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The high frequency of aberrant PI3K pathway activation in hormone receptor–positive (HR+) breast cancer has led to the development, clinical testing, and approval of the p110α-selective PI3K inhibitor alpelisib. The limited clinical efficacy of alpelisib and other PI3K inhibitors is partially attributed to the functional antagonism between PI3K and estrogen receptor (ER) signaling, which is mitigated via combined PI3K inhibition and endocrine therapy. We and others have previously demonstrated chromatin-associated mechanisms by which PI3K supports cancer development and antagonizes ER signaling through the modulation of the H3K4 methylation axis, inhibition of KDM5A promoter H3K4 demethylation and KMT2D/MLL4-directed enhancer H3K4 methylation. Here we show that inhibition of the H3K4 histone methyltransferase MLL1 in combination with PI3K inhibition impairs HR+ breast cancer clonogenicity and cell proliferation. While combined PI3K/MLL1 inhibition reduces PI3K/AKT signaling and H3K4 methylation, MLL1 inhibition increases PI3K/AKT signaling through the dysregulation of gene expression associated with AKT activation. These data reveal a feedback loop between MLL1 and AKT whereby MLL1 inhibition reactivates AKT. We show that combined PI3K and MLL1 inhibition synergizes to cause cell death in in vitro and in vivo models of HR+ breast cancer, which is enhanced by the additional genetic ablation of the H3K4 methyltransferase and AKT target KMT2D/MLL4. Together, our data provide evidence of a feedback mechanism connecting histone methylation with AKT and may support the preclinical development and testing of pan-MLL inhibitors. Significance: Here the authors leverage PI3K/AKT-driven chromatin modification to identify histone methyltransferases as a therapeutic target. Dual PI3K and MLL inhibition synergize to reduce clonogenicity and cell proliferation, and promote in vivo tumor regression. These findings suggest patients with PIK3CA-mutant, HR+ breast cancer may derive clinical benefit from combined PI3K/MLL inhibition.

Vaginal reconstruction with autologous buccal mucosa graft offers a promising alternative to the use of skin grafts and vascularized intestinal segments. Given the novelty of this procedure, the optimal approach to postoperative wound management remains unclear with current practices often requiring many months of vaginal stents/molds. This study aims to evaluate a newly developed negative pressure intravaginal wound vacuum placed at the conclusion of the vaginoplasty with the goals of facilitating graft take and healing.A retrospective review of patients (age 12-21 years) who underwent eight primary and secondary vaginoplasty procedures using autologous buccal mucosa coupled with intravaginal wound vacuum placement was performed.Vaginal reconstruction with fenestrated full-thickness buccal mucosa graft and intravaginal wound vacuum placement was successfully performed eight times in seven patients at a median age of 15.6 years. Four patients underwent robotic vaginal pull-through with buccal mucosa serving as an interposition graft, and four patients underwent vaginoplasty with buccal graft alone. All cases had excellent engraftment at time of wound vacuum removal on postoperative day seven and had healthy-appearing buccal mucosa at a mean follow-up of 148 days. Postoperatively, one patient developed a stricture at the anastomosis between native vagina and buccal mucosa graft, requiring a second buccal mucosa graft six months after the first operation.The use of autologous buccal mucosa graft for primary and secondary vaginal reconstruction coupled with intravaginal wound vacuum therapy offers a promising new approach. Negative pressure wound vacuum therapy may provide a more optimal wound healing environment for improved outcomes.Retrospective Study LEVELS OF EVIDENCE: Level IV.

Figure S2 shows on-target activity of PI3K and MLL1 inhibitors

Supplementary Table 1 shows p values for IHC staining that corresponds to the graphs shown in Figure 5D

The high frequency of aberrant PI3K pathway activation in hormone receptor–positive (HR+) breast cancer has led to the development, clinical testing, and approval of the p110α-selective PI3K inhibitor alpelisib. The limited clinical efficacy of alpelisib and other PI3K inhibitors is partially attributed to the functional antagonism between PI3K and estrogen receptor (ER) signaling, which is mitigated via combined PI3K inhibition and endocrine therapy. We and others have previously demonstrated chromatin-associated mechanisms by which PI3K supports cancer development and antagonizes ER signaling through the modulation of the H3K4 methylation axis, inhibition of KDM5A promoter H3K4 demethylation and KMT2D/MLL4-directed enhancer H3K4 methylation. Here we show that inhibition of the H3K4 histone methyltransferase MLL1 in combination with PI3K inhibition impairs HR+ breast cancer clonogenicity and cell proliferation. While combined PI3K/MLL1 inhibition reduces PI3K/AKT signaling and H3K4 methylation, MLL1 inhibition increases PI3K/AKT signaling through the dysregulation of gene expression associated with AKT activation. These data reveal a feedback loop between MLL1 and AKT whereby MLL1 inhibition reactivates AKT. We show that combined PI3K and MLL1 inhibition synergizes to cause cell death in in vitro and in vivo models of HR+ breast cancer, which is enhanced by the additional genetic ablation of the H3K4 methyltransferase and AKT target KMT2D/MLL4. Together, our data provide evidence of a feedback mechanism connecting histone methylation with AKT and may support the preclinical development and testing of pan-MLL inhibitors.Significance:Here the authors leverage PI3K/AKT-driven chromatin modification to identify histone methyltransferases as a therapeutic target. Dual PI3K and MLL inhibition synergize to reduce clonogenicity and cell proliferation, and promote in vivo tumor regression. These findings suggest patients with PIK3CA-mutant, HR+ breast cancer may derive clinical benefit from combined PI3K/MLL inhibition.

