Developmental origin, functional maintenance and genetic rescue of osteoclasts.

Osteoclasts are multinucleated giant cells that resorb bone, ensuring development and continuous remodelling of the skeleton and the bone marrow haematopoietic niche. Defective osteoclast activity leads to osteopetrosis and bone marrow failure1-9, whereas excess activity can contribute to bone loss and osteoporosis10. Osteopetrosis can be partially treated by bone marrow transplantation in humans and mice11-18, consistent with a haematopoietic origin of osteoclasts13,16,19 and studies that suggest that they develop by fusion of monocytic precursors derived from haematopoietic stem cells in the presence of CSF1 and RANK ligand1,20. However, the developmental origin and lifespan of osteoclasts, and the mechanisms that ensure maintenance of osteoclast function throughout life in vivo remain largely unexplored. Here we report that osteoclasts that colonize fetal ossification centres originate from embryonic erythro-myeloid progenitors21,22. These erythro-myeloid progenitor-derived osteoclasts are required for normal bone development and tooth eruption. Yet, timely transfusion of haematopoietic-stem-cell-derived monocytic cells in newborn mice is sufficient to rescue bone development in early-onset autosomal recessive osteopetrosis. We also found that the postnatal maintenance of osteoclasts, bone mass and the bone marrow cavity involve iterative fusion of circulating blood monocytic cells with long-lived osteoclast syncytia. As a consequence, parabiosis or transfusion of monocytic cells results in long-term gene transfer in osteoclasts in the absence of haematopoietic-stem-cell chimerism, and can rescue an adult-onset osteopetrotic phenotype caused by cathepsin K deficiency23,24. In sum, our results identify the developmental origin of osteoclasts and a mechanism that controls their maintenance in bones after birth. These data suggest strategies to rescue osteoclast deficiency in osteopetrosis and to modulate osteoclast activity in vivo.

Lymphatic endothelial S1P promotes mitochondrial function and survival in naive T cells.

Effective adaptive immune responses require a large repertoire of naive T cells that migrate throughout the body, rapidly identifying almost any foreign peptide. Because the production of T cells declines with age, naive T cells must be long-lived. However, it remains unclear how naive T cells survive for years while constantly travelling. The chemoattractant sphingosine 1-phosphate (S1P) guides T cell circulation among secondary lymphoid organs, including spleen, lymph nodes and Peyer's patches, where T cells search for antigens. The concentration of S1P is higher in circulatory fluids than in lymphoid organs, and the S1P1 receptor (S1P1R) directs the exit of T cells from the spleen into blood, and from lymph nodes and Peyer's patches into lymph. Here we show that S1P is essential not only for the circulation of naive T cells, but also for their survival. Using transgenic mouse models, we demonstrate that lymphatic endothelial cells support the survival of T cells by secreting S1P via the transporter SPNS2, that this S1P signals through S1P1R on T cells, and that the requirement for S1P1R is independent of the established role of the receptor in guiding exit from lymph nodes. S1P signalling maintains the mitochondrial content of naive T cells, providing cells with the energy to continue their constant migration. The S1P signalling pathway is being targeted therapeutically to inhibit autoreactive T cell trafficking, and these findings suggest that it may be possible simultaneously to target autoreactive or malignant cell survival.

Identification of vulnerable plaques and patients by intracoronary near-infrared spectroscopy and ultrasound (PROSPECT II): a prospective natural history study.

