A Bispecific Modeling Framework Enables the Prediction of Efficacy, Toxicity, and Optimal Molecular Design of Bispecific Antibodies Targeting MerTK.

Inhibiting MerTK on macrophages is a promising therapeutic strategy for augmenting anti-tumor immunity. However, blocking MerTK on retinal pigment epithelial cells (RPEs) results in retinal toxicity. Bispecific antibodies (bsAbs) containing an anti-MerTK therapeutic and anti-PD-L1 targeting arm were developed to reduce drug binding to MerTK on RPEs, since PD-L1 is overexpressed on macrophages but not RPEs. In this study, we present a modeling framework using in vitro receptor occupancy (RO) and pharmacokinetics (PK) data to predict efficacy, toxicity, and therapeutic index (TI) of anti-MerTK bsAbs. We first used simulations and in vitro RO data of anti-MerTK monospecific antibody (msAb) to estimate the required MerTK RO for in vivo efficacy and toxicity. Using these estimated RO thresholds, we employed our model to predict the efficacious and toxic doses for anti-MerTK bsAbs with varying affinities for MerTK. Our model predicted the highest TI for the anti-MerTK/PD-L1 bsAb with an attenuated MerTK binding arm, which was consistent with in vivo efficacy and toxicity observations. Subsequently, we used the model, in combination with sensitivity analysis and parameter scans, to suggest an optimal molecular design of anti-MerTK bsAb with the highest predicted TI in humans. Our prediction revealed that this optimized anti-MerTK bsAb should contain a MerTK therapeutic arm with relatively low affinity, along with a high affinity targeting arm that can bind to a low abundance target with slow turnover rate. Overall, these results demonstrated that our modeling framework can guide the rational design of bsAbs.

Therapeutically targeting type I interferon directly to XCR1+ dendritic cells reveals the role of cDC1s in anti-drug antibodies.

Conventional type 1 dendritic cells (cDC1s) are superior in antigen cross-presentation and priming CD8+ T cell anti-tumor immunity and thus, are a target of high interest for cancer immunotherapy. Type I interferon (IFN) is a potent inducer of antigen cross-presentation, but, unfortunately, shows only modest results in the clinic given the short half-life and high toxicity of current type I IFN therapies, which limit IFN exposure in the tumor. CD8+ T cell immunity is dependent on IFN signaling in cDC1s and preclinical studies suggest targeting IFN directly to cDC1s may be sufficient to drive anti-tumor immunity. Here, we engineered an anti-XCR1 antibody (Ab) and IFN mutein (IFNmut) fusion protein (XCR1Ab-IFNmut) to determine whether systemic delivery could drive selective and sustained type I IFN signaling in cDC1s leading to anti-tumor activity and, in parallel, reduced systemic toxicity. We found that the XCR1Ab-IFNmut fusion specifically enhanced cDC1 activation in the tumor and spleen compared to an untargeted control IFN. However, multiple treatments with the XCR1Ab-IFNmut fusion resulted in robust anti-drug antibodies (ADA) and loss of drug exposure. Using other cDC1-targeting Ab-IFNmut fusions, we found that localizing IFN directly to cDC1s activates their ability to promote ADA responses, regardless of the cDC1 targeting antigen. The development of ADA remains a major hurdle in immunotherapy drug development and the cellular and molecular mechanisms governing the development of ADA responses in humans is not well understood. Our results reveal a role of cDC1s in ADA generation and highlight the potential ADA challenges with targeting immunostimulatory agents to this cellular compartment.

Wnt signaling in synaptogenesis of Alzheimer's disease.