Figure S2 shows Iba-1 staining in CD47 WT and CD47 KO xenografts.

Table S1: Primers.

Glioblastomas (GBM) are highly infiltrated by myeloid-derived innate immune cells that contribute to the immunosuppressive nature of the brain tumor microenvironment (TME). CD47 has been shown to mediate immune evasion, as the CD47–SIRPα axis prevents phagocytosis of tumor cells by macrophages and other myeloid cells. In this study, we established CD47 homozygous deletion (CD47−/−) in human and mouse GBM cells and investigated the impact of eliminating the "don't eat me" signal on tumor growth and tumor–TME interactions. CD47 knockout (KO) did not significantly alter tumor cell proliferation in vitro but significantly increased phagocytosis of tumor cells by macrophages in cocultures. Compared with CD47 wild-type xenografts, orthotopic xenografts derived from CD47−/− tumor cells grew significantly slower with enhanced tumor cell phagocytosis and increased recruitment of M2-like tumor-associated microglia/macrophages (TAM). CD47 KO increased tumor-associated extracellular matrix protein tenascin C (TNC) in xenografts, which was further examined in vitro. CD47 loss of function upregulated TNC expression in tumor cells via a Notch pathway–mediated mechanism. Depletion of TNC in tumor cells enhanced the growth of CD47−/− xenografts in vivo and decreased the number of TAM. TNC knockdown also inhibited phagocytosis of CD47−/− tumor cells in cocultures. Furthermore, TNC stimulated release of proinflammatory factors including TNFα via a Toll-like receptor 4 and STAT3-dependent mechanism in human macrophage cells. These results reveal a vital role for TNC in immunomodulation in brain tumor biology and demonstrate the prominence of the TME extracellular matrix in affecting the antitumor function of brain innate immune cells.Significance:These findings link TNC to CD47-driven phagocytosis and demonstrate that TNC affects the antitumor function of brain TAM, facilitating the development of novel innate immune system–based therapies for brain tumors.

Supplementary Figures 1-6, Methods and Materials from CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region

Children with vesicoureteral reflux (VUR) are at an increased risk of recurrent urinary tract infections (UTIs) and renal scarring. Gut microbiota are associated with disease phenotypes, but there has been no study that associates urinary microbiota (uMB) and metabolic profiles with VUR pathology. To identify dominant uMB genera and metabolites associated with UTIs in VUR, urine samples collected under sterile conditions underwent 16S ribosomal RNA sequencing (n = 49) and metabolomic analysis by mass spectrometry (n = 96). Alterations in uMB and metabolomic profiles in VUR patients suggest remodeling of urinary bacterial communities after UTIs: Dorea- and Escherichia-dominant uMB profiles were more frequently identified in participants with VUR. Prevotella- and Lactobacillus-dominant uMB profiles were more prevalent in controls (p 0.001). Microbial composition varied based on recurrent febrile UTI status (p = 0.001). A total of 243 urinary metabolites involved in energy, amino acid, nucleotide, and lipid metabolism were altered in VUR patients with UTIs (p 0.05). Importantly, VUR specimens revealed changes in the bacteria-associated metabolic pathways such as glutamate degradation, methyl-citrate cycle, and bile acid metabolism. PATIENT SUMMARY: Differences in urinary commensal bacteria and metabolites exist between children with and without vesicoureteral reflux (VUR). These changes may be utilized to identify patients at risk of VUR-associated kidney damage.

