Croquemort elicits activation of the immune deficiency pathway in ticks.

The immune deficiency (IMD) pathway directs host defense in arthropods upon bacterial infection. In Pancrustacea, peptidoglycan recognition proteins sense microbial moieties and initiate nuclear factor-κB-driven immune responses. Proteins that elicit the IMD pathway in non-insect arthropods remain elusive. Here, we show that an Ixodes scapularis homolog of croquemort (Crq), a CD36-like protein, promotes activation of the tick IMD pathway. Crq exhibits plasma membrane localization and binds the lipid agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Crq regulates the IMD and jun N-terminal kinase signaling cascades and limits the acquisition of the Lyme disease spirochete B. burgdorferi. Additionally, nymphs silenced for crq display impaired feeding and delayed molting to adulthood due to a deficiency in ecdysteroid synthesis. Collectively, we establish a distinct mechanism for arthropod immunity outside of insects and crustaceans.

Boosting with AIDSVAX B/E Enhances Env Constant Region 1 and 2 Antibody-Dependent Cellular Cytotoxicity Breadth and Potency.

Induction of protective antibodies is a critical goal of HIV-1 vaccine development. One strategy is to induce nonneutralizing antibodies (NNAbs) that kill virus-infected cells, as these antibody specificities have been implicated in slowing HIV-1 disease progression and in protection. HIV-1 Env constant region 1 and 2 (C1C2) monoclonal antibodies (MAbs) frequently mediate potent antibody-dependent cellular cytotoxicity (ADCC), making them an important vaccine target. Here, we explore the effect of delayed and repetitive boosting of RV144 vaccine recipients with AIDSVAX B/E on the C1C2-specific MAb repertoire. It was found that boosting increased clonal lineage-specific ADCC breadth and potency. A ligand crystal structure of a vaccine-induced broad and potent ADCC-mediating C1C2-specific MAb showed that it bound a highly conserved Env gp120 epitope. Thus, boosting to affinity mature these types of IgG C1C2-specific antibody responses may be one method by which to make an improved HIV vaccine with higher efficacy than that seen in the RV144 trial.IMPORTANCE Over one million people become infected with HIV-1 each year, making the development of an efficacious HIV-1 vaccine an important unmet medical need. The RV144 human HIV-1 vaccine regimen is the only HIV-1 clinical trial to date to demonstrate vaccine efficacy. An area of focus has been on identifying ways by which to improve upon RV144 vaccine efficacy. The RV305 HIV-1 vaccine regimen was a follow-up boost of RV144 vaccine recipients that occurred 6 to 8 years after the conclusion of RV144. Our study focused on the effect of delayed boosting in humans on the vaccine-induced Env constant region 1 and 2 (C1C2)-specific antibody repertoire. It was found that boosting with an HIV-1 Env vaccine increased C1C2-specific antibody-dependent cellular cytotoxicity potency and breadth.

Specificity of Molecular Fragments Binding to S100B versus S100A1 as Identified by NMR and Site Identification by Ligand Competitive Saturation (SILCS).

S100B, a biomarker of malignant melanoma, interacts with the p53 protein and diminishes its tumor suppressor function, which makes this S100 family member a promising therapeutic target for treating malignant melanoma. However, it is a challenge to design inhibitors that are specific for S100B in melanoma versus other S100-family members that are important for normal cellular activities. For example, S100A1 is most similar in sequence and structure to S100B, and this S100 protein is important for normal skeletal and cardiac muscle function. Therefore, a combination of NMR and computer aided drug design (CADD) was used to initiate the design of specific S100B inhibitors. Fragment-based screening by NMR, also termed "SAR by NMR," is a well-established method, and was used to examine spectral perturbations in 2D [1H, 15N]-HSQC spectra of Ca2+-bound S100B and Ca2+-bound S100A1, side-by-side, and under identical conditions for comparison. Of the 1000 compounds screened, two were found to be specific for binding Ca2+-bound S100A1 and four were found to be specific for Ca2+-bound S100B, respectively. The NMR spectral perturbations observed in these six data sets were then used to model how each of these small molecule fragments showed specificity for one S100 versus the other using a CADD approach termed Site Identification by Ligand Competitive Saturation (SILCS). In summary, the combination of NMR and computational approaches provided insight into how S100A1 versus S100B bind small molecules specifically, which will enable improved drug design efforts to inhibit elevated S100B in melanoma. Such a fragment-based approach can be used generally to initiate the design of specific inhibitors for other highly homologous drug targets.

Systematic Injection of Low-Dose LPS Transiently Improves the Retina Function and Structure of a Mouse Model of Geographic Atrophy.

