Citations

Abstract Background Chromosomal translocation has been detected in many human cancers including gliomas and is considered a driving force in tumorigenesis. Co-deletion of chromosome arms 1p and 19q is a hallmark for oligodendrogliomas. On the molecular level, 1p/19q co-deletion results from t(1;19)(q10;p10), which leads to the concomitant formation of a hybrid chromosome containing the 1q and 19p arms. A method to generate 1p/19q co-deletion is lacking, which hinders the investigation of how 1p/19q co-deletion contributes to gliomagenesis. Methods We hypothesized that chromosomal translocation, such as t(1;19)(q10;p10) resulting in the 1p/19q co-deletion, may be induced by simultaneously introducing DNA double-strand breaks (DSBs) into chromosomes 1p and 19q using CRISPR/Cas9. We developed a CRISPR/Cas9-based strategy to induce t(1;19)(q10;p10) and droplet digital PCR (ddPCR) assays to detect the hybrid 1q/19p and 1p/19q chromosomes. Results After translocation induction, we detected both 1p/19q and 1q/19p hybrid chromosomes by PCR amplification of the junction regions in HEK 293T, and U-251 and LN-229 glioblastoma cells. Sequencing analyses of the PCR products confirmed DNA sequences matching both chromosomes 1 and 19. Furthermore, the 1p/19q hybrid chromosome was rapidly lost in all tested cell lines. The 1q/19p hybrid chromosome also become undetectable over time likely due to cell survival disadvantage. Conclusion We demonstrated that t(1;19)(q10;p10) may be induced by CRISPR/Cas9-mediated genomic editing. This method represents an important step toward engineering the 1p/19q co-deletion to model oligodendrogliomas. This method may also be generalizable to engineering other cancer-relevant translocations, which may facilitate the understanding of translocation roles in cancer progression.

Orthostatic stress such as head‐up tilt (HUT) elicits a wide range of HR and BP responses among healthy individuals. In this study, we evaluated cardiovascular dynamics of subjects with different HR responses to HUT but without autonomic dysfunction. We measured BP (brachial artery) and HR (ECG) during 5 min of 60° HUT in 76 healthy young individuals, while norepinephrine (NE) was assayed during the final 30 s. We then chose 30 individuals based on the extremes of HR responses to HUT: 15 with the highest (ΔHR≥ 20 bpm) and 15 with the lowest response (ΔHR ≤10 bpm). Age and BMI were not different between groups; however, gender distribution was different (high: 5F, 10 M; low: 10F, 5M). Peak HR during HUT was 87.2±10.4 bpm in the high and 69.1±14.1 bpm in the low group (P< 0.05). Baseline HR was not different between groups. High HR responders had lower SBP at baseline (120.9±9.4 vs. 129.4±11.0 mmHg) and during HUT (119.9±9.7 vs. 131.4±13.1 mmHg). High HR responders also had higher plasma NE response to HUT (ΔNE: 156.9±82.1 vs. 89.0±48.0 pg/ml; P< 0.05). Higher HR with lower BP during HUT is consistent with normal baroreflex mechanisms of integration. Although inter‐individual variability appears to be a fundamental part of cardiovascular regulation, the mechanisms of these differences and the gender discrepancy requires further investigation. Supported by K23 RR17520, HL46493, NS32352, KL2 RR024151 ‐01.

Normotensive individuals who show a robust pressor response to sympathoexcitatory stimuli are at increased risk for developing hypertension. In healthy young adults, we defined a cumulative pressor score (CPS) as the mean increase in systolic arterial pressure across 3 stressors: mental stress, cold pressor test (CPT), and isometric handgrip to fatigue (HG). We hypothesized that individuals with high CPS would possess other physiologic traits associated with cardiovascular reactivity. In 143 subjects (M/F: 57/86, ages 18–40 yr) quartiles were formed with hyper‐responders (CPS ≥ 27.0 mmHg) in the top and hypo‐responders (≤ 16.6 mmHg) in the bottom quartile. Comparisons across quartiles included age, sex, BMI, arterial catecholamines at rest and during stress, HR variability, baroreflex sensitivity by modified Oxford, pulse wave velocity, and 24‐hr ambulatory BP measures. Age was associated with CPS, as hyper‐responders (mean age±SE: 29±1 yr) were approximately 4 yrs older than each quartile (p<0.01). Hypo‐responders had decreased norepinephrine during CPT (p<0.01) and HG (p<0.01), and epinephrine during HG (p<0.01) when compared to each quartile. No other physiologic variable was associated with CPS or quartile. These findings imply that while hyper‐responders may be at greatest cardiovascular risk, it appears to be a consequence of normal aging, as few other intermediate physiologic markers of BP reactivity were associated with pressor scores. Support: HL‐089331, CTSA RR‐024150