To examine the prenatal diagnosis rates of bladder exstrophy (BE) and Omphalocele-Exstrophy-Imperforate anus-Spinal Defect Syndrome (OEIS) in a large cohort of patients over a 20-year period. We hypothesized that prenatal diagnosis rates improved over time due to evolving techniques in fetal imaging.A multi-institutional database was queried to identify BE or OEIS patients who underwent primary closure between 2000 and 2020. We retrospectively determined prenatally or postnatal diagnosis. Those with unknown prenatal history were excluded. Multivariable logistic regression was used to investigate temporal pattern in rate of prenatal diagnosis while adjusting for sex and treating institution.Among 197 BE and 52 OEIS patients, 155 BE and 45 OEIS patients had known prenatal history. Overall prenatal diagnosis rates of BE and OEIS were 47.1% (73/155) and 82.2% (37/45), respectively. Prenatal diagnosis rate was significantly lower in BE versus OEIS (p0.0001). The prenatal diagnosis rate for BE significantly increased over time (OR 1.10; [95%CI: 1.03-1.17]; p=0.003). Between 2000 and 2005, the prenatal diagnosis rate of BE was 30.3% (10/33). Between 2015 and 2020, prenatal diagnosis rate of BE was 61.1% (33/54). Prenatal diagnosis rate for OEIS did not change over time. Rates of prenatal diagnosis did not differ by sex or treating institution.Rates of prenatal diagnosis of BE and OEIS are higher than previously reported. Prenatal diagnosis rate of BE doubled in the last 5 years compared to the first 5 years of the study period. Nonetheless, a significant proportion of both BE and OEIS patients remain undiagnosed prior to delivery.

ContextChronic exposure to glucocorticoids (GCs) or stress increases the risk of medical disorders, including cardiovascular and neuropsychiatric disorders. GCs contribute to accelerated aging; however, while the link between chronic GC exposure and disease onset is well established, the underpinning mechanisms are not clear.ObjectiveWe explored the potential nexus between GCs or stress exposure and telomere length.MethodsIn addition to rats exposed to 3 weeks of chronic stress, an iatrogenic mouse model of Cushing syndrome (CS), and a mouse neuronal cell line, we studied 32 patients with CS and age-matched controls and another cohort of 75 healthy humans.Results(1) Exposure to stress in rats was associated with a 54.5% (P = 0.036) reduction in telomere length in T cells. Genomic DNA (gDNA) extracted from the dentate gyrus of stressed and unstressed rats showed 43.2% reduction in telomere length (P = 0.006). (2) Mice exposed to corticosterone had a 61.4% reduction in telomere length in blood gDNA (P = 5.75 × 10-5) and 58.8% reduction in telomere length in the dentate gyrus (P = 0.002). (3) We observed a 40.8% reduction in the telomere length in patients with active CS compared to healthy controls (P = 0.006). There was a 17.8% reduction in telomere length in cured CS patients, which was not different from that of healthy controls (P = 0.08). For both cured and active CS, telomere length correlated significantly with duration of hypercortisolism (R2 = 0.22, P = 0.007). (4) There was a 27.6% reduction in telomere length between low and high tertiles in bedtime cortisol levels of healthy participants (P = 0.019).ConclusionOur findings demonstrate that exposure to stress and/or GCs is associated with shortened telomeres, which may be partially reversible.

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.

The glycoprotein uromodulin (UMOD) is the most abundant protein in urine, and N-glycans are critical for many biological functions of UMOD. Comprehensive glycan profiling of UMOD provides valuable information to understand the exact mechanisms of glycan-regulated functions. To perform comprehensive glycosylation analysis of UMOD from urine samples with limited volumes, we developed a streamlined workflow that included UMOD isolation from 5 mL of urine from 6 healthy adult donors (3 males and 3 females) and a glycosylation analysis using a highly sensitive and reproducible nanoLC-MS/MS based glycomics approach. In total, 212 N-glycan compositions were identified from the purified UMOD, and 17% were high-mannose glycans, 2% were afucosylated/asialylated, 3% were neutral fucosylated, 28% were sialylated (with no fucose), 46% were fucosylated and sialylated, and 4% were sulfated. We found that isolation of UMOD resulted in a significant decrease in the relative quantity of high-mannose and sulfated glycans with a significant increase of neutral fucosylated glycans in the UMOD-depleted urine relative to the undepleted urine, but depletion had little impact on the sialylated glycans. To our knowledge, this is the first study to perform comprehensive N-glycan profiling of UMOD using nanoLC-MS/MS. This analytical workflow would be very beneficial for studies with limited sample size, such as pediatric studies, and can be applied to larger patient cohorts not only for UMOD interrogation but also for global glycan analysis.

