An assay for aryl radicals using BHAS coupling.

Aryl radicals are intermediates in many reactions, but determining their presence unambiguously is often challenging. As we recently reported, reaction of 2-iodo-1,3-dimethylbenzene (7) in benzene with KOtBu and a suitable organic additive, leads to a base-induced homolytic aromatic substitution (BHAS) coupling reaction giving 2,6-dimethylbiphenyl (12) and biphenyl (3) as coupled products, together with xylene (13). In this case, biphenyl arises from a radical translocation and is the major coupling product. This paper now quantitatively investigates that reaction, which shows a very similar ratio for 3 : 12 [ca. 4 : 1] when using different sources of radical initiation. Deuterium isotope studies provide detailed mechanistic support for the proposed mechanism; when carried out in C6D6vs. C6H6, the reaction is characterised by a strong isotope effect for formation of 3-d10vs. 3, but not for formation of 12-d5vs. 12. These distinctive properties mean that the transformation can act as an assay for aryl radicals. An advantage of such a BHAS process is its sensitivity, since it involves a chain reaction that can amplify radical activity.

A Hierarchy of Ligands Controls Formation and Reaction of Aryl Radicals in Pd-Catalyzed Ground-State Base-Promoted Coupling Reactions.

Palladium salts and complexes were tested separately and in the presence of added ligands as potential sources of aryl radicals in ground-state coupling reactions of aryl halide with arenes under basic conditions (KOtBu). Our recently developed assay for aryl radicals was employed to test for aryl radicals. In this assay, aryl radicals derived from the test substrate, 1-iodo-2,6-dimethylbenzene 7, undergo base-promoted homolytic aromatic substitution (BHAS) with benzene to produce 2,6-dimethylbiphenyl 8 and biphenyl 9 in an approximately 1:4 ratio as well as m-xylene 10. The biphenyl arises from a diagnostic radical transfer reaction with the solvent benzene. Using substrate 7 with a range of Pd sources as potential initiators led to formation of 8, 9, and 10 in varying amounts. However, when any one of a range of diphosphinoferrocenes (e.g., dppf or dippf) or BINAP or the monophosphine, diphenylphosphinoferrocene, was added as a ligand to Pd(OAc)2, the ratio of [2,6-dimethylbiphenyl 8: biphenyl 9] moved decisively to that expected from the BHAS (radical) pathway. Further studies were conducted with dppf. When dppf was added to each of the other Pd sources, the ratio of coupled products was also diverted to that expected for radical BHAS chemistry. Deuterium isotope studies and radical trap experiments provide strong additional support for the involvement of aryl radicals. Accordingly, under these ground-state conditions, palladium sources, in the presence of defined ligands, convert aryl iodides to aryl radicals. A rationale is proposed for these observations.

Lower-limb wearable resistance overloads joint angular velocity during early acceleration sprint running.

Lower-limb wearable resistance (WR) facilitates targeted resistance-based training during sports-specific movement tasks. The purpose of this study was to determine the effect of two different WR placements (thigh and shank) on joint kinematics during the acceleration phase of sprint running. Eighteen participants completed maximal effort sprints while unloaded and with 2% body mass thigh- or shank-placed WR. The main findings were as follows: 1) the increase to 10 m sprint time was small with thigh WR (effect size [ES] = 0.24), and with shank WR, the increase was also small but significant (ES = 0.33); 2) significant differences in peak joint angles between the unloaded and WR conditions were small (ES = 0.23-0.38), limited to the hip and knee joints, and <2° on average; 3) aside from peak hip flexion angles, no clear trends were observed in individual difference scores; and, 4) thigh and shank WR produced similar reductions in average hip flexion and extension angular velocities. The significant overload to hip flexion and extension velocity with both thigh- and shank-placed WR may be beneficial to target the flexion and extension actions associated with fast sprint running.

Disentangling the role of photosynthesis and stomatal conductance on rising forest water-use efficiency.

