Identification of sequence changes in myosin II that adjust muscle contraction velocity.

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (ß/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of ß-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj < 0.05) but not with the clade (padj > 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human ß-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human ß-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of ß-myosin as species mass increased to enable a reduced contraction velocity and heart rate.

Long-acting Injectable PrEP Interest and General PrEP Awareness among People who Inject Drugs in the San Diego-Tijuana Border Metroplex.

Long-acting injectable HIV pre-exposure prophylaxis (LAI-PrEP) could help overcome multilevel challenges to HIV prevention for people who inject drugs (PWID), including those in the binational San Diego-Tijuana metroplex. Yet, general PrEP awareness and interest in LAI-PrEP remain underexplored among PWID. From 2020 to 2021, 562 HIV-negative PWID in San Diego and Tijuana completed surveys assessing general PrEP awareness and interest in oral and LAI-PrEP. Modified Poisson regression examined factors associated with general PrEP awareness. Multinomial logistic regression assessed factors associated with interest in both oral and LAI-PrEP, oral PrEP only, LAI-PrEP only, or neither. General PrEP awareness was low (18%) and associated with experiencing unsheltered homelessness (adjusted prevalence ratio [APR] = 1.50, 95% confidence interval [CI]: 0.96-2.33), past 6-month fentanyl injection (APR = 1.53, 95% CI: 1.04-2.25), and transactional sex (APR = 1.71, 95% CI: 1.06-2.76). Interest in oral PrEP only was most common (44%), followed by LAI-PrEP only (25%) and neither (16%). Compared to the odds of being interested in LAI-PrEP only, the odds of being interested in oral PrEP only were lower among those who were stopped by police (AOR = 0.38, 95% CI: 0.22-0.65), reported past 6-month fentanyl injection (AOR = 0.33, 95% CI: 0.20-0.56), polydrug use (AOR = 0.48, 95% CI: 0.27-0.86), injecting multiple times daily (AOR = 0.26, 95% CI: 0.14-0.46), receptive syringe use (AOR = 0.30, 95% CI: 0.19-0.49), and higher perceived HIV risk (AOR = 0.24, 95% CI: 0.15-0.39). Interest in LAI-PrEP was more common among PWID reporting social and structural factors that could interfere with oral PrEP adherence, suggesting LAI-PrEP implementation could increase PrEP coverage among those most vulnerable to HIV.

Water, sanitation, and hygiene access among people who inject drugs in Tijuana and San Diego in 2020-2021: a cross-sectional study.

BACKGROUND: Water, sanitation, and hygiene (WASH) access is critical to public health and human dignity. People who inject drugs (PWID) experience stigma and structural violence that may limit WASH access. Few studies have assessed WASH access, insecurity, and inequities among PWID. We describe WASH access, social and geographic inequalities, and factors associated with WASH insecurity among PWID in the Tijuana-San Diego metropolitan area. METHODS: In this cross-sectional binational study, we interviewed PWID (age 18+) in 2020-2021 about WASH access and insecurity. City of residence (Tijuana/San Diego) and housing status were considered as independent variables to describe key WASH access outcomes and to assess as factors associated with WASH insecurity outcomes. Measures of association between outcomes and independent variables were assessed using log modified-Poisson regression models adjusting for covariates. RESULTS: Of 586 PWID (202 Tijuana; 384 San Diego), 89% reported basic access to drinking water, 38% had basic hand hygiene, 28% basic sanitation, and 46% access to bathing, and 38% reported recent open defecation. Participants residing in Tijuana reported significantly higher insecurity in accessing basic drinking water (aRR: 1.68, 95%CI: 1.02-2.76), basic hygiene (aRR: 1.45, 95%CI: 1.28-1.64), and bathing (aRR: 1.21, 95%CI: 1.06-1.39) than those living in San Diego. Participants experiencing unsheltered homelessness experienced significantly higher insecurity in accessing basic drinking water (aRR: 2.03, 95%CI: 1.07-3.86), basic sanitation (aRR: 1.68, 95%CI: 1.48, 1.92), bathing (aRR: 1.84, 95%CI: 1.52-2.22), and improved water sources for cleaning wounds (aRR: 3.12, 95%CI: 1.55-6.29) and for preparing drugs (aRR: 2.58, 95%CI: 1.36-4.89) than participants living in permanent housing. CONCLUSION: WASH access among PWID in the Tijuana-San Diego metropolitan area was low by international standards and lower than the national averages in both countries. Homelessness was significantly associated with WASH insecurity in this population. Concentrated efforts are needed to guarantee continuously available WASH services for PWID-especially those who are unsheltered.