Importance Extracellular matrix proteins and enzymes involved in degradation have been found to be associated with tissue fibrosis and ureteropelvic junction obstruction (UPJO). In this study we developed a promising urinary biomarker model which can identify reduced renal function in UPJ obstruction patients. This can potentially serve as a non-invasive way to enhance surgical decision making for patients and urologists. Objective We sought to develop a predictive model to identify UPJO patients at risk for reduced renal function. Design Prospective cohort study Setting Pre-operative urine samples were collected in a prospectively enrolled UPJO biomarker registry at our institution. Urinary MMP-2, MMP-7, TIMP-2, and NGAL were measured as well as clinical characteristics including hydronephrosis grade, differential renal function, t1/2, and UPJO etiology. Participants Children who underwent pyeloplasty for UPJO Main outcome measurement Primary outcome was reduced renal function defined as MAG3 function Results We included 71 patients with UPJO in the original training cohort and 39 in the validation cohort. Median age was 3.3 years (70% male). By univariate analysis, reduced renal function was associated with higher MMP-2 (p = 0.064), MMP-7 (p = 0.047), NGAL (p = 0.001), and lower TIMP-2 (p = 0.033). Combining MMP-7 with TIMP-2, the multivariable logistic regression model predicted reduced renal function with good performance (AUC = 0.830; 95% CI: 0.722–0.938). The independent testing dataset validated the results with good predictive performance (AUC = 0.738). Conclusions and relevance Combination of urinary MMP-7 and TIMP-2 can identify reduced renal function in UPJO patients. With the high sensitivity cutoffs, patients can be categorized into high risk (aggressive management) versus lower risk (observation).

Children born to women who experience stress during pregnancy have an increased risk of atherosclerosis in later life, but few animal models have explored mechanisms. To study this phenomenon, timed-bred ApoE knockout mice were determined pregnant with ultrasound and randomly assigned on gestation day 8.5 to either a control (no stress) or prenatal stress (PS) group using 2 h of restraint for five consecutive days. PS significantly increased plasma corticosterone levels in pregnant mice. The litters from PS mice showed increased neonatal mortality within the first week of life. Body weights (at euthanasia) of adult offspring at 25 wk from the PS group were significantly increased compared with weights of controls. Adult offspring from these pregnancies were serially imaged with ultrasound to measure plaque thickness and were compared with plaque macroscopic and microscopic pathology. PS groups had increased plaque thickness determined by ultrasound, gross, histological evaluation and increased aortic root and valve macrophage infiltration at 25 wk. Five-week-old mice from PS group had significant decrease in mean arterial pressure, yet blood pressure normalized by 10 wk. As prenatal stress induced increased atherosclerosis, and telomeres are susceptible to stress, aortas from 10-wk-old mice were compared for telomere lengths and were found to be significantly shorter in PS mice compared with control mice. These studies support future investigation of how stress impacts telomere shortening in animal models and human aortas. This model could be further used to investigate the role of prenatal stress, telomere biology, and atherosclerosis pathogenesis in adults.

Children born to women who experience stress during pregnancy have an increased risk of cancer in later life, but no previous animal studies have tested such a link. We questioned whether prenatal stress (PS) in A/J mice affected the development of lung tumors after postnatal response to tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Timed-bred A/J mice were randomly assigned on gestation day 12.5 to PS by restraint for 5 consecutive days or control (no restraint). Adult offspring of control and stressed pregnancies were all treated with three NNK injections (50 mg/kg every other day) and euthanized 16 weeks later to examine their lungs. Compared with controls, PS dams exhibited significantly increased levels of plasma corticosterone, increased adrenal weights and decreased fetus weights without fetal loss. Prenatally stressed litters had a significantly higher neonatal death rate within first week of life, and surviving male and female offspring developed lung epithelial proliferations with increase multiplicity, increased area and aggressive morphology. PS also induced more advanced atypical adenomatous hyperplasia lesions. We found no difference in lung NNK-derived methyl DNA adducts, but PS did significantly enhance CD3+ T cell and Foxp3+ T cell tumor infiltration. PS significantly increases multiplicity, area of NNK-induced lung tumors and advanced morphology. PS did not affect production of NNK-derived methyl DNA adducts but did increase lymphocytic infiltration of lung tumors. To our knowledge, this is the first animal model of PS with evaluation of cancer development in offspring.