Multiple lines of evidence suggest that plant water-use efficiency (WUE)-the ratio of carbon assimilation to water loss-has increased in recent decades. Although rising atmospheric CO2 has been proposed as the principal cause, the underlying physiological mechanisms are still being debated, and implications for the global water cycle remain uncertain. Here, we addressed this gap using 30-y tree ring records of carbon and oxygen isotope measurements and basal area increment from 12 species in 8 North American mature temperate forests. Our goal was to separate the contributions of enhanced photosynthesis and reduced stomatal conductance to WUE trends and to assess consistency between multiple commonly used methods for estimating WUE. Our results show that tree ring-derived estimates of increases in WUE are consistent with estimates from atmospheric measurements and predictions based on an optimal balancing of carbon gains and water costs, but are lower than those based on ecosystem-scale flux observations. Although both physiological mechanisms contributed to rising WUE, enhanced photosynthesis was widespread, while reductions in stomatal conductance were modest and restricted to species that experienced moisture limitations. This finding challenges the hypothesis that rising WUE in forests is primarily the result of widespread, CO2-induced reductions in stomatal conductance.

Changes to horizontal force-velocity and impulse measures during sprint running acceleration with thigh and shank wearable resistance.

This study determined the effects of two wearable resistance (WR) placements (i.e. thigh and shank) on horizontal force-velocity and impulse measures during sprint running acceleration. Eleven male athletes performed 50 m sprints either unloaded or with WR of 2% body mass attached to the thigh or shank. In-ground force platforms were used to measure ground reaction forces and determine dependent variables of interest. The main findings were: 1) increases in sprint times and reductions in maximum velocity were trivial to small when using thigh WR (0.00-1.93%) and small to moderate with shank WR (1.56-3.33%); 2) athletes maintained or significantly increased horizontal force-velocity mechanical variables with WR (effect size = 0.32-1.23), except for theoretical maximal velocity with thigh WR, and peak power, theoretical maximal velocity and maximal ratio of force with shank WR; 3) greater increases to braking and vertical impulses were observed with shank WR (2.72-26.3% compared to unloaded) than with thigh WR (2.17-12.1% compared to unloaded) when considering the entire acceleration phase; and, 4) no clear trends were observed in many of the individual responses. These findings highlight the velocity-specific nature of this resistance training method and provide insight into what mechanical components are overloaded by lower-limb WR.

Acute Kinematic Effects of Sprinting With Motorized Assistance.

ABSTRACT: Clark, K, Cahill, M, Korfist, C, and Whitacre, T. Acute kinematic effects of sprinting with motorized assistance. J Strength Cond Res 35(7): 1856-1864, 2021-Although assisted sprinting has become popular for training maximum velocity, the acute effects are not fully understood. To examine this modality, 14 developmental male sprinters (age: 18.0 ± 2.5 years, 100-m personal best: 10.80 ± 0.31 seconds) performed maximal trials, both unassisted and assisted with a motorized towing device using a load of 7 kg (9.9 ± 0.9% body mass). Significant increases in maximum velocity (+9.4%, p ≤ 0.001, d = 3.28) occurred due to very large increases in stride length (+8.7%, p ≤ 0.001, d = 2.04) but not stride rate (+0.7%, p = 0.36, d = 0.11). Stride length increased due to small changes in distance traveled by the center of mass during ground contact (+3.7%, p ≤ 0.001, d = 0.40) combined with very large changes in distance traveled by the center of mass during flight (+13.1%, p ≤ 0.001, d = 2.62). Although stride rate did not demonstrate significant between-condition differences, the combination of contact and flight time was different. Compared to unassisted sprinting, assisted sprinting caused small but significant decreases in contact time (-5.2%, p ≤ 0.001, d = 0.49) and small but significant increases in flight time (+3.4%, p < 0.05, d = 0.58). Sprinting with motorized assistance elicited supramaximal velocities with decreased contact times, which may represent a neuromuscular stimulus for athletes attempting to enhance sprinting performance. Future research is needed to investigate the effects of this modality across various assistive loads and athletic populations, and to determine the longitudinal efficacy as a training method for improving maximum-velocity sprinting performance.

Effects of a Flexible Workout System on Performance Gains in Collegiate Athletes.

