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Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high‐throughput in vivo experimentation leveraged to inform large animal immunology‐based studies. We found that adult rhesus macaque mobilized blood (AMb) CD34+‐enriched hematopoietic stem and progenitor cells (HSPCs) engrafted at low but persistent levels in immune‐deficient mice harboring transgenes for human (NHP cross‐reactive) GM‐CSF and IL3, but did not in mice with wild‐type murine cytokines lacking NHP cross‐reactivity. To enhance engraftment, fetal liver‐derived HSPCs were selected as the infusion product based on an increased CD34hifraction compared with AMb and bone marrow. Coupled with cotransplantation of rhesus fetal thymic fragments beneath the mouse kidney capsule, fetal liver‐derived HSPC infusion in cytokine‐transgenic mice yielded robust multilineage lymphohematopoietic engraftment. The emergent immune system recapitulated that of the fetal monkey, with similar relative frequencies of lymphocyte, granulocyte, and monocyte subsets within the thymic, secondary lymphoid, and peripheral compartments. Importantly, while exhibiting a predominantly naïve phenotype, in vitro functional assays demonstrated robust cellular activation in response to nonspecific and allogenic stimuli. This primatized mouse represents a viable and translatable model for the study of hematopoietic stem cell physiology, immune development, and functional immunology in NHPs. Summary Sentence: Engraftment of rhesus macaque hematopoietic tissues in immune‐deficient mice yields a robust BLT/NeoThy‐type primatized mouse model for studying nonhuman primate hematopoiesis and immune function in vivo. Engraftment of rhesus macaque hematopoietic tissues in immune‐deficient mice yields a robust BLT/NeoThy‐type primatized mouse model for studying non‐human primate hematopoiesis and immune function in vivo.

Administration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear. We evaluated the safety and efficacy of using CD34+CD45+ hematopoietic progenitors derived from MHC homozygous iPSCs (iHPs) to treat cytopenia after myeloablative hematopoietic stem cell (HSC) transplantation in a Mauritian cynomolgus macaque (MCM) nonhuman primate (NHP) model. We demonstrated that infusion of iHPs was well tolerated and safe, observing no teratomas or tumors in the MCMs up to 1 year after HSC transplantation and iHP infusion. Importantly, the iHPs also did not induce significant levels of alloantibodies in MHC-matched or -mismatched immunocompetent MCMs, even after increasing MHC expression on iHPs with interferon-γ. These results support the feasibility of iHP use in the setting of myeloablation and suggest that iHP products pose a low risk of inducing alloreactive antibodies.

Administration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear. We evaluated the safety and efficacy of using CD34+CD45+hematopoietic progenitors derived from MHC homozygous iPSCs (iHPs) to treat cytopenia after myeloablative hematopoietic stem cell (HSC) transplantation in a Mauritian cynomolgus macaque (MCM) nonhuman primate (NHP) model. We demonstrated that infusion of iHPs was well tolerated and safe, observing no teratomas or tumors in the MCMs up to 1 year after HSC transplantation and iHP infusion. Importantly, the iHPs also did not induce significant levels of alloantibodies in MHC-matched or -mismatched immunocompetent MCMs, even after increasing MHC expression on iHPs with interferon-γ. These results support the feasibility of iHP use in the setting of myeloablation and suggest that iHP products pose a low risk of inducing alloreactive antibodies.