BACKGROUND: Near-infrared spectroscopy (NIRS) and intravascular ultrasound are promising imaging modalities to identify non-obstructive plaques likely to cause coronary-related events. We aimed to assess whether combined NIRS and intravascular ultrasound can identify high-risk plaques and patients that are at risk for future major adverse cardiac events (MACEs). METHODS: PROSPECT II is an investigator-sponsored, multicentre, prospective natural history study done at 14 university hospitals and two community hospitals in Denmark, Norway, and Sweden. We recruited patients of any age with recent (within past 4 weeks) myocardial infarction. After treatment of all flow-limiting coronary lesions, three-vessel imaging was done with a combined NIRS and intravascular ultrasound catheter. Untreated lesions (also known as non-culprit lesions) were identified by intravascular ultrasound and their lipid content was assessed by NIRS. The primary outcome was the covariate-adjusted rate of MACEs (the composite of cardiac death, myocardial infarction, unstable angina, or progressive angina) arising from untreated non-culprit lesions during follow-up. The relations between plaques with high lipid content, large plaque burden, and small lumen areas and patient-level and lesion-level events were determined. This trial is registered with ClinicalTrials.gov, NCT02171065. FINDINGS: Between June 10, 2014, and Dec 20, 2017, 3629 non-culprit lesions were characterised in 898 patients (153 [17%] women, 745 [83%] men; median age 63 [IQR 55-70] years). Median follow-up was 3·7 (IQR 3·0-4·4) years. Adverse events within 4 years occurred in 112 (13·2%, 95% CI 11·0-15·6) of 898 patients, with 66 (8·0%, 95% CI 6·2-10·0) arising from 78 untreated non-culprit lesions (mean baseline angiographic diameter stenosis 46·9% [SD 15·9]). Highly lipidic lesions (851 [24%] of 3500 lesions, present in 520 [59%] of 884 patients) were an independent predictor of patient-level non-culprit lesion-related MACEs (adjusted odds ratio 2·27, 95% CI 1·25-4·13) and non-culprit lesion-specific MACEs (7·83, 4·12-14·89). Large plaque burden (787 [22%] of 3629 lesions, present in 530 [59%] of 898 patients) was also an independent predictor of non-culprit lesion-related MACEs. Lesions with both large plaque burden by intravascular ultrasound and large lipid-rich cores by NIRS had a 4-year non-culprit lesion-related MACE rate of 7·0% (95% CI 4·0-10·0). Patients in whom one or more such lesions were identified had a 4-year non-culprit lesion-related MACE rate of 13·2% (95% CI 9·4-17·6). INTERPRETATION: Combined NIRS and intravascular ultrasound detects angiographically non-obstructive lesions with a high lipid content and large plaque burden that are at increased risk for future adverse cardiac outcomes. FUNDING: Abbott Vascular, Infraredx, and The Medicines Company.

Integration and Analysis of CPTAC Proteomics Data in the Context of Cancer Genomics in the cBioPortal.

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) has produced extensive mass spectrometry-based proteomics data for selected breast, colon, and ovarian tumors from The Cancer Genome Atlas (TCGA). We have incorporated the CPTAC proteomics data into the cBioPortal to support easy exploration and integrative analysis of these proteomic datasets in the context of the clinical and genomics data from the same tumors. cBioPortal is an open source platform for exploring, visualizing, and analyzing multidimensional cancer genomics and clinical data. The public instance of the cBioPortal (http://cbioportal.org/) hosts more than 200 cancer genomics studies, including all of the data from TCGA. Its biologist-friendly interface provides many rich analysis features, including a graphical summary of gene-level data across multiple platforms, correlation analysis between genes or other data types, survival analysis, and per-patient data visualization. Here, we present the integration of the CPTAC mass spectrometry-based proteomics data into the cBioPortal, consisting of 77 breast, 95 colorectal, and 174 ovarian tumors that already have been profiled by TCGA for mutations, copy number alterations, gene expression, and DNA methylation. As a result, the CPTAC data can now be easily explored and analyzed in the cBioPortal in the context of clinical and genomics data. By integrating CPTAC data into cBioPortal, limitations of TCGA proteomics array data can be overcome while also providing a user-friendly web interface, a web API, and an R client to query the mass spectrometry data together with genomic, epigenomic, and clinical data.

Vulnerable plaques and patients: state-of-the-art.

Despite advanced understanding of the biology of atherosclerosis, coronary heart disease remains the leading cause of death worldwide. Progress has been challenging as half of the individuals who suffer sudden cardiac death do not experience premonitory symptoms. Furthermore, it is well-recognized that also a plaque that does not cause a haemodynamically significant stenosis can trigger a sudden cardiac event, yet the majority of ruptured or eroded plaques remain clinically silent. In the past 30 years since the term 'vulnerable plaque' was introduced, there have been major advances in the understanding of plaque pathogenesis and pathophysiology, shifting from pursuing features of 'vulnerability' of a specific lesion to the more comprehensive goal of identifying patient 'cardiovascular vulnerability'. It has been also recognized that aside a thin-capped, lipid-rich plaque associated with plaque rupture, acute coronary syndromes (ACS) are also caused by plaque erosion underlying between 25% and 60% of ACS nowadays, by calcified nodule or by functional coronary alterations. While there have been advances in preventive strategies and in pharmacotherapy, with improved agents to reduce cholesterol, thrombosis, and inflammation, events continue to occur in patients receiving optimal medical treatment. Although at present the positive predictive value of imaging precursors of the culprit plaques remains too low for clinical relevance, improving coronary plaque imaging may be instrumental in guiding pharmacotherapy intensity and could facilitate optimal allocation of novel, more aggressive, and costly treatment strategies. Recent technical and diagnostic advances justify continuation of interdisciplinary research efforts to improve cardiovascular prognosis by both systemic and 'local' diagnostics and therapies. The present state-of-the-art document aims to present and critically appraise the latest evidence, developments, and future perspectives in detection, prevention, and treatment of 'high-risk' plaques occurring in 'vulnerable' patients.