ABSTRACT: Walts, CT, Murphy, SM, Stearne, DJ, Rieger, RH, and Clark, KP. Effects of a flexible workout system on performance gains in collegiate athletes. J Strength Cond Res 35(5): 1187-1193, 2021-Although research on the topic of periodization is abundant, investigations into different flexible periodization strategies in collegiate athletes are limited. Furthermore, how state of readiness (SOR) and workout autonomy affect training improvements is largely unknown. Therefore, the purpose of this study was to determine if a flexible periodization (FP) program would elicit significantly greater performance gains compared with a nonflexible periodization (NP) program (significance set p ≤ 0.05). A total of 32 male and female intercollegiate lacrosse players completed performance measures of vertical jump, sprinting speed, change of direction, and strength in bench press and deadlift. After pretesting, subjects were matched and randomly assigned to either FP (n = 17, age = 19.4 ± 1.4 years, height = 1.72 ± 0.10 m, mass = 72.29 ± 13.73 kg) or NP (n = 15, age = 19.9 ± 1.5 years, height = 1.72 ± 0.08 m, mass = 71.68 ± 13.55 kg) training groups. Both groups trained 3 days per week for 8 weeks. The NP group completed all workout volume and intensity as prescribed by a certified strength and conditioning coach. However, the FP group modified workout volume and intensity based on a daily SOR questionnaire. Although appreciable pretest to posttest improvements were observed for the entire subject cohort, multivariate analysis of variance (ANOVA) and a series of ANOVA tests demonstrated no statistically significant between-group differences for pretest to posttest changes on any of the performance tests (range of p values: 0.17-0.95). Although FP does not seem to be more effective than NP for eliciting performance gains, it may provide greater opportunities for autonomy while eliciting equivalent improvement levels. Therefore, flexible periodization based on SOR may be a viable training strategy.

Sled-Push Load-Velocity Profiling and Implications for Sprint Training Prescription in Young Athletes.

ABSTRACT: Cahill, MJ, Oliver, JL, Cronin, JB, Clark, KP, Cross, MR, and Lloyd, RS. Sled-push load-velocity profiling and implications for sprint training prescription in young athletes. J Strength Cond Res 35(11): 3084-3089, 2021-Resisted sled pushing is a popular method of sprint-specific training; however, little evidence exists to support the prescription of resistive loads in young athletes. The purpose of this study was to determine the reliability and linearity of the force-velocity relationship during sled pushing, as well as the amount of between-athlete variation in the load required to cause a decrement in maximal velocity (Vdec) of 25, 50, and 75%. Ninety (n = 90) high school, male athletes (age 16.9 ± 0.9 years) were recruited for the study. All subjects performed 1 unresisted and 3 sled-push sprints with increasing resistance. Maximal velocity was measured with a radar gun during each sprint and the load-velocity (LV) relationship established for each subject. A subset of 16 subjects examined the reliability of sled pushing on 3 separate occasions. For all individual subjects, the LV relationship was highly linear (r > 0.96). The slope of the LV relationship was found to be reliable (coefficient of variation [CV] = 3.1%), with the loads that cause a decrement in velocity of 25, 50, and 75% also found to be reliable (CVs = <5%). However, there was large between-subject variation (95% confidence interval) in the load that caused a given Vdec, with loads of 23-42% body mass (%BM) causing a Vdec of 25%, 45-85 %BM causing a Vdec of 50%, and 69-131 %BM causing a Vdec of 75%. The Vdec method can be reliably used to prescribe sled-push loads in young athletes, but practitioners should be aware that the load required to cause a given Vdec is highly individualized.

Safety and efficacy of intravenous belimumab in children with systemic lupus erythematosus: results from a randomised, placebo-controlled trial.