1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester (DGGR) lipase assays are used to measure lipase activity in the diagnosis of pancreatitis. The effect of hepatic lipases released from damaged hepatocytes on serum DGGR lipase activity has not been reported, to our knowledge. We identified dogs with histologically confirmed liver lesions and concurrent unremarkable pancreatic histology, and dogs with no histologic evidence of hepatic or pancreatic disease. Dogs with relevant comorbidities were excluded. The hepatopathy group(n= 7) included 4 dogs with inflammatory hepatopathies, 2 with hepatic neoplasia, and 1 with unspecified (non-inflammatory, non-neoplastic) hepatopathy. The control group(n= 5) included one dog each with enteritis, subcutaneous hemangiosarcoma, hydrocephalus, myelomalacia, and tetanus. A Mann–Whitney U test compared selected biochemical parameters including serum DGGR lipase, alkaline phosphatase, alanine aminotransferase, and amylase activities, with statistical significance defined as p≤0.05. Data are presented as median and range. Serum DGGR lipase activity (RI: <44 IU/L) was not different between the hepatopathy (52 IU/L; range: 27–85 IU/L) and control (37 IU/L, 25–105 IU/L; p= 0.947) groups. Serum amylase activity (RI: 256–1,610 IU/L) was significantly higher in the hepatopathy group (830 IU/L; 711–1,210 IU/L) than the control group (541 IU/L, 336–695 IU/L; p= 0.028). No association or correlation between serum DGGR lipase activity and hepatic lesions (based on histologic or biochemical findings) was identified, suggesting that clinically relevant changes in serum DGGR lipase activity may not be expected secondary to hepatopathy alone.

The formation of O-acetyl microgrewiapine A is investigated. NMR data for the authentic sample derived from the natural product are corrected. Wholly synthetic samples, produced from reductive N-methylation of synthetic microcosamine A (to give synthetic microgrewiapine A) followed by O-acetylation, exhibit NMR data that are identical to those of the authentic sample. The previous report that this two-step transformation proceeds with epimerization at C-6 is thus shown to be in error: the purported sample of O-acetyl 6-epi-microgrewiapine A is structurally misassigned and is, in fact, O-acetyl microgrewiapine A. A plausible rationale for the structural misassignment is advanced.

Changes in transcriptional regulatory networks can significantly alter cell fate. To gain insight into transcriptional dynamics, several studies have profiled bulk multi-omic data sets with parallel transcriptomic and epigenomic measurements at different stages of a developmental process. However, integrating these data to infer cell type–specific regulatory networks is a major challenge. We present dynamic regulatory module networks (DRMNs), a novel approach to infer cell type–specific cis-regulatory networks and their dynamics. DRMN integrates expression, chromatin state, and accessibility to predict cis-regulators of context-specific expression, where context can be cell type, developmental stage, or time point, and uses multitask learning to capture network dynamics across linearly and hierarchically related contexts. We applied DRMNs to study regulatory network dynamics in three developmental processes, each showing different temporal relationships and measuring a different combination of regulatory genomic data sets: cellular reprogramming, liver dedifferentiation, and forward differentiation. DRMN identified known and novel regulators driving cell type–specific expression patterns, showing its broad applicability to examine dynamics of gene regulatory networks from linearly and hierarchically related multi-omic data sets.

Cardiovascular diseases plague human health because of the lack of transplantable small-diameter blood vessel (SDBV) grafts. Although expanded polytetrafluoroethylene (ePTFE) has the potential to be used as a biocompatible material for SDBV grafts, long-term patency is still the biggest challenge. As discussed in this paper, by virtue of a novel material formulation and a new and benign alcohol/water lubricating agent, biofunctionalized ePTFE blood vessel grafts aimed at providing long-term patency were fabricated. Compared to the most prevalent modification of PTFE, namely surface treatment, this method realized bulk treatment, which could guarantee homogeneous and long-lasting performance throughout PTFE products. These blood vessel grafts included embedded functional biomolecules, such as arginylglycylaspartic acid, heparin, and selenocystamine, using water as a solvent in paste extrusion and in the expansion of ePTFE. Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, and scanning electron microscope results confirmed the existence of these targeting biomolecules in the as-fabricated ePTFE blood vessel grafts. Meanwhile, the greatly improved biological functions of the grafts were demonstrated vialive and dead assays, cell morphology, CD31 staining, nitric oxide (NO) release, and anticoagulation tests. This novel and benign material formulation and fabrication method provides an opportunity to produce multibiofunctional ePTFE blood vessel grafts in a single step, thus yielding a potent product with significant commercial and clinical potential.

