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Host heterogeneity can affect parasite transmission, but determining underlying traits and incorporating them into transmission models remains challenging. Body size is easily measured and affects numerous ecological interactions, including transmission. In the snail-schistosome system, larger snails have a higher exposure to parasites but lower susceptibility to infection per parasite. We quantified the effect of size-based heterogeneity on population-level transmission by conducting transmission trials in differently size-structured snail populations and competing size-dependent transmission models. Populations with greater proportions of large snails had lower prevalence, and small snails were shielded from infection by co-occurring large conspecifics. Furthermore, a fully dependent transmission model that incorporated body size in both exposure and susceptibility outperformed other candidate models considered. Incorporating traits such as body size, which are affected by and directly affect host ecology, into transmission models could yield insights into natural dynamics and disease mitigation in many systems., (© 2023 The Ecological Society of America.)

Myocarditis is an established but rare adverse event following administration of messenger RNA-based coronavirus disease 2019 (COVID-19) vaccines and is most common in male adolescents and young adults. Symptoms typically develop within a few days of vaccine administration. Most patients have mild abnormalities on cardiac imaging with rapid clinical improvement with standard treatment. However, longer term follow-up is needed to determine whether imaging abnormalities persist, to evaluate for adverse outcomes, and to understand the risk associated with subsequent vaccination. The purpose of the review is to evaluate the current literature related to myocarditis following COVID-19 vaccination, including the incidence, risk factors, clinical course, imaging findings, and proposed pathophysiologic mechanisms., Competing Interests: Disclosure Dr K. Hanneman has received speaker’s honorarium from Sanofi-Genzyme, Amicus, and Medscape. Dr P. Thavendiranathan has received speaker’s honorarium from Amgen, Boehringer Ingelheim-Lilly, and Takeda. Dr J.A. Udell has served as a consultant or speaker for AstraZeneca, Bayer, Boehringer Ingelheim-Lilly, Janssen, Merck, Novartis, and Sanofi and has received research grants from AstraZeneca, Amgen, Bayer, Boehringer Ingelheim-Lilly, and Janssen., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Myocarditis is defined as a non-ischemic inflammatory disease of the myocardium. It remains a challenge to diagnose given non-specific symptoms and lack of specific blood biomarkers. Cardiac imaging plays an important role in the evaluation of myocarditis with unique strengths and limitations of different imaging modalities, including cardiac magnetic resonance imaging, echocardiography, cardiac computed tomography, and positron emission tomography. The purpose of this review is to discuss the strengths and limitations of various cardiac imaging techniques in the evaluation of myocarditis, review imaging findings in specific causes of myocarditis including COVID-19 and after vaccination, evaluate the role of imaging in differentiating myocarditis from potential mimics and differential considerations, identify current gaps in knowledge, and propose future directions.

Competing Interests: Funding Support and Author Disclosures Dr Cavalcante has reported research support from Circle Cardiovascular Imaging and Siemens Healthineers. Dr Gössl has reported consulting for Abbott Vascular. Dr Shaw has reported that she has no relationships relevant to the contents of this paper to disclose.

Myocarditis is an established but rare adverse event following administration of messenger RNA-based coronavirus disease 2019 (COVID-19) vaccines and is most common in male adolescents and young adults. Symptoms typically develop within a few days of vaccine administration. Most patients have mild abnormalities on cardiac imaging with rapid clinical improvement with standard treatment. However, longer term follow-up is needed to determine whether imaging abnormalities persist, to evaluate for adverse outcomes, and to understand the risk associated with subsequent vaccination. The purpose of the review is to evaluate the current literature related to myocarditis following COVID-19 vaccination, including the incidence, risk factors, clinical course, imaging findings, and proposed pathophysiologic mechanisms., Competing Interests: Disclosure Dr K. Hanneman has received speaker’s honorarium from Sanofi-Genzyme, Amicus, and Medscape. Dr P. Thavendiranathan has received speaker’s honorarium from Amgen, Boehringer Ingelheim-Lilly, and Takeda. Dr J.A. Udell has served as a consultant or speaker for AstraZeneca, Bayer, Boehringer Ingelheim-Lilly, Janssen, Merck, Novartis, and Sanofi and has received research grants from AstraZeneca, Amgen, Bayer, Boehringer Ingelheim-Lilly, and Janssen., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Our goal was to validate a human hyaluronic acid (HA) ELISA (Hyaluronic acid plus ELISA; TECOmedical Group) for use in feline plasma. Plasma from 5 healthy cats and 5 critically ill cats was used for validation of the assay. Validation methods performed included intra- and inter-assay variability, spike-and-recovery, and dilutional linearity. All measurements were performed in duplicate. The precision study revealed good intra-assay CV of 7.4-8.9%; inter-assay CV was 3.4-4.2%. Extraction efficiency via spiking tests yielded mean recovery of 89.6%. The assay met criteria for acceptable linearity using 3 serial dilutions. Our results demonstrate that this commercial HA ELISA had acceptable analytical performance using feline plasma and could be a useful tool in the veterinary clinical research setting.