OBJECTIVES: This ongoing Phase-2, randomised, placebo-controlled, double-blind study evaluated the efficacy, safety and pharmacokinetics of intravenous belimumab in childhood-onset systemic lupus erythematosus (cSLE). METHODS: Patients (5 to 17 years) were randomised to belimumab 10 mg/kg intravenous or placebo every 4 weeks, plus standard SLE therapy. Primary endpoint: SLE Responder Index (SRI4) response rate (Week 52). Key major secondary endpoints: proportion of patients achieving the Paediatric Rheumatology International Trials Organisation/American College of Rheumatology (PRINTO/ACR) response using 50 and '30 alternative' definitions (Week 52), and sustained response (Weeks 44 to 52) by SRI4 and Parent Global Assessment of well-being (Parent-global). Safety and pharmacokinetics were assessed. Study not powered for statistical testing. RESULTS: Ninety-three patients were randomised (belimumab, n=53; placebo, n=40). At Week 52, there were numerically more SRI4 responders with belimumab versus placebo (52.8% vs 43.6%; OR 1.49 (95% CI 0.64 to 3.46)). PRINTO/ACR 30 alternative (52.8% vs 27.5%; OR 2.92 (95% CI 1.19 to 7.17)) and PRINTO/ACR 50 (60.4% vs 35.0%; OR 2.74 (95% CI 1.15 to 6.54)) responses were more frequent with belimumab than placebo, as were sustained responses for SRI4 (belimumab, 43.4%; placebo, 41.0%; OR 1.08 (95% CI 0.46 to 2.52)) and Parent-global (belimumab, 59.1%; placebo, 33.3%; OR 3.49 (95% CI 1.23 to 9.91)). Serious adverse events were reported in 17.0% of belimumab patients and 35.0% of placebo patients; one death occurred (placebo). Week-52, geometric mean (95% CI) belimumab trough concentration was 56.2 (45.2 to 69.8) µg/mL. CONCLUSION: The belimumab intravenous pharmacokinetics and benefit-risk profile in cSLE are consistent with adult belimumab studies and the 10 mg/kg every 4 weeks dose is appropriate. TRIAL REGISTRATION NUMBER: NCT01649765.

Influence of resisted sled-push training on the sprint force-velocity profile of male high school athletes.

Sled pushing is a commonly used form of resisted sprint training; however, little empirical evidence exists, especially in youth populations. The aim of this study was to assess the effectiveness of unresisted and resisted sled pushing across multiple loads. Fifty high school athletes were assigned to an unresisted (n = 12), or 3 resisted groups; light (n = 14), moderate (n = 13), and heavy (n = 11) resistance that caused a 25%, 50%, and 75% velocity decrement in maximum sprint speed, respectively. All participants performed two sled-push training sessions twice weekly for 8 weeks. Before and after the training intervention, the participants performed a series of jump, strength, and sprint testing to assess athletic performance. Split times between 5 and 20 m improved significantly across all resisted groups (all P < .05, d = 0.34-1.16) but did not improve significantly with unresisted sprinting. For all resisted groups, gains were greatest over the first 5 m (d = 0.67-0.84) and then diminished over each subsequent 5 m split (d = 0.08-0.57). The magnitude of gains in split times was greatest within the heavy group. Small but non-significant within-group effects were found in pre to post force-velocity profiles. There was a main effect of time but no interaction effects as all groups increased force and power, although the greatest increases were observed with the heavy load (d = 0.50-0.51). The results of this study suggest that resisted sled pushing with any load was superior to unresisted sprint training and that heavy loads may elicit the greatest gains in sprint performance over short distances.

Influence of Resisted Sled-Pull Training on the Sprint Force-Velocity Profile of Male High-School Athletes.

Cahill, MJ, Oliver, JL, Cronin, JB, Clark, K, Cross, MR, Lloyd, RS, and Lee, JE. Influence of resisted sled-pull training on the sprint force-velocity profile of male high-school athletes. J Strength Cond Res 34(10): 2751-2759, 2020-Although resisted sled towing is a commonly used method of sprint-specific training, little uniformity exists around training guidelines for practitioners. The aim of this study was to assess the effectiveness of unresisted and resisted sled-pull training across multiple loads. Fifty-three male high-school athletes were assigned to an unresisted (n = 12) or 1 of 3 resisted groups: light (n = 15), moderate (n = 14), and heavy (n = 12) corresponding to loads of 44 ± 4 %BM, 89 ± 8 %BM, and 133 ± 12 %BM that caused a 25, 50, and 75% velocity decrement in maximum sprint speed, respectively. All subjects performed 2 sled-pull training sessions twice weekly for 8 weeks. Split times of 5, 10, and 20 m improved across all resisted groups (d = 0.40-1.04, p < 0.01) but did not improve with unresisted sprinting. However, the magnitude of the gains increased most within the heavy group, with the greatest improvement observed over the first 10 m (d ≥ 1.04). Changes in preintervention to postintervention force-velocity profiles were specific to the loading prescribed during training. Specifically, F0 increased most in moderate to heavy groups (d = 1.08-1.19); Vmax significantly decreased in the heavy group but increased in the unresisted group (d = 012-0.44); whereas, Pmax increased across all resisted groups (d = 0.39-1.03). The results of this study suggest that the greatest gains in short distance sprint performance, especially initial acceleration, are achieved using much heavier sled loads than previously studied in young athletes.