Objective To assess the accuracy of renal ultrasound (RUS) in detecting renal scarring (RS). Methods All initial DMSA scans performed from 2006 to 2009 for history of urinary tract infection (UTI) or vesicoureteral reflux (VUR) in patients under 14 years old were identified, and clinical history obtained via chart review. Patients who had RUS within 4 months of DMSA scan and no documented UTI during that interval were included. Decreased uptake of tracer associated with loss of contours or cortical thinning defined a positive DMSA study. Increased echogenicity/dysplasia, cortical thinning, atrophic kidney and/or abnormal corticomedullary differentiation defined a positive RUS. The sensitivity and specificity of RUS in identifying RS were calculated using DMSA scan as the gold standard. Results A total of 144 patients had initial DMSA scans performed for UTI or VUR, with a RUS within 4 months, and no UTI between the 2 studies. Ninety-five of 144 (66%) had RS on DMSA and 49/144 (34%) did not. Patients with or without RS on DMSA were not different in gender (P = .073), age (P = .432), insurance (P = 1.000) or VUR grade (P = .132). Only 39/144 (27.1%) patients had positive RUS. The sensitivity of RUS for RS was 35.8% and the specificity was 89.8%, leading to an accuracy of 54.2% (95%CI; 45.7-62.5%, P = .999). Conclusion RUS demonstrated poor sensitivity for RS visualized on DMSA scan. This suggests that RUS is a poor screening test for RS or indicators of future renal scar. A normal ultrasound does not rule out RS or risk of future renal scar. Specificity of RUS was excellent.

Recurrent urinary tract infections (UTIs) pose a significant burden on the health care system. Underlying mechanisms predisposing children to UTIs and associated changes in the urinary proteome are not well understood. We aimed to investigate the urinary proteome of a subset of children who have vesicoureteral reflux (VUR) and recurrent UTIs because of their risk of developing infection-related renal damage. Improving diagnostic modalities to identify UTI risk factors would significantly alter the clinical management of children with VUR. We profiled the urinary proteomes of 22 VUR patients with low grade VUR (1–3 out of 5), a history of recurrent UTIs, and renal scarring, comparing them to those obtained from 22 age-matched controls. Urinary proteins were analyzed by mass spectrometry followed by protein quantitation based on spectral counting. Of the 2,551 proteins identified across both cohorts, 964 were robustly quantified, as defined by meeting criteria with spectral count (SC) ≥2 in at least 7 patients in either VUR or control cohort. Eighty proteins had differential expression between the two cohorts, with 44 proteins significantly up-regulated and 36 downregulated (q

Schizophrenia (SCZ) is a complex psychiatric disorder with severe impact on patient's livelihood. In the last years, the importance of neuropeptides in SCZ and other CNS disorders has been recognized, mainly due to their ability to modulate the signaling of classical monoaminergic neurotransmitters as dopamine. In addition, a class of enzymes coined as oligopeptidases are able to cleave several of these neuropeptides, and their potential implication in SCZ was also demonstrated. Interestingly, these enzymes are able to play roles as modulators of neuropeptidergic systems, and they were also implicated in neurogenesis, neurite outgrowth, neuron migration, and therefore, in neurodevelopment and brain formation. Altered activity of oligopeptidases in SCZ was described only more recently, suggesting their possible utility as biomarkers for mental disorders diagnosis or treatment response. We provide here an updated and comprehensive review on neuropeptides and oligopeptidases involved in mental disorders, aiming to attract the attention of physicians to the potential of targeting this system for improving the therapy and for understanding the neurobiology underlying mental disorders as SCZ.

BACKGROUND Evaluation of kidney function in newborns with hydronephrosis is important for clinical decisions. Dynamic contrast-enhanced (DCE) MRI can provide the necessary anatomical and functional information. Golden angle dynamic radial acquisition and compressed sensing reconstruction provides sufficient spatiotemporal resolution to achieve accurate parameter estimation for functional imaging of kidneys. However, bulk motion during imaging (rigid or nonrigid movement of the subject resulting in signal dropout) remains an unresolved challenge. PURPOSE To evaluate a motion-compensated (MoCo) DCE-MRI technique for robust evaluation of kidney function in newborns. Our method includes: 1) motion detection, 2) motion-robust image reconstruction, 3) joint realignment of the volumes, and 4) tracer-kinetic (TK) model fitting to evaluate kidney function parameters. STUDY TYPE Retrospective. SUBJECTS Eleven newborn patients (ages

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life, and in utero obstruction to urine flow is a frequent cause of secondary upper urinary tract malformations. Here, using whole-exome sequencing, we identified three different biallelic mutations in CHRNA3, which encodes the α3 subunit of the nicotinic acetylcholine receptor, in five affected individuals from three unrelated families with functional lower urinary tract obstruction and secondary CAKUT. Four individuals from two families have additional dysautonomic features, including impaired pupillary light reflexes. Functional studies in vitro demonstrated that the mutant nicotinic acetylcholine receptors were unable to generate current following stimulation with acetylcholine. Moreover, the truncating mutations p.Thr337Asnfs(∗)81 and p.Ser340(∗) led to impaired plasma membrane localization of CHRNA3. Although the importance of acetylcholine signaling in normal bladder function has been recognized, we demonstrate for the first time that mutations in CHRNA3 can cause bladder dysfunction, urinary tract malformations, and dysautonomia. These data point to a pathophysiologic sequence by which monogenic mutations in genes that regulate bladder innervation may secondarily cause CAKUT.