Competing Interests: Funding Support and Author Disclosures Dr Cavalcante has received research support from Circle Cardiovascular Imaging and Siemens Healthineers. Dr Gössl has served as a consultant for Abbott Vascular. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Objective: To describe daily changes in serum concentrations of hyaluronic acid (HA), a biomarker of endothelial glycocalyx degradation, in dogs with septic peritonitis and to determine whether relationships exist among serum concentrations of HA and biomarkers of inflammation and patient fluid status., Animals: 8 client-owned dogs., Procedures: Serum samples that had been collected for a previous study and stored at -80°C were used. Blood samples were collected at admission and daily thereafter during hospitalization and were analyzed for concentrations of HA and interleukins 6, 8, and 10. Patient data including acute patient physiologic and laboratory evaluation score, type and amount of fluids administered daily, and daily CBC and lactate concentration results were recorded. To determine the significant predictors of HA concentration, a general linear mixed model for repeated measures was developed., Results: All dogs survived to discharge. Concentrations of HA ranged from 18 to 1,050 ng/mL (interquartile [25th to 75th percentile] range, 49 to 119 ng/mL) throughout hospitalization. Interleukin-6 concentration was a significant predictor of HA concentration as was total administered daily fluid volume when accounting for interleukin-6 concentration. When fluid volume was analyzed independent of inflammatory status, fluid volume was not a significant predictor. Concentrations of HA did not significantly change over time but tended to increase on day 2 or 3 of hospitalization., Conclusions and Clinical Relevance: Results supported the theory that inflammation is associated with endothelial glycocalyx degradation. Dogs recovering from septic peritonitis may become more susceptible to further endothelial glycocalyx damage as increasing fluid volumes are administered.

The majority of single nucleotide variants (SNVs) identified in Genome Wide Association Studies (GWAS) fall within non-protein coding DNA and have the potential to alter gene expression. Non-protein coding DNA can control gene expression by acting as transcription factor (TF) binding sites or by regulating the organization of DNA into chromatin. SNVs in non-coding DNA sequences can disrupt TF binding and chromatin structure and this can result in pathology. Further, environmental health studies have shown that exposure to xenobiotics can disrupt the ability of TFs to regulate entire gene networks and result in pathology. However, there is a large amount of interindividual variability in exposure-linked health outcomes. One explanation for this heterogeneity is that genetic variation and exposure combine to disrupt gene regulation, and this eventually manifests in disease. Many resources exist that annotate common variants from GWAS and combine them with conservation, functional genomics, and TF binding data. These annotation tools provide clues regarding the biological implications of an SNV, as well as lead to the generation of hypotheses regarding potentially disrupted target genes, epigenetic markers, pathways, and cell types. Collectively this information can be used to predict how SNVs can alter an individual's response to exposure and disease risk. A basic understanding of the regulatory information contained within non-protein coding DNA is needed to predict the biological consequences of SNVs, and to determine how these SNVs impact exposure-related disease. We hope that this review will aid in the characterization of disease-associated genetic variation in the non-protein coding genome., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

2'3'-cyclic GMP-AMP (cGAMP) is an intracellular second messenger that is synthesized in response to cytosolic double-stranded DNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, we detected that cGAMP is continuously exported but then efficiently cleared by ENPP1, explaining why it has previously escaped detection. By developing potent, specific, and cell impermeable ENPP1 inhibitors, we found that cancer cells continuously export cGAMP in culture at steady state and at higher levels when treated with ionizing radiation (IR). In mouse tumors, depletion of extracellular cGAMP decreased tumor-associated immune cell infiltration and abolished the curative effect of IR. Boosting extracellular cGAMP with ENPP1 inhibitors synergized with IR to delay tumor growth. In conclusion, extracellular cGAMP is an anti-cancer immunotransmitter that could be harnessed to treat cancers with low immunogenicity., Competing Interests: Competing Interests: M.S. and L.L. are scientific co-founders of Angarus Therapeutics, which has exclusive licensing rights to patent PCT/US2018/50018. J.A.C., V.B., K.E.S., M.S., and L.L. are inventors on patent PCT/US2018/50018.

Objectives: The effect of shoe-worn insoles on biomechanical variables in people with medial knee osteoarthritis has been studied extensively. The majority of research has focused specifically on the effect of lateral wedge insoles at the knee. The aim of this systematic review and meta-analysis was to summarise the known effects of different shoe-worn insoles on all biomechanical variables during level walking in this patient population to date., Methods: Four electronic databases were searched to identify studies containing biomechanical data using shoe-worn insole devices in the knee osteoarthritis population. Methodological quality was assessed and a random effects meta-analysis was performed on biomechanical variables reported in three or more studies for each insole., Results: Twenty-seven studies of moderate-to-high methodological quality were included in this review. The primary findings were consistent reductions in the knee adduction moment with lateral wedge insoles, although increases in ankle eversion with these insoles were also found., Conclusion: Lateral wedge insoles produce small reductions in knee adduction angles and external moments, and moderate increases in ankle eversion. The addition of an arch support to a lateral wedge minimises ankle eversion change, and also minimises adduction moment reductions. The paucity of available data on other insole types and other biomechanical outcomes presents an opportunity for future research., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Speech is a multimodal stimulus, with information provided in both the auditory and visual modalities. The resulting audiovisual signal provides relatively stable, tightly correlated cues that support speech perception and processing in a range of contexts. Despite the clear relationship between spoken language and the moving mouth that produces it, there remains considerable disagreement over how sensitive early language learners-infants-are to whether and how sight and sound co-occur. Here we examine sources of this disagreement, with a focus on how comparisons of data obtained using different paradigms and different stimuli may serve to exacerbate misunderstanding.