Adaptive management as a foundational framework for developing collaborative estuary management programs.

Collaborative nonregulatory programs can benefit the long-term sustainability of environmental resources. Such programs benefit from extensive planning and assessment relative to ecological systems as well as public participation. While many programs use adaptive management as a guiding programmatic framework, few programs successfully integrate social and human context into their adaptive management frameworks. While this adaptive governance framework can be successful, many potential challenges arise when incorporating public stakeholders into the adaptive management framework. To reduce those challenges, programs need participation from diverse stakeholder groups that represent multiple communities of interest, place, and identity. The participatory process benefits from a diverse group of stakeholders and can result in successful management of environmental resources. We highlight the participatory co-management process of three newly developing nonregulatory programs that are modeled after the United States EPA's National Estuary Program in the Perdido and Pensacola Bay systems, Choctawhatchee Bay, and the St. Andrew and St. Joseph Bay systems (Florida USA). This case study illustrates how collaborative nonregulatory programs can be implemented not only in the United States, but also in other regions of the world.

Hybrid intravascular imaging: recent advances, technical considerations, and current applications in the study of plaque pathophysiology.

Cumulative evidence from histology-based studies demonstrate that the currently available intravascular imaging techniques have fundamental limitations that do not allow complete and detailed evaluation of plaque morphology and pathobiology, limiting the ability to accurately identify high-risk plaques. To overcome these drawbacks, new efforts are developing for data fusion methodologies and the design of hybrid, dual-probe catheters to enable accurate assessment of plaque characteristics, and reliable identification of high-risk lesions. Today several dual-probe catheters have been introduced including combined near infrared spectroscopy-intravascular ultrasound (NIRS-IVUS), that is already commercially available, IVUS-optical coherence tomography (OCT), the OCT-NIRS, the OCT-near infrared fluorescence (NIRF) molecular imaging, IVUS-NIRF, IVUS intravascular photoacoustic imaging and combined fluorescence lifetime-IVUS imaging. These multimodal approaches appear able to overcome limitations of standalone imaging and provide comprehensive visualization of plaque composition and plaque biology. The aim of this review article is to summarize the advances in hybrid intravascular imaging, discuss the technical challenges that should be addressed in order to have a use in the clinical arena, and present the evidence from their first applications aiming to highlight their potential value in the study of atherosclerosis.

Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells.

BACKGROUND: Patients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia. OBJECTIVES: Because regulation of the phosphoinositide 3-kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]). METHODS: Patients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse. RESULTS: Autoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T-cell reduction, and changes in T- and B-cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse. CONCLUSION: Heterozygous loss of function of PTEN in human subjects has a significant effect on T- and B-cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T-cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency.

The Lipid-Rich Plaque Study of vulnerable plaques and vulnerable patients: Study design and rationale.

BACKGROUND: It has been hypothesized that the outcome post-PCI could be improved by the detection and subsequent treatment of vulnerable patients and lipid-rich vulnerable coronary plaques (LRP). A near-infrared spectroscopy (NIRS) catheter capable of detecting LRP is being evaluated in The Lipid-Rich Plaque Study. STUDY DESIGN: The LRP Study is an international, multicenter, prospective cohort study conducted in patients with suspected coronary artery disease (CAD) who underwent cardiac catheterization with possible ad hoc PCI for an index event. Patient level and plaque level events were detected by follow-up in the subsequent 2 years. Enrollment began in February 2014 and was completed in March 2016; a total of 1,562 patients were enrolled. Adjudication of new coronary event occurrence and de novo culprit lesion location during the 2-year follow-up is performed by an independent clinical end-points committee (CEC) blinded to NIRS-IVUS findings. The first analysis of the results will be performed when at least 20 de novo events have occurred for which follow-up angiographic data and baseline NIRS-IVUS measurements are available. It is expected that results of the study will be announced in 2018. SUMMARY: The LRP Study will test the hypotheses that NIRS-IVUS imaging to detect LRP in patients can identify vulnerable patients and vulnerable plaques. Identification of vulnerable patients will assist future studies of novel systemic therapies; identification of localized vulnerable plaques would enhance future studies of possible preventive measures.

Saphenous vein graft near-infrared spectroscopy imaging insights from the lipid core plaque association with clinical events near-infrared spectroscopy (ORACLE-NIRS) registry.

