Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in Euphorbia peplus.

Most macro- and polycyclic Euphorbiaceae diterpenoids derive from the common C20 precursor casbene. While the biosynthetic pathway from casbene to the lathyrane jolkinol C is characterized, pathways to other more complex classes of bioactive diterpenoids remain to be elucidated. A metabolomics-guided transcriptomic approach and a genomics approach that led to the discovery of two casbene-derived diterpenoid gene clusters yielded a total of 68 candidate genes that were transiently expressed in Nicotiana benthamiana for activity toward jolkinol C and other lathyranes. We report two short-chain dehydrogenases/reductases (SDRs), identified by RNA sequencing to be highly expressed in Euphorbia peplus latex. One of these, EpSDR-5, is a C3-ketoreductase, converting jolkinol C to the lathyrane jolkinol E. Gene function of EpSDR-5 was further confirmed by heterologous expression in Saccharomyces cerevisiae. To investigate the in vivo role of EpSDR-5, we established virus-induced gene silencing (VIGS) in E. peplus, resulting in a significant reduction in jatrophanes and a corresponding increase in ingenanes. VIGS of Casbene Synthase results in a major reduction in both jatrophanes and ingenanes, the two most abundant classes of E. peplus diterpenoids. VIGS of CYP71D365 had a similar effect, consistent with the previously determined role of this gene in the pathway to jolkinol C. These results point to jolkinol C being a branch point intermediate in the pathways to ingenanes and jatrophanes with EpSDR-5 responsible for the first step from jolkinol C to jatrophane production.

Configurations of mother-child and father-child attachment relationships as predictors of child language competence: An individual participant data meta-analysis.

An individual participant data meta-analysis was conducted to test pre-registered hypotheses about how the configuration of attachment relationships to mothers and fathers predicts children's language competence. Data from seven studies (published between 1985 and 2014) including 719 children (Mage : 19.84 months; 51% female; 87% White) were included in the linear mixed effects analyses. Mean language competence scores exceeded the population average across children with different attachment configurations. Children with two secure attachment relationships had higher language competence scores compared to those with one or no secure attachment relationships (d = .26). Children with two organized attachment relationships had higher language competence scores compared to those with one organized attachment relationship (d = .23), and this difference was observed in older versus younger children in exploratory analyses. Mother-child and father-child attachment quality did not differentially predict language competence, supporting the comparable importance of attachment to both parents in predicting developmental outcomes.

A unified energy-optimality criterion predicts human navigation paths and speeds.

Navigating our physical environment requires changing directions and turning. Despite its ecological importance, we do not have a unified theoretical account of non-straight-line human movement. Here, we present a unified optimality criterion that predicts disparate non-straight-line walking phenomena, with straight-line walking as a special case. We first characterized the metabolic cost of turning, deriving the cost landscape as a function of turning radius and rate. We then generalized this cost landscape to arbitrarily complex trajectories, allowing the velocity direction to deviate from body orientation (holonomic walking). We used this generalized optimality criterion to mathematically predict movement patterns in multiple contexts of varying complexity: walking on prescribed paths, turning in place, navigating an angled corridor, navigating freely with end-point constraints, walking through doors, and navigating around obstacles. In these tasks, humans moved at speeds and paths predicted by our optimality criterion, slowing down to turn and never using sharp turns. We show that the shortest path between two points is, counterintuitively, often not energy-optimal, and, indeed, humans do not use the shortest path in such cases. Thus, we have obtained a unified theoretical account that predicts human walking paths and speeds in diverse contexts. Our model focuses on walking in healthy adults; future work could generalize this model to other human populations, other animals, and other locomotor tasks.

Structure and the Anticancer Activity of Vitamin D Receptor Agonists.