The applicability of combined quantum mechanics/molecular mechanics (QM/MM) methods for the calculation of absolute binding free energies of conserved water molecules in protein/ligand complexes is demonstrated. Here, we apply QM/MM Monte Carlo simulations to investigate binding of water molecules to influenza neuraminidase. We investigate five different complexes, including those with the drugs oseltamivir and peramivir. We investigate water molecules in two different environments, one more hydrophobic and one hydrophilic. We calculate the free-energy change for perturbation of a QM to MM representation of the bound water molecule. The calculations are performed at the BLYP/aVDZ (QM) and TIP4P (MM) levels of theory, which we have previously demonstrated to be consistent with one another for QM/MM modeling. The results show that the QM to MM perturbation is significant in both environments (greater than 1 kcal mol(-1)) and larger in the more hydrophilic site. Comparison with the same perturbation in bulk water shows that this makes a contribution to binding. The results quantify how electronic polarization differences in different environments affect binding affinity and also demonstrate that extensive, converged QM/MM free-energy simulations, with good levels of QM theory, are now practical for protein/ligand complexes.

Invasive fungal infections are very common in solid organ transplants and occur most frequently in the first three months after transplant. A 49-year-old female with a history of two remote heart transplants with the most recent one occurring 5 years ago was admitted for increasing shortness of breath, cough, and fever. Computerized tomography (CT) scan of the chest showed left lower lung ground-glass and tree-in-bud opacities. She was started on broad spectrum antibiotics along with ganciclovir and micafungin. Ganciclovir was added due to the patient's past history of CMV infection and empiric fungal coverage with micafungin. Bronchoalveolar lavage (BAL) was performed as her respiratory status worsened and voriconazole was added for possible aspergillosis in combination therapy with micafungin. BAL galactomannan returned positive which was suggestive of aspergillosis. Patient worsened clinically and subsequently succumbed to cardiorespiratory arrest despite our best efforts. It is important to have a high degree of clinical suspicion for invasive aspergillosis in transplant patients even many years after transplant and initiate aggressive therapy due to poor outcomes.

Buspirone, a 5-HT1A receptor agonist, and environmental enrichment (EE) enhance cognition and reduce histopathology after traumatic brain injury (TBI) in adult rats, but have not been fully evaluated after pediatric TBI, which is the leading cause of death in children. Hence, the aims of this study were to assess the efficacy of buspirone alone (Experiment 1) and in combination with EE (Experiment 2) in TBI postnatal day-17 male rats. The hypothesis was that both therapies would confer cognitive and histological benefits when provided singly, but their combination would be more efficacious. Anesthetized rats received a cortical impact or sham injury and then were randomly assigned to receive intraperitoneal injections of buspirone (0.08 mg/kg, 0.1 mg/kg, and 0.3 mg/kg) or saline vehicle (1.0 mL/kg) 24 h after surgery and once daily for 16 days (Experiment 1). Spatial learning and memory were assessed using the Morris water maze (MWM) on post-operative days 11-16, and cortical lesion volume was quantified on day 17. Sham controls for each condition were significantly better than all TBI groups. In the TBI groups, buspirone (0.1 mg/kg) enhanced MWM performance versus vehicle and buspirone (0.08 mg/kg and 0.3 mg/kg) (p<0.05) and reduced lesion volume relative to vehicle (p=0.038). In Experiment 2, buspirone (0.1 mg/kg) or vehicle was combined with EE after TBI, and the data were compared to the standard (STD)-housed groups from Experiment 1. EE lead to a significant enhancement of spatial learning and a reduction in lesion size versus STD. Moreover, the combined treatment group (buspirone+EE) performed markedly better than the buspirone+STD and vehicle+EE groups, which suggests an additive effect and supports the hypothesis. The data replicate previous studies assessing these therapies in adult rats. These novel findings may have important rehabilitation-relevant implications for clinical pediatric TBI.

Background: The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs., Methods: A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser., Results: Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001). Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454., Conclusions: In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.