Alzheimer's disease (AD), recognized as the leading cause of dementia, occupies a prominent position on the list of significant neurodegenerative disorders, representing a significant global health concern with far-reaching implications at both individual and societal levels. The primary symptom of Alzheimer's disease is a decrease in synaptic potency along with synaptic connection loss. Synapses, which act as important linkages between neuronal units within the cerebral region, are critical in signal transduction processes essential to orchestrating cognitive tasks. Synaptic connections act as critical interconnections between neuronal cells inside the cerebral environment, facilitating critical signal transduction processes required for cognitive functions. The confluence of axonal and dendritic filopodial extensions culminates in the creation of intercellular connections, coordinated by various signals and molecular mechanisms. The progression of synaptic maturation and plasticity is a critical determinant in maintaining mental well-being, and abnormalities in these processes have been linked to the development of neurodegenerative diseases. Wnt signaling pathways are important to the orchestration of synapse development. This review examines the complicated interplay between Wnt signaling and dendritic filopodia, including an examination of the regulatory complexities and molecular machinations involved in synaptogenesis progression. Then, these findings are contextualized within the context of AD pathology, allowing for the consideration of prospective therapeutic approaches based on the findings and development of novel avenues for future scientific research.

Genome expansion by a CRISPR trimmer-integrase.

CRISPR-Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity1. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1-Cas2 integrase is necessary but not sufficient2-5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation6,7, but many CRISPR-Cas systems lack Cas48. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1-Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited9,10 exonucleases for faithful acquisition of new CRISPR immune sequences.

National Institutes of Health research project grant inflation 1998 to 2021.

We analyzed changes in total costs for National Institutes of Health (NIH) awarded Research Project Grants (RPGs) issued from fiscal years (FYs) 1998 to 2021 . Costs are measured in 'nominal' terms, meaning exactly as stated, or in 'real' terms, meaning after adjustment for inflation. The NIH uses a data-driven price index - the Biomedical Research and Development Price Index (BRDPI) - to account for inflation, enabling assessment of changes in real (that is, BRDPI-adjusted) costs over time. The BRDPI was higher than the general inflation rate from FY1998 until FY2012; since then the BRDPI has been similar to the general inflation rate likely due to caps on senior faculty salary support. Despite increases in nominal costs, recent years have seen increases in the absolute numbers of RPG and R01 awards. Real average and median RPG costs increased during the NIH-doubling (FY1998 to FY2003), decreased after the doubling and have remained relatively stable since. Of note, though, the degree of variation of RPG costs has changed over time, with more marked extremes observed on both higher and lower levels of cost. On both ends of the cost spectrum, the agency is funding a greater proportion of solicited projects, with nearly half of RPG money going toward solicited projects. After adjusting for confounders, we find no independent association of time with BRDPI-adjusted costs; in other words, changes in real costs are largely explained by changes in the composition of the NIH-grant portfolio.

CRISPR technology: A decade of genome editing is only the beginning.

The advent of clustered regularly interspaced short palindromic repeat (CRISPR) genome editing, coupled with advances in computing and imaging capabilities, has initiated a new era in which genetic diseases and individual disease susceptibilities are both predictable and actionable. Likewise, genes responsible for plant traits can be identified and altered quickly, transforming the pace of agricultural research and plant breeding. In this Review, we discuss the current state of CRISPR-mediated genetic manipulation in human cells, animals, and plants along with relevant successes and challenges and present a roadmap for the future of this technology.

Child-parent relationship during the Wuhan COVID-19 lockdown: Role of changes in preschool children's daily routines.

Objective: We examined the impact of the strict lockdown on 130 preschool-age children's daily routines and how their routine changes from pre-lockdown were related to child-parent relationship quality during the lockdown. Background: To contain the spread of the COVID-19, the city of Wuhan underwent a strict 76-day lockdown, during which children's routines were drastically altered, yet families did not have a frame of reference to use to determine how changes in children's routines would be related to their family dynamics. Method: Parents provided survey data on the amount of time their children spent daily on learning, screen devices, play and exercise, and nighttime sleep before, during, and after the lockdown. The also described general family functioning, child-parent closeness, and child-parent conflict during the lockdown. Results: The lockdown led to changes in all four routines, but all returned to pre-lockdown level after the lockdown was lifted. Regression analyses showed that decrease in play and exercise time was related to less child-parent closeness, and decrease in learning time and increase in nighttime sleep time were related to more child-parent conflict. Conclusion: Findings suggested changes in the children's play and exercise time, learning time, and nighttime sleep time were related to negative child-parent relationship (i.e., less closeness or more conflict), but favorable general functioning was a protective factor. Implications: Our study highlighted family resilience in restoring the children's routines after the lockdown, as well as family vulnerability during the lockdown, as changes in three of the four routines examined were linked to negative child-parent relationship.