Treatment of cardiovascular diseases suffers from the lack of transplantable small-diameter blood vessel (SDBV) grafts that can prohibit/eliminate thrombosis. Although expanded poly(tetrafluoroethylene) (ePTFE) has the potential to be used for SDBV grafts, recurrence of thrombus remains the biggest challenge. In this study, a reactive oxygen species (ROS)-responsive antithrombogenic drug synthesis and a bulk coating process were employed to fabricate functional ePTFE grafts capable of prohibiting/eliminating blood clots. The synthesized drug that would release antiplatelet ethyl salicylate (ESA), in responding to ROS, was dissolved in a polycaprolactone (PCL) solution, followed by a bulk coating of the as-fabricated ePTFE grafts with the PCL/drug solution. Nuclear magnetic resonance (NMR) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) were employed to investigate and confirm the synthesis and presence of the ROS-responsive drug in the ePTFE grafts. The ESA release functions were demonstrated via the drug-release profile and dynamic anticoagulation tests. The biocompatibility of the ROS-responsive ePTFE grafts was demonstrated via lactate dehydrogenase (LDH) cytotoxicity assays, live and dead cell assays, cell morphology, and cell–graft interactions. The ROS-responsive, antithrombogenic ePTFE grafts provide a feasible way for maintaining long-term patency, potentially solving a critical challenge in SDBV applications.

Derivation and culture of small hepatocyte progenitor cells (SHPCs) capable of proliferating in vitrohas been described in rodents and recently in humans. These cells are capable of engrafting in injured livers, however, they display de-differentiated morphology and reduced xenobiotic metabolism activity in culture over passages. Here we report that SHPCs derived from adult primary human hepatocytes (PHHs) and cultured on mouse embryonic fibroblasts (MEFs) not only display differentiated morphology and exhibit gene expression profiles similar to adult PHHs, but importantly, they retain their phenotype over several passages. Further, unlike previous reports, where extensive manipulations of culture conditions are required to convert SHPCs to metabolically functional hepatocytes, SHPCs in our co-culture system maintain expression of xenobiotic metabolism-associated genes. We show that SHPCs in co-culture are able to perform xenobiotic metabolism at rates equal to their parent PHHs as evidenced by the metabolism of acetaminophen to all of its major metabolites. In summary, we present an improved co-culture system that allows generation of SHPCs from adult PHHs that maintain their differentiated phenotype over multiple passages. Our findings would be useful for expansion of limited PHHs for use in studies of drug metabolism and toxicity testing.

A method to enable the synthesis of conduramines and their N-substituted derivatives (enantiopure or racemic form) in six steps (five steps for N-substituted derivatives) from cyclohexa-1,4-diene is reported. Key features of this reaction sequence include a preparation of benzene oxide that is amenable to multigram scale, and its efficient ring-opening upon treatment with a primary amine. Epoxidation of the resultant amino alcohols (40% aq HBF4then m-CPBA) is accompanied by hydrolytic ring-opening in situ to give the corresponding N-substituted conduramine derivatives directly. These may undergo subsequent N-deprotection to give the parent conduramines, as demonstrated by the preparation of enantiopure (−)-conduramine A1, (−)-conduramine A2, and (−)-conduramine E2 (the latter two for the first time). The selectivity of the epoxidation reaction is proposed to be the result of competitive ammonium-directed and hydroxyl-directed epoxidation processes, followed by either direct (SN2-type) or conjugate (SN2′-type) ring-openings of the intermediate epoxides.

Diabetic retinopathy is the most common microvascular complication of diabetes and is a major cause of blindness, but an understanding of the pathogenesis of the disease has been hampered by a lack of accurate animal models. Here, we explore the dynamics of retinal cellular changes in the Nile rat (Arvicanthis niloticus), a carbohydrate-sensitive model for type 2 diabetes. The early retinal changes in diabetic Nile rats included increased acellular capillaries and loss of pericytes that correlated linearly with the duration of diabetes. These vascular changes occurred in the presence of microglial infiltration but in the absence of retinal ganglion cell loss. After a prolonged duration of diabetes, the Nile rat also exhibits a spectrum of retinal lesions commonly seen in the human condition including vascular leakage, capillary non-perfusion, and neovascularization. Our longitudinal study documents a range and progression of retinal lesions in the diabetic Nile rat remarkably similar to those observed in human diabetic retinopathy, and suggests that this model will be valuable in identifying new therapeutic strategies.

