Exposure to Bacterial CpG DNA Protects from Airway Allergic Inflammation by Expanding Regulatory Lung Interstitial Macrophages.

Living in a microbe-rich environment reduces the risk of developing asthma. Exposure of humans or mice to unmethylated CpG DNA (CpG) from bacteria reproduces these protective effects, suggesting a major contribution of CpG to microbe-induced asthma resistance. However, how CpG confers protection remains elusive. We found that exposure to CpG expanded regulatory lung interstitial macrophages (IMs) from monocytes infiltrating the lung or mobilized from the spleen. Trafficking of IM precursors to the lung was independent of CCR2, a chemokine receptor required for monocyte mobilization from the bone marrow. Using a mouse model of allergic airway inflammation, we found that adoptive transfer of IMs isolated from CpG-treated mice recapitulated the protective effects of CpG when administered before allergen sensitization or challenge. IM-mediated protection was dependent on IL-10, given that Il10-/- CpG-induced IMs lacked regulatory effects. Thus, the expansion of regulatory lung IMs upon exposure to CpG might underlie the reduced risk of asthma development associated with a microbe-rich environment.

Investigating USP42 Mutation as Underlying Cause of Familial Non-Medullary Thyroid Carcinoma.

In a family with Familial Non-Medullary Thyroid Carcinoma (FNMTC), our investigation using Whole-Exome Sequencing (WES) uncovered a novel germline USP42 mutation [p.(Gly486Arg)]. USP42 is known for regulating p53, cell cycle arrest, and apoptosis, and for being reported as overexpressed in breast and gastric cancer patients. Recently, a USP13 missense mutation was described in FNMTC, suggesting a potential involvement in thyroid cancer. Aiming to explore the USP42 mutation as an underlying cause of FNMTC, our team validated the mutation in blood and tissue samples from the family. Using immunohistochemistry, the expression of USP42, Caspase-3, and p53 was assessed. The USP42 gene was silenced in human thyroid Nthy-Ori 3-1 cells using siRNAs. Subsequently, expression, viability, and morphological assays were conducted. p53, Cyclin D1, p21, and p27 proteins were evaluated by Western blot. USP42 protein was confirmed in all family members and was found to be overexpressed in tumor samples, along with an increased expression of p53 and cleaved Caspase-3. siRNA-mediated USP42 downregulation in Nthy-Ori 3-1 cells resulted in reduced cell viability, morphological changes, and modifications in cell cycle-related proteins. Our results suggest a pivotal role of USP42 mutation in thyroid cell biology, and this finding indicates that USP42 may serve as a new putative target in FNMTC.

Introduction to Helping to End Addiction Long-Term Prevention Cooperative: Overview and Strategies.

This supplemental issue describes the individual studies and collaborative efforts of the Helping to End Addiction Long-term Prevention Cooperative's (HPC's) innovative approaches to rapidly develop evidence-based prevention programs for widespread dissemination. This introduction succinctly reviews (1) the context that demands the rapid development of efficacious prevention programs and their scale-ups, (2) the unique objectives of the individual HPC research projects, and (3) collective efforts to harmonize research across studies to advance the prevention of opioid misuse and gain insight into opioid misuse etiology to inform improvements in preventive interventions. At the conclusion of HPC studies, we anticipate the availability of multiple evidence-based programs to prevent opioid misuse and use disorder for persons who experience particular sources of risk and for delivery in settings where prevention has traditionally been lacking. By harmonizing and coordinating efforts across 10 distinct outcomes studies of prevention programs and making data available for analysis by non-HPC researchers, the HPC's efficacy and etiology evidence will far surpass the additive contributions of 10 individual research projects.

Economic Evaluation Design within the HEAL Prevention Cooperative.