PEGylation of anti-MerTK Antibody Modulates Ocular Biodistribution.

Here, we explore whether PEGylation of antibodies can modulate their biodistribution to the eye, an organ once thought to be immune privileged but has recently been shown to be accessible to IV-administered large molecules, such as antibodies. We chose to PEGylate an anti-MerTK antibody, a target with known potential for ocular toxicity, to minimize biodistribution to retinal pigment epithelial cells (RPEs) in the eye by increasing the hydrodynamic volume of the antibody. We used site-specific conjugation to an engineered cysteine on anti-MerTK antibody to chemically attach 40-kDa branched or linear PEG polymers. Despite reduced binding to MerTK on cells, site-specifically PEGylated anti-MerTK retained similar potency in inhibiting MerTK-mediated macrophage efferocytosis of apoptotic cells. Importantly, we found that PEGylation of anti-MerTK significantly reduced MerTK receptor occupancy in RPE cells in both naïve mice and MC-38 tumor-bearing mice, with the branched PEG exhibiting a greater effect than linear PEG. Furthermore, similar to unconjugated anti-MerTK, PEGylated anti-MerTK antibody triggered type I IFN response and exhibited antitumor effect in syngeneic mouse tumor studies. Our results demonstrate the potential of PEGylation to control ocular biodistribution of antibodies.

Structural biology of CRISPR-Cas immunity and genome editing enzymes.

CRISPR-Cas systems provide resistance against foreign mobile genetic elements and have a wide range of genome editing and biotechnological applications. In this Review, we examine recent advances in understanding the molecular structures and mechanisms of enzymes comprising bacterial RNA-guided CRISPR-Cas immune systems and deployed for wide-ranging genome editing applications. We explore the adaptive and interference aspects of CRISPR-Cas function as well as open questions about the molecular mechanisms responsible for genome targeting. These structural insights reflect close evolutionary links between CRISPR-Cas systems and mobile genetic elements, including the origins and evolution of CRISPR-Cas systems from DNA transposons, retrotransposons and toxin-antitoxin modules. We discuss how the evolution and structural diversity of CRISPR-Cas systems explain their functional complexity and utility as genome editing tools.

Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex.

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.

Associations of topic-specific peer review outcomes and institute and center award rates with funding disparities at the National Institutes of Health.

A previous report found an association of topic choice with race-based funding disparities among R01 applications submitted to the National Institutes of Health ('NIH') between 2011 and 2015. Applications submitted by African American or Black ('AAB') Principal Investigators ('PIs') skewed toward a small number of topics that were less likely to be funded (or 'awarded'). It was suggested that lower award rates may be related to topic-related biases of peer reviewers. However, the report did not account for differential funding ecologies among NIH Institutes and Centers ('ICs'). In a re-analysis, we find that 10% of 148 topics account for 50% of applications submitted by AAB PIs. These applications on 'AAB Preferred' topics were funded at lower rates, but peer review outcomes were similar. The lower rate of funding for these topics was primarily due to their assignment to ICs with lower award rates, not to peer-reviewer preferences.

A Motivational Interviewing Intervention for Adolescents in Accelerated High School Curricula: Applicability and Acceptability in a Second Sample.

