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Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

Rhesus cytomegalovirus-based (RhCMV-based) vaccine vectors induce immune responses that protect ~60% of rhesus macaques (RMs) from SIVmac239 challenge. This efficacy depends on induction of effector memory-based (EM-biased) CD8+ T cells recognizing SIV peptides presented by major histocompatibility complex-E (MHC-E) instead of MHC-Ia. The phenotype, durability, and efficacy of RhCMV/SIV-elicited cellular immune responses were maintained when vector spread was severely reduced by deleting the antihost intrinsic immunity factor phosphoprotein 71 (pp71). Here, we examined the impact of an even more stringent attenuation strategy on vector-induced immune protection against SIV. Fusion of the FK506-binding protein (FKBP) degradation domain to Rh108, the orthologue of the essential human CMV (HCMV) late gene transcription factor UL79, generated RhCMV/SIV vectors that conditionally replicate only when the FK506 analog Shield-1 is present. Despite lacking in vivo dissemination and reduced innate and B cell responses to vaccination, Rh108-deficient 68-1 RhCMV/SIV vectors elicited high-frequency, durable, EM-biased, SIV-specific T cell responses in RhCMV-seropositive RMs at doses of ≥ 1 × 106 PFU. Strikingly, elicited CD8+ T cells exclusively targeted MHC-Ia-restricted epitopes and failed to protect against SIVmac239 challenge. Thus, Rh108-dependent late gene expression is required for both induction of MHC-E-restricted T cells and protection against SIV.

Joint attention, or shared attention to an object or event, is a pivotal skill for the development of social interactions and social communication. Joint attention typically develops in natural contexts within the first year of life. Children with autism spectrum disorder (ASD) and related developmental disabilities often experience difficulty developing joint attention. Clinical interventions to increase joint attention exist. However, there are limited studies investigating methods to incorporate joint attention interventions in inclusive preschool classrooms with peers as social partners. For young children in preschool settings, peer-mediated social skills interventions reflect natural contingencies and may promote generalization. In this study, we employed a single-case multiple baseline across child-peer dyads design to evaluate the effects of a peer-mediated joint attention intervention for children with or at risk for ASD on response to joint attention (RJA) from peer bids and initiations of joint attention (IJA) from target children with ASD. The intervention included direct instruction with a social narrative to teach three peers to initiate bids for joint attention to measure target children's responses. Target children later received the same direct instruction to increase IJA to same-age-peers. Results indicate increased target child RJA and IJA to peers. Limitations and implications for research and practice are discussed.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

In an exploratory trial treating "long COVID" with the CCR5-binding antibody leronlimab, we observed significantly increased blood cell surface CCR5 in treated symptomatic responders but not in nonresponders or placebo-treated participants. These findings suggest an unexpected mechanism of abnormal immune downmodulation in some persons that is normalized by leronlimab. Clinical Trials Registration. NCT04678830.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

With the increasing use of triploid fish for sport fisheries management, biologists, researchers, and managers working in field environments need practical methods to determine ploidy. Cytological methods (e.g., flow cytometry, Coulter counter) using erythrocytes are the most common for ploidy determination in fishes. However, collecting and storing erythrocytes can be logistically challenging during field work, and donor fish need to be alive or freshly killed. With rapid advances in molecular genetics, biologists, researchers, and managers may be unaware of molecular approaches for ploidy determination that could alleviate the difficulties associated with cytological methods and allow ploidy determination from archived samples (e.g., fin clips, scales, otoliths). In this study, we analyzed the agreement between molecular-based (using fin tissue) and Coulter counter-based (using blood) ploidy determinations for Walleyes Stizostedion vitreum—the first assessment of concordance between molecular and cytological methods for determining ploidy in the family Percidae. We found that agreement between these two methods was >98%. The high degree of agreement and greater ease of collecting and storing samples for molecular-based approaches relative to the traditional cytological ones support the utility of molecular methods for ploidy determination.

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and β-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.

Children with autism spectrum disorder (ASD) and other developmental disabilities are at risk of isolation from same-aged peers. Furthermore, research indicates that even in inclusive settings, children with ASD benefit from targeted interventions and support. Among the social communication skills that are frequently absent in children with ASD is joint attention. Joint attention can be defined as shared engagement between two individuals on a third object or event and has been identified as potential pivotal skill for later development of age-appropriate social skills and communication. A growing body of literature documents effective intervention on joint attention skill for young children with ASD; however, few studies document the effects of an intervention mediated by a natural change agent. In the present study, independent concurrent multiple-baseline designs were used to evaluate the impact of a joint attention intervention combined with peer training on the response to joint attention (RJA) behaviors exhibited by seven preschoolers with ASD. Results indicated that RJA behaviors increased overall, the seven peers were able to carry out a simple intervention with fidelity, and that both bids from peers and responses to those bids increased following intervention. Limitations and opportunities for future research are discussed.