Vitamin D is a group of seco-steroidal fat-soluble compounds. The two basic forms, vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), do not have biological activity. They are converted in the body by a two-step enzymatic hydroxylation into biologically active forms, 1α,25-dihydroxyvitamin D2 [ercalcitriol, 1,25(OH)2D2] and 1α,25-dihydroxyvitamin D3 [calcitriol, 1,25(OH)2D3], which act as classical steroid hormones. 1,25(OH)2D3 exerts most of its physiological functions by binding to the nuclear vitamin D receptor (VDR), which is present in most body tissues to provide support to a broad range of physiological processes. Vitamin D-liganded VDR controls the expression of many genes. High levels of 1,25(OH)2D3 cause an increase in calcium in the blood, which can lead to harmful hypercalcemia. Several analogs of 1,25(OH)2D3 and 1,25(OH)2D2 have been designed and synthesized with the aim of developing compounds that have a specific therapeutic function, for example, with potent anticancer activity and a reduced toxic calcemic effect. Particular structural modifications to vitamin D analogs have led to increased anticancer activity and reduced calcemic action with the prospect of extending work to provide future innovative therapies.

Molecular Interactions of Selective Agonists and Antagonists with the Retinoic Acid Receptor γ.

All-trans retinoic acid (ATRA), the major active metabolite of all-trans retinol (vitamin A), is a key hormonal signaling molecule. In the adult organism, ATRA has a widespread influence on processes that are crucial to the growth and differentiation of cells and, in turn, the acquisition of mature cell functions. Therefore, there is considerable potential in the use of retinoids to treat diseases. ATRA binds to the retinoic acid receptors (RAR) which, as activated by ATRA, selectively regulate gene expression. There are three main RAR isoforms, RARα, RARß, and RARγ. They each have a distinct role, for example, RARα and RARγ regulate myeloid progenitor cell differentiation and hematopoietic stem cell maintenance, respectively. Hence, targeting an isoform is crucial to developing retinoid-based therapeutics. In principle, this is exemplified when ATRA is used to treat acute promyelocytic leukemia (PML) and target RARα within PML-RARα oncogenic fusion protein. ATRA with arsenic trioxide has provided a cure for the once highly fatal leukemia. Recent in vitro and in vivo studies of RARγ have revealed the potential use of agonists and antagonists to treat diseases as diverse as cancer, heterotopic ossification, psoriasis, and acne. During the final drug development there may be a need to design newer compounds with added modifications to improve solubility, pharmacokinetics, or potency. At the same time, it is important to retain isotype specificity and activity. Examination of the molecular interactions between RARγ agonists and the ligand binding domain of RARγ has revealed aspects to ligand binding that are crucial to RARγ selectivity and compound activity and key to designing newer compounds.

Life Stress and Unmarried, Black Fathers' Attitudes Toward Attachment: The Moderating Role of Shift-and-Persist.

Unmarried, Black fathers' positive engagement contributes to children's health and development beginning in early infancy. For many men, preparations for parenthood begin before birth as expectant fathers formulate parenting attitudes that can promote secure infant-father attachment relationships. This study examined aspects of life stress as predictors of prenatal attitudes toward attachment -- the extent to which expectant fathers endorsed promoting attachment security in their infants. Further, we considered whether shift-and-persist cognitive strategies -- a psychological resilience factor focused on shifting to positive focus and future-orientation -- moderated these associations. A sample of 121 unmarried, Black men expecting the birth of a child were recruited during the 2nd or 3rd trimester of their partner's pregnancy. Expectant fathers reported on childhood trauma, recent negative life experiences, and depressive symptomology. Fathers also completed a survey assessment of shift-and-persist strategies, as well as a newly developed scale assessing attitudes toward attachment. Depressive symptoms and negative life events were directly, positively related to attitudes toward attachment. The association between positive attitudes toward attachment and both negative life events and depressive symptomology was moderated by fathers' ability to shift-and-persist. Specifically, aspects of life stress were generally unrelated to attitudes toward attachment when shift-and-persist was low, but related to more positive attitudes toward attachment when shift-and-persist was high. Preliminary findings point to the potential steeling effects of shift-and-persist strategies for expectant fathers facing moderate levels of life stress.

People adapt a consistent center-of-mass trajectory in a novel force field.