The acetylcholinesterase (AChE) inhibitor donepezil is used as a therapy for Alzheimer's disease and has been recommended as a treatment for enhancing attention and memory after traumatic brain injury (TBI). Although select clinical case studies support the use of donepezil for enhancing cognition, there is a paucity of experimental TBI studies assessing the potential efficacy of this pharmacotherapy. Hence, the aim of this pre-clinical study was to evaluate several doses of donepezil to determine its effect on functional outcome after TBI. Ninety anesthetized adult male rats received a controlled cortical impact (CCI; 2.8 mm cortical depth at 4 m/sec) or sham injury, and then were randomly assigned to six TBI and six sham groups (donepezil 0.25, 0.5, 1.0, 2.0, or 3.0 mg/kg, and saline vehicle 1.0 mL/kg). Treatments began 24 h after surgery and were administered i.p. once daily for 19 days. Function was assessed by motor (beam balance/walk) and cognitive (Morris water maze) tests on days 1-5 and 14-19, respectively. No significant differences were observed among the sham control groups in any evaluation, regardless of dose, and therefore the data were pooled. Furthermore, no significant differences were revealed among the TBI groups in acute neurological assessments (e.g., righting reflex), suggesting that all groups received the same level of injury severity. None of the five doses of donepezil improved motor or cognitive function relative to vehicle-treated controls. Moreover, the two highest doses significantly impaired beam-balance (3.0 mg/kg), beam-walk (2.0 mg/kg and 3.0 mg/kg), and cognitive performance (3.0 mg/kg) versus vehicle. These data indicate that chronic administration of donepezil is not only ineffective in promoting functional improvement after moderate CCI injury, but depending on the dose is actually detrimental to the recovery process. Further work is necessary to determine if other AChE inhibitors exert similar effects after TBI.

It is well established that a relatively brief exposure to environmental enrichment (EE) enhances motor and cognitive performance after experimental traumatic brain injury (TBI), but it is not known whether the benefits can be sustained after EE is discontinued. To address this important rehabilitation-relevant concern, anesthetized rats received a controlled cortical impact (CCI) or sham injury, and for phase 1 of the experiment were randomly assigned to either 3 weeks of EE or standard (STD) housing. Neurobehavioral outcome was assessed by established motor and cognitive tests on postoperative days 1-5 and 14-18, respectively. Beam-balance and spatial learning were facilitated in the TBI + EE more than the TBI + STD group (p<0.0001). In phase 2 of the experiment, half of the rats in EE were transferred to STD conditions (TBI + EE + STD and sham + EE + STD), and neurobehavior was re-assessed once per month for 6 months. The TBI + EE and TBI + EE + STD groups performed markedly better in the water maze than the TBI + STD group (p<0.0001), and did not differ from one another (p=0.53). These data replicate those of several studies from our laboratory showing that EE enhances recovery after CCI injury, and extend those findings by demonstrating that the cognitive benefits are maintained for at least 6 months post-rehabilitation. The persistent benefits shown with this paradigm provide further support for EE as a pre-clinical model of rehabilitation that can be further explored, either alone or in combination with pharmacotherapies, for optimal neurorehabilitation after TBI.

Objective: This study evaluated the effects of three post-brushing mouthwashes containing 0 ppm F, 225 ppm F, and 500 ppm F, respectively, on salivary fluoride retention after brushing with 1450 ppm fluoride (as NaF) toothpaste and rinsing with water immediately after brushing., Methods: In this three-phase, randomized, cross-over study, an ion-specific electrode was used to measure salivary F levels in thirty trial participants before brushing (Time 0), and after brushing, rinsing with water, and then rinsing with one of the three mouthwashes. Time points evaluated after brushing were one, three, five, 10, 20, 30, 45, and 60 minutes. For saliva sample collections, subjects were asked to pool saliva in their mouths for 10 seconds before spitting out into a container for each of the time points., Results: The AUC0-60 means for F in saliva were 554, 252, and 20 for the 500, 225, and 0 ppm F mouthwash groups, respectively. The 500 ppm F mouthwash resulted in a 2660% increase in total fluoride salivary retention over 60 minutes when compared with the 0 ppm F group, and a 120% increase when compared with the 225 ppm F group. A significant difference (p < 0.001) in the AUC0-60 means between the three groups was observed using analysis of variance (ANOVA). Paired t-tests also showed significant differences in the mean fluoride retention over 60 minutes for all three pair-wise group comparisons (p < 0.001)., Conclusion: Use of a fluoride mouthwash containing 225 ppm F or 500 ppm F produced a significant increase in salivary fluoride retention following brushing with a 1450 ppm F toothpaste and rinsing with water compared to rinsing without fluoride. The use of the 500 ppm F mouthwash may be of particular benefit to those at high caries risk.