The paper describes the applicability and acceptability of a selective intervention-Motivation, Assessment, and Planning (MAP)-for high school students that was developed based on the principles of motivational interviewing (MI) and tailored to the unique needs and strengths of students taking accelerated coursework, specifically Advanced Placement (AP) and International Baccalaureate (IB) classes. In addition to detailing the intervention in terms of MI spirit, processes, and relational and technical skills, we report applicability and acceptability data from a second iteration of MAP implementation in eight AP/IB programs in a Southeastern state during spring 2018. We analyzed quantitative and qualitative acceptability data from 121 high school freshmen (97 from AP and 24 from IB courses), as well as the seven MAP coaches who were trained using the Motivational Interview Training and Assessment System (Frey et al. 2017). To gain perspectives from the intended end users of the refined MAP, 12 school counselors and school psychologists who were not trained in MAP evaluated the intervention and provided qualitative and quantitative data on applicability and acceptability. All three stakeholder groups (students, coaches, and school mental health staff) rated and described the intervention as highly acceptable and appropriate for addressing the social-emotional needs of adolescents in AP/IB classes.

Lactic Acid and an Acidic Tumor Microenvironment suppress Anticancer Immunity.

Immune evasion and altered metabolism, where glucose utilization is diverted to increased lactic acid production, are two fundamental hallmarks of cancer. Although lactic acid has long been considered a waste product of this alteration, it is now well accepted that increased lactic acid production and the resultant acidification of the tumor microenvironment (TME) promotes multiple critical oncogenic processes including angiogenesis, tissue invasion/metastasis, and drug resistance. We and others have hypothesized that excess lactic acid in the TME is responsible for suppressing anticancer immunity. Recent studies support this hypothesis and provide mechanistic evidence explaining how lactic acid and the acidic TME impede immune cell functions. In this review, we consider lactic acid's role as a critical immunoregulatory molecule involved in suppressing immune effector cell proliferation and inducing immune cell de-differentiation. This results in the inhibition of antitumor immune responses and the activation of potent, negative regulators of innate and adaptive immune cells. We also consider the role of an acidic TME in suppressing anticancer immunity. Finally, we provide insights to help translate this new knowledge into impactful anticancer immune therapies.

Effects of Social Media and Smartphone Use on Body Esteem in Female Adolescents: Testing a Cognitive and Affective Model.

We examined the predictive relations of social media and smartphone use to body esteem in female adolescents and the mechanism that underlies these relations. As a result of frequent social media and smartphone use, adolescents are continually exposed to appearance-related media content. This likely reinforces a thin ideal and fosters appearance-based comparison and increases fear of external evaluation. Hence, we investigated a cognitive-affective framework in which the associations of social media and smartphone use with body esteem are serially mediated by cognitive internalization of an ideal body image, appearance comparisons, and social appearance anxiety. By testing female adolescents (N = 100) aged 13 to 18, we found that excessive social media use leads to unhealthy body esteem via intensified cognitive internalization, which aggravates appearance comparisons and anxiety regarding negative appearance evaluation. Further, we found that screen time for specific smartphone activities also harmed body esteem, independent of social media use. However, overall smartphone screen time did not affect body esteem when social media use was taken into consideration. Our findings underscore the multifactor mechanism that elucidates the negative impacts of social media and smartphone activities on body esteem in female adolescents, who are developmentally susceptible to poor body esteem.

PEEK Maxillary Obturator Prosthesis Fabrication Using Intraoral Scanning, 3D Printing, and CAD/CAM.

This report presents a digital technique for the fabrication of a two-piece hollow bulb maxillary obturator prosthesis. The procedure described resulted in an accurate prosthesis while avoiding the discomfort associated with analog impressions. The manipulation of a routine postoperative CT scan in conjunction with a 3D printer allowed for the fabrication of a 3D-printed anatomical cast, from which the two-piece hollow bulb obturator was fabricated. The obturator prosthesis framework was digitally designed and milled from a modified PEEK material, resulting in a lightweight prosthesis with excellent biocompatibility. The clinical and laboratory steps involved are described.