WaSH insecurity and anxiety among people who inject drugs in the Tijuana-San Diego border region.

BACKGROUND: Water, sanitation, and hygiene (WaSH) insecurity increases the risk of water-related diseases. However, limited research has been conducted on psychosocial distress as it relates to WaSH insecurity, especially among people who inject drugs (PWID). We examined the relationship between WaSH insecurity and related anxiety among PWID living in different housing conditions along the US-Mexico border region. METHODS: From 2020-2021, a cross-sectional study was conducted among 585 people who injected drugs within the last month in Tijuana (N = 202), San Diego (N = 182), and in both Tijuana and San Diego (N = 201). Participants underwent interviewer-administered surveys related to WaSH access, substance use, and generalized anxiety disorder (GAD-7). Quasi-Poisson regressions were used to assess associations between WaSH insecurity and anxiety in the prior 6-months. RESULTS: Participants were 75% male, 42% were unhoused and 91% experienced WaSH insecurity in the prior 6-months. After adjusting for housing status, gender, and age, lack of access to basic drinking water (Adj RR: 1.28; 95% CI: 1.02-1.58), sanitation (Adj RR:1.28; 95% CI: 1.07-1.55), and a daily bath/shower (Adj RR: 1.38; 95% CI: 1.15-1.66) were associated with mild-severe anxiety. The number of WaSH insecurities was independently associated with a 20% increased risk of experiencing anxiety per every additional insecurity experienced (Adj RR: 1.20; CI: 1.12-1.27). We also found a significant interaction between gender and housing status (p = 0.003), indicating that among people experiencing sheltered/unsheltered homelessness, women had a higher risk of mild-severe anxiety compared to men (Adj RR: 1.55; 95% CI: 1.27-1.89). At the same time, among women, those who are unhoused have 37% increased risk of anxiety than those who live in stable housing conditions (Adj RR: 1.37; 95% CI: 1.01-1.89). CONCLUSION: The lack of specific WaSH services, particularly lack of drinking water, toilets, and daily showers were associated with higher levels of anxiety among PWID in the Tijuana-San Diego border region. Women experiencing homelessness were especially vulnerable. WaSH interventions that provide safe, 24-h access may help to reduce anxiety and health risks associated with WaSH insecurity.

Willingness to use and distribute HIV self-testing kits among people who inject drugs in the San Diego-Tijuana border region.

BACKGROUND: HIV self-testing (HIVST) could increase HIV testing access among people who inject drugs (PWID), and secondary distribution (i.e., peer-delivery) of HIVST kits in PWID social networks could further expand coverage. We assessed willingness to use and distribute HIVST kits among PWID in the San Diego-Tijuana border region. METHODS: From 2020 to 2021, HIV-negative PWID in San Diego, USA, and Tijuana, Mexico, completed surveys and provided data on individual (N = 539) and social network (N = 366) characteristics. We used modified Poisson regression to examine the effects of individual and social network characteristics on willingness to use and distribute HIVST kits. RESULTS: Most participants were willing to use (81%) and distribute (81%) HIVST kits. At the individual level, prior HIV testing was positively associated with willingness to use (adjusted prevalence ratio [aPR] = 1.24, 95% confidence interval [CI] 1.10-1.40) and distribute (aPR = 1.27, 95% CI 1.12-1.43) HIVST kits, while perceiving oneself to be at higher HIV risk than others was negatively associated with willingness to use HIVST kits (aPR = 0.83, 95% CI 0.74-0.93). At the network level, willingness to distribute HIVST kits was positively associated with network size (aPR = 1.04 per member, 95% CI 1.01-1.08) and greater proportions of one's network encouraging them to use drugs (aPR = 1.29, 95% CI 1.16-1.44) and having a history of homelessness (aPR = 1.51, 95% CI 1.31-1.74) or detention/arrest (aPR = 1.57, 95% CI 1.36-1.82), and negatively associated with a greater proportion of one's network including "very close" persons (aPR = 0.80, 95% CI 0.69-0.94). CONCLUSIONS: We found high potential for HIVST kits and their secondary distribution to increase HIV testing among PWID who face the greatest barriers to facility-based testing.

