Computational and AI-driven 3D structural analysis of human papillomavirus (HPV) oncoproteins E5, E6, and E7 reveal significant divergence of HPV E5 between low-risk and high-risk genotypes.

There are over 220 identified genotypes of Human papillomavirus (HPV), and the HPV genome encodes 3 major oncogenes, E5, E6, and E7. Conservation and divergence in protein sequence and function between low-risk versus high-risk oncogenic HPV genotypes has not been fully characterized. Here, we used modern computational and structural folding algorithms to perform a comparative analysis of HPV E5, E6, and E7 between multiple low risk and high risk genotypes. We first identified significantly greater sequence divergence in E5 between low- and high-risk genotypes compared to E6 and E7. Next, we used AlphaFold to model the structure of papillomavirus proteins and complexes with high confidence, including some with no established consensus structure. We observed that HPV E5, but not E6 or E7, had a dramatically different 3D structure between low-risk and high-risk genotypes. To our knowledge, this is the first comparative analysis of HPV proteins using Alphafold artificial intelligence (AI) system. The marked differences in E5 sequence and structure in high-risk HPVs may contribute in important and underappreciated ways to the development of HPV-associated cancers.

Effects of adolescent alcohol exposure on stress-induced reward deficits, brain CRF, monoamines and glutamate in adult rats.

BACKGROUND: Adolescent alcohol exposure may increase depression vulnerability in adulthood by increasing the anhedonic response to stress. METHODS: Male Wistar rats (postnatal days 28-53) were exposed to binge-like adolescent intermittent ethanol (AIE) or water. In adulthood, rats were exposed to social defeat, consisting of daily confrontations with an aggressive conspecific, followed by testing of brain reward function in a discrete-trial current-intensity intracranial self-stimulation procedure for 10 consecutive days. Neurochemistry and corticotropin-releasing factor (CRF) and CRF receptor 1 (CRFR1) mRNA levels were assessed in corticolimbic brain areas on day 11 of social defeat stress. RESULTS: Social defeat elevated reward thresholds in both AIE- and water-exposed rats indicating stress-induced anhedonia. However, AIE-exposed rats were more likely to show threshold elevations after repeated stress compared to water-exposed rats. AIE exposure decreased CRF mRNA levels in the nucleus accumbens and increased CRFR1 mRNA levels in the prefrontal cortex, while stress increased CRF mRNA levels in the central amygdala. In the caudate putamen, AIE exposure decreased dopamine turnover, while stress increased glutamate and serotonin metabolism and turnover. CONCLUSIONS: These results demonstrate increased risk of repeated stress-induced anhedonia after AIE exposure, an effect that may be due to alterations in brain CRF and dopamine systems. These results suggest that the increased rates of depression reported in people with a history of adolescent alcohol exposure may be related to alterations in brain reward and stress systems that may contribute to increased stress-induced anhedonia.

Effects of early life stress and adolescent ethanol exposure on adult cognitive performance in the 5-choice serial reaction time task in Wistar male rats.

RATIONALE: Early life stress combined with heavy adolescent alcohol use predicts impaired neuropsychological functioning in adulthood. We investigated whether adolescent intermittent ethanol (AIE) exposure combined with neonatal maternal separation in rats altered attentional processes and impulsivity in adulthood. METHODS: Male Wistar rat pups were exposed to maternal separation (postnatal days (PNDs) 1-14) and moderate AIE exposure (PNDs 28-57). Adult rats were tested in the five-choice serial reaction time task, which provides separate measures of attention, motor impulsivity, and compulsivity. Rats were tested under baseline conditions and in response to task manipulations that increased attentional load and impulsive behaviors, and after acute ethanol administration. RESULTS: Short stimulus and short intertrial interval (ITI) durations disrupted attention while long ITI durations impaired impulsivity in all rats. Moderate- and high-dose ethanol challenges impaired attention in all rats. Rats exposed to maternal separation and/or AIE exposure had significantly decreased omissions than non-handled water-exposed rats at baseline and tended to retain this effect in response to task challenges (i.e., the shorter stimulus and ITI durations, longer test session) and ethanol challenges, indicating moderate improvement of attentional performance. Maternal separation significantly increased perseverative responses at baseline and in response to decreased stimulus duration challenge, suggesting increased compulsivity. CONCLUSIONS: Separate or combined exposure to early life stress and AIE exposure moderately disrupts some aspects of adult executive control functions (e.g., increased compulsivity) but improves others (e.g., increased attention). The relative intensity of either manipulation during neonatal and adolescent periods may influence the direction in which cognitive behaviors are affected in adulthood.

Risky choice and brain CRF after adolescent ethanol vapor exposure and social stress in adulthood.