OBJECTIVES: We sought to examine near-infrared spectroscopy (NIRS) imaging findings of aortocoronary saphenous vein grafts (SVGs). BACKGROUND: SVGs are prone to develop atherosclerosis similar to native coronary arteries. They have received little study using NIRS. METHODS: We examined the clinical characteristics and imaging findings from 43 patients who underwent NIRS imaging of 45 SVGs at our institution between 2009 and 2016. RESULTS: The mean patient age was 67 ± 7 years and 98% were men, with high prevalence of diabetes mellitus (56%), hypertension (95%), and dyslipidemia (95%). Mean SVG age was 7 ± 7 years, mean SVG lipid core burden index (LCBI) was 53 ± 60 and mean maxLCBI4 mm was 194 ± 234. Twelve SVGs (27%) had lipid core plaques (2 yellow blocks on the block chemogram), with a higher prevalence in SVGs older than 5 years (46% vs. 5%, P = 0.002). Older SVG age was associated with higher LCBI (r = 0.480, P < 0.001) and higher maxLCBI4 mm (r = 0.567, P < 0.001). On univariate analysis, greater annual total cholesterol exposure was associated with higher SVG LCBI (r = 0.30, P = 0.042) and annual LDL-cholesterol and triglyceride exposure were associated with higher SVG maxLCBI4 mm (LDL-C: r = 0.41, P = 0.020; triglycerides: r = 0.36, P = 0.043). On multivariate analysis, the only independent predictor of SVG LCBI and maxLCBI4mm was SVG age. SVG percutaneous coronary intervention was performed in 63% of the patients. An embolic protection device was used in 96% of SVG PCIs. Periprocedural myocardial infarction occurred in one patient. CONCLUSIONS: Older SVG age and greater lipid exposure are associated with higher SVG lipid burden. © 2016 Wiley Periodicals, Inc.

Confirmation of the Intracoronary Near-Infrared Spectroscopy Threshold of Lipid-Rich Plaques That Underlie ST-Segment-Elevation Myocardial Infarction.

OBJECTIVE: In a previous exploratory analysis, intracoronary near-infrared spectroscopy (NIRS) found the majority of culprit lesions in ST-segment-elevation myocardial infarction (STEMI) to contain a maximum lipid core burden index in 4 mm (maxLCBI4mm) of >400. This initial study was limited by a small sample size, enrollment at a single center, and post hoc selection of the maxLCBI4mm ≥400 threshold. This study was designed a priori to substantiate the ability of NIRS to discriminate STEMI culprit from nonculprit segments and to confirm the performance of the maxLCBI4mm ≥400 threshold. APPROACH AND RESULTS: At 2 centers in the United States and Sweden, 75 STEMI patients underwent intracoronary NIRS imaging after establishing thrombolysis in myocardial infarction 3 flow, but before stenting. Blinded core laboratory analysis defined the culprit segment as the 10-mm segment distal to the proximal angiographic culprit margin. The remaining vessel was divided into contiguous 10-mm nonculprit segments. The maxLCBI4mm of culprit segments (median [interquartile range]: 543 [273-756]) was 4.4-fold greater than nonculprit segments (median [interquartile range]: 123 [0-307]; P<0.001). Receiver-operating characteristic analysis demonstrated that maxLCBI4mm differentiated culprit from nonculprit segments with high accuracy (c-statistic=0.83; P<0.001). A threshold maxLCBI4mm ≥400 identified STEMI culprit segments with a sensitivity of 64% and specificity of 85%. CONCLUSIONS: This study substantiates the ability of NIRS to accurately differentiate STEMI culprit from nonculprit segments and confirms that a threshold maxLCBI4mm ≥400 is detected by NIRS in the majority of STEMI culprits.

Near-Infrared Spectroscopy Enhances Intravascular Ultrasound Assessment of Vulnerable Coronary Plaque: A Combined Pathological and In Vivo Study.