Geographic atrophy (GA), the advanced form of AMD, has been linked to oxidative stress within the RPE and with low-grade inflammation. The RPE-specific Sod2 knockout mouse model of GA develops increase oxidative stress and slow retinal degeneration. Mice of the SOD2floxed::VMD2Cre+ genotype were injected subcutaneously with either saline or 3 mg/kg of lipopolysaccharide (LPS) at 8 weeks of age. Mice were evaluated by electroretinography (ERG) and spectral domain optical coherence tomography. Inflammatory cells within the retina were studied by CD45 immunofluorescence. Systemic low-dose LPS transiently, but significantly, improved both function and structure of RPE-specific Sod2 KO mice retina when compared to saline-injected mice. There was no difference in CD45 positive cells between saline and LPS treatment. Low-grade activation of the immune system leads to a preconditioning effect that transiently protects the retina of a mouse model of geographic atrophy.

Decay of Coliphages in Sewage-Contaminated Freshwater: Uncertainty and Seasonal Effects.

Understanding the fate of enteric viruses in water is vital for protection of water quality. However, the decay of enteric viruses is not well characterized, and its uncertainty has not been examined yet. In this study, the decay of coliphages, an indicator for enteric viruses, was investigated in situ under both sunlit and shaded conditions as well as in summer and winter. The decay rates of coliphages and their uncertainties were analyzed using a Bayesian approach. The results from the summer experiments revealed that the decay rates of somatic coliphages were significantly higher in sunlight (1.29 ± 0.06 day-1) than in shade (0.96 ± 0.04 day-1), but the decay rates of male-specific (F+) coliphages were not significantly different between sunlight (1.09 ± 0.09 day-1) and shaded treatments (1.11 ± 0.08 day-1). The decay rates of both F+ coliphages (0.25 ± 0.02 day-1) and somatic coliphages (0.12 ± 0.01 day-1) in winter were considerably lower than those in summer. Temperature and chlorophyll a (chla) concentration varied significantly (p < 0.001) between the two seasons, suggesting that these parameters might be important contributors to the seasonal variation of coliphage decay. Additionally, the Bayesian approach provided full distributions of decay rates and reduced the uncertainty, offering useful information for comparing decay rates under different conditions.

Addressing fears of children with Williams syndrome: therapist and child behavior in the context of a novel play-and humor-infused exposure therapy approach.

Many children with Williams syndrome struggle with fears and phobias that significantly impact their daily lives. Yet, there is sparse literature about the impact of behavioral interventions to treat anxiety and phobias among children with Williams syndrome. Using observational coding of intervention videos, the current study examines patterns of the therapist's use of play and humor and relations to child behavioral responses for four children with Williams syndrome who were identified as treatment responders to humor- and play-infused exposure therapy for fears and anxieties. Sessions were coded for therapist behaviors (exposure with or without play/humor, stimulus type used during exposure, passive or invited attention to feared stimulus, and spontaneous parent participation in exposure) as well as positive, negative, and neutral child behaviors (verbalizations and behaviors). Temporal patterns between therapist and child behaviors were analyzed using lag sequential analyses. The results showed that tolerance of feared stimuli improved for two of the four children following this play- and humor-infused exposure therapy approach, and the remaining two participants demonstrated progress beyond tolerating the feared stimulus and showed increased positive behaviors with the feared stimulus across sessions. Findings also showed patterns of therapist attunement to the child's anxiety level demonstrated through efforts to flexibly adjust the degrees of exposure. Therapist-initiated invited attention behaviors, indicative of the therapist's use of narration and priming, were associated with child tolerance and positive behaviors during exposure to the feared stimulus. Limitations of this study include a very small sample size, short duration of intervention, and a single-subject research design, which limit the generalizability of findings. Implications and future directions of this research are discussed.

Evaluating the feasibility, acceptability, and preliminary effectiveness of tele-comprehensive behavior therapy for tics (teleCBIT) for Tourette syndrome in youth and adults.