Adolescent ethanol exposure increases risky choice and alters corticotropin releasing factor (CRF) systems in adulthood. The impact of stress on risky choice after adolescent intermittent ethanol (AIE) exposure is not known. We investigated time-specific effects of AIE vapor exposure during early adolescence on risky choice after stress or no stress in adulthood. Male Wistar rats were exposed to air or AIE vapor on postnatal days 28-42 (adolescence) and were exposed to 10days of social defeat or no stress on postnatal days 172-181 (adulthood). Risky choice was assessed in the probability discounting task under baseline conditions and after days 1 and 10 of social defeat. CRF and CRF receptor 1 (CRFR1) mRNA levels were assessed in the prefrontal cortex (PFC) and the central nucleus of the amygdala (CeA) 24h post-stress to evaluate persistent effects of stress on the brain. AIE exposure had no effect on risky choice either at baseline or after social defeat. Additionally, neither acute nor chronic social defeat affected risky choice in air-exposed rats. In the PFC, chronic social defeat selectively decreased CRF mRNA levels in air-exposed rats and increased CRFR1 mRNA levels in all rats. AIE exposure increased CRF mRNA levels in the CeA with no effect of social stress. Our results indicate no effect of ethanol exposure via vapor during early adolescence on risky choice, while our previous findings indicated that AIE exposure via gavage affected risky choice. Both AIE exposure and social defeat altered CRF and CRFR1 mRNA levels in the brain.

Adolescent alcohol exposure decreased sensitivity to nicotine in adult Wistar rats.

Many adolescents engage in heavy alcohol use. Limited research in humans indicates that adolescent alcohol use predicts adult tobacco use. The present study investigated whether adolescent intermittent ethanol (AIE) exposure alters nicotine sensitivity in adulthood. Adolescent male Wistar rats (postnatal day 28-53) were exposed to AIE exposure that consisted of 5 g/kg of 25 percent ethanol three times per day in a 2 days on/2 days off regimen. Control rats received water with the same exposure regimen. In adulthood, separate groups of rats were tested for nicotine intravenous self-administration (IVSA), drug discrimination and conditioned taste aversion (CTA). The dose-response function for nicotine IVSA under a fixed-ratio schedule of reinforcement was similar in AIE-exposed and control rats. However, AIE-exposed rats self-administered less nicotine at the lowest dose, suggesting that low-dose nicotine was less reinforcing in AIE-exposed, compared with control rats. AIE-exposed rats self-administered less nicotine under a progressive-ratio schedule, suggesting decreased motivation for nicotine after AIE exposure. The discriminative stimulus effects of nicotine were diminished in AIE-exposed rats compared with control rats. No group differences in nicotine CTA were observed, suggesting that AIE exposure had no effect on the aversive properties of nicotine. Altogether, these results demonstrate that AIE exposure decreases sensitivity to the reinforcing, motivational and discriminative properties of nicotine while leaving the aversive properties of nicotine unaltered in adult rats. These findings suggest that drinking during adolescence may result in decreased sensitivity to nicotine in adult humans, which may in turn contribute to the higher rates of tobacco smoking.

Adolescent intermittent ethanol exposure diminishes anhedonia during ethanol withdrawal in adulthood.

Adolescent alcohol use may interfere with neurodevelopment, increasing the likelihood of adult alcohol use disorders (AUDs). We investigated whether adolescent intermittent ethanol (AIE) exposure alters the adult reward response to ethanol. Adolescent rats were administered ethanol once (moderate exposure; Cohort 1) or three times per day (severe exposure; Cohort 2) in a 2 days on/2 days off pattern. In adulthood, subjects responded for electrical stimulation directed at the posterior lateral hypothalamus in a discrete-trial intracranial self-stimulation (ICSS) procedure that provides current-intensity thresholds as a measure of brain reward function. The effects of ethanol administration and withdrawal were assessed. Control rats showed dose-dependent threshold elevations after acute ethanol, indicating reward deficits. A majority of moderately AIE-exposed rats (Cohort 1) showed threshold lowering after ethanol, suggesting ethanol-induced reward enhancement in this sub-set of rats. Rats exposed to severe AIE (Cohort 2) showed no threshold elevation or lowering, suggesting a blunted affective ethanol response. Daily ethanol induced threshold elevations 24h after administration in control rats but not in either group of AIE-exposed rats, suggesting decreased sensitivity to the negative affective state of ethanol withdrawal. Withdrawal from a 4-day ethanol binge produced robust and enduring threshold elevations in all rats, although threshold elevations were diminished in rats exposed to severe AIE. These results indicate that AIE exposure diminished reward deficits associated with ethanol intoxication and withdrawal and may have increased ethanol-induced reward enhancement in a sub-set of rats. In humans, enhanced ethanol reward accompanied by reduced withdrawal severity may contribute to the development of AUDs.