The rapid rise in opioid misuse, disorder, and opioid-involved deaths among older adolescents and young adults is an urgent public health problem. Prevention is a vital part of the nation's response to the opioid crisis, yet preventive interventions for those at risk for opioid misuse and opioid use disorder are scarce. In 2019, the National Institutes of Health (NIH) launched the Preventing Opioid Use Disorder in Older Adolescents and Young Adults cooperative as part of its broader Helping to End Addiction Long-term (HEAL) Initiative ( https://heal.nih.gov/ ). The HEAL Prevention Cooperative (HPC) includes ten research projects funded with the goal of developing effective prevention interventions across various settings (e.g., community, health care, juvenile justice, school) for older adolescent and young adults at risk for opioid misuse and opioid use disorder (OUD). An important component of the HPC is the inclusion of an economic evaluation by nine of these research projects that will provide information on the costs, cost-effectiveness, and sustainability of these interventions. The HPC economic evaluation is integrated into each research project's overall design with start-up costs and ongoing delivery costs collected prospectively using an activity-based costing approach. The primary objectives of the economic evaluation are to estimate the intervention implementation costs to providers, estimate the cost-effectiveness of each intervention for reducing opioid misuse initiation and escalation among youth, and use simulation modeling to estimate the budget impact of broader implementation of the interventions within the various settings over multiple years. The HPC offers an extraordinary opportunity to generate economic evidence for substance use prevention programming, providing policy makers and providers with critical information on the investments needed to start-up prevention interventions, as well as the cost-effectiveness of these interventions relative to alternatives. These data will help demonstrate the valuable role that prevention can play in combating the opioid crisis.

Multidisciplinary Strategies for Preventing Opioid Misuse and Escalation by Targeting Mental Health Symptoms and Conditions.

We aim to review the association between childhood-onset mental health conditions and increased risk for early substance use including opioid misuse and opioid use disorders (OUD). The association between mental health conditions and opioid misuse suggests youth with mental health conditions may benefit from opioid prevention efforts that concurrently address mental health. To aid in the identification of youth with mental health conditions who could benefit from interventions, we will review opportunities and challenges associated with screening for mental health symptoms or substance use in settings where youth at high risk for mental health conditions present. We will also review how research projects within the National Institutes of Health's Helping to End Addiction Long-term (HEAL) Prevention Cooperative are addressing mental health within opioid misuse and OUD prevention interventions for youth.

Methodological Strategies for Prospective Harmonization of Studies: Application to 10 Distinct Outcomes Studies of Preventive Interventions Targeting Opioid Misuse.

The Helping to End Addiction Long-Term (HEAL) Prevention Cooperative (HPC) is rapidly developing 10 distinct evidence-based interventions for implementation in a variety of settings to prevent opioid misuse and opioid use disorder. One HPC objective is to compare intervention impacts on opioid misuse initiation, escalation, severity, and disorder and identify whether any HPC interventions are more effective than others for types of individuals. It provides a rare opportunity to prospectively harmonize measures across distinct outcomes studies. This paper describes the needs, opportunities, strategies, and processes that were used to harmonize HPC data. They are illustrated with a strategy to measure opioid use that spans the spectrum of opioid use experiences (termed involvement) and is composed of common "anchor items" ranging from initiation to symptoms of opioid use disorder. The limitations and opportunities anticipated from this approach to data harmonization are reviewed. Lastly, implications for future research cooperatives and the broader HEAL data ecosystem are discussed.

Digoxin in Patients With Advanced Heart Failure and Sinus Rhythm Submitted to Cardiac Resynchronization Therapy-Is There Any Benefit?

ABSTRACT: Digoxin (DG) use in patients with heart failure with reduced ejection fraction (HFrEF) and sinus rhythm remains controversial. We aimed to assess the prognostic effect of DG in patients in sinus rhythm submitted to cardiac resynchronization therapy (CRT). Retrospective study including 297 consecutive patients in sinus rhythm, with advanced HFrEF submitted to CRT. Patients were divided into 2 groups: with DG and without DG (NDG). During a mean follow-up of 4.9 ± 3.4 years, we evaluated the effect of DG on the composite end point defined as cardiovascular hospitalization, progression to heart transplantation, and all-cause mortality. Previous to CRT, 104 patients (35%) chronically underwent DG and 193 patients (65%) underwent NDG treatment. The 2 groups did not differ significantly regarding HF functional class, HF etiology, QRS, and baseline left ventricular ejection fraction. The proportion of responders to CRT was similar in both groups (54% in DG vs. 56% in NDG; P = 0.78). During the long-term follow-up period, the primary end point occurred in a higher proportion in DG patients (67 vs. 48%; P = 0.002). After adjustment for potential confounders, DG use remained as an independent predictor of the composite end point of CV hospitalization, heart transplantation, and all-cause mortality [hazards ratio = 1.58; confidence interval, 95 (1.01-2.46); P = 0.045]. In conclusion, in patients in sinus rhythm with HFrEF submitted to CRT, DG use was associated with CV hospitalization, progression to heart transplant, and all-cause mortality.