INTRODUCTION: Comprehensive behavioral intervention for tics (CBIT) is an efficacious, first-line treatment for Tourette syndrome (TS) and other chronic or persistent tic disorders. However, CBIT's public health impact has been limited by suboptimal treatment access. Preliminary research has shown that providing CBIT over videoconference (teleCBIT) is a promising delivery method for patients who cannot access in-person care. However, extant studies have been small efficacy trials focused only on pediatric patients. Replication of these studies is needed in additional treatment settings and across a wider age range of patients, especially in light of advances in telehealth technology and increasing telehealth adoption among practitioners. METHODS: We conducted a single-arm trial to evaluate the feasibility, acceptability, and effectiveness of teleCBIT embedded in comprehensive, medical tic specialty clinics. From October 2016 to September 2018, patients were offered teleCBIT at their usual care appointments. Those who were interested and met inclusion/exclusion criteria received 8 sessions of CBIT guided by a manualized protocol. An independent evaluator, masked to treatment progress, administered assessments at baseline, post-treatment, and 3 and 6 months after treatment. RESULTS: Twenty-five percent of patients who were offered treatment initiated teleCBIT through the study, and all treatment initiators completed treatment. From pre- to post-treatment, decreases in Yale Global Tic Severity Scale (YGTSS) total tic severity scores showed a large effect size among pediatric patients (n = 19; t = 5.72, P < 0.001, d = 1.31) and a medium-to-large effect size for adult patients (n = 10, t = 1.41, P = 0.096, d = 0.664). Thirteen of 19 pediatric patients (68%) and 6 of 10 adult patients (60%) had a positive global treatment response at post-treatment. Patients rated the treatment as highly satisfactory. Ninety-three percent of sessions were free of substantial technical problems. DISCUSSION: Within the context of medical tic specialty clinics, teleCBIT demonstrated strong evidence of feasibility, acceptability, and preliminary effectiveness comparable to in-person treatment for both pediatric and adult patients. TeleCBIT warrants study in future research on enhancing care systems for patients with TS. TRIAL REGISTRY: https://clinicaltrials.gov/ct2/keydates/NCT04007913.

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery.

EF-hand Ca2+-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca2+. Upon binding Ca2+, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca2+-binding affinity of CaM and most S100s in the absence of target is weak (CaKD > 1 µM). However, upon effector protein binding, the Ca2+ affinity of these proteins increases via heterotropic allostery (CaKD < 1 µM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca2+ homeostasis and (ii) strict maintenance of Ca2+-signaling within a narrow dynamic range of free Ca2+ ion concentrations, [Ca2+]free. In this review, mechanisms of allostery are coalesced into an empirical "binding and functional folding (BFF)" physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca2+ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.

Physiologically Relevant Free Ca2+ Ion Concentrations Regulate STRA6-Calmodulin Complex Formation via the BP2 Region of STRA6.

The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 [1]). In the absence of Ca2+, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg2+-bound CaM (MgCaM). Upon titration of Ca2+ into MgCaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca2+-binding domains, EF3 and EF4 (CaKD = 60 ± 7 nM). As higher concentrations of free Ca2+ were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (CaKD = 1000 ± 160 nM). Thermodynamic and kinetic Ca2+ binding studies showed that BP2 addition increased the Ca2+-binding affinity of CaM and slowed its Ca2+ dissociation rates (koff) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca2+ concentrations (<100 nM) like those found at resting intracellular levels. As free Ca2+ levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca2+-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca2+-loaded CaM (CaCaM-BP2). Because this structural rearrangement observed for the CaCaM-BP2 complex occurs as intracellular free Ca2+ concentrations approach those typical of a Ca2+-signaling event (CaKD = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length CaCaM-STRA6.

Sectoral activation of glia in an inducible mouse model of autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of blinding disorders caused by diverse mutations, including in rhodopsin (RHO). Effective therapies have yet to be discovered. The I307N Rho mouse is a light-inducible model of autosomal dominant RP. Our purpose was to describe the glial response in this mouse model to educate future experimentation. I307N Rho mice were exposed to 20,000 lx of light for thirty minutes to induce retinal degeneration. Immunofluorescence staining of cross-sections and flat-mounts was performed to visualize the response of microglia and Müller glia. Histology was correlated with spectral-domain optical coherence tomography imaging (SD-OCT). Microglia dendrites extended between photoreceptors within two hours of induction, withdrew their dendrites between twelve hours and one day, appeared ameboid by three days, and assumed a ramified morphology by one month. Glial activation was more robust in the inferior retina and modulated across the boundary of light damage. SD-OCT hyper-reflectivity overlapped with activated microglia. Finally, microglia transiently adhered to the RPE before which RPE cells appeared dysmorphic. Our data demonstrate the spatial and temporal pattern of glial activation in the I307N Rho mouse, and correlate these patterns with SD-OCT images, assisting in interpretation of SD-OCT images in preclinical models and in human RP.

Co-Delivery of a Short-Hairpin RNA and a shRNA-Resistant Replacement Gene with Adeno-Associated Virus: An Allele-Independent Strategy for Autosomal-Dominant Retinal Disorders.

Recombinant adeno-associated virus (rAAV) has become an important gene delivery vector for the treatment of inherited retinal degenerative diseases. Many of the mutations leading to retinal degeneration are inherited in an autosomal-dominant pattern and can produce toxic gain-of-function and/or dominant-negative effects. Here we describe an allele-independent gene therapy strategy with rAAV to treat autosomal-dominant retinal degenerative diseases. In this methodology, we co-deliver a short-hairpin RNA (shRNA) to inhibit expression of both the toxic and (WT) copies of the gene as well as an shRNA-resistant cDNA for functional gene replacement with a rAAV.