Osteoradionecrosis in osseous free flap reconstruction: Risk factors and treatment.

OBJECTIVE: To determine the rate and risk factors for osteoradionecrosis (ORN) in osseous free flaps after postoperative radiation therapy (PORT). To describe the treatment of free flap ORN. METHODS: Seventy-four patients undergoing osseous free flap reconstruction were analyzed. Thirty-eight completed PORT. Patients were followed for ≥6 months. RESULTS: The rate of ORN was 34% overall; 0% with 50 to 59.9 Gy; 8% with 60 Gy; 40% with 66 Gy; 56% with 70 to 74.4 Gy. Mean time to ORN was 13.1 months. 0/28 patients without PORT developed free flap osteonecrosis. Multivariate analysis found the only factor predicting ORN: PORT >60 Gy, which increased the risk 21-fold. Treatment included PENTACLO, hyperbaric oxygen, and surgical debridement with 75% within 2 years. CONCLUSION: PORT >60 Gy is significantly associated with free flap ORN. As the dose of adjuvant RT increases beyond 60 Gy, the risk of ORN in free flaps rises. Consideration should be given to lower PORT doses or delaying free flap reconstruction when feasible.

DABs are inorganic carbon pumps found throughout prokaryotic phyla.

Bacterial autotrophs often rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins-which we name DabA and DabB, for DABs accumulate bicarbonate-assemble into a heterodimeric complex, which contains a putative ß-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (Ci) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized Ci pumps and play a critical role in the metabolism of Ci throughout prokaryotic phyla.

Fabrication of a 3D-printed interim obturator prosthesis: A contemporary approach.

This report describes the combination of analog and digital techniques for the fabrication of a 2-piece hollow bulb maxillary obturator. The procedure described provides an accurate representation of the surgical defect while avoiding the discomfort associated with analog impressions. The manipulation of a routine postoperative computed tomography (CT) scanner in conjunction with a 3D printer allowed for the fabrication of a 3D-printed anatomic cast from which the 2-piece hollow bulb obturator was fabricated. The clinical and laboratory steps involved are described in this article.

A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System.

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.

Protocol for the systematic review of research on professional learning to promote implementation of a multitiered system of support in education.

INTRODUCTION: A multitiered system of supports (MTSS) represents a widely adopted public health approach to education in the USA. Researchers agree professional learning is critical for educators to implement the critical components of MTSS; however, professional learning approaches vary in their designs and targeted outcomes. While researchers increasingly focus their inquiries on professional learning for MTSS, no systematic research review exists. OBJECTIVES: The primary objectives for this mixed-methods review are to (1) understand how professional learning focused on MTSS has been operationalised (2) determine the impact of professional learning on educator (eg, knowledge) and implementation (eg, data-based decision-making processes) outcomes and (3) understand the contextual variables that influence professional learning in the USA. We aim to determine which elements of professional learning improve educators' capacity to implement MTSS. METHODS AND ANALYSIS: We will include studies that use quantitative and qualitative methods. PsycInfo, PubMed, CIHAHL and ERIC will be the primary research databases used to search for studies published from January 1997 to May 2018. We also will search the US Institute for Educational Sciences and Office of Special Education Programs websites, ProQuest, Google Scholar, Science Watch and MSN. Finally, we will search the proceedings of relevant conferences, examine the reference lists of studies that pass full screening and contact authors for additional work. Data extraction will include participant demographics, intervention details, study design, outcomes, analyses and key findings. We will conduct a quality assessment and analyse the data using effect size and thematic analyses. ETHICS AND DISSEMINATION: Institutional review board or ethics approval is not needed for this review of already published works. We will disseminate the findings through presentations at state, national and international conferences; presentations to stakeholders and agencies; publication in peer-reviewed journals; and posts to organisational and agency websites.