Induction of osteoblast apoptosis stimulates macrophage efferocytosis and paradoxical bone formation.

Apoptosis is crucial for tissue homeostasis and organ development. In bone, apoptosis is recognized to be a main fate of osteoblasts, yet the relevance of this process remains underexplored. Using our murine model with inducible Caspase 9, the enzyme that initiates intrinsic apoptosis, we triggered apoptosis in a proportion of mature osteocalcin (OCN+) osteoblasts and investigated the impact on postnatal bone development. Osteoblast apoptosis stimulated efferocytosis by osteal macrophages. A five-week stimulation of OCN+ osteoblast apoptosis in 3-week-old male and female mice significantly enhanced vertebral bone formation while increasing osteoblast precursors. A similar treatment regimen to stimulate osterix+ cell apoptosis had no impact on bone volume or density. The vertebral bone accrual following stimulation of OCN+ osteoblast apoptosis did not translate in improved mechanical strength due to disruption of the lacunocanalicular network. The observed bone phenotype was not influenced by changes in osteoclasts but was associated with stimulation of macrophage efferocytosis and vasculature formation. Phenotyping of efferocytic macrophages revealed a unique transcriptomic signature and expression of factors including VEGFA. To examine whether macrophages participated in the osteoblast precursor increase following osteoblast apoptosis, macrophage depletion models were employed. Depletion of macrophages via clodronate-liposomes and the CD169-diphtheria toxin receptor mouse model resulted in marked reduction in leptin receptor+ and osterix+ osteoblast precursors. Collectively, this work demonstrates the significance of osteoblast turnover via apoptosis and efferocytosis in postnatal bone formation. Importantly, it exposes the potential of targeting this mechanism to promote bone anabolism in the clinical setting.

Life stage-specific poly(A) site selection regulated by Trypanosoma brucei DRBD18.

The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process.

RESC14 and RESC8 cooperate to mediate RESC function and dynamics during trypanosome RNA editing.

Mitochondrial transcripts in Trypanosoma brucei require extensive uridine insertion/deletion RNA editing to generate translatable open reading frames. The RNA editing substrate binding complex (RESC) serves as the scaffold that coordinates the protein-protein and protein-RNA interactions during editing. RESC broadly contains two modules termed the guide RNA binding complex (GRBC) and the RNA editing mediator complex (REMC), as well as organizer proteins. How the protein and RNA components of RESC dynamically interact to facilitate editing is not well understood. Here, we examine the roles of organizer proteins, RESC8 and RESC14, in facilitating RESC dynamics. High-throughput sequencing of editing intermediates reveals an overlapping RESC8 and RESC14 function during editing progression across multiple transcripts. Blue native PAGE analysis demonstrates that RESC14 is essential for incorporation of RESC8 into a large RNA-containing complex, while RESC8 is important in recruiting a smaller ribonucleoprotein complex (RNP) to this large complex. Proximity labeling shows that RESC14 is important for stable RESC protein-protein interactions, as well as RESC-RECC associations. Together, our data support a model in which RESC14 is necessary for assembly of editing competent RESC through recruitment of an RNP containing RESC8, GRBC and gRNA to REMC and mRNA.

Estimating the Prevalence of Injection Drug Use Among Acute Hepatitis C Cases From a National Surveillance System: Application of Random Forest-Based Multiple Imputation.

