Purinergic Receptor Functionality Is Necessary for Infection of Human Hepatocytes by Hepatitis Delta Virus and Hepatitis B Virus

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) are major sources of acute and chronic hepatitis. HDV requires the envelope proteins of HBV for the processes of assembly and infection of new cells. Both viruses are able to infect hepatocytes though previous studies have failed to determine the mechanism of entry into such cells. This study began with evidence that suramin, a symmetrical hexasulfated napthylurea, could block HDV entry into primary human hepatocytes (PHH) and was then extrapolated to incorporate findings of others that suramin is one of many compounds that can block activation of purinergic receptors. Thus other inhibitors, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS) and brilliant blue G (BBG), both structurally unrelated to suramin, were tested and found to inhibit HDV and HBV infections of PHH. BBG, unlike suramin and PPADS, is known to be more specific for just one purinergic receptor, P2X7. These studies provide the first evidence that purinergic receptor functionality is necessary for virus entry. Furthermore, since P2X7 activation is known to be a major component of inflammatory responses, it is proposed that HDV and HBV attachment to susceptible cells, might also contribute to inflammation in the liver, that is, hepatitis

Targeting Hepatitis B Virus with Zinc Finger Nucleases

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy

INFLUENCE OF BODY POSITION ON VALSALVA RESPONSES IN YOUNG BLACK AND AFRICAN AMERICAN ADULTS

Ryan Aultman, Marshall Dearmon, Kenneth R. Ladner, Barry Faulkner, Ta\u27quoris Newsome, Jon Stavres. The University of Southern Mississippi, Hattiesburg, MS. BACKGROUND: Evidence suggests that Black and African American (BAA) individuals are more susceptible to autonomic dysfunction compared to White individuals. However, it remains unclear if these findings extend to the autonomic responses to the Valsalva (VL) maneuver. Accordingly, this study compared blood pressure and heart rate responses to the VL maneuver between BAA and White individuals. METHODS: Twenty individuals (10 BAA, 10 White) completed two VLs, each separated by 45 seconds, in both the seated and supine positions (4 VLs in total). VLs were maintained at 45 mmHg, and the order of conditions was counterbalanced between participants. Cardiac rhythm and beat-by-beat blood pressure were continuously recorded throughout each VL, and the relative changes in mean arterial pressure (ΔMAP), systolic blood pressure (ΔSBP), diastolic blood pressure (ΔDBP), pulse pressure (ΔPP), and heart rate (ΔHR) were recorded for phases I (initial hypertensive response), IIa (early phase II depressor response), IIb (late phase II blood pressure recovery), III (rebound hypotensive response after exhalation), and IV (45 second period following exhalation) of each VL. Responses were averaged across both VLs performed within each condition (seated vs. supine), and compared across conditions and between races using repeated measures analyses of variance. RESULTS: At baseline, the BAA group demonstrated a significantly lower mean PP compared to White individuals (-18 ± 8 mmHg, P=0.01), and tended to demonstrate an elevated mean DBP (+10 ± 4 mmHg, P=0.05). When VL responses were compared between groups and across conditions, a significant race by condition interaction was observed for the ΔMAP response during phase IIa (F1,18=6.80, P=0.01). This was explained by a significant exaggeration of the phase IIa ΔMAP response in White individuals during the seated condition (-9 ± 2 ΔmmHg, P\u3c0.01), which was not observed in the BAA group (-1 ± 2 ΔmmHg, P=0.64). Significant main effects of race were also observed for the absolute DBP responses, such that DBP was higher in the BAA group compared to White individuals during all phases (all P\u3c0.04), with the exception of phase IIb (P=0.34). CONCLUSIONS: These findings suggest that phase IIa VL responses are altered in BAA individuals compared to White individuals. This may have important implications for better understanding autonomic dysfunction in this population

COMBINED INFLUENCE OF POSTURE AND ISOMETRIC HANDGRIP ON PHASE IIA RESPONSES TO THE VALSALVA MANEUVER

Barry Faulkner, Marshall Dearmon, Kenneth R. Ladner, Ta’Quoris Newsome, Ryan Aultman, Jon Stavres. University of Southern Mississippi, Hattiesburg, MS. BACKGROUND: The Valsalva maneuver (VL) is a well-documented assessment of autonomic function, and is characterized by 5 distinct blood pressure phases (Phases I, IIa, IIb, III, and IV). While body position is known to influence the early phase II (a.k.a. “phase IIa”) blood pressure responses, the influence of sympathoexcitation is less clear. The aim of this study was to examine the independent and combined effects of sympathoexcitation (via isometric handgrip) and body position on the phase IIa blood pressure responses to VLs in young healthy adults. METHODS: Seventeen subjects (9 male) participated in 4 separate experimental trials (T1-T4) repeated across 2 different conditions (supine and seated). Trials were conducted in sequential order, and included VLs performed without handgrip (Trials 1 [T1] and 4 [T4]), five minutes of isometric handgrip (HG; 35% MVC; Trial 2 [T2]), and VLs performed with superimposed HG (Trial 3 [T3]). Cardiac rhythm (electrocardiography) and beat-by-beat blood pressure data were collected continuously during each trial, and the relative changes in heart rate (ΔHR), mean arterial pressure (ΔMAP), systolic blood pressure (ΔSBP), and diastolic blood pressure (ΔDBP) were compared between conditions and across trials using analyses of variance with repeated measures. RESULTS: Phase IIa ΔSBP responses were significantly blunted during T3 compared to T1 in the supine condition (+6 ± 3 ΔmmHg, P=0.03), and ΔMAP, ΔSBP, and ΔDBP responses were significantly augmented during T1 in the seated condition compared to T1 in the supine condition (-9 ± 2 ΔmmHg, -15 ± 4 ΔmmHg, -8 ± 2 ΔmmHg, respectively, all P\u3c0.01). However, no significant differences were observed for ΔMAP, ΔSBP, or ΔDBP between T3 in the seated condition compared to T1 in the supine condition (-6 ± 3 ΔmmHg, -8 ± 4 ΔmmHg, and -3 ± 2 ΔmmHg, respectively, all P\u3e0.10), suggesting that handgrip mitigated the influence of orthostasis. No significant differences were observed for ΔHR responses during phase IIa for any comparison (all P\u3e0.40). CONCLUSIONS: Handgrip blunts the phase IIa blood pressure responses to VLs performed in the supine condition, and also appears to mitigate the exaggerated phase IIa responses observed during orthostasis. This information has important implications for understanding the physiological factors contributing to abnormal VL responses in young healthy adults