Background: Hypertensive patients have blunted arterial baroreflex sensitivity (BRS). In these patients, BRS is positively related to heart rate variability and inversely related to blood pressure (BP) variability. We hypothesized that this relationship would also be evident in healthy normotensives, and that individuals with higher BRS would have lower ambulatory 24‐hour BP. Methods: 24‐hour ambulatory BP and heart rate was recorded using Spacelabs recorders in 53 healthy, normotensive, non‐obese individuals (32 women, 21 men). Furthermore, BRS (baroreflex control of RR‐interval) was assessed with the modified Oxford technique (sequential bolus administration of sodium nitroprusside and phenylephrine). Results: BRS as well as heart rate and BP variabilities varied greatly among participants. Whereas no relation was observed between BRS and 24‐hour heart rate and BP variabilities, BRS was positively correlated to daytime heart rate variability (R=0.324, p=0.018) and inversely to 24‐hour average mean arterial pressure (R=0.400, p=0.003). Conclusion: In normotensive individuals, higher BRS is related to higher heart rate variability during daytime. Furthermore, BRS was inversely related to 24‐hour mean arterial pressure, consistent with the idea that the baroreflex may have more long‐term influences than previously thought. Supported by NIH grants K23 RR17520, HL 46493, DFG grant He 4605/1‐1.

Head up tilt (HUT) is a meaningful diagnostic tool for baroreflex dysfunction or autonomic failure. We sought to determine if baroreflex sensitivity (BRS), the rise in plasma catecholamines, or 24‐hr ambulatory HR variability (HRV) would correlate with HR and BP during HUT. We performed 60° HUT for 5 min followed by 2 modified Oxford trials in 46 young healthy males and females. Blood pressure (brachial artery) and HR (ECG) were recorded, while arterial epinephrine (Epi) and norepinephrine (NE) were assayed during the final 30 s of HUT. HUT increased HR (mean ± SD: 63 ± 9 to 79 ± 12), MAP (89 ± 7 to 93 ± 8), Epi (28 ± 13 to 61 ± 45), and NE (111 ± 57 to 242 ± 85) (p< 0.05 for all). Individuals with a high 24‐hr HRV had a greater increase in HR during HUT (r= 0.41, P<0.05). Interestingly, a negative correlation was observed between BRS and the NE response to HUT (r= −0.36, p<0.05), such that individuals with low NE response to tilt also had a high BRS. The Epi response was not correlated with any of the measures. These findings suggest that subjects with high vagal activity (high BRS) require a lower sympathetic response (NE) to maintain BP during orthostatic stress. Supported by: K23 RR17520, HL 46493, K12 RR023263 ‐03

Patients with hypertension have a blunted sensitivity of baroreflex control of heart period. In these patients, baroreflex sensitivity is positively related to heart rate variability and inversely related to blood pressure variability. We hypothesized that this relationship would also be evident in healthy normotensive subjects and that individuals with higher baroreflex sensitivity would have lower ambulatory 24-hour blood pressure. Twenty-four-hour ambulatory blood pressure and heart rate were recorded in 50 healthy, normotensive, nonobese individuals (31 women and 19 men). The baroreflex was assessed using sequential bolus administration of sodium nitroprusside and phenylephrine, and baroreflex sensitivity was calculated as the slope of the relation between systolic blood pressure and R-R interval during the resulting blood pressure transients. Baroreflex sensitivity was inversely correlated to 24-hour average mean arterial pressure ( R =0.49; P <0.001) and positively related to daytime heart rate variability ( R =0.33; P =0.02). In contrast, no relationship was found between baroreflex sensitivity and 24-hour heart rate or blood pressure variabilities. We conclude that the relationship between baroreflex sensitivity and daytime heart rate variability was similar to that reported previously in hypertensive subjects. Furthermore, the inverse relation between baroreflex sensitivity and mean arterial pressure supports the idea that the baroreflex may exert longer-term effects on blood pressure than thought previously.