BACKGROUND: Injection drug use (IDU) is a major contributor to the syndemic of viral hepatitis, human immunodeficiency virus, and drug overdose. However, information on IDU is frequently missing in national viral hepatitis surveillance data, which limits our understanding of the full extent of IDU-associated infections. Multiple imputation by chained equations (MICE) has become a popular approach to address missing data, but its application for IDU imputation is less studied. METHODS: Using the 2019-2021 National Notifiable Diseases Surveillance System acute hepatitis C case data and publicly available county-level measures, we evaluated listwise deletion (LD) and 3 models imputing missing IDU data through MICE: parametric logistic regression, semi-parametric predictive mean matching (PMM), and nonparametric random forest (RF) (both standard RF [sRF] and fast implementation of RF [fRF]). RESULTS: The estimated IDU prevalence among acute hepatitis C cases increased from 63.5% by LD to 65.1% by logistic regression, 66.9% by PMM, 76.0% by sRF, and 85.1% by fRF. Evaluation studies showed that RF-based MICE imputation, especially fRF, has the highest accuracy (as measured by smallest raw bias, percent bias, and root mean square error) and highest efficiency (as measured by smallest 95% confidence interval width) compared to LD and other models. Sensitivity analyses indicated that fRF remained robust when data were missing not at random. CONCLUSION: Our analysis suggested that RF-based MICE imputation, especially fRF, could be a valuable approach for addressing missing IDU data in the context of population-based surveillance systems like National Notifiable Diseases Surveillance System. The inclusion of imputed IDU data may enhance the effectiveness of future surveillance and prevention efforts for the IDU-driven syndemic.

Investigating phenotypic plasticity due to toxicants with exposure disparities in primary human breast cells in vitro.

Introduction: Breast cancer is the second most diagnosed cancer, as well as the primary cause of cancer death in women worldwide. Of the different breast cancer subtypes, triple-negative breast cancer (TNBC) is particularly aggressive and is associated with poor prognosis. Black women are two to three times more likely to be diagnosed with TNBCs than white women. Recent experimental evidence suggests that basal-like TNBCs may derive from luminal cells which acquire basal characteristics through phenotypic plasticity, a newly recognized hallmark of cancer. Whether chemical exposures can promote phenotypic plasticity in breast cells is poorly understood. Methods: To investigate further, we developed a high-content immunocytochemistry assay using normal human breast cells to test whether chemical exposures can impact luminal/basal plasticity by unbiased quantification of keratin 14 (KRT14), a basal-myoepithelial marker; keratin 8 (KRT8), a luminal-epithelial marker; and Hoechst 33342, a DNA marker. Six cell lines established from healthy tissue from donors to the Susan G. Komen Normal Tissue Bank were exposed for 48 hours to three different concentrations (0.1µM, 1µM, and 10µM) of eight ubiquitous chemicals (arsenic, BPA, BPS, cadmium, copper, DDE, lead, and PFNA), with documented exposure disparities in US Black women, in triplicate. Automated fluorescence image quantification was performed using Cell Profiler software, and a random-forest classifier was trained to classify individual cells as KRT8 positive, KRT14 positive, or hybrid (both KRT8 and KRT14 positive) using Cell Profiler Analyst. Results and discussion: Results demonstrated significant concentration-dependent increases in hybrid populations in response to BPA, BPS, DDE, and PFNA. The increase in hybrid populations expressing both KRT14 and KRT8 is indicative of a phenotypically plastic progenitor-like population in line with known theories of carcinogenesis. Furthermore, BPA, BPS, DDE, and copper produced significant increases in cell proliferation, which could be indicative of a more malignant phenotype. These results further elucidate the relationship between chemical exposure and breast phenotypic plasticity and highlight potential environmental factors that may impact TNBC risk.

Effects of Developmental Lead and Phthalate Exposures on DNA Methylation in Adult Mouse Blood, Brain, and Liver: A Focus on Genomic Imprinting by Tissue and Sex.