During human walking the whole body center-of-mass (COM) trajectory may be a control objective, a goal the central nervous system uses to plan and regulate movement. Our previous observation, that after practice walking in a novel laterally directed force field people adapt a COM trajectory similar to their normal trajectory, supports this idea. However, our prior work only presented data demonstrating changes in COM trajectory in response to a single force field. To evaluate whether this phenomena is robust, in the present study we present new data demonstrating that people adapt their COM trajectory in a similar manner when the direction of the external force field is changed resulting in drastically different lower limb joint dynamics. Specifically, we applied a continuous, left-directed force field (in the previous experiment the force field was applied to the right) to the COM as participants performed repeated trials of a discrete walking task. We again hypothesized that with practice walking in the force field people would adapt a COM trajectory that was similar to their baseline performance and exhibit aftereffects, deviation of their COM trajectory in the opposite direction of force field, when the field was unexpectedly removed. These hypotheses were supported and suggest that participants formed an internal model to control their COM trajectory. Collectively these findings demonstrate that people adapt their gait patterns to anticipate consistent aspects of the external environment. These findings suggest that this response is robust to force fields applied in multiple directions that may require substantially different neural control.NEW & NOTEWORTHY With experience people adapted a predictive internal model to control their whole body center-of-mass walking trajectory that anticipated the disruptive laterally directed forces of a novel and consistent external environment. Collectively these findings demonstrate that adaptation of gait to anticipate consistent aspects of the external environment is a response that is robust to force fields in multiple directions that require substantially different lower limb dynamics and neural control.

Chemical Descriptors for a Large-Scale Study on Drop-Weight Impact Sensitivity of High Explosives.

The drop-weight impact test is an experiment that has been used for nearly 80 years to evaluate handling sensitivity of high explosives. Although the results of this test are known to have large statistical uncertainties, it is one of the most common tests due to its accessibility and modest material requirements. In this paper, we compile a large data set of drop-weight impact sensitivity test results (mainly performed at Los Alamos National Laboratory), along with a compendium of molecular and chemical descriptors for the explosives under test. These data consist of over 500 unique explosives, over 1000 repeat tests, and over 100 descriptors, for a total of about 1500 observations. We use random forest methods to estimate a model of explosive handling sensitivity as a function of chemical and molecular properties of the explosives under test. Our model predicts well across a wide range of explosive types, spanning a broad range of explosive performance and sensitivity. We find that properties related to explosive performance, such as heat of explosion, oxygen balance, and functional group, are highly predictive of explosive handling sensitivity. Yet, models that omit many of these properties still perform well. Our results suggest that there is not one or even several factors that explain explosive handling sensitivity, but that there are many complex, interrelated effects at play.

Targeting the Retinoic Acid Pathway to Eradicate Cancer Stem Cells.

All-trans retinoic acid is a morphogen during embryogenesis and a teratogen. Cancer is an error of development, and the retinoic acid receptors (RAR) for all-trans retinoic acid play a role in cancer. Expression of the cytosolic aldehyde dehydrogenases, which mediate the last step to the synthesis of all-trans retinoic acid, is deregulated in various human cancers. Inhibiting these enzymes using a variety of agents reduced the proliferation of lung cancer cells, reduced the proliferation and induced apoptosis of ovarian, prostate, squamous, and uterine cancer cells, and sensitised breast, colorectal and ovarian cancer cells to chemotherapeutic agents. RARγ is an oncogene within some cases of AML, cholangiocarcinoma, colorectal cancer, clear cell renal cell carcinoma, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, prostate cancer, and ovarian cancer. Pan-RAR and RARγ antagonist inhibition of the action of RARγ led to necroptosis of human prostate and pediatric brain tumour cancer stem cells. Treatment of hepatocellular carcinoma cells with the flavenoid acacetin, which interferes with the action of RARγ, decreased cell growth and induced apoptosis. Targeting the retinoic acid pathway is promising regarding the development of new drugs to eradicate cancer stem cells.