Diabetic retinopathy is the most common microvascular complication of diabetes and is a major cause of blindness, but an understanding of the pathogenesis of the disease has been hampered by a lack of accurate animal models. Here, we explore the dynamics of retinal cellular changes in the Nile rat (Arvicanthis niloticus), a carbohydrate-sensitive model for type 2 diabetes. The early retinal changes in diabetic Nile rats included increased acellular capillaries and loss of pericytes that correlated linearly with the duration of diabetes. These vascular changes occurred in the presence of microglial infiltration but in the absence of retinal ganglion cell loss. After a prolonged duration of diabetes, the Nile rat also exhibits a spectrum of retinal lesions commonly seen in the human condition including vascular leakage, capillary non-perfusion, and neovascularization. Our longitudinal study documents a range and progression of retinal lesions in the diabetic Nile rat remarkably similar to those observed in human diabetic retinopathy, and suggests that this model will be valuable in identifying new therapeutic strategies.

The reported characterization data for the allocolchicinoid alkaloid suhailamine, isolated from Colchicum decaisneiand known to have an erroneous structure, have been reanalyzed. This analysis has led to the current proposal that suhailamine has the same structure as N-acetylcolchinol methyl ether (NCME), an assertion that is supported by comparison with previously reported data for NCME. Suhailamine is therefore a phantom natural product, while NCME represents a naturally occurring allocolchicinoid rather than a purely synthetic entity.

The diastereoselectivities and rates of epoxidation (upon treatment with Cl3CCO2H then m-CPBA) of a range of cis- and trans-4-aminocycloalk-2-en-1-ol derivatives (containing five-, six-, and seven-membered rings) have been investigated. In all cases where the two potential directing groups can promote epoxidation on opposite faces of the ring scaffold, evidence of competitive epoxidation pathways, promoted by hydrogen-bonding to either the in situ formed ammonium moiety or the hydroxyl group, was observed. In contrast to the relative directing group abilities already established for the six-membered ring system (NHBn ≫ OH > NBn2), an N,N-dibenzylammonium moiety appeared more proficient than a hydroxyl group at directing the stereochemical course of the epoxidation reaction in a five- or seven-membered system. In the former case, this was rationalized by the drive to minimize torsional strain in the transition state being coupled with assistance from hydrogen-bonding to the ammonium moiety. In the latter case, this was ascribed to the steric bulk of the ammonium moiety disfavoring conformations in which hydrogen-bonding to the hydroxyl group results in direction of the epoxidation to the synface. In cases where the two potential directing groups can promote epoxidation on the same face of the ring scaffold, an enhancement of epoxidation diastereoselectivity was not observed, while introduction of a second, allylic heteroatom to the substrate results in diminishment of the rate of epoxidation in all cases. Presumably, reduction of the nucleophilicity of the olefin by the second, inductively electron-withdrawing heteroatom is the dominant factor, and any assistance to the epoxidation reaction by the potential to form hydrogen-bonds to two directing groups rather than one is clearly unable to overwhelm it.

The 1H and 13C NMR data of synthetic samples of (S)-N(1)-methyl-2-[2′-(3″-hydroxy-4″-methoxyphenyl)ethyl]-1,2,3,4-tetrahydroquinoline, the originally proposed structure of the Hancock alkaloid (−)-galipeine, do not match those of the natural product. Herein, the preparation of the regioisomer (S)-N(1)-methyl-2-[2′-(3″-methoxy-4″-hydroxyphenyl)ethyl]-1,2,3,4-tetrahydroquinoline is reported, the 1H and 13C NMR data of which are in excellent agreement with those of (−)-galipeine. Comparison of specific rotation data enables assignment of the absolute (S)-configuration of the alkaloid, and together, these data engender the structural revision of (−)-galipeine to (S)-N(1)-methyl-2-[2′-(3″-methoxy-4″-hydroxyphenyl)ethyl]-1,2,3,4-tetrahydroquinoline.