BACKGROUND: Maternal exposure to environmental chemicals can cause adverse health effects in offspring. Mounting evidence supports that these effects are influenced, at least in part, by epigenetic modifications. It is unknown whether epigenetic changes in surrogate tissues such as the blood are reflective of similar changes in target tissues such as cortex or liver. OBJECTIVE: We examined tissue- and sex-specific changes in DNA methylation (DNAm) associated with human-relevant lead (Pb) and di(2-ethylhexyl) phthalate (DEHP) exposure during perinatal development in cerebral cortex, blood, and liver. METHODS: Female mice were exposed to human relevant doses of either Pb (32 ppm) via drinking water or DEHP (5mg/kg-day) via chow for 2 weeks prior to mating through offspring weaning. Whole genome bisulfite sequencing (WGBS) was utilized to examine DNAm changes in offspring cortex, blood, and liver at 5 months of age. Metilene and methylSig were used to identify differentially methylated regions (DMRs). Annotatr and ChIP-enrich were used for genomic annotations and gene set enrichment tests of DMRs, respectively. RESULTS: The cortex contained the majority of DMRs associated with Pb (66%) and DEHP (57%) exposure. The cortex also contained the greatest degree of overlap in DMR signatures between sexes (n=13 and 8 DMRs with Pb and DEHP exposure, respectively) and exposure types (n=55 and 39 DMRs in males and females, respectively). In all tissues, detected DMRs were preferentially found at genomic regions associated with gene expression regulation (e.g., CpG islands and shores, 5' UTRs, promoters, and exons). An analysis of GO terms associated with DMR-containing genes identified imprinted genes to be impacted by both Pb and DEHP exposure. Of these, Gnas and Grb10 contained DMRs across tissues, sexes, and exposures, with some signatures replicated between target and surrogate tissues. DMRs were enriched in the imprinting control regions (ICRs) of Gnas and Grb10, and we again observed a replication of DMR signatures between blood and target tissues. Specifically, we observed hypermethylation of the Grb10 ICR in both blood and liver of Pb-exposed male animals. CONCLUSIONS: These data provide preliminary evidence that imprinted genes may be viable candidates in the search for epigenetic biomarkers of toxicant exposure in target tissues. Additional research is needed on allele- and developmental stage-specific effects, as well as whether other imprinted genes provide additional examples of this relationship. https://doi.org/10.1289/EHP14074.

Efferocytosis and Bone Dynamics.

PURPOSE OF REVIEW: This review summarizes the recently published scientific evidence regarding the role of efferocytosis in bone dynamics and skeletal health. RECENT FINDINGS: Several types of efferocytes have been identified within the skeleton, with macrophages being the most extensively studied. Efferocytosis is not merely a 'clean-up' process vital for maintaining skeletal homeostasis; it also plays a crucial role in promoting resolution pathways and orchestrating bone dynamics, such as osteoblast-osteoclast coupling during bone remodeling. Impaired efferocytosis has been associated with aging-related bone loss and various skeletal pathologies, including osteoporosis, osteoarthritis, rheumatoid arthritis, and metastatic bone diseases. Accordingly, emerging evidence suggests that targeting efferocytic mechanisms has the potential to alleviate these conditions. While efferocytosis remains underexplored in the skeleton, recent discoveries have shed light on its pivotal role in bone dynamics, with important implications for skeletal health and pathology. However, there are several knowledge gaps and persisting technical limitations that must be addressed to fully unveil the contributions of efferocytosis in bone.

Estimating hepatitis C prevalence in the United States, 2017-2020.

BACKGROUND AND AIMS: The National Health and Nutrition Examination Survey (NHANES) underestimates the true prevalence of HCV infection. By accounting for populations inadequately represented in NHANES, we created 2 models to estimate the national hepatitis C prevalence among US adults during 2017-2020. APPROACH AND RESULTS: The first approach (NHANES+) replicated previous methodology by supplementing hepatitis C prevalence estimates among the US noninstitutionalized civilian population with a literature review and meta-analysis of hepatitis C prevalence among populations not included in the NHANES sampling frame. In the second approach (persons who injected drugs [PWID] adjustment), we developed a model to account for the underrepresentation of PWID in NHANES by incorporating the estimated number of adult PWID in the United States and applying PWID-specific hepatitis C prevalence estimates. Using the NHANES+ model, we estimated HCV RNA prevalence of 1.0% (95% CI: 0.5%-1.4%) among US adults in 2017-2020, corresponding to 2,463,700 (95% CI: 1,321,700-3,629,400) current HCV infections. Using the PWID adjustment model, we estimated HCV RNA prevalence of 1.6% (95% CI: 0.9%-2.2%), corresponding to 4,043,200 (95% CI: 2,401,800-5,607,100) current HCV infections. CONCLUSIONS: Despite years of an effective cure, the estimated prevalence of hepatitis C in 2017-2020 remains unchanged from 2013 to 2016 when using a comparable methodology. When accounting for increased injection drug use, the estimated prevalence of hepatitis C is substantially higher than previously reported. National action is urgently needed to expand testing, increase access to treatment, and improve surveillance, especially among medically underserved populations, to support hepatitis C elimination goals.