OBJECTIVES: Pathological studies demonstrate the dual significance of plaque burden (PB) and lipid composition for mediating coronary plaque vulnerability. We evaluated relationships between intravascular ultrasound (IVUS)-derived PB and arterial remodeling with near-infrared spectroscopy (NIRS)-derived lipid content in ex vivo and in vivo human coronary arteries. APPROACH AND RESULTS: Ex vivo coronary NIRS and IVUS imaging was performed through blood in 116 coronary arteries of 51 autopsied hearts, followed by 2-mm block sectioning (n=2070) and histological grading according to modified American Heart Association criteria. Lesions were defined as the most heavily diseased 2-mm block per imaged artery on IVUS. IVUS-derived PB and NIRS-derived lipid core burden index (LCBI) of each block and lesion were analyzed. Block-level analysis demonstrated significant trends of increasing PB and LCBI across more complex atheroma (Ptrend <0.001 for both LCBI and PB). Lesion-based analyses demonstrated the highest LCBI and remodeling index within coronary fibroatheroma (Ptrend <0.001 and 0.02 versus all plaque groups, respectively). Prediction models demonstrated similar abilities of PB, LCBI, and remodeling index for discriminating fibroatheroma (c indices: 0.675, 0.712, and 0.672, respectively). A combined PB+LCBI analysis significantly improved fibroatheroma detection accuracy (c index 0.77, P=0.028 versus PB; net-reclassification index 43%, P=0.003), whereas further adding remodeling index did not (c index 0.80, P=0.27 versus PB+LCBI). In vivo comparisons of 43 age- and sex-matched patients (to the autopsy cohort) undergoing combined NIRS-IVUS coronary imaging yielded similar associations to those demonstrated ex vivo. CONCLUSIONS: Adding NIRS to conventional IVUS-derived PB imaging significantly improves the ability to detect more active, potentially vulnerable coronary atheroma.

TCR Microclusters pre-exist and contain molecules necessary for TCR signal transduction.

TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These pre-existing microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters linker for activation of T cells and growth factor receptor-bound protein 2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could explain, in part, the rapid kinetics with which TCR signal transduction occurs.

Risk of myocardial infarction immediately after alcohol consumption.

BACKGROUND: Habitual moderate alcohol consumption is associated with a lower risk of acute myocardial infarction (MI), whereas heavy (binge) drinking is associated with higher cardiovascular risk. However, less is known about the immediate effects of alcohol consumption on the risk of acute MI and whether any association differs by beverage type or usual drinking patterns. METHODS: We conducted a case-crossover analysis of 3869 participants from the Determinants of Myocardial Infarction Onset Study who were interviewed during hospitalization for acute MI in one of the 64 medical centers across the United States in 1989-1996. We compared the observed number of times that each participant consumed wine, beer, or liquor in the hour preceding MI symptom onset with the expected frequency based on each participant's control information, defined as the number of times the participant consumed alcohol in the past year. RESULTS: Among 3869 participants, 2119 (55%) reported alcohol consumption in the past year, including 76 within 1 hour before acute MI onset. The incidence rate of acute MI onset was elevated 1.72-fold (95% confidence interval [CI] = 1.37-2.16) within 1 hour after alcohol consumption. The association was stronger for liquor than for beer or wine. The higher rate was not apparent for daily drinkers. For the 24 hours after consumption, there was a 14% lower rate (relative risk = 0.86 [95% CI = 0.79-0.95]) of MI compared with periods with no alcohol consumption. CONCLUSIONS: Alcohol consumption is associated with an acutely higher risk of MI in the subsequent hour among people who do not typically drink alcohol daily.

Rates of chemical cleavage of DNA and RNA oligomers containing guanine oxidation products.

The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design.

Spirodi(iminohydantoin) products from oxidation of 2'-deoxyguanosine in the presence of NH4Cl in nucleoside and oligodeoxynucleotide contexts.

Upon oxidation of the heterocyclic ring in 2'-deoxyguanosine (dG), the initial electrophilic intermediate displays a wide range of reactivities with nucleophiles leading to many downstream products. In the present study, the product profiles were mapped when aqueous solutions of dG were allowed to react with NH4Cl in the presence of the photooxidants riboflavin and Rose Bengal as well as the diffusible one-electron oxidant Na2IrCl6. Product characterization identified the 2'-deoxyribonucleosides of spiroiminodihydantoin, 5-guanidinohydantoin, and oxazolone resulting from H2O as the nucleophile. When NH3 was the nucleophile, a set of constitutional isomers that are diastereotopic were also observed, giving characteristic masses of dG + 31. ESI(+)-MS/MS of these NH3 adducts identified them to be spirocycles with substitution of either the C5 or C8 carbonyl with an amine. The NH3 adducts exhibit acid-catalyzed hydrolysis to spiroiminodihydantoin. Quantification of the NH3 and H2O adducts resulting from oxidation of dG in the nucleoside, single-stranded, and duplex oligodeoxynucleotide contexts were monitored allowing mechanisms for product formation to be proposed. These data also provide a cautionary note to those who purify their oligonucleotide samples with ammonium salts before oxidation because this will lead to unwanted side reactions in which ammonia participates in product formation.

Generation of human cerebral organoids with a structured outer subventricular zone.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.