Impulsive choice and anxiety-like behavior in adult rats exposed to chronic intermittent ethanol during adolescence and adulthood.

Binge drinking during adolescence and adulthood may have differential long-term effects on the brain. We investigated the long-term effects of chronic intermittent ethanol (CIE) exposure during adolescence and adulthood on impulsivity and anxiety-like behavior. Adolescent (adolescent-exposed) and adult (adult-exposed) rats were exposed to CIE/water on postnatal days (PND) 28-53 and PND146-171, respectively, and a 4-day ethanol/water binge on PND181-184 and PND271-274, respectively. During withdrawal from CIE and 4-day binge exposures, anxiety-like behavior and arousal were measured in the light-potentiated startle (LPS) and acoustic startle (ASR) procedures, respectively. Impulsive choice was evaluated in the delay discounting task (DDT) at baseline and after ethanol challenges. Independent of age, ASR and LPS were decreased during withdrawal from CIE exposure. In contrast, LPS was increased in adult-exposed, but not adolescent-exposed, rats during withdrawal from the 4-day ethanol binge. CIE exposure had no effect on preference for the large delayed reward at baseline, independent of age. During DDT acquisition, CIE-exposed, compared with water-exposed rats, omitted more responses, independent of age, suggesting the CIE-induced disruption of cognitive processes. Ethanol challenges decreased preference for the large reward in younger adolescent-exposed rats but had no effect in older adult-exposed rats, independent of previous CIE/water exposure. Taken together, the present studies demonstrate that CIE withdrawal-induced decreases in anxiety and arousal were not age-specific. CIE exposure had no long-term effects on baseline impulsive choice. Subsequent ethanol exposure produced age-dependent effects on impulsivity (increased impulsivity in younger adolescent-exposed rats) and anxiety-like behavior (increased anxiety-like behavior in older adult-exposed rats).

Adolescent intermittent ethanol exposure is associated with increased risky choice and decreased dopaminergic and cholinergic neuron markers in adult rats.

BACKGROUND: Binge drinking is prevalent during adolescence and may have effects on the adult brain and behavior. The present study investigated whether adolescent intermittent ethanol exposure alters adult risky choice and prefrontal dopaminergic and forebrain cholinergic neuronal marker levels in male Wistar rats. METHODS: Adolescent (postnatal day 28-53) rats were administered 5 g/kg of 25% (vol/vol) ethanol 3 times/d in a 2-days-on/2-days-off exposure pattern. In adulthood, risky choice was assessed in the probability discounting task with descending and ascending series of large reward probabilities and after acute ethanol challenge. Immunohistochemical analyses assessed tyrosine hydroxylase, a marker of dopamine and norepinephrine in the prelimbic and infralimbic cortices, and choline acetyltransferase, a marker of cholinergic neurons, in the basal forebrain. RESULTS: All of the rats preferred the large reward when it was delivered with high probability. When the large reward became unlikely, control rats preferred the smaller, safe reward, whereas adolescent intermittent ethanol-exposed rats continued to prefer the risky alternative. Acute ethanol had no effect on risky choice in either group of rats. Tyrosine hydroxylase (prelimbic cortex only) and choline acetyltransferase immunoreactivity levels were decreased in adolescent intermittent ethanol-exposed rats compared with controls. Risky choice was negatively correlated with choline acetyltransferase, implicating decreased forebrain cholinergic activity in risky choice. CONCLUSIONS: The decreases in tyrosine hydroxylase and choline acetyltransferase immunoreactivity suggest that adolescent intermittent ethanol exposure has enduring neural effects that may lead to altered adult behaviors, such as increased risky decision making. In humans, increased risky decision making could lead to maladaptive, potentially harmful consequences.

Long-term effects of chronic intermittent ethanol exposure in adolescent and adult rats: radial-arm maze performance and operant food reinforced responding.