Sex-Specific Deflection of Age-Related DNA Methylation and Gene Expression in Mouse Heart by Perinatal Toxicant Exposures.

Background: Global and site-specific changes in DNA methylation and gene expression are associated with cardiovascular aging and disease, but how toxicant exposures during early development influence the normal trajectory of these age-related molecular changes, and whether there are sex differences, has not yet been investigated. Objectives: We used an established mouse model of developmental exposures to investigate the effects of perinatal exposure to either lead (Pb) or diethylhexyl phthalate (DEHP), two ubiquitous environmental contaminants strongly associated with CVD, on age-related cardiac DNA methylation and gene expression. Methods: Dams were randomly assigned to receive human physiologically relevant levels of Pb (32 ppm in water), DEHP (25 mg/kg chow), or control water and chow. Exposures started two weeks prior to mating and continued until weaning at postnatal day 21 (3 weeks of age). Approximately one male and one female offspring per litter were followed to 3 weeks, 5 months, or 10 months of age, at which time whole hearts were collected (n ≥ 5 per sex per exposure). Enhanced reduced representation bisulfite sequencing (ERRBS) was used to assess the cardiac DNA methylome at 3 weeks and 10 months, and RNA-seq was conducted at all 3 time points. MethylSig and edgeR were used to identify age-related differentially methylated regions (DMRs) and differentially expressed genes (DEGs), respectively, within each sex and exposure group. Cell type deconvolution of bulk RNA-seq data was conducted using the MuSiC algorithm and publicly available single cell RNA-seq data. Results: Thousands of DMRs and hundreds of DEGs were identified in control, DEHP, and Pb-exposed hearts across time between 3 weeks and 10 months of age. A closer look at the genes and pathways showing differential DNA methylation revealed that the majority were unique to each sex and exposure group. Overall, pathways governing development and differentiation were most frequently altered with age in all conditions. A small number of genes in each group showed significant changes in DNA methylation and gene expression with age, including several that were altered by both toxicants but were unchanged in control. We also observed subtle, but significant changes in the proportion of several cell types due to age, sex, and developmental exposure. Discussion: Together these data show that perinatal Pb or DEHP exposures deflect normal age-related gene expression, DNA methylation programs, and cellular composition across the life course, long after cessation of exposure, and highlight potential biomarkers of developmental toxicant exposures. Further studies are needed to investigate how these epigenetic and transcriptional changes impact cardiovascular health across the life course.

PAPP-A as a Potential Target in Thyroid Eye Disease.

CONTEXT: Proptosis in Thyroid Eye Disease (TED) can result in facial disfigurement and visual dysfunction. Treatment with Insulin-like growth factor I receptor (IGF-IR) inhibitors has been shown to be effective in reducing proptosis but with side effects. OBJECTIVE: To test the hypothesis that inhibition of IGF-IR indirectly and more selectively with PAPP-A inhibitors attenuates IGF-IR signaling in TED. DESIGN: Informed consent was obtained from TED patients undergoing surgery, and retro-orbital tissue collected for fibroblast isolation and culture. SETTING: Surgeries were performed in Mayo Clinic operating suites. Cell culture was performed in a sterile tissue culture facility. PATIENT SAMPLES: Retro-orbital tissue was collected from 19 TED patients. INTERVENTIONS: Treatment of TED fibroblasts with pro-inflammatory cytokines. Flow separation of CD34- and CD34+ orbital fibroblasts, the latter representing infiltrating fibrocytes into the orbit in TED. MAIN OUTCOME MEASURES: PAPP-A expression and proteolytic activity, IGF-I stimulation of phosphatidylinositol 3 kinase/Akt pathway and inhibition by immuno-neutralizing antibodies against PAPP-A, CD34+ status and associated PAPP-A and IGF-IR expression. RESULTS: Pro-inflammatory cytokines markedly increased PAPP-A expression in TED fibroblasts. IGF-IR expression was not affected by cytokine treatment. Inhibition of PAPP-A's proteolytic activity suppressed IGF-IR activation in orbital fibroblasts from TED patients. TED fibroblasts that were CD34+ represented ∼80% of the cells in culture and accounted for ∼70% of PAPP-A and IGF-IR expressing cells. CONCLUSIONS: These results support a role for PAPP-A in TED pathogenesis and indicate the potential for novel therapeutic targeting of the IGF axis.