Feeling Invisible and Unheard: A Qualitative Exploration of Gendered-Racist Stereotypes Influence on Sexual Decision Making and Mistreatment of Black Teen Girls.

Gendered racism can impact how Black teen girls perceive themselves in relation to the world and influence their behaviors. This form of discrimination tends to manifest in stereotypes that promote the victimization and mistreatment of Black teen girls. This qualitative study, using Black feminist thought through a Black Girlhood lens as a guiding framework, aims to understand how Black teen girls are affected by gendered-racist stereotypes and how these stereotypes impact sexual decision making among this group. Using a sample of (N = 27) Black teen girls, three major themes arose: (1) experiencing the effects of racist-sexist stereotypes, (2) feeling powerless and invisible due to stereotypes, and (3) navigating the pressure to have sex due to stereotypes. Implications for this study include incorporating elements of Black Feminist Thought through a Black girlhood lens within prevention programing while also providing Black teen girls with the tools to challenge negative stereotypes with support from adult allies in school and family settings.

Equine cervical remodeling during placentitis and the prepartum period: a transcriptomic approach.

Cervical remodeling is a critical component in both term and preterm labor in eutherian mammals. However, the molecular mechanisms underlying cervical remodeling remain poorly understood in the mare. The current study compared the transcriptome of the equine cervix (cervical mucosa (CM) and stroma (CS)) during placentitis (placentitis group, n = 5) and normal prepartum mares (prepartum group, n = 3) to normal pregnant mares (control group, n = 4). Transcriptome analysis identified differentially expressed genes (DEGs) during placentitis (5310 in CM and 907 in CS) and during the normal prepartum period (189 in CM and 78 in CS). Our study revealed that cervical remodeling during placentitis was dominated by inflammatory signaling as reflected by the overrepresented toll-like receptor signaling, interleukin signaling, T cell activation, and B cell activation pathways. These pathways were accompanied by upregulation of several proteases, including matrix metalloproteinases (MMP1, MMP2, and MMP9), cathepsins (CTSB, CTSC, and CTSD) and a disintegrin and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1, ADAMTS4, and ADAMTS5), which are crucial for degradation of cervical collagens during remodeling. Cervical remodeling during placentitis was also associated with upregulation of water channel-related transcripts (AQP9 and RLN), angiogenesis-related transcripts (NOS3, ENG1, THBS1, and RAC2), and aggrecan (ACAN), a hydrophilic glucosaminoglycan, with subsequent cervical hydration. The normal prepartum cervix was associated with upregulation of ADAMTS1, ADAMTS4, NOS3 and THBS1, which might reflect an early stage of cervical remodeling taking place in preparation for labor. In conclusion, our findings revealed the possible key regulators and mechanisms underlying equine cervical remodeling during placentitis and the normal prepartum period.

Saliva pools for screening of human cytomegalovirus using real-time PCR.