Background: Environmental enrichment (EE) is a complex living milieu that has been shown to enhance functional recovery versus standard (STD) housing after experimental traumatic brain injury (TBI) and therefore may be considered a rodent correlate of rehabilitation. However, the typical EE paradigm consists of continuous exposure to enrichment after TBI, which is inconsistent with the limited time frame in clinical rehabilitation., Objective: To determine whether abbreviated EE (ie, rehabilitation-relevant dose response) confers benefits similar to typical EE after TBI., Methods: Adult male rats received either a controlled cortical impact (2.8 mm depth at 4 m/s) or sham injury and were then randomly assigned to TBI + EE, TBI + EE (2 hours), TBI + EE (4 hours), TBI + EE (6 hours), TBI + STD, and respective sham controls. Motor (beam balance/beam walk) and cognitive (Morris water maze) performance was assessed on postoperative days 1 to 5 and 14 to 19, respectively., Results: The TBI + EE (2 hours) and TBI + EE (4 hours) groups were not statistically different from the TBI + STD group in any behavioral assessment. In contrast, the TBI + EE (6 hours) group exhibited significant enhancement of motor and cognitive performance when compared with the TBI + STD group, as well as the TBI + EE (2 hours) and TBI + EE (4 hours) groups (P < .003), and did not differ from the TBI + EE (typical) group., Conclusions: These data demonstrate that abbreviated EE (6 hours) produces motor and cognitive benefits similar to continuous EE after TBI and thus may be considered a dose-relevant rehabilitation paradigm.

Combined quantum mechanics/molecular mechanics (QM/MM) methods allow computations on chemical events in large molecular systems. Here, we have tested the suitability of the standard CHARMM27 forcefield Lennard-Jones van der Waals (vdW) parameters for the treatment of nucleic acid bases in QM/MM calculations at the B3LYP/6-311+G(d,p)-CHARMM27 level. Alternative parameters were also tested by comparing the QM/MM hydrogen bond lengths and interaction energies with full QM [B3LYP/6-311+G(d,p)] results. The optimization of vdW parameters for nucleic acid bases is challenging because of the likelihood of multiple hydrogen bonds between the nucleic acid base and a water molecule. Two sets of optimized atomic vdW parameters for polar hydrogen, carbonyl carbon, and aromatic nitrogen atoms for nucleic acid bases are reported: base-dependent and base-independent. The results indicate that, for QM/MM investigations of nucleic acids, the standard forcefield vdW parameters may not be appropriate for atoms treated by QM. QM/MM interaction energies calculated with standard CHARMM27 parameters are found to be too large, by around 3 kcal/mol. This is because of overestimation of electrostatic interactions. Interaction energies closer to the full QM results are found using the optimized vdW parameters developed here. The optimized vdW parameters [developed by reference to B3LYP/6-311+G(d,p) results] were also tested at the B3LYP/6-31G(d) QM/MM level and were found to be transferable to the lower level. The optimized parameters also model the interaction energies of charged nucleic acid bases and deprotonation energies reasonably well.

Molecular simulation is increasingly demonstrating its practical value in the investigation of biological systems. Computational modelling of biomolecular systems is an exciting and rapidly developing area, which is expanding significantly in scope. A range of simulation methods has been developed that can be applied to study a wide variety of problems in structural biology and at the interfaces between physics, chemistry and biology. Here, we give an overview of methods and some recent developments in atomistic biomolecular simulation. Some recent applications and theoretical developments are highlighted.

Aims: To investigate the thermal biology of entomopathogenic fungi being examined as potential microbial control agents of Varroa destructor, an ectoparasite of the European honey bee Apis mellifera., Methods and Results: Colony extension rates were measured at three temperatures (20, 30 and 35 degrees C) for 41 isolates of entomopathogenic fungi. All of the isolates grew at 20 and 30 degrees C but only 11 isolates grew at 35 degrees C. Twenty-two isolates were then selected on the basis of appreciable growth at 30-35 degrees C (the temperature range found within honey bee colonies) and/or infectivity to V. destructor, and their colony extension rates were measured at 10 temperatures (12.5-35 degrees C). This data were then fitted to Schoolfield et al. [J Theor Biol (1981)88:719-731] re-formulation of the Sharpe and DeMichele [J Theor Biol (1977)64:649-670] model of poikilotherm development. Overall, this model accounted for 87.6-93.9% of the data variance. Eleven isolates exhibited growth above 35 degrees C. The optimum temperatures for extension rate ranged from 22.9 to 31.2 degrees C. Only three isolates exhibited temperature optima above 30 degrees C. The super-optimum temperatures (temperature above the optimum at which the colony extension rate was 10% of the maximum rate) ranged from 31.9 to 43.2 degrees C., Conclusions: The thermal requirements of the isolates examined against V. destructor are well matched to the temperatures in the broodless areas of honey bee colonies, and a proportion of isolates, should also be able to function within drone brood areas., Significance and Impact of the Study: Potential exists for the control of V. destructor with entomopathogenic fungi in honey bee colonies. The methods employed in this study could be utilized in the selection of isolates for microbial control prior to screening for infectivity and could help in predicting the activity of a fungal control agent of V. destructor under fluctuating temperature conditions.