A role for a Trypanosoma brucei cytosine RNA methyltransferase homolog in ribosomal RNA processing.

In Trypanosoma brucei, gene expression is primarily regulated posttranscriptionally making RNA metabolism critical. T. brucei has an epitranscriptome containing modified RNA bases. Yet, the identity of the enzymes catalyzing modified RNA base addition and the functions of the enzymes and modifications remain unclear. Homology searches indicate the presence of numerous T. brucei cytosine RNA methyltransferase homologs. One such homolog, TbNop2 was studied in detail. TbNop2 contains the six highly conserved motifs found in cytosine RNA methyltransferases and is evolutionarily related to the Nop2 protein family required for rRNA modification and processing. RNAi experiments targeting TbNop2 resulted in reduced levels of TbNop2 RNA and protein, and a cessation of parasite growth. Next generation sequencing of bisulfite-treated RNA (BS-seq) detected the presence of two methylation sites in the large rRNA; yet TbNop2 RNAi did not result in a significant reduction of methylation. However, TbNop2 RNAi resulted in the retention of 28S internal transcribed spacer RNAs, indicating a role for TbNop2 in rRNA processing.

High rates of International Code violations: a cross-sectional study in a region of Canada with low breastfeeding rates.

BACKGROUND: Exposure to marketing and promotion of commercial milk formula is associated with an increased likelihood of formula-feeding. In 1981, the International Code (IC) of Marketing of Breastmilk Substitutes was adopted by the 34th World Health Assembly to restrict the promotion, marketing and advertising of commercial milk formula and protect breastfeeding. RESEARCH AIM: The current study examines mothers' exposure to violations of the IC in Newfoundland and Labrador, a province of Canada with low breastfeeding rates. METHODS: A cross-sectional online survey measured exposure to IC violations (e.g., marketing, advertising and promotion of commercial milk formula) by mothers of infants less than two years old (n = 119). Data were collected on type, frequency, and location of violation. RESULTS: Most participants (87%, n = 104/119) reported exposure to at least one IC violation. Of this group (n = 104): 94% received coupons or discount codes for the purchase of commercial milk formula; 88% received free samples of commercial milk formula from manufacturers, and 79% were contacted directly by commercial milk formula companies via email, text message, mail or phone for advertising purposes. One-third (n = 28/104, 27%) observed commercial milk formula promotional materials in health care facilities. The most frequent locations were violations occurred were doctors' offices (79%), supermarkets(75%), and pharmacies (71%). CONCLUSION: The majority of mothers of young infants were exposed to violations of the IC involving the marketing, advertising and promotion of commercial milk formula. Companies producing commercial milk formula reached out directly to new mothers to offer unsolicited promotions and free samples of commercial milk formula.

A cross-sectional study of breastfed infants referred for tongue tie assessment and frenotomy in one Canadian health region.