INFLUENCE OF ISOMETRIC HANDGRIP AND BODY POSITION ON BAROREFLEX SENSITIVITY IN BLACK AND WHITE ADULTS

Ta\u27Quoris Newsome, Marshall Dearmon, Kenneth R. Ladner, Barry Faulkner, Hunter Haynes, Ryan Aultman, Jon Stavres. University of Southern Mississippi, Hattiesburg, MS. BACKGROUND: Resting sympathetic tone has been shown to be elevated in Black and African American (BAA) individuals compared to White individuals, but differences in Baroreflex Sensitivity (BRS) are less clear. Considering that exercise and orthostasis are both known to elicit acute changes in sympathetic tone, evaluating changes in BRS during handgrip exercise or during an orthostatic challenge may unmask any potential differences in BRS between BAA and White individuals. As such, the purpose of this study was to examine the relative influences of isometric handgrip exercise and body position on BRS in a sample of BAA and White participants. METHODS: Cardiac rhythm (via electrocardiography) and beat-by-beat blood pressure (via finger photoplethysmography) were continuously recorded in twenty participants (10 BAA, 10 White) during 3 minutes of rest and 3 minutes of isometric handgrip exercise (35% maximal voluntary isometric contraction) in the supine and seated positions. After manual exclusion of cardiac arrhythmias, BRS was evaluated for each 3-minute period using the sequence method (seq. length \u3e 3, r \u3e 0.8, delay = 0 beats). BRS was quantified as the total baroreflex gain of up-ramping sequences (BRSup), the gain of down-ramping sequences (BRSdown), and total gain (BRStotal). Each value was compared across conditions (supine rest vs. supine handgrip exercise vs. seated rest) and between races using analyses of variance with repeated measures. RESULTS: At baseline, no significant differences were observed for BRSup (-0.12 ± 10.97 ms/mmHg, P=0.99), BRSdown(-2.06 ± 8.24 ms/mmHg, P=0.80), or BRStotal (-3.14 ± 9.35 ms/mmHg, P=0.74) between BAA or White participants. When the influence of handgrip and body position were examined, no significant race by condition interactions were observed for any value (all P\u3e0.31). Instead, significant main effects of condition were observed for the entire sample (F1,32=9.75, P\u3c0.01), which was explained by significant decreases in BRSup (-21.82 ± 5.14 ms/mmHg, P\u3c0.01) and BRStotal (-13.41 ± 4.54 ms/mmHg, P=0.02) during handgrip compared to supine rest. BRSup was also significantly lower during handgrip exercise compared to seated rest (-10.43 ± 3.85 ms/mmHg, P=0.04). CONCLUSIONS: These results suggest that handgrip exercise decreases BRS similarly between BAA and White individuals, supporting the notion that BRS may not be significantly altered in healthy BAA adults

Mating patterns and post-mating isolation in three cryptic species of the Engystomops petersi species complex

Determining the extent of reproductive isolation in cryptic species with dynamic geographic ranges can yield important insights into the processes that generate and maintain genetic divergence in the absence of severe geographic barriers. We studied mating patterns, propensity to hybridize in nature and subsequent fertilization rates, as well as survival and development of hybrid F1 offspring for three nominal species of the Engystomops petersi species complex in Yasuní National Park, Ecuador. We found at least two species in four out of six locations sampled, and 14.3% of the wild pairs genotyped were mixed-species (heterospecific) crosses. We also found reduced fertilization rates in hybrid crosses between E. petersi females and E. "magnus" males, and between E. "magnus" females and E. "selva" males but not in the reciprocal crosses, suggesting asymmetric reproductive isolation for these species. Larval development times decreased in F1 hybrid crosses compared to same species (conspecific) crosses, but we did not find significant reduction in larval survival or early metamorph survival. Our results show evidence of post-mating isolation for at least two hybrid crosses of the cryptic species we studied. The general decrease in fertilization rates in heterospecific crosses suggests that sexual selection and reinforcement might have not only contributed to the pattern of call variation and behavioral isolation we see between species today, but they may also contribute to further signal divergence and behavioral evolution, especially in locations where hybridization is common and fertilization success is diminished