in a non-radiation stent population

Intimal hyperplasia within the body of the stent is the primary mechanism for in-stent restenosis; however, stent edge restenosis has been described after brachytherapy. Our current understanding about the magnitude of in vivo intimal hyperplasia and edge restenosis is limited to data obtained primarily from select, symptomatic patients requiring repeat angiography. The purpose of this study was to determine the extent and distribution of intimal hyperplasia both within the stent and along the stent edge in relatively nonselect, asymptomatic patients scheduled for 6-month intravascular ultrasound (IVUS) as part of a multicenter trial: Heparin Infusion Prior to Stenting. Planar IVUS measurements 1 mm apart were obtained throughout the stent and over a length of 10 mm proximal and distal to the stent at index and follow-up. Of the 179 patients enrolled, 140 returned for repeat angiography and IVUS at 6.4 +/- 1.9 months and had IVUS images adequate for analysis. Patients had 1.2 +/- 0.6 Palmaz-Schatz stents per vessel. There was a wide individual variation of intimal hyperplasia distribution within the stent and no mean predilection for any location. At 6 months, intimal hyperplasia occupied 29.3 +/- 16.2% of the stent volume on average. Lumen loss within 2 mm of the stent edge was due primarily to intimal proliferation. Beyond 2 mm, negative remodeling contributed more to lumen loss. Gender, age, vessel location, index plaque burden, hypercholesterolemia, diabetes, and tobacco did not predict luminal narrowing at the stent edges, but diabetes, unstable angina at presentation, and lesion length were predictive of in-stent intimal hyperplasia. In a non-radiation stent population, 29% of the stent volume is filled with intimal hyperplasia at 6 months. Lumen loss at the stent edge is due primarily to intimal proliferation.     

HLA-DRB3*0101 is associated with Graves' disease in Jamaicans

OBJECTIVES: Graves' disease is associated with different human leucocyte antigen (HLA) genes in different populations. This studywasdesigned to examinethe HLA class II associations with Graves' disease in Jamaicans. PATIENTS: One hundred and six Jamaicans with Graves' disease and 104 controls. DESIGN: Oligotyping for HLA-DRB1, DRB3, DQA1 and DQB1 alleles was performed using the polymerase chain reaction sequence specific oligonucleotide probe (PCR-SSOP) technique. RESULTS The frequency of HLA-DRB3 *0101 was increased significantly in the patients compared to controls (38.7% vs. 19.2%; RR = 2.72; Pc < 0.015). The protective alleles for Graves' disease were DRB1 *0901 (0.9% vs. 20.2%; RR = 0.04; Pc < 0.001), DRB1*1001 (0.0% vs. 11%; RR = 0.0%; Pc < 0.01) and DRB4 *0101 (0.0% vs. 12.5%; RR = 0.0; Pc < 0.05). A high female to male ratio of Graves' disease, 25 :1, was observed. Other associated autoimmune diseases were rare and no significant HLA class II associations were found with clinical markers of disease. CONCLUSIONS: Jamaican patients with Graves' disease share the DRB3 *0101 susceptible allele and the DRB4 *01 protective allele but not the susceptible haplotype DRB1 *0301, DRB3*0101, DQA1*0501 with Caucasians.     

Improving postprandial hyperglycemia in patients with type 2 diabetes already on basal insulin therapy: Review of current strategies

A large number of patients with type 2 diabetes (T2D) on basal insulin do not reach their HbA1c goals and require additional therapy to address postprandial hyperglycemia. Guidelines from expert bodies have outlined several approaches to accomplish postprandial glucose (PPG) control, and recent literature suggests several more. This article provides strategies for primary care physicians caring for patients with T2D who do not achieve glycemic control with basal insulin alone. Current treatment guidelines and strategies for improving PPG control are reviewed, including the efficacy, safety, and cost‐effectiveness of rapid‐acting insulin (RAI) analogs, premixed insulin, glucagon‐like peptide‐1 (GLP‐1) receptor agonists (RAs), dipeptidyl peptidase 4 inhibitors, sodium–glucose cotransporter 2 inhibitors, and α‐glucosidase inhibitors. Other approaches, such as combinations of newer basal insulin plus RAI and a fixed‐ratio combination of basal insulin and a GLP‐1 RA, are also described.