The asymmetric syntheses of all members of the Hancock alkaloid family based upon a 2-substituted N-methyl-1,2,3,4-tetrahydroquinoline core are delineated. The conjugate addition of enantiopure lithium N-benzyl-N-(α-methyl-p-methoxybenzyl)amide to 5-(o-bromophenyl)-N-methoxy-N-methylpent-2-enamide is used to generate the requisite C-2 stereogenic center of the targets, while an intramolecular Buchwald–Hartwig coupling is used to form the 1,2,3,4-tetrahydroquinoline ring. Late-stage diversification completes construction of the C-2 side chains. Thus, (−)-cuspareine, (−)-galipinine, (−)-galipeine, and (−)-angustureine were prepared in overall yields of 30%, 28%, 15%, and 39%, respectively, in nine steps from commercially available 3-(o-bromophenyl)propanoic acid in all cases. Unambiguously corrected 1H and 13C NMR data for the originally isolated samples of (−)-cuspareine, (−)-galipinine, and (−)-angustureine are also reported, representing a valuable reference resource for these popular synthetic targets.

As one of Earth’s most carbon-dense regions, tropical forests are central to climate change mitigation efforts. Their unparalleled species richness also makes them vital for safeguarding biodiversity. However, because research has not been conducted at management-relevant scales and has often not accounted for forest disturbance, the biodiversity implications of carbon conservation strategies remain poorly understood. We investigated tropical carbon–biodiversity relationships and trade-offs along a forest-disturbance gradient, using detailed and extensive carbon and biodiversity datasets. Biodiversity was positively associated with carbon in secondary and highly disturbed primary forests. Positive carbon–biodiversity relationships dissipated at around 100 MgC ha–1, meaning that in less disturbed forests more carbon did not equal more biodiversity. Simulated carbon conservation schemes therefore failed to protect many species in the most species-rich forests. These biodiversity shortfalls were sensitive to opportunity costs and could be decreased for small carbon penalties. To ensure that the most ecologically valuable forests are protected, biodiversity needs to be incorporated into carbon conservation planning.

Epoxidations (40% aq HBF4then m-CPBA) of racemic cis-2-(N-benzylamino)cyclohex-3-en-1-ol and racemic cis-2-(N,N-dibenzylamino)cyclohex-3-en-1-ol proceed with very high levels of diastereoselectivity (>95:5 dr). The latter is in direct contrast to the epoxidation of the corresponding trans-diastereoisomer (which proceeds with essentially no selectivity), showing that the relative configuration of the substrate dramatically influences the diastereoselectivity in these instances. Meanwhile, epoxidations of enantiopure (1R,2S,αR)-2-[(N-α-methylbenzyl)amino]cyclohex-3-en-1-ol and (1S,2R,αR)-2-[(N-α-methylbenzyl)amino]cyclohex-3-en-1-ol [surrogates for the enantiomers of cis-2-(N-benzylamino)cyclohex-3-en-1-ol] proceed with complete diastereoselectivity (>95:5 dr) under the same conditions, showing that neither the presence of the α-methyl group nor the relative configuration of the α-methylbenzyl stereocenter have an effect upon the established level of diastereoslectivity in these cases. In contrast, epoxidations of enantiopure (1R,2S,αR)-2-[N-benzyl-N-(α-methylbenzyl)amino]cyclohex-3-en-1-ol and (1S,2R,αR)-2-[N-benzyl-N-(α-methylbenzyl)amino]cyclohex-3-en-1-ol [surrogates for the enantiomers of cis-2-(N,N-dibenzylamino)cyclohex-3-en-1-ol] proceed with lower diastereoselectivity (∼70:30 dr). Thus, the presence of the α-methyl group has a detrimental effect on the established level of diastereoselectivity in these cases (although again the relative configuration of the α-methylbenzyl stereocenter is unimportant). The diastereoselective epoxidation pathway is used to enable the asymmetric syntheses of six hitherto unknown, enantiopure dihydroconduramines (+)-C-2, (−)-C-2, (+)-D-2, (+)-E-2, (+)-F-2, and (−)-F-2 (>99% ee in each case).