Importance: Tongue tie (TT) is a condition that can cause infant feeding difficulties due to restricted tongue movement. When TT presents as a significant barrier to breastfeeding, a frenotomy may be recommended. Universally accepted diagnostic criteria for TT are lacking and wide prevalence estimates are reported. New referral processes and a Frenotomy Assessment Tool were implemented in one Canadian health region to connect breastfeeding dyads with a provider for TT evaluation and frenotomy. Objective: To determine the proportion of babies with TT as well as the frequency of frenotomy. Methods: This cross-sectional study included infants who initiated breastfeeding at birth and were referred for TT evaluation over a 14-month period. Data were collected retrospectively by chart review and analyzed using SPSS. Factors associated with frenotomy were examined using logistic regression. Results: Two hundred and forty-one babies were referred. Ninety-two percent (n = 222) were diagnosed with TT and 66.0% (n = 159) underwent frenotomy. In the multivariate model, nipple pain/trauma, inability to latch, inability to elevate tongue, and dimpling of tongue on extension were associated with frenotomy (P < 0.05). Most referrals in our region resulted in a diagnosis of TT; however, the number of referrals was lower than expected, and of these two-thirds underwent frenotomy. Interpretation: TT is a relatively common finding among breastfed infants. Future research should examine whether a simplified assessment tool containing the four items associated with frenotomy in our multivariate model can identify breastfed infants with TT who require frenotomy.

Translational toxicoepigenetic Meta-Analyses identify homologous gene DNA methylation reprogramming following developmental phthalate and lead exposure in mouse and human offspring.

Although toxicology uses animal models to represent real-world human health scenarios, a critical translational gap between laboratory-based studies and epidemiology remains. In this study, we aimed to understand the toxicoepigenetic effects on DNA methylation after developmental exposure to two common toxicants, the phthalate di(2-ethylhexyl) phthalate (DEHP) and the metal lead (Pb), using a translational paradigm that selected candidate genes from a mouse study and assessed them in four human birth cohorts. Data from mouse offspring developmentally exposed to DEHP, Pb, or control were used to identify genes with sex-specific sites with differential DNA methylation at postnatal day 21. Associations of human infant DNA methylation in homologous mouse genes with prenatal DEHP or Pb were examined with a meta-analysis. Differential methylation was observed on 6 cytosines (adjusted-p < 0.05) and 90 regions (adjusted-p < 0.001). This translational approach offers a unique method that can detect conserved epigenetic differences that are developmentally susceptible to environmental toxicants.

A skeleton in a huff: insights in etiologies of osteosclerosis.

A 30-yr-old man developed right lower leg pain and a palpable solid mass. Radiographic imaging revealed a periosteal reaction with an exostotic mass arising from the right distal fibula. Generalized skeletal osteosclerosis with periosteal reaction was discovered on a radiographic skeletal survey. A biopsy of the right fibular mass revealed reactive woven bone. The patient was referred to a metabolic bone disease clinic, where laboratory values were consistent with secondary hyperparathyroidism and increased bone turnover. A DXA bone density scan revealed high bone density, with an L1-4 spine Z-score of +9.3, a left femoral neck Z-score of +8.5, and a total hip Z-score of +6.5. A dental exam revealed generalized gingival inflammation, teeth mobility, generalized horizontal alveolar bone loss and widening of the periodontal ligament space, increased bone density around the teeth, and thickening of the radicular lamina dura. An extensive evaluation was performed, with the result of a single test revealing the diagnosis. The differential diagnoses of osteosclerosis affecting the skeleton, teeth, and oral cavity are discussed.

A 30-yr-old man developed, over a short period, pain in his lower right leg accompanied by a hard mass. He also reported weight loss and night sweats for the past 6 months. After evaluation by his primary physician, an X-ray was ordered that reported a bony mass arising from the right fibula bone. A biopsy was performed of the mass, but no evidence of cancer or any other specific abnormality was found. The patient was then referred to a bone disease specialty clinic. Laboratory tests revealed a large increase in how quickly the patient's skeleton was remodeling, affecting the balance of bone formation and removal involved in maintaining a healthy skeleton. A bone density scan reported that the patient had very dense bones. Other unusual changes were also discovered in a dental exam, suggesting bone thickening. After an extensive evaluation, a single blood test revealed the cause of the fibular bone mass and dense bones.

The Lighter Touch: Less-Restriction in Sequentially Implemented Behavioral Sleep Interventions for Children with Rare Genetic Neurodevelopmental Conditions.