Health, harm reduction, and social service providers' perspectives on the appropriateness and feasibility of peer distribution of HIV self-test kits among people who use drugs.

BACKGROUND: People who use drugs (PWUD) experience elevated HIV risk and numerous barriers to facility-based HIV testing. HIV self-testing (HIVST) could circumvent many of those barriers and is acceptable among PWUD, yet HIVST implementation for PWUD is limited. Service providers' perspectives on specific HIVST delivery strategies could help increase availability for PWUD. METHODS: From April-November 2021, we interviewed 16 health, harm reduction, and social service providers working with PWUD in San Diego, CA. Interviews and rapid thematic analysis explored perspectives on HIVST's utility and appropriateness, as well as the feasibility of and anticipated challenges with specific HIVST delivery strategies, including peer or secondary distribution. RESULTS: Participants viewed HIV as a significant threat to PWUD health and confirmed the presence of numerous barriers to local facility-based HIV testing. Participants viewed HIVST as a promising and potentially empowering solution. Based on community familiarity with secondary distribution of harm reduction supplies (i.e., naloxone) and information, participants viewed secondary distribution of HIVST kits as an appropriate and feasible strategy for increasing the reach of HIVST, but also described potential barriers (e.g., engaging socially disconnected individuals, ensuring linkages to services following HIVST) and provided suggestions for alternative HIVST kit delivery models (e.g., harm reduction vending machines). CONCLUSIONS: Service providers viewed secondary distribution of HIVST kits among PWUD as promising, appropriate, and feasible, yet specialized efforts may be needed to reach the most marginalized individuals and ensure consistent provision of educational information and referral supports that maximize the impact of this approach.

Preliminary Efficacy of a Theory-Informed Intervention to Increase COVID-19 Testing Uptake Among People Who Inject Drugs in San Diego County: Findings From a Pilot Randomized Controlled Trial.

BACKGROUND: People who inject drugs (PWID) have low rates of COVID-19 testing yet are vulnerable to severe disease. In partnership with a mobile syringe service program (SSP) in San Diego County, CA, we developed the evidence-, community-, and Social Cognitive Theory-informed "LinkUP" intervention (tailored education, motivational interviewing, problem-solving, and planning) to increase COVID-19 testing uptake among PWID. PURPOSE: To assess preliminary efficacy of LinkUP in increasing PWID COVID-19 testing in a pilot randomized controlled trial (RCT). METHODS: We referred participants (PWID, ≥18 years old, San Diego County residents who had not recently undergone voluntary COVID-19 testing) to mobile SSP sites that had been randomized (by week) to offer the active LinkUP intervention or didactic attention-control conditions delivered by trained peer counselors. Following either condition, counselors offered on-site rapid COVID-19 antigen testing. Analyses estimated preliminary intervention efficacy and explored potential moderation. RESULTS: Among 150 participants, median age was 40.5 years, 33.3% identified as Hispanic/Latinx, 64.7% were male, 73.3% were experiencing homelessness, and 44.7% had prior mandatory COVID-19 testing. The LinkUP intervention was significantly associated with higher COVID-19 testing uptake (p < .0001). Homelessness moderated intervention effects; LinkUP increased COVID-19 testing uptake more among participants experiencing homelessness (adjusted risk ratio [aRR]: 1.80; 95% CI: 1.56-2.09; p < .0001) than those not experiencing homelessness (aRR: 1.20; 95% CI: 1.01-1.43; p = .04). CONCLUSIONS: Findings from this pilot RCT support the preliminary efficacy of the "LinkUP" intervention to increase COVID-19 testing among PWID and underscore the importance of academic-community partnerships and prevention service delivery through SSPs and other community-based organizations serving vulnerable populations.

People who inject drugs (PWID) are vulnerable to severe COVID-19 disease yet have low rates of COVID-19 testing. We partnered with a syringe service program (SSP) in San Diego County, CA, to develop "LinkUP," an evidence- and community-informed intervention. Specifically, LinkUP used tailored education, motivational interviewing, and problem-solving and planning strategies to increase COVID-19 testing uptake among PWID. This study was a pilot randomized controlled trial (RCT) designed to assess the preliminary efficacy of LinkUP in increasing PWID COVID-19 testing. We referred participants (PWID, ≥18 years old, San Diego residents without recent voluntary COVID-19 testing) to mobile SSP sites that had been randomized (by week). Trained peer counselors then offered LinkUP or an educational control condition lasting the same length (~30 minutes). After either condition, counselors offered on-site rapid COVID-19 antigen testing. Among 150 participants, our analyses found that the LinkUP intervention was associated with higher COVID-19 testing uptake, especially for participants experiencing homelessness. In summary, our findings from this pilot RCT support the preliminary efficacy of the "LinkUP" intervention in increasing COVID-19 testing among PWID. This study also underscores the importance of academic-community partnerships and prevention service delivery through SSPs and other community-based organizations serving vulnerable populations.