The National Football League Combine 40-yd Dash: How Important is Maximum Velocity?

Clark, KP, Rieger, RH, Bruno, RF, and Stearne, DJ. The NFL combine 40-yard dash: how important is maximum velocity? J Strength Cond Res 33(6): 1542-1550, 2019-This investigation analyzed the sprint velocity profiles for athletes who completed the 40-yard (36.6 m) dash at the 2016 National Football League (NFL) Combine. The purpose was to evaluate the relationship between maximum velocity and sprint performance, and to compare acceleration patterns for fast and slow athletes. Using freely available online sources, data were collected for body mass and sprint performance (36.6 m time with split intervals at 9.1 and 18.3 m). For each athlete, split times were used to generate modeled curves of distance vs. time, velocity vs. time, and velocity vs. distance using a monoexponential equation. Model parameters were used to quantify acceleration patterns as the ratio of maximum velocity to maximum acceleration (vmax/amax, or τ). Linear regression was used to evaluate the relationship between maximum velocity and sprint performance for the entire sample. In addition, athletes were categorized into fast and slow groups based on maximum velocity, with independent t-tests and effect size statistics used to evaluate between-group differences in sprint performance and acceleration patterns. Results indicated that maximum velocity was strongly correlated with sprint performance across 9.1, 18.3, and 36.6 m (r of 0.72, 0.83, and 0.94, respectively). However, both fast and slow groups accelerated in a similar pattern relative to maximum velocity (τ = 0.768 ± 0.068 seconds for the fast group and τ = 0.773 ± 0.070 seconds for the slow group). We conclude that maximum velocity is of critical importance to 36.6 m time, and inclusion of more maximum velocity training may be warranted for athletes preparing for the NFL Combine.

Defining the Early, Mid, and Late Subsections of Sprint Acceleration in Division I Men's Soccer Players.

Bellon, CR, DeWeese, BH, Sato, K, Clark, KP, and Stone, MH. Defining the early, mid, and late subsections of sprint acceleration in Division I men's soccer players. J Strength Cond Res 33(4): 1001-1006, 2019-The purpose of this study was to investigate whether the acceleration phase of sprinting could be split into subphases specific to the competitive demands of a soccer match by comparing sprint metrics at various sprint distances in Division I men's soccer players. Twenty-three Division I men's soccer athletes completed 2 maximal-effort 20-m sprints from a standing start position through an optical measurement system. Sprint metrics measured included sprint velocity (SV), step length (SL), step frequency (SF), and ground contact time (GCT). Each metric was recorded at approximately 2.5, 6, and 12 m. Sprint metrics at each distance were compared using a 2-tailed, 1-way repeated-measures analysis of variance. The results indicated that SV, SL, and SF were statistically greater at 12 m in comparison with 6 m (p < 0.001) and 2.5 m (p < 0.001), whereas GCT was statistically shorter at 12 m compared with 6 m (p < 0.001) and 2.5 m (p < 0.001). In addition, sprint metrics at 6 m also displayed the same relationships when compared to 2.5 m, with SV, SL, and SF being statistically greater (p < 0.001) at this distance, and GCT being statistically shorter (p < 0.001) as well. These results suggest that the acceleration phase may effectively be differentiated into early, mid, and late subphases based on differences in key sprint metrics at distances of 2.5, 6, and 12 m, respectively, in Division I men's soccer athletes.

Assessing the interplay between canopy energy balance and photosynthesis with cellulose δ18O: large-scale patterns and independent ground-truthing.