Human cytomegalovirus (HCMV) is the leading congenital infection agent in the world. The importance of screening this infection has been debated, as 10-15% of the asymptomatic newborns with HCMV at birth will present late sequelae. The aim of this study was to test the feasibility of using saliva pools from newborns in a screening program for congenital HCMV infection, in two Portuguese hospitals. The screening was based on the use of pools of 10 saliva samples for detection of viral DNA by real-time PCR. Whenever there was a positive pool, the samples were tested individually, and for each positive sample the result was confirmed with a urine sample collected in the first 2 weeks of life. The study involved 1492 newborns. One hundred and fifty pools were screened, with 14 positive results in saliva, but only 10 were confirmed in urine samples, giving a prevalence of congenital HCMV infection in both hospitals of 0.67% (CI95% 0.36 to 1.23%).Conclusion: The overall prevalence of congenital HCMV infection in both hospitals was 0.67%. The use of saliva pools proved to be effective for the screening of this congenital infection, allowing timely screening and confirmation in a large population, with associated cost reduction. What is Known: • Newborn screening for HCMV is desirable. • Saliva is a good and practical sample. What is New: • The feasibility of using saliva pools for a large-scale screening. • The cost reduction of this strategy.

Effectiveness of a web-based tobacco product use prevention videogame intervention on young adolescents' beliefs and knowledge.

BACKGROUND: Preventing tobacco product initiation in youth is a critical need. While cigarette smoking among youth has been on the decline, tobacco use in other forms, such as e-cigarettes and vaping, continue to be a major concern. The purpose of this study was to conduct a real-world, quasi-experimental test of the effectiveness of a web-based videogame, smokeSCREEN, aimed at developing healthy beliefs and knowledge associated with tobacco product use prevention, including electronic cigarettes. Methods: Adolescents (N = 560) aged 10-16 years were enrolled from schools and afterschool programs in a single-group pre-post study. Measures included a pre- and post-survey of beliefs and knowledge about tobacco product use. At post-survey, participants were asked questions regarding their gameplay experience. Paired responses for the tobacco product use in the beliefs and knowledge survey before and after the smokeSCREEN videogame intervention were compared using McNemar's test. Descriptive statistics were generated to assess overall participant gameplay experience. Results: McNemar's test showed significant differences in the proportions of correct answers before (pre-survey) and after (post-survey) the intervention in seven out of eight belief questions (p < . 0001). It also suggested significant differences in the proportions of correct answers before (pre-survey) and after (post-survey) the intervention in all six knowledge questions (p < . 0001). Several gender and age differences were noted for belief and knowledge questions related to e-cigarettes and vaping. There was no association between gameplay duration at post-survey or to the answers of the beliefs or knowledge questions. Overall, participants reported that they enjoyed playing the game. Conclusions: Findings suggest that the videogame intervention, smokeSCREEN, has a promising effect on participants' beliefs and knowledge about tobacco product use, including electronic cigarettes and vaping, and is well accepted by adolescents.

Natural Multimerization Rules the Performance of Affinity-Based Physical Hydrogels for Stem Cell Encapsulation and Differentiation.

Tissue engineering and stem cell research greatly benefit from cell encapsulation within hydrogels as it promotes cell expansion and differentiation. Affinity-triggered hydrogels, an appealing solution for mild cell encapsulation, rely on selective interactions between the ligand and target and also on the multivalent presentation of these two components. Although these hydrogels represent a versatile option to generate dynamic, tunable, and highly functional materials, the design of hydrogel properties based on affinity and multivalency remains challenging and unstudied. Here, the avidin-biotin affinity pair, with the highest reported affinity constant, is used to address this challenge. It is demonstrated that the binding between the affinity hydrogel components is influenced by the multivalent display selected. In addition, the natural multivalency of the interaction must be obeyed to yield robust multicomponent synthetic protein hydrogels. The hydrogel's resistance to erosion depends on the right stoichiometric match between the hydrogel components. The developed affinity-triggered hydrogels are biocompatible and support encapsulation of induced pluripotent stem cells and their successful differentiation into a neural cell line. This principle can be generalized to other affinity pairs using multimeric proteins, yielding biomaterials with controlled performance.

Elevated blood urea nitrogen alters the transcriptome of equine embryos.