Network-level HIV risk norms are associated with individual-level HIV risk and harm reduction behaviors among people who inject drugs: a latent profile analysis.

The COVID-19 related U.S.-Mexico border-crossing restrictions disrupted social networks and HIV harm reduction services among people who inject drugs (PWID) in San Diego and Tijuana. We assessed associations of descriptive network norms on PWID's HIV vulnerability during this period. Between 10/2020 and 10/2021, 399 PWID completed a behavioral and egocentric questionnaire. We used Latent Profile Analysis to categorize PWID into network norm risk profiles based on proportions of their network (n = 924 drug use alters) who injected drugs and engaged in cross-border drug use (CBDU), among other vulnerabilities. We used logistic and linear regressions to assess network profile associations with individual-level index of HIV vulnerability and harm reduction behaviors. Fit indices specified a 4-latent profile solution of descriptive network risk norms: lower (n = 178), moderate with (n = 34) and without (n = 94) CBDU and obtainment, and higher (n = 93). Participants in higher risk profiles reported more HIV vulnerability behaviors and fewer harm reduction behaviors. PWID's gradient of HIV risk was associated with network norms, warranting intervention on high-vulnerability networks when services are limited.

The Leu/Val6.51 Side Chain of Cannabinoid Receptors Regulates the Binding Mode of the Alkyl Chain of Δ9-Tetrahydrocannabinol.

(-)-Δ9-trans-tetrahydrocannabinol (THC), which is the principal psychoactive constituent of Cannabis, mediates its action by binding to two members of the G-protein-coupled receptor (GPCR) family: the cannabinoid CB1 (CB1R) and CB2 (CB2R) receptors. Molecular dynamics simulations showed that the pentyl chain of THC could adopts an I-shape conformation, filling an intracellular cavity between Phe3.36 and Trp6.48 for initial agonist-induced receptor activation, in CB1R but not in CB2R. This cavity opens to the five-carbon chain of THC by the conformational change of the γ-branched, flexible, Leu6.51 side chain of CB1R, which is not feasible by the ß-branched, mode rigid, Val6.51 side chain of CB2R. In agreement with our computational results, THC could not decrease the forskolin-induced cAMP levels in cells expressing mutant CB1RL6.51V receptor but could activate the mutant CB2RV6.51L receptor as efficiently as wild-type CB1R. Additionally, JWH-133, a full CB2R agonist, contains a branched dimethyl moiety in the ligand chain that bridges Phe3.36 and Val6.51 for receptor activation. In this case, the substitution of Val6.51 to Leu in CB2R makes JWH-133 unable to activate CB2RV6.51L. In conclusion, our combined computational and experimental results have shown that the amino acid at position 6.51 is a key additional player in the initial mechanism of activation of GPCRs that recognize signaling molecules derived from lipid species.

EVALUATION OF THE INCLUSION OF SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY IN A TELEMEDICINE DIABETIC RETINOPATHY SCREENING PROGRAM: A Real Clinical Practice.

PURPOSE: To evaluate whether combining spectral domain optical coherence tomography with monoscopic fundus photography using a nonmydriatic camera (MFP-NMC) improves the accuracy of diabetic macular edema (DME) referrals in a teleophthalmology diabetic retinopathy screening program. METHODS: We conducted a cross-sectional study with all diabetic patients aged 18 years or older who attended screening from September 2016 to December 2017. We assessed DME according to the three MFP-NMC and the four spectral domain optical coherence tomography criteria. The sensitivity and specificity obtained for each criterion were estimated by comparing them with the ground truth of DME. RESULTS: This study included 3,918 eyes (1,925 patients; median age, 66 years; interquartile range, 58-73; females, 40.7%; once-screened, 68.1%). The prevalence of DME ranged from 1.22% to 1.83% and 1.54% to 8.77% on MFP-NMC and spectral domain optical coherence tomography, respectively. Sensitivity barely reached 50% in MFP-NMC and less for the quantitative criteria of spectral domain optical coherence tomography. When macular thickening and anatomical signs of DME were considered, sensitivity increased to 88.3% and the false DMEs and non-gradable images were reduced. CONCLUSION: Macular thickening and anatomical signs showed the highest suitability for screening, with a sensitivity of 88.3% and a specificity of 99.8%. Notably, MFP-NMC alone missed half of the true DMEs that lacked indirect signs.