There are few whole-canopy or ecosystem scale assessments of the interplay between canopy temperature and photosynthesis across both spatial and temporal scales. The stable oxygen isotope ratio (δ18O) of plant cellulose can be used to resolve a photosynthesis-weighted estimate of canopy temperature, but the method requires independent confirmation. We compare isotope-resolved canopy temperatures derived from multi-year homogenization of tree cellulose δ18O to canopy-air temperatures weighted by gross primary productivity (GPP) at multiple sites, ranging from warm temperate to boreal and subalpine forests. We also perform a sensitivity analysis for isotope-resolved canopy temperatures that showed errors in plant source water δ18O lead to the largest errors in canopy temperature estimation. The relationship between isotope-resolved canopy temperatures and GPP-weighted air temperatures was highly significant across sites (p < 0.0001, R2 = 0.82), thus offering confirmation of the isotope approach. The previously observed temperature invariance from temperate to boreal biomes was confirmed, but the greater elevation of canopy temperature above air temperature in the boreal forest was not. Based on the current analysis, we conclude that canopy temperatures in the boreal forest are as warm as those in temperate systems because day-time-growing-season air temperatures are similarly warm.

A general relationship links gait mechanics and running ground reaction forces.

The relationship between gait mechanics and running ground reaction forces is widely regarded as complex. This viewpoint has evolved primarily via efforts to explain the rising edge of vertical force-time waveforms observed during slow human running. Existing theoretical models do provide good rising-edge fits, but require more than a dozen input variables to sum the force contributions of four or more vague components of the body's total mass (mb). Here, we hypothesized that the force contributions of two discrete body mass components are sufficient to account for vertical ground reaction force-time waveform patterns in full (stance foot and shank, m1=0.08mb; remaining mass, m2=0.92mb). We tested this hypothesis directly by acquiring simultaneous limb motion and ground reaction force data across a broad range of running speeds (3.0-11.1 m s-1) from 42 subjects who differed in body mass (range: 43-105 kg) and foot-strike mechanics. Predicted waveforms were generated from our two-mass model using body mass and three stride-specific measures: contact time, aerial time and lower limb vertical acceleration during impact. Measured waveforms (N=500) differed in shape and varied by more than twofold in amplitude and duration. Nonetheless, the overall agreement between the 500 measured waveforms and those generated independently by the model approached unity (R2=0.95±0.04, mean±s.d.), with minimal variation across the slow, medium and fast running speeds tested (ΔR2≤0.04), and between rear-foot (R2=0.94±0.04, N=177) versus fore-foot (R2=0.95±0.04, N=323) strike mechanics. We conclude that the motion of two anatomically discrete components of the body's mass is sufficient to explain the vertical ground reaction force-time waveform patterns observed during human running.

Identification of selective 8-(piperidin-4-yloxy)quinoline sulfone and sulfonamide histamine H1 receptor antagonists for use in allergic rhinitis.

A series of potent, selective and long-acting quinoline-based sulfonamide human H1 histamine receptor antagonists, designed for once-daily intranasal administration for the treatment of rhinitis were developed. Sulfonamide 33b had a slightly lower affinity for the H1 receptor than azelastine, had low oral bioavailability in the rat and dog, and was turned over to five major metabolites. Furthermore, 33b had longer duration of action than azelastine in guinea pigs, lower rat brain-penetration, and did not cause time dependent inhibition of CYP2D6 or CYP3A4. The clinical dose in humans is expected to be low (approximately 0.5mg per day) based on the clinical dose used for azelastine and a comparison of efficacy data from animal models for 33b and azelastine.

Loperamide Abuse Associated With Cardiac Dysrhythmia and Death.

Loperamide is an over-the-counter antidiarrheal with µ-opioid agonist activity. Central nervous system opioid effects are not observed after therapeutic oral dosing because of poor bioavailability and minimal central nervous system penetration. However, central nervous system opioid effects do occur after supratherapeutic oral doses. Recently, oral loperamide abuse as an opioid substitute has been increasing among patients attempting to self-treat their opioid addiction. Ventricular dysrhythmias and prolongation of the QRS duration and QTc interval have been reported after oral loperamide abuse. We describe 2 fatalities in the setting of significantly elevated loperamide concentrations.

In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization.

With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by 'third generation chemistries' may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.