Understanding the pathways guiding the development of definitive hematopoiesis with lymphoid potential is essential for advancing human pluripotent stem cell (hPSC) technologies for the treatment of blood diseases and immunotherapies. In the embryo, lymphoid progenitors and hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries but not veins. Here, we show that activation of the arterial program through ETS1 overexpression or by modulating MAPK/ERK signaling pathways at the mesodermal stage of development dramatically enhanced the formation of arterial-type HE expressing DLL4 and CXCR4. Blood cells generated from arterial HE were more than 100-fold enriched in T cell precursor frequency and possessed the capacity to produce B lymphocytes and red blood cells expressing high levels of BCL11a and β-globin. Together, these findings provide an innovative strategy to aid in the generation of definitive lymphomyeloid progenitors and lymphoid cells from hPSCs for immunotherapy through enhancing arterial programming of HE.

The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (∼6–8 weeks), and cost-effective.

In einer Zeit, in der sich die internationalen Beziehungen in eine Richtung entwickeln, die stärker von strategischer Konfrontation gekennzeichnet ist, nimmt Abschreckung wieder einen zentralen Stellenwert ein. Der Artikel gibt einen Überblick über die Kernfragen westlicher Abschreckungspolitik während des Kalten Krieges und in der heutigen Zeit. Während sich die Umstände erheblich geändert haben, bleibt auch heute das zentrale Anliegen von Abschreckung dasselbe wie im Kalten Krieg: Wie hält man angesichts von Gegnern, die ihrerseits mit verheerenden Atomangriffen Vergeltungsschläge gegen die Vereinigten Staaten ausführen können, die Glaubwürdigkeit der US-amerikanischen Drohung aufrecht, auch zur Verteidigung von Alliierten Atomwaffen einzusetzen? Es besteht kaum ein Zweifel an der Glaubwürdigkeit der Drohung der Vereinigten Staaten oder anderer Atommächte, im Fall eines Atomangriffs auf ihre eigenen Staatsgebiete Vergeltung zu üben, sofern die Vergeltungskapazitäten den Erstangriff überstehen. Das Problem liegt in der Glaubwürdigkeit der erweiterten US-amerikanischen Abschreckung.

Against the backdrop of an international system becoming more confrontational in nature, the subject of deterrence is back again. This article provides an overview of the nature of the deterrence problem during the Cold War period and today. While the broader circumstances have changed markedly, today, the central issue of deterrence remains the same as in the Cold War: how to maintain the credibility of the American threat to employ nuclear weapons in the defense of allies in the face of adversaries that can retaliate with devastating nuclear attacks against the US itself. There is little doubt about the threat of the US or other nuclear powers to retaliate in the event of a nuclear attack against their own homelands, so long as those retaliatory forces can survive the initial attack. The problem is the credibility of US extended deterrence.

Cell type‐specific investigations commonly use gene reporters or single‐cell analytical techniques. However, reporter line development is arduous and generally limited to a single gene of interest, while single‐cell RNA (scRNA)‐sequencing (seq) frequently yields equivocal results that preclude definitive cell identification. To examine gene expression profiles of multiple retinal cell types derived from human pluripotent stem cells (hPSCs), we performed scRNA‐seq on optic vesicle (OV)‐like structures cultured under cGMP‐compatible conditions. However, efforts to apply traditional scRNA‐seq analytical methods based on unbiased algorithms were unrevealing. Therefore, we developed a simple, versatile, and universally applicable approach that generates gene expression data akin to those obtained from reporter lines. This method ranks single cells by expression level of a bait gene and searches the transcriptome for genes whose cell‐to‐cell rank order expression most closely matches that of the bait. Moreover, multiple bait genes can be combined to refine datasets. Using this approach, we provide further evidence for the authenticity of hPSC‐derived retinal cell types. StemCells2018;36:313–324 Schematic of a novel single cell RNA‐seq analysis method using Spearman's rank correlation coefficient analysis (SRCCA). By selecting “bait” genes indicative of individual cell types, subsequent application of SRCCA allows rapid, in‐depth examination of stem cell‐derived retinal cell progeny and gene profiling of individual retinal cell types. This simple and versatile analytical method is applicable to any complex culture or tissue.