BACKGROUND: Adolescence is not only a critical period of late-stage neurological development in humans, but is also a period in which ethanol consumption is often at its highest. Given the prevalence of ethanol use during this vulnerable developmental period we assessed the long-term effects of chronic intermittent ethanol (CIE) exposure during adolescence, compared to adulthood, on performance in the radial-arm maze (RAM) and operant food-reinforced responding in male rats. METHODOLOGY/PRINCIPAL FINDINGS: Male Sprague Dawley rats were exposed to CIE (or saline) and then allowed to recover. Animals were then trained in either the RAM task or an operant task using fixed- and progressive- ratio schedules. After baseline testing was completed all animals received an acute ethanol challenge while blood ethanol levels (BECs) were monitored in a subset of animals. CIE exposure during adolescence, but not adulthood decreased the amount of time that animals spent in the open portions of the RAM arms (reminiscent of deficits in risk-reward integration) and rendered animals more susceptible to the acute effects of an ethanol challenge on working memory tasks. The operant food reinforced task showed that these effects were not due to altered food motivation or to differential sensitivity to the nonspecific performance-disrupting effects of ethanol. However, CIE pre-treated animals had lower BEC levels than controls during the acute ethanol challenges indicating persistent pharmacokinetic tolerance to ethanol after the CIE treatment. There was little evidence of enduring effects of CIE alone on traditional measures of spatial and working memory. CONCLUSIONS/SIGNIFICANCE: These effects indicate that adolescence is a time of selective vulnerability to the long-term effects of repeated ethanol exposure on neurobehavioral function and acute ethanol sensitivity. The positive and negative findings reported here help to further define the nature and extent of the impairments observed after adolescent CIE and provide direction for future research.

Examining the discriminative and strengthening effects of reinforcers in concurrent schedules.

Reinforcers may increase operant responding via a response-strengthening mechanism whereby the probability of the preceding response increases, or via some discriminative process whereby the response more likely to provide subsequent reinforcement becomes, itself, more likely. We tested these two accounts. Six pigeons responded for food reinforcers in a two-alternative switching-key concurrent schedule. Within a session, equal numbers of reinforcers were arranged for responses to each alternative. Those reinforcers strictly alternated between the two alternatives in half the conditions, and were randomly allocated to the alternatives in half the conditions. We also varied, across conditions, the alternative that became available immediately after a reinforcer. Preference after a single reinforcer always favored the immediately available alternative, regardless of the local probability of a reinforcer on that alternative (0 or 1 in the strictly alternating conditions, .5 in the random conditions). Choice then reflected the local reinforcer probabilities, suggesting some discriminative properties of reinforcement. At a more extended level, successive same-alternative reinforcers from an alternative systematically shifted preference towards that alternative, regardless of which alternative was available immediately after a reinforcer. There was no similar shift when successive reinforcers came from alternating sources. These more temporally extended results may suggest a strengthening function of reinforcement, or an enhanced ability to respond appropriately to "win-stay" contingencies over "win-shift" contingencies.

Contingent stimuli signal subsequent reinforcer ratios.

Conditioned reinforcer effects may be due to the stimulus' discriminative rather than its strengthening properties. While this was demonstrated in a frequently-changing choice procedure, a single attempt to replicate in a relatively static choice environment failed. We contend that this was because the information provided by the stimuli was nonredundant in the frequently-changing preparation, and redundant in the steady-state arrangement. In the present experiments, 6 pigeons worked in a steady-state concurrent schedule procedure with nonredundant informative stimuli (red keylight illuminations). When a response-contingent red keylight signaled that the next food delivery was more likely on one of the two alternatives, postkeylight choice responding was reliably for that alternative. This effect was enhanced after a history of extended informative red keylight presentation (Experiment 2). These results lend support to recent characterizations of conditioned reinforcer effects as reflective of a discriminative, rather than a reinforcing, property of the stimulus.

Time versus response indices affect conclusions about preference pulses.

Using data from two conditions of an experiment that arranged response-contingent foods and red keylight illuminations, we compared time-indexed and response-indexed preference pulses. The interpretation of red keylight effects (relative to food effects) differed according to whether time or behaviour was used to index choice in the post-event period: preference after a red keylight was more extreme than after a food in the response pulses while preference after a food was more extreme than after a keylight in the time pulses. This was due to different response latencies following the two events in combination with a tendency for initial preference to decrease as latency increased: responding began sooner after non-foods and responding that began later, after any event, tended towards indifference. Our finding suggests that preference pulses ought to be indexed according to time since the last event delivery, particularly if the response-contingent events may result in systematically different response latencies.

Conditional reinforcers and informative stimuli in a constant environment.

Five pigeons responded on steady-state concurrent variable-interval variable-interval schedules of food presentation in which half of the foods were removed and replaced with nonfood stimuli. Across conditions, the stimuli were either paired or unpaired with food, and the correlation between the ratio of food deliveries on the two alternatives and the ratio of nonfood stimuli was either -1, 0, or +1. Neither the pairing of stimuli with food, nor the correlation between stimuli and food, affected generalized-matching performance, but paired stimuli had a demonstrable effect at a local level of analysis. This effect was independent of the food-stimulus correlation. These results differ from results previously obtained in a frequently changing environment. We attribute this difference in results to differences in the information value of response-contingent stimuli in frequently changing versus relatively constant environments, as well as to differences between forward pairing and simultaneous pairing of the stimuli with food.