PURPOSE: The prevalence of sleep difficulties among children with rare genetic neurodevelopmental conditions (RGNC) is high. Behavioral interventions are commonly used in the treatment of sleep difficulties in children with neurodevelopmental conditions such as autism, however, research is scarce in children with RGNC. The range of co-occurring complexities within this population, means there is a need for research to not only determine the effectiveness of behavioral sleep interventions, but also which components might be the least restrictive (i.e., intensive/aversive) and minimally sufficient. METHODS: This study used a single-case multiple baseline design to investigate the effectiveness and acceptability of behavioral sleep interventions, indicated within a Functional Behavior formulation in eight children with RGNC (M = 7.3 years). Intervention components were sequentially administered across up to three phases, based on the principle of less restriction (from least to relatively more intensive) to determine what might be minimally sufficient. RESULTS: Results showed an improvement in sleep onset latency, night wakings, early morning waking and unwanted bed-sharing for 7/7, 6/7, 3/3 and 3/3 children respectively. Improvement was observed for most participants following the less restrictive phases of intervention (circadian modifications, antecedent modifications and positive reinforcement), however, more restrictive, albeit modified, extinction procedures were still implemented for five participants. Improvements were maintained at follow-up and interventions were deemed acceptable to parents. CONCLUSIONS: Less restrictive function-based behavioral strategies are an effective, and in some cases sufficient, contribution to a sequence of interventions for a range of sleep difficulties. They should be implemented first, before more restrictive strategies.

piOxi database: a web resource of germline and somatic tissue piRNAs identified by chemical oxidation.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that are highly expressed and extensively studied from the germline. piRNAs associate with PIWI proteins to maintain DNA methylation for transposon silencing and transcriptional gene regulation for genomic stability. Mature germline piRNAs have distinct characteristics including a 24- to 32-nucleotide length and a 2'-O-methylation signature at the 3' end. Although recent studies have identified piRNAs in somatic tissues, they remain poorly characterized. For example, we recently demonstrated notable expression of piRNA in the murine soma, and while overall expression was lower than that of the germline, unique characteristics suggested tissue-specific functions of this class. While currently available databases commonly use length and association with PIWI proteins to identify piRNA, few have included a chemical oxidation method that detects piRNA based on its 3' modification. This method leads to reproducible and rigorous data processing when coupled with next-generation sequencing and bioinformatics analysis. Here, we introduce piOxi DB, a user-friendly web resource that provides a comprehensive analysis of piRNA, generated exclusively through sodium periodate treatment of small RNA. The current version of piOxi DB includes 435 749 germline and 9828 somatic piRNA sequences robustly identified from M. musculus, M. fascicularis and H. sapiens. The database provides species- and tissue-specific data that are further analyzed according to chromosome location and correspondence to gene and repetitive elements. piOxi DB is an informative tool to assist broad research applications in the fields of RNA biology, cancer biology, environmental toxicology and beyond. Database URL:  https://pioxidb.dcmb.med.umich.edu/.

The effects of preosteoblast-derived exosomes on macrophages and bone in mice.

The effect of preosteoblast-derived exosomes on bone marrow macrophages (BMMΦ) and calvarial osteoblasts (cOB) was evaluated in vitro, and bone formation studies were performed in vivo in mice. Preosteoblastic MC3T3-E1 clone 4 (MC4) cell-derived exosomes (MC4exo) were characterized with particle tracking, transmission electron microscopy and western blot analysis to validate size, number, shape and phenotypic exosome markers. Exosomes pre-labelled with PKH67 were incubated with BMMΦ and phagocytosis of exosomes was confirmed. To examine the effect of MC4exo on macrophage polarization, BMMΦ were treated with MC4exo and the expression of pro- and anti-inflammatory cytokines was determined by qPCR. MC4exo treatment upregulated mRNA expression of Cd86, Il1ß, Ccl2, Rankl and Nos, and downregulated Cd206, Il10 and Tnfα, suggesting a shift towards pro-inflammatory 'M1-like' macrophage polarization. Combination of RANKL and MC4exo increased osteoclast differentiation of BMMΦ in comparison to RANKL alone as analysed by TRAP staining. MC4exo treatment showed no significant effect on calvarial osteoblast mineralization. For in vivo studies, intratibial inoculation of MC4exo (2 × 109 particles in PBS, n = 12) and vehicle control (PBS only, n = 12) was performed in C57Bl/6 mice (8 weeks, male). Micro-CT analyses of the trabecular and cortical bone compartments were assessed at 4 weeks post-injection. Tibial sections were stained for TRAP activity to determine osteoclast presence and immunofluorescence staining was performed to detect osteocalcin (Ocn), osterix (Osx) and F4/80 expression. Intratibial inoculation of MC4exo increased the diaphyseal bone mineral density and trabecular bone volume fraction due to increased trabecular number. This increase in bone was accompanied by a reduction in bone marrow macrophages and osteoclasts at the experimental endpoint. Together, these findings suggest that preosteoblast-derived exosomes enhanced bone formation by influencing macrophage responses.