Metrological Qualification of PD Analysers for Insulation Diagnosis of HVDC and HVAC Grids.

On-site partial discharge (PD) measurements have turned out to be a very efficient technique for determining the insulation condition in high-voltage electrical grids (AIS, cable systems, GIS, HVDC converters, etc.); however, there is not any standardised procedure for determining the performances of PD measuring systems. In on-line and on-site PD measurements, high-frequency current transformers (HFCTs) are commonly used as sensors as they allow for monitoring over long distances in high-voltage installations. To ensure the required performances, a metrological qualification of the PD analysers by applying an evaluation procedure is necessary. A novel evaluation procedure was established to specify the quantities to be measured (electrical charge and PD repetition rate) and to describe the evaluation tests considering the measured influence parameters: noise, charge amplitude, pulse width and time interval between consecutive pulses. This procedure was applied to different types of PD analysers used for off-line measurements, sporadic on-line measurements and continuous PD monitoring. The procedure was validated in a round-robin test involving two metrological institutes (RISE from Sweden and FFII from Spain) and three universities (TUDelft from the Netherlands, TAU from Finland and UPM from Spain). With this round-robin test, the effectiveness of the proposed qualification procedure for discriminating between efficient and inappropriate PD analysers was demonstrated. Furthermore, it was shown that the PD charge quantity can be properly determined for on-line measurements and continuous monitoring by integrating the pulse signals acquired with HFCT sensors. In this case, these sensors must have a flat frequency spectrum in the range between several tens of kHz and at least two tens of MHz, where the frequency pulse content is more significant. The proposed qualification procedure can be useful for improving the future versions of the technical specification TS IEC 62478 and the standard IEC 60270.

Cannabidiol at Nanomolar Concentrations Negatively Affects Signaling through the Adenosine A2A Receptor.

Cannabidiol (CBD) is a phytocannabinoid with potential as a therapy for a variety of diseases. CBD may act via cannabinoid receptors but also via other G-protein-coupled receptors (GPCRs), including the adenosine A2A receptor. Homogenous binding and signaling assays in Chinese hamster ovary (CHO) cells expressing the human version of the A2A receptor were performed to address the effect of CBD on receptor functionality. CBD was not able to compete for the binding of a SCH 442416 derivative labeled with a red emitting fluorescent probe that is a selective antagonist that binds to the orthosteric site of the receptor. However, CBD reduced the effect of the selective A2A receptor agonist, CGS 21680, on Gs-coupling and on the activation of the mitogen activated kinase signaling pathway. It is suggested that CBD is a negative allosteric modulator of the A2A receptor.

Correlates of Coronavirus Disease 2019 (COVID-19) Vaccine Hesitancy Among People Who Inject Drugs in the San Diego-Tijuana Border Region.

BACKGROUND: People who inject drugs (PWID) are vulnerable to acquiring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examined correlates of coronavirus disease 2019 (COVID-19) vaccine hesitancy among PWID in the US-Mexico border region, of whom only 7.6% had received ≥ 1 COVID-19 vaccine dose by September 2021. METHODS: Between October 2020 and September 2021, participants aged ≥ 18 years from San Diego, California, USA, and Tijuana, Baja California, Mexico, who injected drugs within the last month completed surveys and SARS-CoV-2, human immunodeficiency virus (HIV), and hepatitis C virus (HCV) serologic testing. Logistic regressions with robust standard error estimation via generalized estimating equations identified factors associated with being unsure or unwilling to receive COVID-19 vaccines. RESULTS: Of 393 participants, 266 (67.7%) were willing to receive COVID-19 vaccines and 127 (32.3%) were hesitant (23.4% unwilling and 8.9% unsure). Older participants, those with greater food insecurity, and those with greater concern about acquiring SARS-CoV-2 were more willing to be vaccinated. Higher numbers of chronic health conditions, having access to a smart phone or computer, and citing social media as one's most important source of COVID-19 information were independently associated with vaccine hesitancy. COVID-19-related disinformation was independently associated with vaccine hesitancy (adjusted odds ratio: 1.51 per additional conspiracy theory endorsed; 95% confidence interval: 1.31-1.74). CONCLUSIONS: Nearly one third of people injecting drugs in the US-Mexico border region were COVID-19 vaccine hesitant, which was significantly associated with exposure to social media, disinformation and co-morbidities and inversely associated with food security and high perceived threat of COVID-19. Interventions that improve accurate knowledge of and trust in COVID-19 vaccines are needed in this vulnerable population.