Fingerspelling and its relationship with literacy skills among deaf and hard of hearing children who use American Sign Language is an increasingly popular research topic. However, there is limited research on whether reading interventions that systematically include fingerspelling are more effective for improving literacy skills than reading interventions that do not. In an adapted alternating-treatment single-case study, the authors contrasted the number of words learned under three conditions: a productive fingerspelling condition, in which word reading was taught through activities that emphasized productive fingerspelling; a chaining condition, in which teachers chained written words with receptive fingerspelling; and a sign-to-print condition, in which fingerspelling was not used. Five of the 6 participants learned most of the words taught with no differentiation by condition. Participants could recognize and fingerspell taught words, even if those words were not taught via fingerspelling.

Children on the autism spectrum often experience difficulty generalizing social skills across environments and contexts, which can make developing friendships challenging in early childhood. This means that, in addition to initial social skills instruction, children with autism may need specialized supports to promote the generalization of newly learned skills to natural inclusive play routines such as unstructured social centers and playdates. In this paper, we describe strategies teachers can employ to promote the generalization of newly learned social skills. Specifically, we describe how social narratives, visual supports, and environmental arrangement, prompting, and praise can be used during social centers and playdates to facilitate setting generalization. When teachers systematically support generalized social skills and behaviors, children will have more opportunities to develop meaningful friendships.

Numerous developmental difficulties differentiate children with autism from typically developing children, including delays in social communication skills. In this study, we used a multiple baseline design across three behaviors (i.e., imitation, behavioral requests, and response to joint attention) for two parent-child dyads to investigate the effectiveness of a parent-implemented social communication intervention. Parents received training on least to most prompting, and coaching while practicing with their child until they reached the intervention fidelity criterion. Results indicated increased parent fidelity for one parent in two strategies and in all strategies for one parent. Environmental arrangement training was not provided for one parent due to increases in baseline. In addition, all child behaviors increased. The results and implications for future research and practice are discussed.

Given the continued changes in demographic diversity of students in the United States, it is important to ensure that participants included in special education research reflect the diversity of the classroom. We examined 16 years of intervention research across 12 special education journals to evaluate the extent to which diverse student populations (e.g., race, ethnicity, disability, sexual orientation, English language learner status) were included in published intervention research. We analyzed 495 intervention articles (9.6%) out of 5,180 total articles. Results revealed that progress has been made in the inclusion of diverse participants in special education intervention research compared with previously conducted reviews, yet some racial and ethnic populations are still underrepresented. We discuss strategies for recruitment and retention of underrepresented diverse populations.

Problem-based learning (PBL) is widely recognized as a pedagogical approach across disciplines. However, the relevance and application of PBL in psychology has received limited attention. Therefore, this article presents a PBL-based master degree programme for psychologists. The article is divided into three sections. First, we present the rationale and need for developing this programme. Second, the programme curriculum is described in detail and, third, the programme's practical and theoretical aspects and potentials are discussed in light of PBL principles.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

We have formatted an assay to detect Mycobacterium tuberculosis complex infections of non-human primates. Commercially available reagents were used to elicit a specific immune response that was measured by interferon-gamma release. Initial evaluation using blood samples from Rhesus macaques experimentally infected with M tuberculosis distinguished infected versus uninfected animals.

Herpes simplex virus 2 (HSV-2) is a common sexually transmitted infection with a highly variable clinical course. Many infections quickly become subclinical, with episodes of spontaneous virus reactivation. To study host-HSV-2 interactions, an animal model of subclinical HSV-2 infection is needed. In an effort to develop a relevant model, rhesus macaques (RM) were inoculated intravaginally with two or three HSV-2 strains (186, 333, and/or G) at a total dose of 1 × 107 PFU of HSV-2 per animal. Infectious HSV-2 and HSV-2 DNA were consistently shed in vaginal swabs for the first 7 to 14 days after each inoculation. Proteins associated with wound healing, innate immunity, and inflammation were significantly increased in cervical secretions immediately after HSV-2 inoculation. There was histologic evidence of acute herpesvirus pathology, including acantholysis in the squamous epithelium and ballooning degeneration of and intranuclear inclusion bodies in epithelial cells, with HSV antigen in mucosal epithelial cells and keratinocytes. Further, an intense inflammatory infiltrate was found in the cervix and vulva. Evidence of latent infection and reactivation was demonstrated by the detection of spontaneous HSV-2 shedding post-acute inoculation (102 to 103 DNA copies/swab) in 80% of RM. Further, HSV-2 DNA was detected in ganglia in most necropsied animals. HSV-2-specifc T-cell responses were detected in all animals, although antibodies to HSV-2 were detected in only 30% of the animals. Thus, HSV-2 infection of RM recapitulates many of the key features of subclinical HSV-2 infection in women but seems to be more limited, as virus shedding was undetectable more than 40 days after the last virus inoculation.IMPORTANCE Herpes simplex virus 2 (HSV-2) infects nearly 500 million persons globally, with an estimated 21 million incident cases each year, making it one of the most common sexually transmitted infections (STIs). HSV-2 is associated with increased human immunodeficiency virus type 1 (HIV-1) acquisition, and this risk does not decline with the use of antiherpes drugs. As initial acquisition of both HIV and HSV-2 infections is subclinical, study of the initial molecular interactions of the two agents requires an animal model. We found that HSV-2 can infect RM after vaginal inoculation, establish latency in the nervous system, and spontaneously reactivate; these features mimic some of the key features of HSV-2 infection in women. RM may provide an animal model to develop strategies to prevent HSV-2 acquisition and reactivation.