Attenuated molecular clones of simian immunodeficiency virus (SIVmac) are important tools for studying the correlates of protective immunity to lentivirus infection in nonhuman primates. The most highly attenuated SIVmac mutants fail to induce disease but also fail to induce immune responses capable of protecting macaques from challenge with pathogenic virus. We recently described a novel attenuated virus, SIVmac-M4, containing multiple mutations in the transmembrane protein (TM) intracytoplasmic domain. This domain has been implicated in viral assembly, infectivity, and cytopathogenicity. Whereas parental SIVmac239-Nef(+) induced persistent viremia and simian AIDS in rhesus macaques, SIVmac-M4 induced transient viremia in juvenile and neonatal macaques, with no disease for at least 1 year postinfection. In this vaccine study, 8 macaques that were infected as juveniles (n = 4) or neonates (n = 4) with SIVmac-M4 were challenged with pathogenic SIVmac251 administered through oral mucosa. At 1 year postchallenge, six of the eight macaques had low to undetectable plasma viremia levels. Assays of cell-mediated immune responses to SIVmac Gag, Pol, Env, and Nef revealed that all animals developed strong CD8(+) T-cell responses to Gag after challenge but not before. Unvaccinated control animals challenged with SIVmac251 developed persistent viremia, had significantly weaker SIV-specific T-cell responses, and developed AIDS-related symptoms. These findings demonstrate that SIVmac-M4, which contains a full-length Nef coding region and multiple point mutations in the TM, can provide substantial protection from mucosal challenge with pathogenic SIVmac251.

The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames for tat, rev, and nef are not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques.

Rhesus macaques infected with simian immunodeficiency virus (SIV) containing either a large nef deletion (SIVmac239Delta(152)nef) or interleukin-2 in place of nef developed high virus loads and progressed to simian AIDS. Viruses recovered from both juvenile and neonatal macaques with disease produced a novel truncated Nef protein, tNef. Viruses recovered from juvenile macaques infected with serially passaged virus expressing tNef exhibited a pathogenic phenotype. These findings demonstrated strong selective pressure to restore expression of a truncated Nef protein, and this reversion was linked to increased pathogenic potential in live attenuated SIV vaccines.

SIV/HIV-1 (SHIV) chimeric clones, constructed by substituting portions of the pathogenic molecular clone SIVmac239 with counterpart portions from HIV-1 clones, provide a means to analyze functions of selected HIV-1 genes in vivo in nonhuman primates. Our studies focused on SHIVSF33, which contains the vpu, tat, rev, and env genes of the cytopathic, T-cell line tropic clone HIV-1sf33 (subtype-B); this clone has a premature stop codon in the vpu gene. In three juvenile macaques inoculated intravenously with SHIVSF33, low-level persistent infection was established; no disease was observed for a period of >2 years. However, at approximately 16 months p.i., one of four SHIVSF33-infected juvenile macaques exhibited an increase in virus load, depletion of CD4(+) T cells in peripheral blood and lymph nodes, and other symptoms of simian AIDS (SAIDS). Virus recovered from this animal in the symptomatic stage was designated SHIVSF33a (A, adapted); this virus displayed multiple amino acid sequence changes throughout the HIV-1 env gene compared with the input SHIVSF33 clone. Additionally, a mutation in all clones from SHIVSF33a restored the open reading frame for the vpu gene. In vitro evaluations in tissue-culture systems revealed that SHIVSF33a replicated to higher levels and exhibited greater cytopathicity than SHIVSF33. Furthermore cloned env genes for SHIVSF33a were more fusogenic in a cell-fusion assay compared with the env gene of the SHIVSF33. Intravenous inoculation of SHIVsf33a into juvenile and newborn macaques resulted in a rapid decline in CD4(+) T cells to very low levels and development of a fatal AIDS-like disease. A cell-free preparation of this pathogenic chimeric virus also established persistent infection when applied to oral mucosal membranes of juvenile macaques and produced a fatal AIDS-like disease. These studies on pathogenic SHIVSF33a establish the basis for further investigations on the role of the HIV-1 env gene in virus adaptation and in mechanism(s) of immunodeficiency in primates; moreover, the chimeric virus SHIVSF33a can play a role in elucidating mucosal membrane transmission and development of antiviral vaccines in newborns as well as juvenile and adult macaques., (Copyright 1999 Academic Press.)

SIVmac1A11 and SIVmac239 are nonpathogenic and pathogenic molecular clones in rhesus macaques, respectively. Although these viruses exhibit approximately 98% nucleotide and amino acid sequence homology, differences are found in the length of the translation frames for several genes. SIVmac239 has a premature stop codon in nef, whereas SIVmac1A11 has a premature stop codon in vpr and two premature stop codons in the intracytoplasmic domain of the env-transmembrane (TM) subunit. Recombinant viruses, constructed through reciprocal exchange of large DNA restriction enzyme fragments between SIVmac1A11 and SIVmac239, were evaluated in adult rhesus macaques. This in vivo analysis revealed that two or more regions of the SIVmac genome were essential for high virus load and disease progression (Marthas et al., 1993. J. Virol. 67, 6047-6055). An important gap in knowledge remaining from this study was whether the premature stop codons in env-TM of recombinant virus SIV1A11/239gag-env/1A11 (Full-length vpr and nef, two stop codons in env-TM) reverted to coding triplets in vivo. Here, we report that viral sequences in macaques, which succumbed to an AIDS-like disease after infection with SIV1A11/239gag-env/1A11, exhibited reversion of both env-TM stop codons. In addition, antibodies to the intracytoplasmic domain of env-TM were detected in macaques containing revertant virus and showing disease; this finding indicates that this domain of the env glycoprotein was expressed in vivo. Thus selection for viral variants with full-length env-TM demonstrated that the cytoplasmic domain of the SIVmac env glycoprotein plays a role in viral persistence and immunodeficiency in primates.