We performed a retrospective, single-institution study to characterize the pathological findings of testis tissue specimens from older boys and adolescents with cryptorchidism.With institutional review board approval, pathology reports were obtained for testicular specimens from patients age 10 years or older at a pediatric hospital from 1994 to 2016. Reports were excluded if they lacked clinical records, lacked testicular parenchyma, were from a descended testis or were from a patient with differences of sexual development. Variables of interest included age, testis location, procedure and pathological findings. Presence of malignancy among intra-abdominal versus extra-abdominal undescended testes was compared using Fisher's Exact Test.Seventy-one patients met inclusion criteria. The median age was 15.3 years (range 10.1-27.7). None had a history of testicular malignancy. Forty-five unilateral orchiectomies, 22 unilateral orchiopexies with biopsy and 4 bilateral procedures were performed. Seventeen testes (22.7%) were intra-abdominal, 42 (56.0%) were in the inguinal canal, 9 (12.0%) were at the external inguinal ring, 3 (4.0%) were in the superficial inguinal pouch and 4 (5.3%) were in the scrotum. Malignancy was detected in 2/71 patients (2.8%). By location, 2/16 patients (12.5%) with intra-abdominal testis and 0/55 patients (0%) with extra-abdominal testis demonstrated malignancy (p=0.048).Among males with cryptorchidism ages 10 years and older without differences of sexual development, 2/16 patients with intra-abdominal testis and 0/55 patients with extra-abdominal testis demonstrated malignancy. In older boys and adolescents, orchiectomy or biopsy is indicated for intra-abdominal testes but may not be necessary for extra-abdominal undescended testes.

Somatic missense mutations in histone genes turn these essential proteins into oncohistones, which can drive oncogenesis. Understanding how missense mutations alter histone function is challenging in mammals as mutations occur in a single histone gene. For example, described oncohistone mutations predominantly occur in the histoneH3.3gene, despite the human genome encoding 15 H3 genes. To understand how oncogenic histone missense mutations alter histone function, we leveraged the budding yeast model, which contains only two H3 genes, to explore the functional consequences of oncohistones H3K36M, H3G34W, H3G34L, H3G34R, and H3G34V. Analysis of cells that express each of these variants as the sole copy of H3 reveals that H3K36 mutants show different drug sensitivities compared to H3G34 mutants. This finding suggests that changes to proximal amino acids in the H3 N-terminal tail alter distinct biological pathways. We exploited the caffeine sensitive growth of H3K36 mutant cells to perform a high copy suppressor screen. This screen identified genes linked to histone function and transcriptional regulation, including Esa1, a histone H4/H2A acetyltransferase; Tos4, a forkhead-associated domain-containing gene expression regulator; Pho92, an N6-methyladenosine RNA binding protein and Sgv1/Bur1, a cyclin-dependent kinase. We show that the Esa1 lysine acetyltransferase activity is critical for suppression of the caffeine sensitive growth of H3K36R mutant cells while the previously characterized binding interactions of Tos4 and Pho92 are not required for suppression. This screen identifies pathways that could be altered by oncohistone mutations and highlights the value of yeast genetics to identify pathways altered by such mutations.

Allostatic load (AL) refers to the cumulative "wear and tear" on an organism throughout its lifetime. One of the primary contributing factors to AL is prolonged exposure to stress or its primary catabolic agent cortisol. Chronic exposure to stress or cortisol is associated with numerous diseases, including cardiovascular disease, metabolic disorders, and psychiatric disorders. Therefore, a molecular marker capable of integrating a past history of cortisol exposure would be of great utility for assessing disease risk. To this end, we recruited 87 healthy males and females of European ancestry between 18 and 60 years old, extracted genomic DNA and RNA from leukocytes, and implemented a gene-centric DNA enrichment method coupled with bisulfite sequencing and RNA-Seq of total RNA for the determination of genome-wide methylation and gene transcription, respectively. Sequencing data were analyzed against awakening and bedtime cortisol data to identify differentially methylated regions (DMRs) and CpGs (DMCs) and differentially expressed genes (DEGs). Six candidate DMCs (punadjusted < 0.005) and nine DEGs (punadjusted < 0.0005) were used to construct a prediction model that could capture past 30+ days of both bedtime and awakening cortisol levels. Utilizing a cross-validation approach, we obtained a regression coefficient of R2 = 0.308 for predicting continuous awakening cortisol and an area under the curve (AUC) = 0.753 for dichotomous (high vs. low tertile) awakening cortisol, and R2 = 0.224 and AUC = 0.723 for continuous and dichotomous bedtime cortisol levels, respectively. To our knowledge, the current study represents the first attempt to identify genome-wide predictors of cortisol exposure that utilizes both methylation and transcription targets. The utility of our approach needs to be replicated in an independent cohort of samples for which similar cortisol metrics are available.