A cross-sectional study of factors associated with COVID-19 testing among people who inject drugs: missed opportunities for reaching those most at risk.

BACKGROUND: People who inject drugs (PWID) are vulnerable to SARS-CoV-2 infection. We examined correlates of COVID-19 testing among PWID in the U.S.-Mexico border region and described encounters with services representing potential opportunities (i.e., 'touchpoints') where COVID-19 testing could have been offered. METHODS: Between October, 2020 and September, 2021, participants aged ≥18 years from San Diego, California, USA and Tijuana, Baja California, Mexico who injected drugs within the last month completed surveys and SARS-CoV-2, HIV, and HCV serologic testing. Logistic regression identified factors associated with COVID-19 testing including potential touchpoints, comorbidities and COVID-19 related misinformation and disinformation. RESULTS: Of 583 PWID, 30.5% previously had a COVID-19 test. Of 172 PWID who tested SARS-CoV-2 seropositive (30.1%), 50.3% encountered at least one touchpoint where COVID-19 testing could have been offered within the prior six months. Factors independently associated with at least two fold higher odds of COVID-19 testing were living in San Diego, recent incarceration, receiving substance use treatment, and experiencing ≥1 chronic health condition. Homelessness, having received ≥1 dose of COVID-19 vaccine, and having a HIV or HCV test since the COVID-19 epidemic began were also independently associated with having had a prior COVID-19 test. CONCLUSION: We identified several factors independently associated with COVID-19 testing and multiple touchpoints where COVID-19 testing could be scaled up for PWID, such as SUD treatment programs and syringe service programs. Integrated health services are needed to improve access to rapid, free COVID-19 testing in this vulnerable population.

Regulation of Expression of Cannabinoid CB2 and Serotonin 5HT1A Receptor Complexes by Cannabinoids in Animal Models of Hypoxia and in Oxygen/Glucose-Deprived Neurons.

Background: Cannabidiol (CBD) is a phytocannabinoid with potential in one of the most prevalent syndromes occurring at birth, the hypoxia of the neonate. CBD targets a variety of proteins, cannabinoid CB2 and serotonin 5HT1A receptors included. These two receptors may interact to form heteromers (CB2-5HT1A-Hets) that are also a target of CBD. Aims: We aimed to assess whether the expression and function of CB2-5HT1A-Hets is affected by CBD in animal models of hypoxia of the neonate and in glucose- and oxygen-deprived neurons. Methods: We developed a quantitation of signal transduction events in a heterologous system and in glucose/oxygen-deprived neurons. The expression of receptors was assessed by immuno-cyto and -histochemistry and, also, by using the only existing technique to visualize CB2-5HT1A-Hets fixed cultured cells and tissue sections (in situ proximity ligation PLA assay). Results: CBD and cannabigerol, which were used for comparative purposes, affected the structure of the heteromer, but in a qualitatively different way; CBD but not CBG increased the affinity of the CB2 and 5HT1A receptor-receptor interaction. Both cannabinoids regulated the effects of CB2 and 5HT1A receptor agonists. CBD was able to revert the upregulation of heteromers occurring when neurons were deprived of oxygen and glucose. CBD significantly reduced the increased expression of the CB2-5HT1A-Het in glucose/oxygen-deprived neurons. Importantly, in brain sections of a hypoxia/ischemia animal model, administration of CBD led to a significant reduction in the expression of CB2-5HT1A-Hets. Conclusions: Benefits of CBD in the hypoxia of the neonate are mediated by acting on CB2-5HT1A-Hets and by reducing the aberrant expression of the receptor-receptor complex in hypoxic-ischemic conditions. These results reinforce the potential of CBD for the therapy of the hypoxia of the neonate.