Children with autism spectrum disorder (ASD) present with social skills difficulties, which can create barriers for them to develop friendships with their peers. Playdates are a common way that young children practice play and friendship skills in home and community environments. Few studies have trained parents to embed social skills instruction into playdates for children with ASD, and such studies have been comprised of children with mild to moderate needs. In the current study, we employed a concurrent single-case multiple probe across three parent-child-peer triads design to evaluate the effects of training and coaching in a parent-implemented playdate intervention on parental strategy use and cascading effects on child-peer social interactions during playdates. Parents also rated the social validity of the intervention. Results indicated that two of the three parents reached criterion on the strategies quickly, and the third triad required a procedural modification. Challenging behavior appeared to be a barrier to implementation. Implications and future directions are discussed.

Sleep problems in children with autism spectrum disorder are prevalent and persistent but also treatable. Little is known about how and why parents of such children seek help for sleep disturbance. Via an online survey (n = 244 respondents), we gathered information about parents' attributions about children's sleep problems and beliefs about causes and on sources of information about, and their decisions regarding, help-seeking. Eighty-two percent of parents reported seeking some kind of help for their child's sleep disturbance, and the average parent had tried six different treatment strategies, most commonly medical. Alignment of parents' treatment choices with empirical evidence about treatment efficacy was poor, but belief in effectiveness was closely related to frequency of use of a treatment. In a Principal Components Analysis, parental attributions loaded on two factors: one which suggests the sleep problems are viewed as intrinsic to autism and stable (factor one) and the other as located within the child, stable, and treatment resistant (factor two). These findings have important implications for parental education and clinical practice in the treatment of sleep problems in children with autism spectrum disorder.

Paraeducators are ideal candidates for delivering communication interventions to children with developmental disabilities and delays (DD) because they spend a significant amount of time with these children. However, professional development is often inadequate and limited research supports best practices. Additionally, paraeducators work with multiple children with varying skill levels. Little research has been conducted on the use of existing strategies with multiple children. This single-case study examines the effect of a training package on paraeducators' fidelity of intervention implementation with a child dyad and subsequent child outcomes. Results suggest that formal coaching contributed to improved fidelity of intervention implementation. Furthermore, paraeducators were able to use intervention strategies with children with varying communication skills and goals. Variable increases in child communication were also detected.

Children with autism spectrum disorder (ASD) display social-communication deficits and present with rigid and repetitive patterns of behavior and/or interests (RRBIs). Compared to interventions for social-communication skills, less attention has been given to RRBIs, especially with regard to interventions for young children. We surveyed 128 behavior analysts who implemented interventions for young children with ASD on their use of 16 practices and one assessment for the treatment of RRBIs. The majority of our sample perceived the practices to be effective in producing sustainable behavior change. Behavior analysts generally responded in the same way to items about reinforcement-based practices, punishment-based practices, and a group of commonly packaged antecedent and consequence-based package components. Implications and future directions are discussed.

This review discusses the evolution of preoperative fasting guidelines and examines the incidence of pulmonary aspiration of gastric contents and suggested treatments. Nine guidelines developed by professional societies and published in peer-reviewed journals since 1994 were identified. The recommendations on preoperative fasting for various categories have undergone only small adaptations in the following three decades in pediatric anesthesia. We found twelve published studies of the incidence of pulmonary aspiration, which ranges from 0.6 to 12 in 10,000 anesthetics in children. However, this variation reflects differences in the definition of aspiration as well as differences in study design. The main risk factors identified are emergency surgery, ASA physical status, and patient age. Several additional risk factors have been suggested, including non-compliance to fasting guidelines. The duration of clear fluid fasting is not associated with an increased risk of pulmonary aspiration which may be reflected in future guideline updates in pediatric anesthesia. [ABSTRACT FROM AUTHOR]

HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.

The Hippo pathway plays a central role in tissue homoeostasis, and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include a kinase cascade of MST1/2 and LATS1/2 and the transcription co-activators YAP/TAZ. In response to stimulation, LATS1/2 phosphorylate and inhibit YAP/TAZ, the main effectors of the Hippo pathway. Accumulating evidence suggests that MST1/2 are not required for the regulation of YAP/TAZ. Here we show that deletion of LATS1/2 but not MST1/2 abolishes YAP/TAZ phosphorylation. We have identified MAP4K family members--Drosophila Happyhour homologues MAP4K1/2/3 and Misshapen homologues MAP4K4/6/7-as direct LATS1/2-activating kinases. Combined deletion of MAP4Ks and MST1/2, but neither alone, suppresses phosphorylation of LATS1/2 and YAP/TAZ in response to a wide range of signals. Our results demonstrate that MAP4Ks act in parallel to and are partially redundant with MST1/2 in the regulation of LATS1/2 and YAP/TAZ, and establish MAP4Ks as components of the expanded Hippo pathway.