Detection of Telomeric DNA:RNA Hybrids Using TeloDRIP-qPCR.

Because of their intrinsic characteristics, telomeres are genomic loci that pose significant problems during the replication of the genome. In particular, it has been observed that telomeres that are maintained in cancer cells by the alternative mechanism of the lengthening of telomeres (ALT) harbor higher levels of replicative stress compared with telomerase-positive cancer cells. R-loops are three-stranded structures formed by a DNA:RNA hybrid and a displaced ssDNA. Emerging evidence suggests that controlling the levels of R-loops at ALT telomeres is critical for telomere maintenance. In fact, on the one hand, they favor telomere recombination, but on the other, they are a source of detrimental replicative stress. DRIP (DNA:RNA immunoprecipitation) is the main technique used for the detection of R-loops, and it is based on the use of the S9.6 antibody, which recognizes preferentially DNA:RNA hybrids in a sequence-independent manner. The detection of DNA:RNA hybrids in repetitive sequences such as telomeres requires some additional precautions as a result of their repetitive nature. Here, we share an optimized protocol for the detection of telomeric DNA:RNA hybrids, and we demonstrate its application in an ALT and in a telomerase-positive cell line. We demonstrate that ALT telomeres bear higher levels of DNA:RNA hybrids, and we propose this method as a reliable way to detect them in telomeres.

Alternative lengthening of telomeres (ALT) cells viability is dependent on C-rich telomeric RNAs.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism activated in ~10-15% of cancers, characterized by telomeric damage. Telomeric damage-induced long non-coding RNAs (dilncRNAs) are transcribed at dysfunctional telomeres and contribute to telomeric DNA damage response (DDR) activation and repair. Here we observed that telomeric dilncRNAs are preferentially elevated in ALT cells. Inhibition of C-rich (teloC) dilncRNAs with antisense oligonucleotides leads to DNA replication stress responses, increased genomic instability, and apoptosis induction selectively in ALT cells. Cell death is dependent on DNA replication and is increased by DNA replication stress. Mechanistically, teloC dilncRNA inhibition reduces RAD51 and 53BP1 recruitment to telomeres, boosts the engagement of BIR machinery, and increases C-circles and telomeric sister chromatid exchanges, without increasing telomeric non-S phase synthesis. These results indicate that teloC dilncRNA is necessary for a coordinated recruitment of DDR factors to ALT telomeres and it is essential for ALT cancer cells survival.

Is vitamin D status known among children living in Northern Italy?

PURPOSE: To assess vitamin D status in children aged 2-220 months in northeastern Italy (latitude 46°). Serum 25-hydroxyvitamin D (25OHD) concentration was assessed in 93 children afferent to the Pediatric Department of the Hospital of Udine. METHODS: Vitamin D status was defined as follows: sufficient with serum 25OHD between 50 and 250 nmol/l (level 4); insufficient between 37.5 and 50 nmol/l (level 3); deficient less than 37.5 nmol/l (level 2); severely deficient if less than 12.5 nmol/l (level 1). We investigated the potential risk factors of vitamin D deficit. RESULTS: We found that six children (6.4%) had level 1, 36 (38.7%) had level 2, 9 (9.7%) had level 3, and only 45.2% had sufficient level of 25OHD. Immigrate children had a higher risk for vitamin D deficiency if compared with Italians: 75% of non-Italian children had an insufficient 25OHD level compared with 47.0% of Italian children (p = 0.0036). There was a marked seasonal effect on 25OHD level: when plasma sample was withdrawn between November and May, only 29.4% of children showed sufficient vitamin D level, while 70.5% was insufficient (p < 0.0001). Among the obese children, 9.0% had sufficient level of 25OHD with 90% being deficient (p = 0.01). We did not find any significant difference in vitamin D status among children in different age groups. CONCLUSION: Vitamin D deficiency is common in children living in northeastern Italy. The risk factors were winter season for blood withdrawal, non-Caucasian race, and obesity. These high-risk groups should be targeted for screening and educated about the need of sunlight exposure.

Influence of the patellar button thickness on the knee flexion after total knee arthroplasty.

PURPOSE: One of the main problems of knee replacement is the limit of knee flexion. This study focuses on the knee implant and the patellar component currently in use in total knee arthroplasty, analyzing the influence of patellar thickness on the degree of knee flexion following surgery. METHODS: A kinematics study was performed to evaluate whether an optimal patellar thickness can be identified, which enables the maximum flexion angle to be achieved. Using TC images, a healthy model was built. On this basis, a model of a knee joint which had undergone total knee arthroplasty using a Legion PS prosthesis was constructed. Initially, the standard thickness of patellar implant (9 mm) was used to build the model; then several different patellar implant thicknesses (in the range of 5-15 mm) were analyzed. RESULTS: The results show a non-linear trend: a button thickness of less than 9 mm does not change the flexion angle, whereas a button thickness of over 9 mm results in a loss of flexion. The flexion loss is significant in the first two additions of thicknesses but negligible in the last ones. CONCLUSIONS: In the case studied, flexion reduction is not linearly proportional to the patellar thickness. The outcome of total knee arthroplasty is considered to be satisfactory with the standard patellar button. The results of this study could be used to compare the kinematics with other total prosthesis and patellar implants, and should enable the optimization of the patellar residue bone thickness to obtain deep flexion.