High blood urea nitrogen (BUN) in cows and ewes has a negative effect on embryo development; however, no comparable studies have been published in mares. The aims of the present study were to evaluate the effects of high BUN on blastocoele fluid, systemic progesterone and Day 14 equine embryos. When a follicle with a mean (±s.e.m.) diameter of 25±3mm was detected, mares were administered urea (0.4g kg-1) with sweet feed and molasses (n=9) or sweet feed and molasses alone (control; n=10). Blood samples were collected every other day. Mares were subjected to AI and the day ovulation was detected was designated as Day 0. Embryos were collected on Day 14 (urea-treated, n=5 embryos; control, n=7 embryos). There was an increase in systemic BUN in the urea-treated group compared with control (P<0.05), with no difference in progesterone concentrations. There were no differences between the two groups in embryo recovery or embryo size. Urea concentrations in the blastocoele fluid tended to be higher in the urea-treated mares, with a strong correlation with plasma BUN. However, there was no difference in the osmolality or pH of the blastocoele fluid between the two groups. Differentially expressed genes in Day 14 embryos from urea-treated mares analysed by RNA sequencing were involved in neurological development, urea transport, vascular remodelling and adhesion. In conclusion, oral urea treatment in mares increased BUN and induced transcriptome changes in Day 14 equine embryos of genes important in normal embryo development.

Small RNA (sRNA) expression in the chorioallantois, endometrium and serum of mares following experimental induction of placentitis.

Intrauterine infection and inflammation remain a major cause of preterm labour in women and mares, with little known about small RNA (sRNA) expression in tissue or circulation. To better characterise placental inflammation (placentitis), we examined sRNA expression in the endometrium, chorioallantois and serum of mares with and without placentitis. Disease was induced in 10 mares via intracervical inoculation of Streptococcus equi ssp. zooepidemicus, either with moderate or high levels of inoculum; three uninoculated gestationally matched mares were used as controls. Matched chorioallantois and endometrium were sampled in two locations: Region 1, gross inflammation near cervical star with placental separation and Region 2, gross inflammation without placental separation. In Region 1, 26 sRNAs were altered in chorioallantois, while 20 were altered in endometrium. Within Region 2, changes were more subdued in both chorioallantois (10 sRNAs) and endometrium (two sRNAs). Within serum, we identified nine significantly altered sRNAs. In summary, we have characterised the expression of sRNA in the chorioallantois, the endometrium and the serum of mares with experimentally induced placentitis using next-generation sequencing, identifying significant changes within each tissue examined. These data should provide valuable information about the physiology of placental inflammation to clinicians and researchers alike.

Are mule pregnancies really longer than equine pregnancies? Comparison between mule and equine pregnancies.

In equine management, it is important to predict the approximate foaling date of mares to monitor parturition and allow early identification and intervention of problems during the perinatal period. There are no studies comparing accurate gestational length (GL) when mares are carrying mule foals and no controlled comparisons between GL of mares pregnant with equine or mule foals. Therefore, the objectives of this study were to compare GL of mares pregnant with equine or mule foals and establish normal reference values for GL of mares pregnant with male and female mules. Gestational length of Mangalarga Paulista breed mares pregnant with equine (n = 54) or with mule (n = 54) foals during the breeding seasons of 2007 to 2016 was evaluated. The mean GL was 347.2 ± 1.4 days (range of 326-368 days) and 341.1 ± 1.6 days (range of 307-360 days) for equine and mule pregnancies, respectively. The normal GL reference for mule pregnancies was 316.9-365.3 days. Therefore, GL of equine pregnancies was longer than of mule pregnancies. Gestational length was not different when pregnancies resulted in females or males within each group. This study established an important reference value for normal GL of mule pregnancies, which can be used by practitioners to estimate and predict foaling dates more accurately.

Structural glycobiology of human α1-acid glycoprotein and its implications for pharmacokinetics and inflammation.