To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman primate model, we have constructed infectious recombinant viruses (chimeras) between the pathogenic molecular clone of simian immunodeficiency virus (SIV) SIVmac239 and molecular clones of HIV-1 that differ in phenotypic properties controlled by the env gene. HIV-1SF33 is a T-cell-line-tropic virus which induces syncytia, and HIV-1SF162 is a macrophage-tropic virus that does not induce syncytia. A DNA fragment encoding tat, rev, and env (gp160) of SIVmac239 has been replaced with the counterpart genetic region of HIV-1SF33 and HIV-1SF162 to derive chimeric recombinant simian/human immunodeficiency virus (SHIV) strains SHIVSF33 and SHIVSF162, respectively. In the acute infection stage, macaques inoculated with SHIVSF33 had levels of viremia similar to macaques infected with SIVmac239, whereas virus loads were 1/10th to 1/100th those in macaques infected with SHIVSF162. Of note is the relatively small amount of virus detected in lymph nodes of SHIVSF162-infected macaques. In the chronic infection stage, macaques infected with SHIVSF33 also showed higher virus loads than macaques infected with SHIVSF162. Virus persists for over 1 year, as demonstrated by PCR for amplification of viral DNA in all animals and by virus isolation in some animals. Antiviral antibodies, including antibodies to the HIV-1 env glycoprotein (gp160), were detected; titers of antiviral antibodies were higher in macaques infected with SHIVSF33 than in macaques infected with SHIVSF162. Although virus has persisted for over 1 year after inoculation, these animals have remained healthy with no signs of immunodeficiency. These findings demonstrate the utility of the SHIV/macaque model for analyzing HIV-1 env gene functions and for evaluating vaccines based on HIV-1 env antigens.

Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).

Simian foamy virus type 1 (SFV-1), a member of the Spumavirinae subfamily of retroviruses, encodes a transcriptional transactivator (taf) that strongly augments gene expression directed by the viral long terminal repeat (LTR) (A. Mergia, K. E. S. Shaw, E. Pratt-Lowe, P. A. Barry, and P. A. Luciw, J. Virol. 65:2903-2909, 1991). This report describes cis-acting regulatory elements in the LTR that control viral gene expression. A series of LTR mutants and hybrid promoter constructs have been analyzed in transient expression assays for responsiveness to Taf. The targets for transactivation have been mapped to two regions of the U3 domain of the LTR, between positions -1196 and -880 and between positions -403 and -125 (+1 represents the transcription initiation site). No significant nucleotide sequence homology between these two regions is noted; thus, the SFV-1 taf gene acts through at least two distinct sequence elements in the LTR. The target contained between positions -403 and -125 acts independently of orientation, in different cell types and species, and in the context of a heterologous promoter. Thus, the target element between positions -403 and -125 has properties of a transcriptional enhancer. The observation that two distinct elements in the SFV-1 LTR are targets for transcriptional transactivation is novel with respect to observations for other retroviral systems. The R-U5 region of the SFV-1 LTR down-regulates transactivation by severalfold. Computer analysis of the R-U5 region revealed a secondary structure with a free-energy level of -74 kcal (ca. -310,000 J); this structural feature may account for the inhibitory effect on gene expression directed by the LTR. Taf of SFV-1 had no effect on gene expression directed by the LTR of the related human foamy virus, whereas Taf transactivates gene expression directed by the LTRs of the human and simian immunodeficiency viruses. Comparative functional analysis of Taf on homologous and heterologous LTRs may facilitate elucidation of the mechanism of transactivation of foamy viruses.

Simian foamy virus type 1 (SFV-1), a member of spumavirus subfamily of retroviruses, encodes a transcriptional transactivator that functions to strongly augment gene expression directed by the viral long terminal repeat (LTR). The objective of this study was to identify the viral gene responsible for transactivation. Nucleotide sequences between the env gene and the LTR of SFV-1 were determined. The predicted amino acid sequence revealed two large open reading frames (ORFs), designated ORF-1 (311 amino acids) and ORF-2 (422 amino acids). In the corresponding region of the human foamy virus, three ORFs (bel-1, bel-2, and bel-3) have been identified (R. M. Flugel, A. Rethwilm, B. Maurer, and G. Darai, EMBO J. 6:2077-2084, 1987). Pairwise comparisons of the ORF-1 and ORF-2 with bel-1 and bel-2 show small clusters of homology; less than 39% overall homology of conserved amino acids is observed. A counterpart for human foamy virus bel-3 is not present in the SFV-1 sequence. Three species of viral RNA have been identified in cells infected with SFV-1; an 11.5-kb RNA representing full-length transcripts, a 6.5-kb RNA representing the env message, and a 2.8-kb RNA from the ORF region. Analysis of a cDNA clone encoding the ORF region of SFV-1 reveals that the 2.8-kb message is generated by complex splicing events involving the 3' end of the env gene. In transient expression assays in cell lines representing several species. ORF-1 was shown to be necessary and sufficient for transactivating viral gene expression directed by the SFV-1 LTR. The target for transactivation is located in the U3 domain of the LTR, upstream from position - 125 (+ 1 represents the transcription initiation site). We propose that OFF-1 of SFV-1 be designated the transcriptional transactivator of foamy virus (taf).