BackgroundEqual dosage of X-linked genes between males and females is maintained by the X-inactivation of the second X chromosome in females through epigenetic mechanisms. Boys with aneuploidy of the X chromosome exhibit a host of symptoms such as low fertility, musculoskeletal anomalies, and cognitive and behavioral deficits that are presumed to be caused by the abnormal dosage of these genes. The objective of this pilot study is to assess the relationship between CpG methylation, an epigenetic modification, at several genes on the X chromosome and behavioral dysfunction in boys with supernumerary X chromosomes.ResultsTwo parental questionnaires, the Behavior Rating Inventory of Executive Function (BRIEF) and Child Behavior Checklist (CBCL), were analyzed, and they showed expected differences in both internal and external behaviors between neurotypical (46,XY) boys and boys with 49,XXXXY. There were several CpGs inARandMAOAof boys with 49,XXXXY whose methylation levels were skewed from levels predicted from having one active (Xa) and three inactive (Xi) X chromosomes. Further, methylation levels of multiple CpGs inMAOAshowed nominally significant association with externalizing behavior on the CBCL, and the methylation level of one CpG inARshowed nominally significant association with the BRIEF Regulation Index.ConclusionsBoys with 49,XXXXY displayed higher levels of CpG methylation at regulatory intronic regions in X-linked genes encoding the androgen receptor (AR) and monoamine oxidase A (MAOA), compared to that in boys with 47,XXY and neurotypical boys. Our pilot study results suggest a link between CpG methylation levels and behavior in boys with 49,XXXXY.

Morbidity and mortality associated with pediatric necrotizing fasciitis are strongly dependent on early diagnosis and timely intervention. Yet, the lack of early cutaneous findings and nonspecific symptoms may result in initial delayed diagnosis or misdiagnosis. Infants may be particularly at risk of missed or delayed diagnosis due to inherent barriers in communication and rarity of the condition, especially among healthy patients. We describe two cases of necrotizing fasciitis following routine genitourinary surgery in healthy infants.

Objective Evaluate renal outcomes and early predictive factors in infants with congenital posterior urethral valves who required catheter or surgical urinary tract decompression within the first 7 days of life. Study design A 10-year retrospective study at a single hospital. Primary outcomes were estimated glomerular filtration rate (eGFR) and development of end stage renal disease (ESRD). Results Of 35 infants, 50% developed eGFR

Purpose:The Urinary Tract Dilation classification system was designed to be more objective and reproducible than currently available grading systems. We evaluated the reliability and consistency of...

Glioblastomas (GBM) are highly infiltrated by myeloid-derived innate immune cells that contribute to the immunosuppressive nature of the brain tumor microenvironment (TME). CD47 has been shown to mediate immune evasion, as the CD47–SIRPα axis prevents phagocytosis of tumor cells by macrophages and other myeloid cells. In this study, we established CD47 homozygous deletion (CD47−/−) in human and mouse GBM cells and investigated the impact of eliminating the "don't eat me" signal on tumor growth and tumor–TME interactions. CD47 knockout (KO) did not significantly alter tumor cell proliferation in vitro but significantly increased phagocytosis of tumor cells by macrophages in cocultures. Compared with CD47 wild-type xenografts, orthotopic xenografts derived from CD47−/− tumor cells grew significantly slower with enhanced tumor cell phagocytosis and increased recruitment of M2-like tumor-associated microglia/macrophages (TAM). CD47 KO increased tumor-associated extracellular matrix protein tenascin C (TNC) in xenografts, which was further examined in vitro. CD47 loss of function upregulated TNC expression in tumor cells via a Notch pathway–mediated mechanism. Depletion of TNC in tumor cells enhanced the growth of CD47−/− xenografts in vivo and decreased the number of TAM. TNC knockdown also inhibited phagocytosis of CD47−/− tumor cells in cocultures. Furthermore, TNC stimulated release of proinflammatory factors including TNFα via a Toll-like receptor 4 and STAT3-dependent mechanism in human macrophage cells. These results reveal a vital role for TNC in immunomodulation in brain tumor biology and demonstrate the prominence of the TME extracellular matrix in affecting the antitumor function of brain innate immune cells. Significance: These findings link TNC to CD47-driven phagocytosis and demonstrate that TNC affects the antitumor function of brain TAM, facilitating the development of novel innate immune system–based therapies for brain tumors.