Human α1-acid glycoprotein (AGP) is an abundant human plasma glycoprotein that may be N-glycosylated at five positions. AGP plays important roles on pharmacokinetics and can rise up to 5-fold in inflammatory events. In such events, the glycan chains attached to Asn54, Asn75 and Asn85 may become fucosylated, originating a sialyl-Lewis X epitope. This epitope, in turn, can bind selectin proteins. Such interplay is important for immunomodulation. While the X-ray structure of unglycosylated AGP has been reported, the absence of the glycan chains hampered the further insights into its structural biology and, ultimately, into its biological function. Thus, the current work intends to contribute in the characterization of the structural glycobiology and function of AGP by building a structural model of its fully glycosylated form, taking into account the different glycoforms that are found in vivo. The obtained data points to the absence of a major influence of glycosylation on AGP's secondary structure, in agreement with crystallography observations. However, the glycan chains seem able to interfere with the protein dynamics, mainly at the AGP-ligand-binding site, indicating a possible role in its complexation to drugs and other bioactive compounds. By examining the influence of fucosylation on AGP structure and binding to selectins, it is proposed that the latter may bind to glycan chains linked to Asn54 and Asn75, and that this binding may involve other glycans, such as the one attached to Asn15. These results point to an increased participation of carbohydrates on the observed AGP roles in pharmacokinetics and inflammation.

Diversification and distinctive structural features of S-RNase alleles in the genus Solanum.

The multigenic and multiallelic S-locus in plants is responsible for the gametophytic self-incompatibility system, which is important to prevent the detrimental effects of self-fertilization and inbreeding depression. Several studies have discussed the importance of punctual mutations, recombination, and natural selection in the generation of allelic diversity in the S-locus. However, there has been no wide-ranging study correlating the molecular evolution and structural aspects of the corresponding proteins in Solanum. Therefore, we evaluated the molecular evolution of one gene in this locus and generated a statistically well-supported phylogenetic tree, as well as evidence of positive selection, helping us to understand the diversification of S alleles in Solanum. The three-dimensional structures of some of the proteins corresponding to the major clusters of the phylogenetic tree were constructed and subsequently submitted to molecular dynamics to stabilize the folding and obtain the native structure. The positively selected amino acid residues were predominantly located in the hyper variable regions and on the surface of the protein, which appears to be fundamental for allele specificity. One of the positively selected residues was identified adjacent to a conserved strand that is crucial for enzymatic catalysis. Additionally, we have shown significant differences in the electrostatic potential among the predicted molecular surfaces in S-RNases. The structural results indicate that local changes in the three-dimensional structure are present in some regions of the molecule, although the general structure seems to be conserved. No previous study has described such structural variations in S-RNases.

A tailor-made "tag-receptor" affinity pair for the purification of fusion proteins.

A novel affinity "tag-receptor" pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet-21c expression vector and expressed in E. coli host as soluble protein. A solid-phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96-well format for binding to the RKRKRK-tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK-tagged GFP with A7C1 emerged as a promising solution (Ka of 2.45×10(5) M(-1) ). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.

Identification of Synergistetes in endodontic infections.

The bacterial phylum Synergistetes consists of Gram-negative anaerobes. Oral Synergistetes are divided in two main clusters, namely A and B. Increasing evidence demonstrates their involvement in etiology of oral infections, including apical periodontitis. This condition causes bone loss around the apex of the tooth, subsequent to pulp inflammation (pulpitis). Although the presence of Synergistetes has been confirmed in endodontic infections by molecular methods, these have not been morphologically identified in the affected apical region, and their prevalence among different endodontic infections has not been determined. Therefore, the aim of this study was to evaluate the prevalence, levels and morphology of oral Synergistetes clusters A and B, in apical root canal samples obtained of teeth with irreversible pulpitis, pulp necrosis and apical periodontitis, or previously root-filled teeth with apical periodontitis. For their detection, fluorescence in situ hybridization and epifluorescence microscopy were used. Synergistetes cluster A was not detected in pulpitis, but was found in both apical periodontitis groups, more frequently and at higher ranges in teeth which were previously root-filled. Microscopically, they appeared as straight or slightly curved long rods. Synergistetes cluster B was not detected in any of the cases. Fusobacteria and Actinomyces, which are well-established taxa in endodontic infections, were detected more frequently and at higher ranges than Synergistetes. In conclusion, Synergistetes cluster A constitutes part of the mixed apical microbiota in apical periodontitis, and may be involved in its pathogenesis.