A patient developed isolated numbness, 1st confined to the lateral nose and upper lip, but later involving the cheek, lower lip, upper gingiva, and the palate. This numbness was later associated with paresis of the muscles of the upper lip and angle of the mouth and with ipsilateral lower lid droop (the "numb cheek-limp lower lid" syndrome). Squamous cell carcinoma was discovered infiltrating the infraorbital nerve and distal branches of the facial nerve. Cheek numbness associated with lower eyelid or upper lip weakness may herald a neoplasm affecting the infraorbital nerve and distal facial nerve branches.

Simian foamy viruses, members of the spumavirus subfamily of retroviruses, are found in a variety of nonhuman primates and, as yet, remain to be characterized with respect to genetic structure and regulation of viral gene expression. The genome of simian foamy virus type 1 (SFV-1), an isolate from rhesus macaques, has been molecularly cloned, and the role of the viral long terminal repeat (LTR) in transcriptional control has been investigated. The SFV-1 LTR is 1,621 base pairs long, and sequence comparisons with human foamy virus revealed a pattern of clustered homology. A cap site in the LTR was identified by analysis of SFV-1 transcripts in infected cells. Transient expression assays in cell lines representing several species and different cell types showed that the SFV-1 LTR has low basal activity in uninfected cells, whereas LTR-directed expression is greatly increased in cells infected with SFV-1. This transactivation is mediated by a mechanism involving increases in steady-state levels of viral transcripts. Thus, the SFV-1 genome encodes a transactivator that functions on the LTR at the transcriptional level.

We have molecularly cloned and sequenced a portion of the simian foamy virus type 1 (SFV-1); open reading frames representing the endonuclease domain of the polymerase (pol) and the envelope (env) genes were identified by comparison with the human foamy virus (HFV). Unlike the HFV genomic organization, the SFV-1 pol gene overlaps the env gene; thus, the open reading frames reported for HFV between pol and env is not present in SFV-1. Comparisons of predicted amino acid sequences of HFV and SFV-1 reveal that the endonuclease domains of the pol genes are about 84% related. The region predicted to encode the SFV-1 extracellular env domain is 569 codons; SFV-1 and HFV have 64% amino acid similarity in this env domain. The predicted hydrophobic transmembrane env proteins of both HFV and SFV-1 show about 73% similarity. A total of 16 potential glycosylation sites are found in SFV-1 env, and 15 are found in HFV; 11 are shared. SFV-1 has 25 cysteine residues, and HFV has 23 residues; all 23 cysteine residues of HFV are conserved in SFV-1. This sequence analysis reveals that the human and simian foamy viruses are highly related.

Mutants of Drosophila melanogaster which are defective in DNA synthesis have been identified among mutagen-sensitive stocks through analysis of both organ and cell cultures. A new procedure employing larval brain ganglia allows poorly fertile or sterile mutants to be analyzed for the first time. Parallel studies were performed in both tissues to establish the sensitivity of the new assay relative to that of the proven cell-culture assay. Damage was induced in the DNA of cultured cells with UV irradiation and in that of ganglial cells with the carcinogen N-acetoxy-2-acetylaminofluorene. Cultures were then pulse-labeled with 3H-thymidine, incubated in the absence of thymidine, and the newly synthesized DNA was analyzed by alkaline sucrose gradient centrifugation. The molecular weight of labeled DNA from mutant cells was compared with that from control cells to assess the effect of the mutant on DNA synthesis. Among 16 mutant stocks that were scanned in either or both tissues, seven show reductions in DNA synthesis using an undamaged template. Mutants at five different genetic loci [mus(2)205, mus(3)304, mus(3)308, mus(3)310 and mus(3)311] possess a reduced capacity to synthesize DNA on a UV-damaged template in primary cell cultures. Four of these five defects can also be detected in carcinogen-treated organ cultures. Two additional defects in postreplication repair were observed with the brain-ganglia assay in strains that cannot be assayed in cell culture [mus(1)108, mus(2)206].

A total of 34 third chromosomes of Drosophila melanogaster that render homozygous larvae hypersensitive to killing by chemical mutagens have been isolated. Genetic analyses have placed responsible mutations in more than eleven complementation groups. Mutants in three complementation groups are strongly sensitive to methyl methanesulfonate, those in one are sensitive to nitrogen mustard, and mutants in six groups are hypersensitive to both mutagens. Eight of the ten loci mapped fall within 15% of the genetic map that encompasses the centromere of chromosome 3. Mutants from four of the complementation groups are associated with moderate to strong meiotic effects in females. Preliminary biochemical analyses have implicated seven of these loci in DNA metabolism.