The glycoprotein uromodulin (UMOD) is the most abundant protein in urine, and

Background and ObjectivesSport-related concussions affect millions of individuals across the United States each year, and current techniques to diagnose and monitor them rely largely on subjective measures. Our goal was to discover and validate objective, quantifiable noninvasive biomarkers with the potential to be used in sport-related concussion diagnosis.MethodsUrine samples from a convenience series of healthy control collegiate athletes who had not sustained a concussion and athletes who sustained a concussion as diagnosed by a sports medicine physician within 7 days were collected prospectively and studied. Participants also completed an instrumented single-task gait analysis as a functional measure. Participants were recruited from a single collegiate athletic program and were ≥18 years of age and were excluded if they had a concomitant injury, active psychiatric conditions, or preexisting neurologic disorders. Using Tandem Mass Tags (TMT) mass spectroscopy and ELISA, we identified and validated urinary biomarkers of concussion.ResultsForty-eight control and 47 age- and sex-matched athletes with concussion were included in the study (51.6% female, 48.4% male, average age 19.6 years). Participants represented both contact and noncontact sports. All but 1 of the postconcussion participants reported experiencing symptoms at the time of data collection. Insulin-like growth factor 1 (IGF-1) and IGF binding protein 5 (IGFBP5) were downregulated in the urine of athletes with concussions compared to healthy controls. Multivariable risk algorithms developed to predict the probability of sport-related concussion showed that IGF-1 multiplexed with single-task gait velocity predicts concussion risk across a range of postinjury time points (area under the curve [AUC] 0.786, 95% confidence interval [CI] 0.690–0.884). When IGF-1 and IGFBP5 are multiplexed with single-task gait velocity, they accurately distinguish between healthy controls and individuals with concussion at acute time points (AUC 0.835, 95% CI 0.701–0.968, p < 0.001).DiscussionThese noninvasive biomarkers, discovered in an objective and validated manner, may be useful in diagnosing and monitoring sport-related concussions in both acute phases of injury and several days after injury.Trial Registration InformationClinicalTrials.gov Identifier: NCT02354469 (submitted February 2015, first patient enrolled August 2015).Classification of EvidenceThis study provides Class III evidence that urinary IGF-1 and IGFBP5 multiplexed with single-task gait velocity may be useful in diagnosing sport-related concussion.

Exposure to stress or glucocorticoids (GCs) is associated with epigenetic and transcriptional changes in genes that either mediate or are targets of GC signalling. FKBP5 (FK506 binding protein 5) is one such gene that also plays a central role in negative feedback regulation of GC signalling and several stress-related psychiatric disorders. In this study, we sought to examine how the mouse Fkbp5 gene is regulated in a neuronal context and identify requisite factors that can mediate the epigenetic sequelae of excess GC exposure. Mice treated with GCs were used to establish the widespread changes in DNA methylation (DNAm) and expression of Fkbp5 across four brain regions. Then two cell lines were used to test the persistence, decay, and functional significance of GC-induced methylation changes near two GC response elements (GREs) in the fifth intron of Fkbp5. We also tested the involvement of DNMT1, cell proliferation, and MeCP2 in mediating the effect of GCs on DNAm and gene activation. DNAm changes at some CpGs persist while others decay, and reduced methylation states are associated with a more robust transcriptional response. Importantly, the ability to undergo GC-induced DNAm loss is tied to DNMT1 function during cell division. Further, GC-induced DNAm loss is associated with reduced binding of MeCP2 at intron 5 and a physical interaction between the fifth intron and promoter of Fkbp5. Our results highlight several key factors at the Fkbp5 locus that may have important implications for GC- or stress-exposure during early stages of neurodevelopment.

This introductory chapter provides a brief review of the current state of genetics research on psychiatric disorders. It reports on the progress that has been accomplished in the last few years which has led to the identification of new genes and genetic variants for many disorders, with schizophrenia being in the lead. It describes the different approaches that have been used and often points out how the genetics can predict an important role for epigenetics. This is followed by a quick overview of evidence for the importance of epigenetics in psychiatric disease and the links with many epigenetically relevant environmental factors. Finally, after a comparison highlighting similarities and differences between epigenetic and genetic approaches, the chapter provides some insights into moving psychiatric epigenetic research forward.

A new synthetic approach to 1,2,4-thiadiazoles using tetra(n-butyl)ammonium peroxydisulfate via oxidative dimerization of thioamides is described. This new oxidative protocol is simple, highly efficient, and allows the practical synthesis of 1,2,4-thiadiazoles from various primary thioamides in good to excellent yields.