Deregulation of All-Trans Retinoic Acid Signaling and Development in Cancer.

Cancer stem cells are the root cause of cancer, which, in essence, is a developmental disorder. All-trans retinoic acid (ATRA) signaling via ligand-activation of the retinoic acid receptors (RARs) plays a crucial role in tissue patterning and development during mammalian embryogenesis. In adults, active RARγ maintains the pool of hematopoietic stem cells, whereas active RARα drives myeloid cell differentiation. Various findings have revealed that ATRA signaling is deregulated in many cancers. The enzymes for ATRA synthesis are downregulated in colorectal, gastric, lung, and oropharyngeal cancers. ATRA levels within breast, ovarian, pancreatic, prostate, and renal cancer cells were lower than within their normal counterpart cells. The importance is that 0.24 nM ATRA activates RARγ (for stem cell stemness), whereas 100 times more is required to activate RARα (for differentiation). Moreover, RARγ is an oncogene regarding overexpression within colorectal, cholangiocarcinoma, hepatocellular, ovarian, pancreatic, and renal cancer cells. The microRNA (miR) 30a-5p downregulates expression of RARγ, and miR-30a/miR-30a-5p is a tumor suppressor for breast, colorectal, gastric, hepatocellular, lung, oropharyngeal, ovarian, pancreatic, prostate, and renal cancer. These complementary findings support the view that perturbations to ATRA signaling play a role in driving the abnormal behavior of cancer stem cells. Targeting ATRA synthesis and RARγ has provided promising approaches to eliminating cancer stem cells because such agents have been shown to drive cell death.

Novel Strategies in the Development of New Therapies, Drug Substances, and Drug Carriers Volume II.

The highly successful previous Volume 1 [...].

Editorial: Special Issue "Stem Cell Biology and Cancer".

Cancer stem cells (CSCs) are now well-established as key players in tumor initiation, progression, and therapy resistance [...].

Description of collaborative, fee-for-service, office-based, pharmacist-directed medical cannabis therapy management service for patients with chronic pain.

BACKGROUND: Treatment with medical cannabis (MC) in the United States tends to be patient-driven in nature despite evidence that suggests that patients have remarkably poor knowledge on the medical use of this treatment modality. OBJECTIVE: To develop and pilot a collaborative, fee-for-service (FFS), office-based, pharmacist-directed MC therapy management (MCTM) service for patients suffering chronic pain. PRACTICE DESCRIPTION: A collaborative, FFS, office-based, pharmacist-directed MCTM service where patients are seen after a physician deems them suitable for treatment with MC. The pharmacist designs the initial treatment regimen by selecting a formulation, dose, route, and frequency of administration and then manages ongoing therapy by making regimen changes based on the patient's response, adverse effects, and financial concerns. PRACTICE INNOVATION: The creation of a specialized service where a registered MC pharmacist is positioned in a collaborating provider's office and sees patients face-to-face for the provision of MCTM services. EVALUATION METHODS: Patient retention, revenue generated, and ability to replicate the service were evaluated. Patient satisfaction was assessed by collecting subjective feedback on the service. RESULTS: The pilot site that developed the service has seen 133 patients from 2016 to 2021 and has retained 89% of patients after 5 years of quarterly appointments. Patients appear willing to pay out of pocket for the service, and the revenue generated covers the pharmacist's and collaborating physician's time as well as additional overhead. The service has been replicated at 2 additional sites, and patient feedback has been positive. CONCLUSIONS: MCTM is another useful pharmacist service that patients are willing to pay for. MCTM services decrease the collaborating provider's workload while still allowing them to offer their patients personalized treatment with MC. In our experience, the service retains patients, generates enough revenue to cover costs, can be replicated, and is well received by patients.

Antagonizing RARγ Drives Necroptosis of Cancer Stem Cells.