Parathyroid hormone and trabectedin have differing effects on macrophages and stress fracture repair.

Stress fractures occur as a result of repeated mechanical stress on bone and are commonly found in the load-bearing lower extremities. Macrophages are key players in the immune system and play an important role in bone remodeling and fracture healing. However, the role of macrophages in stress fractures has not been adequately addressed. We hypothesize that macrophage infiltration into a stress fracture callus site promotes bone healing. To test this, a unilateral stress fracture induction model was employed in which the murine ulna of four-month-old, C57BL/6 J male mice was repeatedly loaded with a pre-determined force until the bone was displaced a distance below the threshold for complete fracture. Mice were treated daily with parathyroid hormone (PTH, 50 µg/kg/day) starting two days before injury and continued until 24 h before euthanasia either four or six days after injury, or treated with trabectedin (0.15 mg/kg) on the day of stress fracture and euthanized three or seven days after injury. These treatments were used due to their established effects on macrophages. While macrophages have been implicated in the anabolic effects of PTH, trabectedin, an FDA approved chemotherapeutic, compromises macrophage function and reduces bone mass. At three- and four-days post injury, callus macrophage numbers were analyzed histologically. There was a significant increase in macrophages with PTH treatment compared to vehicle in the callus site. By one week of healing, treatments differentially affected the bony callus as analyzed by microcomputed tomography. PTH enhanced callus bone volume. Conversely, callus bone volume was decreased with trabectedin treatment. Interestingly, concurrent treatment with PTH and trabectedin rescued the reduction observed in the callus with trabectedin treatment alone. This study reports on the key involvement of macrophages during stress fracture healing. Given these observed outcomes on macrophage physiology and bone healing, these findings may be important for patients actively receiving either of these FDA-approved therapeutics.

Exploiting Differences in Heme Biosynthesis between Bacterial Species to Screen for Novel Antimicrobials.

The final three steps of heme biogenesis exhibit notable differences between di- and mono-derm bacteria. The former employs the protoporphyrin-dependent (PPD) pathway, while the latter utilizes the more recently uncovered coproporphyrin-dependent (CPD) pathway. In order to devise a rapid screen for potential inhibitors that differentiate the two pathways, the genes associated with the protoporphyrin pathway in an Escherichia coli YFP strain were replaced with those for the CPD pathway from Staphylococcus aureus (SA) through a sliding modular gene replacement recombineering strategy to generate the E. coli strain Sa-CPD-YFP. Potential inhibitors that differentially target the pathways were identified by screening compound libraries against the YFP-producing Sa-CPD-YFP strain in comparison to a CFP-producing E. coli strain. Using a mixed strain assay, inhibitors targeting either the CPD or PPD heme pathways were identified through a decrease in one fluorescent signal but not the other. An initial screen identified both azole and prodigiosin-derived compounds that were shown to specifically target the CPD pathway and which led to the accumulation of coproheme, indicating that the main target of inhibition would appear to be the coproheme decarboxylase (ChdC) enzyme. In silico modeling highlighted that these inhibitors are able to bind within the active site of ChdC.