Similarities and differences upon binding of naturally occurring Δ9-tetrahydrocannabinol-derivatives to cannabinoid CB1 and CB2 receptors.

We have here assessed, using Δ9-tetrahydrocannabinol (Δ9-THC) for comparison, the effect of Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and of Δ9-tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB1, CB2, and CB1-CB2 receptor functional units, expressed in a heterologous system. Binding to the CB1 and CB2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB2 receptor, was obtained for Δ9-THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ9-THCA and Δ9-THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB1, CB2 or CB1-CB2) and on the compound used as reference to calculate the bias factor (Δ9-THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ9-THCV and Δ9-THCA and Δ9-THCV were able to revert the effect of a selective CB2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2'-chloroethylamide (ACEA 100 nM) a selective agonist of the CB1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activation.

The ATPase cycle of human muscle myosin II isoforms: Adaptation of a single mechanochemical cycle for different physiological roles.

Striated muscle myosins are encoded by a large gene family in all mammals, including humans. These isoforms define several of the key characteristics of the different striated muscle fiber types, including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of seven different human myosin isoforms to define the actin·myosin cross-bridge cycle in solution. Here, we present data on an eighth isoform, the perinatal, which has not previously been characterized. The perinatal is distinct from the embryonic isoform, appearing to have features in common with the adult fast-muscle isoforms, including weak affinity of ADP for actin·myosin and fast ADP release. We go on to use a recently developed modeling approach, MUSICO, to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction, including duty ratio, shortening velocity, ATP economy, and load dependence of these parameters. The work shows that the parameters of the cross-bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics.

Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties.

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the ß-cardiac myosin heavy chain gene (ß-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any ß-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.

Calculation of accurate interatomic contact surface areas for the quantitative analysis of non-bonded molecular interactions.

SUMMARY: Intra- and intermolecular contact surfaces are routinely calculated for a large array of applications in bioinformatics but are typically approximated from differential solvent accessible surface area calculations and not calculated directly. These approximations do not properly take the effects of neighboring atoms into account and tend to deviate considerably from the true contact surface. We implemented an extension of the original Shrake-Rupley algorithm to accurately estimate interatomic contact surface areas of molecular structures and complexes. Our extended algorithm is able to calculate the contact area of an atom to all nearby atoms by directly calculating overlapping surface patches, taking into account the possible shielding effects of neighboring atoms. Here, we present a versatile software tool and web server for the calculation of contact surface areas, as well as buried surface areas and solvent accessible surface areas (SASA) for different types of biomolecules, such as proteins, nucleic acids and small organic molecules. Detailed results are provided in tab-separated values format for analysis and Protein Databank files for visualization. Direct contact surface area calculation resulted in improved accuracy in a benchmark with a non-redundant set of 245 protein-DNA complexes. SASA-based approximations underestimated protein-DNA contact surfaces on average by 40%. This software tool may be useful for surface-based intra- and intermolecular interaction analyses and scoring function development. AVAILABILITY AND IMPLEMENTATION: A web server, stand-alone binaries for Linux, MacOS and Windows and C++ source code are freely available from http://schuellerlab.org/dr_sasa/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pharmacological potential of varinic-, minor-, and acidic phytocannabinoids.

While natural Δ9-tetrahidrocannabinol (Δ9THC), cannabidiol (CBD), and their therapeutic potential have been extensively researched, some cannabinoids have been less extensively investigated. The present article compiles data from the literature that highlight the health benefits and therapeutic potential of lesser known phytocannabinoids, which we have divided into varinic, acidic, and "minor" (i.e., cannabinoids that are not present in high quantities in common varieties of Cannabis sativa L). A growing interest in these compounds, which are enriched in some cannabis varieties, has already resulted in enough preclinical information to show that they are promising therapeutic agents for a variety of diseases. Every phytocannabinoid has a "preferential" mechanism of action, and often targets the cannabinoid receptors, CB1 and/or CB2. The recent resolution of the structure of cannabinoid receptors demonstrates the atypical nature of cannabinoid binding, and that different binding modes depend on the agonist or partial agonist/inverse agonist, which allows for differential signaling, even acting on the same cannabinoid receptor. In addition, other players and multiple signaling pathways may be targeted/engaged by phytocannabinoids, thereby expanding the mechanistic possibilities for therapeutic use.