There is a need for agents that eliminate cancer stem cells, which sustain cancer and are also largely responsible for disease relapse and metastasis. Conventional chemotherapeutics and radiotherapy are often highly effective against the bulk of cancer cells, which are proliferating, but spare cancer stem cells. Therapeutics that target cancer stem cells may also provide a bona fide cure for cancer. There are two rationales for targeting the retinoic acid receptor (RAR)γ. First, RARγ is expressed selectively within primitive cells. Second, RARγ is a putative oncogene for a number of human cancers, including cases of acute myeloid leukemia, cholangiocarcinoma, and colorectal, renal and hepatocellular carcinomas. Prostate cancer cells depend on active RARγ for their survival. Antagonizing all RARs caused necroptosis of prostate and breast cancer stem cell-like cells, and the cancer stem cells that gave rise to neurospheres from pediatric patients' primitive neuroectodermal tumors and an astrocytoma. As tested for prostate cancer, antagonizing RARγ was sufficient to drive necroptosis. Achieving cancer-selectively is a longstanding paradigm for developing new treatments. The normal prostate epithelium was less sensitive to the RARγ antagonist and pan-RAR antagonist than prostate cancer cells, and fibroblasts and blood mononuclear cells were insensitive. The RARγ antagonist and pan-RAR antagonist are promising new cancer therapeutics.

The Social Norm of Hematopoietic Stem Cells and Dysregulation in Leukemia.

The hematopoietic cell system is a complex ecosystem that meets the steady-state and emergency needs of the production of the mature blood cell types. Steady-state hematopoiesis replaces worn out cells, and the hematopoietic system is highly adaptive to needs during, for example, an infection or bleeding. Hematopoiesis is highly integrated and the cell hierarchy behaves in a highly social manner. The social tailoring of hematopoietic stem cells to needs includes the generation of cells that are biased towards a cell lineage; these cells remain versatile and can still adopt a different pathway having made a lineage "choice", and some cytokines instruct the lineage fate of hematopoietic stem and progenitor cells. Leukemia stem cells, which may well often arise from the transformation of a hematopoietic stem cell, sustain the hierarchy of cells for leukemia. Unlike hematopoietic stem cells, the offspring of leukemia stem cells belongs to just one cell lineage. The human leukemias are classified by virtue of their differentiating or partially differentiating cells belonging to just one cell lineage. Some oncogenes set the fate of leukemia stem cells to a single lineage. Therefore, lineage restriction may be largely an attribute whereby leukemia stem cells escape from the normal cellular society. Additional antisocial behaviors are that leukemia cells destroy and alter bone marrow stromal niches, and they can create their own niches.

Oncogenes and the Origins of Leukemias.

Self-maintaining hematopoietic stem cells are a cell population that is primarily 'at risk' to malignant transformation, and the cell-of-origin for some leukemias. Tissue-specific stem cells replenish the different types of functional cells within a particular tissue to meet the demands of an organism. For hematopoietic stem cells, this flexibility is important to satisfy the changing requirements for a certain type of immune cell, when needed. From studies of the natural history of childhood acute lymphoblastic leukemia, an initial oncogenic and prenatal insult gives rise to a preleukemic clone. At least a second genomic insult is needed that gives rise to a leukemia stem cell: this cell generates a hierarchy of leukemia cells. For some leukemias, there is evidence to support the concept that one of the genomic insults leads to dysregulation of the tissue homeostatic role of hematopoietic stem cells so that the hierarchy of differentiating leukemia cells belongs to just one cell lineage. Restricting the expression of particular oncogenes in transgenic mice to hematopoietic stem and progenitor cells led to different human-like lineage-restricted leukemias. Lineage restriction is seen for human leukemias by virtue of their sub-grouping with regard to a phenotypic relationship to just one cell lineage.

Hematopoietic and Chronic Myeloid Leukemia Stem Cells: Multi-Stability versus Lineage Restriction.