This review covers further applications of the conjugate addition of enantiomerically pure lithium amides as chiral ammonia equivalents in asymmetric synthesis and provides an update since our last review of this area, which was published in 2012.

The asymmetric syntheses of the N-terminal syn-α-hydroxy-β-amino acid components of microginins 612, 646 and 680, and the corresponding C(2)-epimeric anti-α-hydroxy-β-amino acids, are reported using diastereoselective aminohydroxylation of the requisite (E)-α,β-unsaturated esters as the key step.

Pluripotent stem (PS)-cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous; therefore, clinical translation requires the development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment. We identify CD54, integrin α9β1, and Syndecan2 (SDC2) as surface markers on PAX7-induced myogenic progenitors. We show that these markers allow for the isolation of myogenic progenitors using both fluorescent- and CGMP-compatible magnetic-based sorting technologies and that CD54+α9β1+SDC2+ cells contribute to long-term muscle regeneration in vivo. These findings represent a critical step toward enabling the translation of PS-cell-based therapies for muscle diseases.

Elucidating the pathways that lead to vasculogenic cells, and being able to identify their progenitors and lineage-restricted cells, is critical to the establishment of human pluripotent stem cell (hPSC) models for vascular diseases and development of vascular therapies. Here, we find that mesoderm-derived pericytes (PCs) and smooth muscle cells (SMCs) originate from a clonal mesenchymal progenitor mesenchymoangioblast (MB). In clonogenic cultures, MBs differentiate into primitive PDGFRβ+CD271+CD73−mesenchymal progenitors, which give rise to proliferative PCs, SMCs, and mesenchymal stem/stromal cells. MB-derived PCs can be further specified to CD274+capillary and DLK1+arteriolar PCs with a proinflammatory and contractile phenotype, respectively. SMC maturation was induced using a MEK inhibitor. Establishing the vasculogenic lineage tree, along with identification of stage- and lineage-specific markers, provides a platform for interrogating the molecular mechanisms that regulate vasculogenic cell specification and diversification and manufacturing well-defined mural cell populations for vascular engineering and cellular therapies from hPSCs.

ABSTRACTThe level of anthocyanins in plants vary widely among cultivars, developmental stages and environmental stimuli. Previous studies have reported that the expression of various MYBs regulate anthocyanin pigmentation during growth and development. Here we examine the activity of 3 novel R2R3-MYB transcription factor (TF) genes, PamMybA.1, PamMybA.3and PamMybA.5from Prunus americana. The anthocyanin accumulation patterns mediated by CaMV double35Spromoter (db35Sp) controlled expression of the TFs in transgenic tobacco were compared with citrus-MoroMybA, Arabidopsis-AtMybA1and grapevine-VvMybA1transgenics during their entire growth cycles. The db35Sp-PamMybA.1and db35Sp-PamMybA.5 constructs induced high levels of anthocyanin accumulation in both transformed tobacco calli and the regenerated plants. The red/purple color pigmentation induced in the PamMybA.1and PamMybA.5 lines was not uniformly distributed, but appeared as patches in the leaves, whereas the flowers showed intense uniform pigmentation similar to the VvMybA1expressing lines. MoroMybAand AtMybA1showed more uniform pink coloration in both vegetative and reproductive tissues. Plant morphology, anthocyanin content, seed viability, and transgene inheritance were examined for the PamMybA.5transgenic plants and compared with the controls. We conclude that these TFs alone are sufficient for activating anthocyanin production in plants and may be used as visible reporter genes for plant transformation. Evaluating these TFs in a heterologous crop species such as citrus further validated that these genes can be useful for the metabolic engineering of anthocyanin production and cultivar enhancement.