Expression of a CARD Slows the Retinal Degeneration of a Geographic Atrophy Mouse Model.

Age-related macular degeneration (AMD) has been linked to oxidative damage and para-inflammation, an activation of inflammasome signaling in the retinal pigment epithelium (RPE) and the underlying choriocapillaris. Herein, we tested the efficacy of a gene-delivered caspase-1 inhibitor in controlling the retinal degeneration observed in two models of RPE-choroid oxidative damage. In an acute model of oxidative stress (NaIO3 injection), eyes pre-treated with the sGFP-TatCARD (trans-activator of transcription; caspase activation and recruitment domain) vector demonstrated a recovery of retinal function and partial protection of RPE structure 1 month after damage, in contrast with control-treated eyes. In a model of chronic oxidative stress (RPE-specific deletion of Sod2), eyes treated with the sGFP-TatCARD vector after the onset of degeneration had a significantly slower decline in retinal function when compared to control-treated eyes. Earlier treatment of this model with the same adeno-associated virus (AAV) vector resulted in a greater protection of RPE function in eyes treated with the TatCARD when compared to control-treated eyes. Our results demonstrate that intravitreal delivery of sGFP-TatCARD reduces inflammation and can protect the retina from both acute and sustained oxidative damage within the RPE and choroid. Therefore, gene therapy with a cell-penetrating inflammasome inhibitor such as CARD may stem the progression of AMD.

AAV Mediated Delivery of Myxoma Virus M013 Gene Protects the Retina against Autoimmune Uveitis.

Uveoretinitis is an ocular autoimmune disease caused by the activation of autoreactive T- cells targeting retinal antigens. The myxoma M013 gene is known to block NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) and inflammasome activation, and its gene delivery has been demonstrated to protect the retina against lipopolysaccharide (LPS)-induced uveitis. In this report we tested the efficacy of M013 in an experimental autoimmune uveoretinitis (EAU) mouse model. B10RIII mice were injected intravitreally with AAV (adeno associated virus) vectors delivering either secreted GFP (sGFP) or sGFP-TatM013. Mice were immunized with interphotorecptor retinoid binding protein residues 161-180 (IRBP161-180) peptide in complete Freund's adjuvant a month later. Mice were evaluated by fundoscopy and spectral domain optical coherence tomography (SD-OCT) at 14 days post immunization. Eyes were evaluated by histology and retina gene expression changes were measured by reverse transcribed quantitative PCR (RT-qPCR). No significant difference in ERG or retina layer thickness was observed between sGFP and sGFP-TatM013 treated non-uveitic mice, indicating safety of the vector. In EAU mice, expression of sGFP-TatM013 strongly lowered the clinical score and number of infiltrative cells within the vitreous humor when compared to sGFP treated eyes. Retina structure was protected, and pro-inflammatory genes expression was significantly decreased. These results indicate that gene delivery of myxoma M013 could be of clinical benefit against autoimmune diseases.

Clinically Relevant Outcome Measures for the I307N Rhodopsin Mouse: A Model of Inducible Autosomal Dominant Retinitis Pigmentosa.

Purpose: The I307N rhodopsin (Rho) mouse is a light-inducible model of autosomal dominant retinitis pigmentosa (adRP) that may be useful in testing therapies. We investigated the time-course of retinal changes of the I307N Rho mouse with spectral-domain optical coherence tomography (SD-OCT). Methods: SD-OCT was performed up to day 30 after light damage; electroretinography (ERG) was employed to evaluate photoreceptor function. We utilized ImageJ to analyze reflectivity of the retina. We used light and electron microscopy to assess retinal organization. We stained synaptophysin and zonula occludins-1 with immunohistochemistry to determine injury to the plexiform layers and retinal pigment epithelium (RPE). We performed lectin staining to evaluate retinal blood vessels. Results: Retinal degeneration increased with longer exposures to light. An increase in retinal thickness was detected by SD-OCT on day 1 after light challenge followed by loss of the outer nuclear layer (ONL) by day 8. Degeneration was most severe in the nasal and inferior retina. Hyper-reflectivity on SD-OCT developed as early as 1 day after light exposure. Disorganization of the ONL, condensation of photoreceptor chromatin, disruption of the outer limiting membrane, and disarray of outer segments were associated with the hyper-reflectivity. Retraction of the outer plexiform synapses and resorption of the subretinal detachment contributed to retinal thinning. The RPE remained intact, whereas atrophied major retinal vessels were evident after light damage. Conclusions: Our time-course analysis of retinal degeneration in the I307N Rho mouse with SD-OCT and other outcome measures should enable the use of the mouse model in preclinical efficacy studies and mechanistic studies.