There is compelling evidence to support the view that the cell-of-origin for chronic myeloid leukemia is a hematopoietic stem cell. Unlike normal hematopoietic stem cells, the progeny of the leukemia stem cells are predominantly neutrophils during the disease chronic phase and there is a mild anemia. The hallmark oncogene for chronic myeloid leukemia is the BCR-ABLp210 fusion gene. Various studies have excluded a role for BCR-ABLp210 expression in maintaining the population of leukemia stem cells. Studies of BCR-ABLp210 expression in embryonal stem cells that were differentiated into hematopoietic stem cells and of the expression in transgenic mice have revealed that BCR-ABLp210 is able to veer hematopoietic stem and progenitor cells towards a myeloid fate. For the transgenic mice, global changes to the epigenetic landscape were observed. In chronic myeloid leukemia, the ability of the leukemia stem cells to choose from the many fates that are available to normal hematopoietic stem cells appears to be deregulated by BCR-ABLp210 and changes to the epigenome are also important. Even so, we still do not have a precise picture as to why neutrophils are abundantly produced in chronic myeloid leukemia.

Novel Strategies in the Development of New Therapies, Drug Substances, and Drug Carriers Volume I.

At present, there is a strong need for new therapies that are effective and safe for widespread diseases [...].

In Silico Prediction of the Metabolic Resistance of Vitamin D Analogs against CYP3A4 Metabolizing Enzyme.

The microsomal cytochrome P450 3A4 (CYP3A4) and mitochondrial cytochrome P450 24A1 (CYP24A1) hydroxylating enzymes both metabolize vitamin D and its analogs. The three-dimensional (3D) structure of the full-length native human CYP3A4 has been solved, but the respective structure of the main vitamin D hydroxylating CYP24A1 enzyme is unknown. The structures of recombinant CYP24A1 enzymes have been solved; however, from studies of the vitamin D receptor, the use of a truncated protein for docking studies of ligands led to incorrect results. As the structure of the native CYP3A4 protein is known, we performed rigid docking supported by molecular dynamic simulation using CYP3A4 to predict the metabolic conversion of analogs of 1,25-dihydroxyvitamin D2 (1,25D2). This is highly important to the design of novel vitamin D-based drug candidates of reasonable metabolic stability as CYP3A4 metabolizes ca. 50% of the drug substances. The use of the 3D structure data of human CYP3A4 has allowed us to explain the substantial differences in the metabolic conversion of the side-chain geometric analogs of 1,25D2. The calculated free enthalpy of the binding of an analog of 1,25D2 to CYP3A4 agreed with the experimentally observed conversion of the analog by CYP24A1. The metabolic conversion of an analog of 1,25D2 to the main vitamin D hydroxylating enzyme CYP24A1, of unknown 3D structure, can be explained by the binding strength of the analog to the known 3D structure of the CYP3A4 enzyme.

Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.

To engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed to increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by up-regulation of key genes of the non-mevalonate (MEP) pathway sourced from Arabidopsis thaliana. We used transient expression to evaluate combinations of the eight MEP pathway genes plus GGPP synthase and a Jatropha curcas casbene synthase (JcCAS) to identify an optimal combination for production of casbene from GGPP. AtDXS and AtHDR together with AtGGPPS and JcCAS gave a 410% increase in casbene production compared to transient expression of JcCAS alone. This combination was cloned into a single construct using the MoClo toolkit, and stably integrated into the N. benthamiana genome. We also created multigene constructs for stable transformation of two J. curcas cytochrome P450 genes, JcCYP726A20 and JcCYP71D495 that produce the more complex diterpenoid jolkinol C from casbene when expressed transiently with JcCAS in N. benthamiana. Stable transformation of JcCYP726A20, JcCYP71D495 and JcCAS did not produce any detectable jolkinol C until these genes were co-transformed with the optimal set of precursor-pathway genes. One such stable homozygous line was used to evaluate by transient expression the involvement of an 'alkenal reductase'-like family of four genes in the further conversion of jolkinol C, leading to the demonstration that one of these performs reduction of the 12,13-double bond in jolkinol C. This work highlights the need to optimize precursor supply for production of complex diterpenoids in stable transformants and the value of such lines for novel gene discovery.